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Casey, Patrick John

Overview:

Research in this laboratory focuses on the area of transmembrane signaling mediated through guanine nucleotide-binding regulatory proteins (G proteins). Many of these signaling pathways are involved in control of cell growth; this property is highlighted by discoveries over the past decade that mutations in G proteins can lead to cell transformation. There are two major areas of research ongoing in the lab. The first is the covalent modification of G proteins by isoprenoid lipids and the role this modification, termed protein prenylation, plays in the membrane targeting and function of G proteins. Prenylation plays a crucial role oncogenic transformation by one class of G proteins, the Ras proteins. The enzymes that catalyze these modifications have been isolated and cloned and are being used to develop in vitro systems to both define the enzymes’ structures and molecular mechanisms and elucidate the role of prenylation in G protein function. The importance of this work is highlighted by the fact that several of these enzymes, most notably protein farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase-1), a prenyl protein-specific protease termed Rce1, and a specific methyltransferase termed Icmt have become major targets in the development of anti-cancer therapeutics.

The second general area of research involves identification of the signaling pathways controlled by specific types of G proteins. One such protein, termed Gz, exhibits very limited tissue distribution that includes primarily neuronal and neuroendocrine cells. Gz exhibits several biochemical properties that suggest that this protein controls a unique signaling pathway, and we have recently linked Gz to control of important aspects of pancreatic beta-cell function. We have also have a program to identify molecular targets of G12 proteins. We have linked the G12 proteins to cell-surface cadherins and to activation of the GTPase Rho, and have obtained evidence that activation of G12 impacts on the cellular processes of of adhesion and migration and that aberrant activation of G12 contributes to metastatic progression of breast and prostate cancer.

Positions:

James B. Duke Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Professor of Biochemistry

Biochemistry
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Director of the Center for Chemical Biology

Pharmacology & Cancer Biology
School of Medicine

Education:

Ph.D. 1987

Ph.D. — Brandeis University

Grants:

G(alpha)Z signaling in insulin secretion and glucose tolerance

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
April 01, 2008
End Date
August 07, 2010

Nonclassical signaling of the androgen receptor polyproline domain

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
July 01, 2009
End Date
June 30, 2010

Regulation of G-Protein Signaling

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 25, 1998
End Date
June 30, 2010

G Protein Involvement in Oncogenesis and Metastasis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2004
End Date
July 31, 2009

Developing inhibitors of RalA function for the treatment of pancreatic cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Significant Contributor
Start Date
March 12, 2007
End Date
February 28, 2009

Biomarker Studies for Novel Anti-Cancer Agents

Administered By
Medicine, Medical Oncology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
May 28, 2003
End Date
February 29, 2008

Molecular Mechanisms of Protein Prenylation

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 1998
End Date
March 31, 2007

Structure and Function of ICMT Methyltransferase

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2004
End Date
January 31, 2007

Role of G(alpha)Z in Pancreatic Islet Beta-cell Biology

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2004
End Date
March 31, 2006

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1993
End Date
February 28, 2005

Interaction of Galpha12 with cadherin in oncogenesis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 09, 2001
End Date
February 28, 2004

Regulation Of Cyclic Gmp Phosphodiesterase By Gz

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 20, 1997
End Date
July 31, 2001

Regulation Of G Protein Mediated Signaling

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1997
End Date
December 31, 1999

Biology Of The Testis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 1997
End Date
February 28, 1999

Mechanism Of Pkc Regulation

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 1995
End Date
September 30, 1995

Molecular Mechanisms Of Protein Prenylation

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
August 01, 1991
End Date
January 31, 1995
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Awards:

AAAS Fellows. American Association for the Advancement of Science, The.

Type
National
Awarded By
American Association for the Advancement of Science, The
Date
January 01, 2012

Publications:

Isoprenylcysteine carboxylmethyltransferase is critical for malignant transformation and tumor maintenance by all RAS isoforms.

Despite extensive effort, there has been limited progress in the development of direct RAS inhibitors. Targeting isoprenylcysteine carboxylmethyltransferase (ICMT), a unique enzyme of RAS post-translational modification, represents a promising strategy to inhibit RAS function. However, there lacks direct genetic evidence on the role of ICMT in RAS-driven human cancer initiation and maintenance. Using CRISPR/Cas9 genome editing, we have created Icmt loss-of-function isogenic cell lines for both RAS-transformed human mammary epithelial cells (HME1) and human cancer cell lines MiaPaca-2 and MDA-MB-231 containing naturally occurring mutant KRAS. In both in vitro and in vivo tumorigenesis studies, Icmt loss-of-function abolishes the tumor initiation ability of all major isoforms of mutant RAS in HME1 cells, and the tumor maintenance capacity of MiaPaca-2 and MDA-MB-231 cells, establishing the critical role of ICMT in RAS-driven cancers.Oncogene advance online publication, 13 February 2017; doi:10.1038/onc.2016.508.

Authors
Lau, HY; Tang, J; Casey, PJ; Wang, M
MLA Citation
Lau, HY, Tang, J, Casey, PJ, and Wang, M. "Isoprenylcysteine carboxylmethyltransferase is critical for malignant transformation and tumor maintenance by all RAS isoforms." Oncogene (February 13, 2017).
PMID
28192404
Source
epmc
Published In
Oncogene: Including Oncogene Reviews
Publish Date
2017
DOI
10.1038/onc.2016.508

Inhibition of Isoprenylcysteine carboxylmethyltransferase induces cell cycle arrest and apoptosis through p21 and p21-regulated BNIP3 induction in pancreatic cancer.

Pancreatic cancer remains one of the most difficult to treat human cancers despite recent advances in targeted therapy. Inhibition of Isoprenylcysteine carboxylmethyltransferase (ICMT), an enzyme that post-translationally modifies a group of proteins including several small GTPases, suppresses proliferation of some human cancer cells. However, the efficacy of ICMT inhibition on human pancreatic cancer has not been evaluated. In this study we have evaluated a panel of human pancreatic cancer cell lines and identified those that are sensitive to ICMT inhibition. In these cells, ICMT suppression inhibited proliferation and induced apoptosis. This responsiveness to ICMT inhibition was confirmed in in vivo xenograft tumor mouse models using both a small molecule inhibitor and shRNA targeting Icmt. Mechanistically, we found that that, in sensitive pancreatic cancer cells, ICMT inhibition induced mitochondrial respiratory deficiency and cellular energy depletion, leading to significant up-regulation of p21. Further, we characterized the role of p21 as a regulator and coordinator of cell signaling that responds to cell energy depletion. Apoptosis, but not autophagy, that is induced via p21-activated BNIP3 expression accounts for the efficacy of ICMT inhibition in sensitive pancreatic cancer cells in both in vitro and in vivo models. In contrast, cells resistant to ICMT inhibition-demonstrated no mitochondria dysfunction or p21 signaling changes under ICMT suppression. These findings not only identify pancreatic cancers as potential therapeutic targets for ICMT suppression, but also provide an avenue for identifying those subtypes that would be most responsive to agents targeting this critical enzyme.

Authors
Manu, KA; Chai, TF; Teh, JT; Zhu, WL; Casey, PJ; Wang, M
MLA Citation
Manu, KA, Chai, TF, Teh, JT, Zhu, WL, Casey, PJ, and Wang, M. "Inhibition of Isoprenylcysteine carboxylmethyltransferase induces cell cycle arrest and apoptosis through p21 and p21-regulated BNIP3 induction in pancreatic cancer." Molecular cancer therapeutics (February 6, 2017).
PMID
28167504
Source
epmc
Published In
Molecular cancer therapeutics
Publish Date
2017
DOI
10.1158/1535-7163.mct-16-0703

The GNA13-RhoA signaling axis suppresses expression of tumor protective Kallikreins.

Gα13 (encoded by GNA13 gene) is the alpha subunit of a heterotrimeric G-protein that mediates signaling through specific G-protein-coupled receptors (GPCRs). Increased GNA13 expression has been observed in metastatic breast cancer cells. Recently, we have shown that enhanced GNA13 signaling in MCF-10a cells, a benign breast cancer cell line increased its invasiveness. Previous studies have reported that Kallikrein-related peptidases (KLKs 1-15) are down-regulated in breast tumors and may have a tumor protective function. However, the mechanisms that lead to the down-regulation of KLK genes in breast cancer are yet to be elucidated. We found that enhanced GNA13 signaling represses KLK gene expression in breast cancer, and undertook examination of the mechanisms involved. A microarray analysis revealed down-regulation of several members of the Kallikrein-related peptidases (KLK) gene family, namely KLK5, KLK6, KLK7, KLK8 and KLK10, in MCF-10a lines with enhanced GNA13 protein expression. Using real-time PCR and promoter analysis, we identified that the mRNA expression and promoter activities of these KLKs are suppressed upon enforced expression of GNA13 in MCF-10a cells. Using Rhotekin pull-down assays, we identified that GNA13 suppressed Rho-A activation and protein levels in MCF-10a cells. Blocking Rho-A activation using C3-toxin or by inhibiting its down-stream effector, Rho-associated kinase (ROCK), reduced the above-mentioned KLK mRNAs in MCF-10A cells. Importantly, in a metastatic breast cancer cell line MDA-MB-157, knock down of GNA13 alone was sufficient to induce the expression KLK mRNAs. Taken together, our findings suggested that enhanced GNA13 signaling down-regulates KLK gene transcription. The ability of enhanced GNA13 signaling to suppress KLK gene expression appears at least in part due to the ability of enhanced GNA13 signaling to negatively impact Rho/ROCK-signaling.

Authors
Teo, CR; Casey, PJ; Rasheed, SAK
MLA Citation
Teo, CR, Casey, PJ, and Rasheed, SAK. "The GNA13-RhoA signaling axis suppresses expression of tumor protective Kallikreins." Cellular signalling 28.10 (October 2016): 1479-1488.
PMID
27424208
Source
epmc
Published In
Cellular Signalling
Volume
28
Issue
10
Publish Date
2016
Start Page
1479
End Page
1488
DOI
10.1016/j.cellsig.2016.07.001

Inhibition of isoprenylcysteine carboxylmethyltransferase augments BCR-ABL1 tyrosine kinase inhibition-induced apoptosis in chronic myeloid leukemia.

Despite the success of BCR-ABL1 tyrosine kinase inhibitors in patients with chronic myeloid leukemia (CML), resistance to tyrosine kinase inhibitors remains a therapeutic challenge. One strategy used to overcome resistance is combination of existing BCR-ABL1 tyrosine kinase inhibitors with agents that target alternative pathways. We report that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), a key enzyme in the protein prenylation pathway, with the selective inhibitor cysmethynil enhances the effect of BCR-ABL1 tyrosine kinase inhibitors in killing CML cells. Cysmethynil augments tyrosine kinase inhibitor-induced apoptosis in both BCR-ABL1 wild type and BCR-ABL1 kinase domain mutant-expressing cell lines. Importantly, the enhanced apoptosis observed with the combination of cysmethynil and imatinib is significant only in primary CML CD34+ progenitor cells, not normal cord blood progenitor cells. The combination was also selective in inhibiting colony formation in CML CD34+ cells. The enhanced apoptosis appears to be due to combination of immediate and persistent inhibition of MAPK signaling. Consistent with in vitro studies, cysmethynil and imatinib, in combination, enhance the in vivo effects of either drug used alone. We found that simultaneous inhibition of BCR-ABL1 and Icmt may represent a potential therapeutic strategy for CML.

Authors
Sun, WT; Xiang, W; Ng, BL; Asari, K; Bunte, RM; Casey, PJ; Wang, M; Chuah, C
MLA Citation
Sun, WT, Xiang, W, Ng, BL, Asari, K, Bunte, RM, Casey, PJ, Wang, M, and Chuah, C. "Inhibition of isoprenylcysteine carboxylmethyltransferase augments BCR-ABL1 tyrosine kinase inhibition-induced apoptosis in chronic myeloid leukemia." Experimental hematology 44.3 (March 2016): 189-93.e2.
PMID
26706195
Source
epmc
Published In
Experimental Hematology
Volume
44
Issue
3
Publish Date
2016
Start Page
189
End Page
93.e2
DOI
10.1016/j.exphem.2015.12.002

Protein prenylation: unique fats make their mark on biology.

The modification of eukaryotic proteins by isoprenoid lipids, which is known as prenylation, controls the localization and activity of a range of proteins that have crucial functions in biological regulation. The roles of prenylated proteins in cells are well conserved across species, underscoring the biological and evolutionary importance of this lipid modification pathway. Genetic suppression and pharmacological inhibition of the protein prenylation machinery have provided insights into several cellular processes and into the aetiology of diseases in which prenylation is involved. The functional dependence of prenylation substrates, such as RAS proteins, on this modification and the therapeutic potential of targeting the prenylation process in pathological conditions accentuate the need to fully understand this form of post-translational modification.

Authors
Wang, M; Casey, PJ
MLA Citation
Wang, M, and Casey, PJ. "Protein prenylation: unique fats make their mark on biology." Nature reviews. Molecular cell biology 17.2 (February 2016): 110-122.
PMID
26790532
Source
epmc
Published In
Nature Reviews Molecular Cell Biology
Volume
17
Issue
2
Publish Date
2016
Start Page
110
End Page
122
DOI
10.1038/nrm.2015.11

Protein Geranylgeranyltransferase Type 1 as a Target in Cancer.

The process of protein prenylation involves the covalent linkage of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoid lipds to conserved cysteine residues in the carboxyl-terminus of proteins. Protein geranylgeranyltransferase I (GGTase-I) is the enzyme that catalyzes the addition of the geranylgeranyl moiety from geranylgeranyl pyrophosphate to the target protein, which contains a Cterminal consensus sequence termed a CaaX motif. Geranylgeranylation is important to the function of a number of proteins, including the majority of Rho GTPases, G protein gamma subunits, and several other regulatory proteins. Studies over the past two decades have revealed that many of these proteins contribute to tumor development and metastasis. Blocking Rho GTPase activity through inhibition of GGTase-I in particular has been advanced as a potential strategy for disease therapy. This review will provide an overview of the CaaX prenyltransferases, the rationale for targeting GGTase-I in cancer in particular, and the current status of GGTase-I inhibitor (GGTI) development.

Authors
Ullah, N; Mansha, M; Casey, PJ
MLA Citation
Ullah, N, Mansha, M, and Casey, PJ. "Protein Geranylgeranyltransferase Type 1 as a Target in Cancer." Current cancer drug targets 16.7 (January 2016): 563-571.
PMID
26648485
Source
epmc
Published In
Current cancer drug targets
Volume
16
Issue
7
Publish Date
2016
Start Page
563
End Page
571
DOI
10.2174/1568009616666151203224603

Isoprenylcysteine carboxylmethyltransferase regulates mitochondrial respiration and cancer cell metabolism

© 2015 Macmillan Publishers Limited All rights reserved.Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last of the three-step posttranslational protein prenylation process for the so-called CaaX proteins, which includes many signaling proteins, such as most small GTPases. Despite extensive studies on Icmt and its regulation of cell functions, the mechanisms of much of the impact of Icmt on cellular functions remain unclear. Our recent studies demonstrated that suppression of Icmt results in induction of autophagy, inhibition of cell growth and inhibition of proliferation in various cancer cell types, prompting this investigation of potential metabolic regulation by Icmt. We report here the findings that Icmt inhibition reduces the function of mitochondrial oxidative phosphorylation in multiple cancer cell lines. In-depth oximetry analysis demonstrated that functions of mitochondrial complex I, II and III are subject to Icmt regulation. Consistently, Icmt inhibition decreased cellular ATP and depleted critical tricarboxylic acid cycle metabolites, leading to suppression of cell anabolism and growth, and marked autophagy. Several different approaches demonstrated that the impact of Icmt inhibition on cell proliferation and viability was largely mediated by its effect on mitochondrial respiration. This previously unappreciated function of Icmt, which can be therapeutically exploited, likely has a significant role in the impact of Icmt on tumorigenic processes.

Authors
Teh, JT; Zhu, WL; Ilkayeva, OR; Li, Y; Gooding, J; Casey, PJ; Summers, SA; Newgard, CB; Wang, M
MLA Citation
Teh, JT, Zhu, WL, Ilkayeva, OR, Li, Y, Gooding, J, Casey, PJ, Summers, SA, Newgard, CB, and Wang, M. "Isoprenylcysteine carboxylmethyltransferase regulates mitochondrial respiration and cancer cell metabolism." Oncogene 34.25 (June 20, 2015): 3296-3304.
Source
scopus
Published In
Oncogene: Including Oncogene Reviews
Volume
34
Issue
25
Publish Date
2015
Start Page
3296
End Page
3304
DOI
10.1038/onc.2014.260

Isoprenylcysteine carboxylmethyltransferase regulates mitochondrial respiration and cancer cell metabolism.

Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last of the three-step posttranslational protein prenylation process for the so-called CaaX proteins, which includes many signaling proteins, such as most small GTPases. Despite extensive studies on Icmt and its regulation of cell functions, the mechanisms of much of the impact of Icmt on cellular functions remain unclear. Our recent studies demonstrated that suppression of Icmt results in induction of autophagy, inhibition of cell growth and inhibition of proliferation in various cancer cell types, prompting this investigation of potential metabolic regulation by Icmt. We report here the findings that Icmt inhibition reduces the function of mitochondrial oxidative phosphorylation in multiple cancer cell lines. In-depth oximetry analysis demonstrated that functions of mitochondrial complex I, II and III are subject to Icmt regulation. Consistently, Icmt inhibition decreased cellular ATP and depleted critical tricarboxylic acid cycle metabolites, leading to suppression of cell anabolism and growth, and marked autophagy. Several different approaches demonstrated that the impact of Icmt inhibition on cell proliferation and viability was largely mediated by its effect on mitochondrial respiration. This previously unappreciated function of Icmt, which can be therapeutically exploited, likely has a significant role in the impact of Icmt on tumorigenic processes.

Authors
Teh, JT; Zhu, WL; Ilkayeva, OR; Li, Y; Gooding, J; Casey, PJ; Summers, SA; Newgard, CB; Wang, M
MLA Citation
Teh, JT, Zhu, WL, Ilkayeva, OR, Li, Y, Gooding, J, Casey, PJ, Summers, SA, Newgard, CB, and Wang, M. "Isoprenylcysteine carboxylmethyltransferase regulates mitochondrial respiration and cancer cell metabolism." Oncogene 34.25 (June 2015): 3296-3304.
PMID
25151967
Source
epmc
Published In
Oncogene: Including Oncogene Reviews
Volume
34
Issue
25
Publish Date
2015
Start Page
3296
End Page
3304
DOI
10.1038/onc.2014.260

MicroRNA-31 controls G protein alpha-13 (GNA13) expression and cell invasion in breast cancer cells.

Gα13 (GNA13) is the α subunit of a heterotrimeric G protein that mediates signaling through specific G protein-coupled receptors (GPCRs). Our recent study showed that control of GNA13 expression by specific microRNAs (miRNAs or miRs) is important for prostate cancer cell invasion. However, little is known about the control of GNA13 expression in breast cancers. This project was carried out to determine (i) whether enhanced GNA13 expression is important for breast cancer cell invasion, and (ii) if so, the mechanism of deregulation of GNA13 expression in breast cancers.To determine the probable miRNAs regulating GNA13, online miRNA target prediction tool Targetscan and Luciferase assays with GNA13-3'-UTR were used. Effect of miRNAs on GNA13 mRNA, protein and invasion was studied using RT-PCR, western blotting and in vitro Boyden chamber assay respectively. Cell proliferation was done using MTT assays.Overexpression of GNA13 in MCF-10a cells induced invasion, whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited invasion. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3'-UTR-reporter activity. Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3'-UTR reporter activity. Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels.These data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression.

Authors
Rasheed, SAK; Teo, CR; Beillard, EJ; Voorhoeve, PM; Zhou, W; Ghosh, S; Casey, PJ
MLA Citation
Rasheed, SAK, Teo, CR, Beillard, EJ, Voorhoeve, PM, Zhou, W, Ghosh, S, and Casey, PJ. "MicroRNA-31 controls G protein alpha-13 (GNA13) expression and cell invasion in breast cancer cells." Molecular cancer 14 (January 2015): 67-.
PMID
25889182
Source
epmc
Published In
Molecular Cancer
Volume
14
Publish Date
2015
Start Page
67
DOI
10.1186/s12943-015-0337-x

An improved isoprenylcysteine carboxylmethyltransferase inhibitor induces cancer cell death and attenuates tumor growth in vivo.

Inhibitors of isoprenylcysteine carboxylmethyltransferase (Icmt) are promising anti-cancer agents, as modification by Icmt is an essential component of the protein prenylation pathway for a group of proteins that includes Ras GTPases. Cysmethynil, a prototypical indole-based inhibitor of Icmt, effectively inhibits tumor cell growth. However, the physical properties of cysmethynil, such as its low aqueous solubility, make it a poor candidate for clinical development. A novel amino-derivative of cysmethynil with superior physical properties and marked improvement in efficacy, termed compound 8.12, has recently been reported. We report here that Icmt (-/-) mouse embryonic fibroblasts (MEFs) are much more resistant to compound 8.12-induced cell death than their wild-type counterparts, providing evidence that the anti-proliferative effects of this compound are mediated through an Icmt specific mechanism. Treatment of PC3 prostate and HepG2 liver cancer cells with compound 8.12 resulted in pre-lamin A accumulation and Ras delocalization from the plasma membrane, both expected outcomes from inhibition of the Icmt-catalyzed carboxylmethylation. Treatment with compound 8.12 induced cell cycle arrest, autophagy and cell death, and abolished anchorage-independent colony formation. Consistent with its greater in vitro efficacy, compound 8.12 inhibited tumor growth with greater potency than cysmethynil in a xenograft mouse model. Further, a drug combination study identified synergistic antitumor efficacy of compound 8.12 and the epithelial growth factor receptor (EGFR)-inhibitor gefitinib, possibly through enhancement of autophagy. This study establishes compound 8.12 as a pharmacological inhibitor of Icmt that is an attractive candidate for further preclinical and clinical development.

Authors
Lau, HY; Ramanujulu, PM; Guo, D; Yang, T; Wirawan, M; Casey, PJ; Go, M-L; Wang, M
MLA Citation
Lau, HY, Ramanujulu, PM, Guo, D, Yang, T, Wirawan, M, Casey, PJ, Go, M-L, and Wang, M. "An improved isoprenylcysteine carboxylmethyltransferase inhibitor induces cancer cell death and attenuates tumor growth in vivo." Cancer biology & therapy 15.9 (September 2014): 1280-1291.
PMID
24971579
Source
epmc
Published In
Cancer Biology and Therapy
Volume
15
Issue
9
Publish Date
2014
Start Page
1280
End Page
1291
DOI
10.4161/cbt.29692

Inhibitory G proteins and their receptors: emerging therapeutic targets for obesity and diabetes.

The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.

Authors
Kimple, ME; Neuman, JC; Linnemann, AK; Casey, PJ
MLA Citation
Kimple, ME, Neuman, JC, Linnemann, AK, and Casey, PJ. "Inhibitory G proteins and their receptors: emerging therapeutic targets for obesity and diabetes." Experimental & molecular medicine 46 (June 20, 2014): e102-. (Review)
PMID
24946790
Source
epmc
Published In
Experimental & molecular medicine
Volume
46
Publish Date
2014
Start Page
e102
DOI
10.1038/emm.2014.40

Gαz regulates BDNF-induction of axon growth in cortical neurons.

The disruption of neurotransmitter and neurotrophic factor signaling in the central nervous system (CNS) is implicated as the root cause of neuropsychiatric disorders, including schizophrenia, epilepsy, chronic pain, and depression. Therefore, identifying the underlying molecular mechanisms by which neurotransmitter and neurotrophic factor signaling regulates neuronal survival or growth may facilitate identification of more effective therapies for these disorders. Previously, our lab found that the heterotrimeric G protein, Gz, mediates crosstalk between G protein-coupled receptors and neurotrophin signaling in the neural cell line PC12. These data, combined with Gαz expression profiles--predominantly in neuronal cells with higher expression levels corresponding to developmental times of target tissue innervation--suggested that Gαz may play an important role in neurotrophin signaling and neuronal development. Here, we provide evidence in cortical neurons, both manipulated ex vivo and those cultured from Gz knockout mice, that Gαz is localized to axonal growth cones and plays a significant role in the development of axons of cortical neurons in the CNS. Our findings indicate that Gαz inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for Gαz in regulating neurotrophin signaling in the CNS.

Authors
Hultman, R; Kumari, U; Michel, N; Casey, PJ
MLA Citation
Hultman, R, Kumari, U, Michel, N, and Casey, PJ. "Gαz regulates BDNF-induction of axon growth in cortical neurons." Mol Cell Neurosci 58 (January 2014): 53-61.
PMID
24321455
Source
pubmed
Published In
Molecular and Cellular Neuroscience
Volume
58
Publish Date
2014
Start Page
53
End Page
61
DOI
10.1016/j.mcn.2013.12.004

Breast cancer cell invasion mediated by Gα12 signaling involves expression of interleukins-6 and -8, and matrix metalloproteinase-2.

BACKGROUND: Recent studies on the involvement of the G12 family of heterotrimeric G proteins (Gα12 and Gα13, the products of the GNA12 and GNA13 genes, respectively) in oncogenic pathways have uncovered a link between G12 signaling and cancer progression. However, despite a well characterized role of Rho GTPases, the potential role of secreted factors in the capacity of G12 signaling to promote invasion of cancer cells is just beginning to be addressed. METHODS: MDA-MB-231 and MCF10A breast cancer cell lines were employed as a model system to explore the involvement of secreted factors in G12-stimulated cell invasion. Factors secreted by cells expressing dominant-active Gα12 were identified by protein array, and their involvement in breast cancer cell invasion was assessed through both RNAi-mediated knockdown and antibody neutralization approaches. Bioinformatics analysis of the promoter elements of the identified factors suggested NF-κB elements played a role in their enhanced expression, which was tested by chromatin immunoprecipitation. RESULTS: We found that signaling through the Gα12 in MDA-MB-231 and MCF10A breast cancer cell lines enhances expression of interleukins (IL)-6 and -8, and matrix metalloproteinase (MMP)-2, and that these secreted factors play a role in G12-stimulated cell invasion. Furthermore, the enhanced expression of these secreted factors was found to be facilitated by the activation of their corresponding promoters, where NF-κB seems to be one of the major regulators. Inhibition of IL-6 and IL-8, or MMP-2 activity significantly decreased Gα12-mediated cell invasion. CONCLUSIONS: These studies confirm and extend findings that secreted factors contribute to the oncogenic potential of G12 signaling, and suggest potential therapeutic targets to control this process.

Authors
Chia, CY; Kumari, U; Casey, PJ
MLA Citation
Chia, CY, Kumari, U, and Casey, PJ. "Breast cancer cell invasion mediated by Gα12 signaling involves expression of interleukins-6 and -8, and matrix metalloproteinase-2." Journal of molecular signaling 9 (January 2014): 6-.
PMID
24976858
Source
epmc
Published In
Journal of Molecular Signaling
Volume
9
Publish Date
2014
Start Page
6
DOI
10.1186/1750-2187-9-6

Control of RhoA methylation by carboxylesterase I.

A number of proteins that play key roles in cell signaling are post-translationally modified by the prenylation pathway. The final step in this pathway is methylation of the carboxyl terminus of the prenylated protein by isoprenylcysteine carboxylmethyltransferase. Due to the impact of methylation on Rho function, we sought to determine if the process was reversible and hence could control Rho function in a dynamic fashion. Elevating isoprenylcysteine carboxylmethyltransferase activity in cells has profound effects on MDA-MB-231 cell morphology, implying the presence of a pool of unmethylated prenyl proteins in these cells under normal conditions. Using a knockdown approach, we identified a specific esterase, carboxylesterase 1, whose function had a clear impact not only on the methylation status of RhoA but also RhoA activation and cell morphology. These data provide compelling evidence that C-terminal modification of prenyl proteins, rather than being purely a constitutive process, can serve as a point of regulation of function for this important class of protein.

Authors
Cushman, I; Cushman, SM; Potter, PM; Casey, PJ
MLA Citation
Cushman, I, Cushman, SM, Potter, PM, and Casey, PJ. "Control of RhoA methylation by carboxylesterase I." J Biol Chem 288.26 (June 28, 2013): 19177-19183.
PMID
23658012
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
26
Publish Date
2013
Start Page
19177
End Page
19183
DOI
10.1074/jbc.M113.467407

Deciphering the signaling networks underlying simvastatin-induced apoptosis in human cancer cells: evidence for non-canonical activation of RhoA and Rac1 GTPases

Authors
Zhu, Y; Casey, PJ; Kumar, AP; Pervaiz, S
MLA Citation
Zhu, Y, Casey, PJ, Kumar, AP, and Pervaiz, S. "Deciphering the signaling networks underlying simvastatin-induced apoptosis in human cancer cells: evidence for non-canonical activation of RhoA and Rac1 GTPases." CELL DEATH & DISEASE 4 (April 2013).
PMID
23559002
Source
wos-lite
Published In
Cell Death and Disease
Volume
4
Publish Date
2013
DOI
10.1038/cddis.2013.103

MicroRNA-182 and microRNA-200a control G-protein subunit α-13 (GNA13) expression and cell invasion synergistically in prostate cancer cells.

G protein-coupled receptors (GPCRs) and their ligands have been implicated in progression and metastasis of several cancers. GPCRs signal through heterotrimeric G proteins, and among the different types of G proteins, GNA12/13 have been most closely linked to tumor progression. In this study, we explored the role of GNA13 in prostate cancer cell invasion and the mechanism of up-regulation of GNA13 in these cells. An initial screen for GNA13 protein expression showed that GNA13 is highly expressed in the most aggressive cancer cell lines. Knockdown of GNA13 in highly invasive PC3 cells revealed that these cells depend on GNA13 expression for their invasion, migration, and Rho activation. As mRNA levels in these cells did not correlate with protein levels, we assessed the potential involvement of micro-RNAs (miRNAs) in post-transcriptional control of GNA13 expression. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-182 and miR-141/200a showed an inverse correlation to the protein expression in LnCAP and PC3 cells. Ectopic expression of miR-182 and miR-141/200a in PC3 cells significantly reduced protein levels, GNA13-3'-UTR reporter activity and in vitro invasion of these cells. This effect was blocked by restoration of GNA13 expression in these cells. Importantly, inhibition of miR-182 and miR-141/200a in LnCAP cells using specific miRNA inhibitors elevated the expression of GNA13 and enhanced invasion of these cells. These data provide strong evidence that GNA13 is an important mediator of prostate cancer cell invasion, and that miR-182 and miR-200 family members regulate its expression post-transcriptionally.

Authors
Rasheed, SAK; Teo, CR; Beillard, EJ; Voorhoeve, PM; Casey, PJ
MLA Citation
Rasheed, SAK, Teo, CR, Beillard, EJ, Voorhoeve, PM, and Casey, PJ. "MicroRNA-182 and microRNA-200a control G-protein subunit α-13 (GNA13) expression and cell invasion synergistically in prostate cancer cells." J Biol Chem 288.11 (March 15, 2013): 7986-7995.
PMID
23329838
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
11
Publish Date
2013
Start Page
7986
End Page
7995
DOI
10.1074/jbc.M112.437749

Functionalized indoleamines as potent, drug-like inhibitors of isoprenylcysteine carboxyl methyltransferase (Icmt)

The enzyme isoprenylcysteine carboxyl methyltransferase (Icmt) plays an important role in the post-translational modification of proteins involved in the regulation of cell growth and oncogenesis. The biological consequences of Icmt inhibition strongly implicate the enzyme as a potential therapeutic target for cancer and provide a compelling rationale for developing specific Icmt inhibitors as anti-cancer agents. We report here the systematic modification of the known Icmt inhibitor cysmethynil to give an analog 15 with greatly improved solubility and PAMPA permeability which was achieved with concurrent gains in Icmt inhibitory and cell-based antiproliferative activities. The modifications involved replacing the amide side chain of cysmethynil with a tertiary amine, and introducing an aminopyrimidine ring in place of m-tolyl. The presence of the weakly basic and polar aminopyrimidine ring contributed significantly to the potency and drug-like profile of the final compound. © 2013 Elsevier Masson SAS. All rights reserved.

Authors
Ramanujulu, PM; Yang, T; Yap, S-Q; Wong, F-C; Casey, PJ; Wang, M; Go, M-L
MLA Citation
Ramanujulu, PM, Yang, T, Yap, S-Q, Wong, F-C, Casey, PJ, Wang, M, and Go, M-L. "Functionalized indoleamines as potent, drug-like inhibitors of isoprenylcysteine carboxyl methyltransferase (Icmt)." European Journal of Medicinal Chemistry 63 (2013): 378-386.
PMID
23514631
Source
scival
Published In
European Journal of Medicinal Chemistry
Volume
63
Publish Date
2013
Start Page
378
End Page
386
DOI
10.1016/j.ejmech.2013.02.007

Deciphering the signaling networks underlying simvastatin-induced apoptosis in human cancer cells: Evidence for non-canonical activation of RhoA and Rac1 GTPases

Although statins are known to inhibit proliferation and induce death in a number of cancer cell types, the mechanisms through which downregulation of the mevalonate (MVA) pathway activates death signaling remain poorly understood. Here we set out to unravel the signaling networks downstream of the MVA pathway that mediate the death-inducing activity of simvastatin. Consistent with previous reports, exogenously added geranylgeranylpyrophosphate, but not farnesylpyrophosphate, prevented simvastatin's growth-inhibitory effect, thereby suggesting the involvement of geranylgeranylated proteins such as Rho GTPases in the anticancer activity of simvastatin. Indeed, simvastatin treatment led to increased levels of unprenylated Ras homolog gene family, member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42). Intriguingly, instead of inhibiting the functions of Rho GTPases as was expected with loss of prenylation, simvastatin caused a paradoxical increase in the GTP-bound forms of RhoA, Rac1 and Cdc42. Furthermore, simvastatin disrupted the binding of Rho GTPases with the cytosolic inhibitor Rho GDIa, which provides a potential mechanism for GTP loading of the cytosolic Rho GTPases. We also show that the unprenylated RhoA- and Rac1-GTP retained at least part of their functional activities, as evidenced by the increase in intracellular superoxide production and JNK activation in response to simvastatin. Notably, blocking superoxide production attenuated JNK activation as well as cell death induced by simvastatin. Finally, we provide evidence for the involvement of the B-cell lymphoma protein 2 family, Bcl-2-interacting mediator (Bim), in a JNK-dependent manner, in the apoptosis-inducing activity of simvastatin. Taken together, our data highlight the critical role of non-canonical regulation of Rho GTPases and involvement of downstream superoxide-mediated activation of JNK pathway in the anticancer activity of simvastatin, which would have potential clinical implications. © 2013 Macmillan Publishers Limited All rights reserved.

Authors
Zhu, Y; Casey, PJ; Kumar, AP; Pervaiz, S
MLA Citation
Zhu, Y, Casey, PJ, Kumar, AP, and Pervaiz, S. "Deciphering the signaling networks underlying simvastatin-induced apoptosis in human cancer cells: Evidence for non-canonical activation of RhoA and Rac1 GTPases." Cell Death and Disease 4.4 (2013).
Source
scival
Published In
Cell Death and Disease
Volume
4
Issue
4
Publish Date
2013
DOI
10.1038/cddis.2013.103

Deletion of GαZ protein protects against diet-induced glucose intolerance via expansion of β-cell mass.

Insufficient plasma insulin levels caused by deficits in both pancreatic β-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed β-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating β-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric G(z) protein, Gα(z), impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gα(z)-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gα(z) is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gα(z) are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in β-cell proliferation is correlated with significantly increased β-cell mass. Further, an endogenous Gα(z) signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gα(z) to both major aspects of β-cell decompensation: insufficient β-cell function and mass.

Authors
Kimple, ME; Moss, JB; Brar, HK; Rosa, TC; Truchan, NA; Pasker, RL; Newgard, CB; Casey, PJ
MLA Citation
Kimple, ME, Moss, JB, Brar, HK, Rosa, TC, Truchan, NA, Pasker, RL, Newgard, CB, and Casey, PJ. "Deletion of GαZ protein protects against diet-induced glucose intolerance via expansion of β-cell mass." J Biol Chem 287.24 (June 8, 2012): 20344-20355.
PMID
22457354
Source
pubmed
Published In
The Journal of biological chemistry
Volume
287
Issue
24
Publish Date
2012
Start Page
20344
End Page
20355
DOI
10.1074/jbc.M112.359745

A role for Rac3 GTPase in the regulation of autophagy.

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.

Authors
Zhu, WL; Hossain, MS; Guo, DY; Liu, S; Tong, H; Khakpoor, A; Casey, PJ; Wang, M
MLA Citation
Zhu, WL, Hossain, MS, Guo, DY, Liu, S, Tong, H, Khakpoor, A, Casey, PJ, and Wang, M. "A role for Rac3 GTPase in the regulation of autophagy." J Biol Chem 286.40 (October 7, 2011): 35291-35298.
PMID
21852230
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
40
Publish Date
2011
Start Page
35291
End Page
35298
DOI
10.1074/jbc.M111.280990

Prenylated C17orf37 Induces Filopodia Formation to Promote Cell Migration and Metastasis

Authors
Dasgupta, S; Cushman, I; Kpetemey, M; Casey, PJ; Vishwanatha, JK
MLA Citation
Dasgupta, S, Cushman, I, Kpetemey, M, Casey, PJ, and Vishwanatha, JK. "Prenylated C17orf37 Induces Filopodia Formation to Promote Cell Migration and Metastasis." JOURNAL OF BIOLOGICAL CHEMISTRY 286.29 (July 22, 2011): 25935-25946.
PMID
21628459
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
286
Issue
29
Publish Date
2011
Start Page
25935
End Page
25946
DOI
10.1074/jbc.M111.254599

Site-specific analysis of protein S-acylation by resin-assisted capture.

Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells.

Authors
Forrester, MT; Hess, DT; Thompson, JW; Hultman, R; Moseley, MA; Stamler, JS; Casey, PJ
MLA Citation
Forrester, MT, Hess, DT, Thompson, JW, Hultman, R, Moseley, MA, Stamler, JS, and Casey, PJ. "Site-specific analysis of protein S-acylation by resin-assisted capture." J Lipid Res 52.2 (February 2011): 393-398.
PMID
21044946
Source
pubmed
Published In
Journal of lipid research
Volume
52
Issue
2
Publish Date
2011
Start Page
393
End Page
398
DOI
10.1194/jlr.D011106

RHO methylation matters: a role for isoprenylcysteine carboxylmethyltransferase in cell migration and adhesion.

Numerous proteins involved in diverse aspects of cell biology undergo a process of post-translational modification termed prenylation. The prenylation pathway consists of three enzymatic steps, the final of which is methylation of the carboxyl-terminal prenylcysteine formed in the first two steps by the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt). Due to the prevalence of prenylated proteins in cancer biology, and the findings that several of the proteins are involved in processes controlling cell migration and adhesion, we sought to examine the role of Icmt - mediated methylation on cell behavior associated with metastasis. We found that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading were also impaired by Icmt inhibition. Further investigation revealed that inhibition of Icmt significantly decreased the activation of both RhoA and Rac1, which are both prenylated proteins. The data obtained were consistent with the decreased activation being due to an increase in Rho GDP-dissociation inhibitor (GDI) binding by both proteins in the absence of their methylation. Importantly, the addition of exogenous RhoA or Rac1 to cells in which Icmt was inhibited was able to partially, but selectively, rescue directed and random migration, respectively. These results establish a role for Icmt-mediated methylation in cell migration, and point to specific prenylated proteins involved in this biology. The prenylation pathway has been targeted for oncogenic therapies, but the role of methylation in cell motility had been largely unexplored until now. The finding that methylation of Rho family members impacts on a specific component of their function provides an additional avenue through which to interrogate the biology of this important class of regulatory proteins.

Authors
Cushman, I; Casey, PJ
MLA Citation
Cushman, I, and Casey, PJ. "RHO methylation matters: a role for isoprenylcysteine carboxylmethyltransferase in cell migration and adhesion." Cell Adh Migr 5.1 (January 2011): 11-15.
PMID
20798596
Source
pubmed
Published In
Cell adhesion & migration
Volume
5
Issue
1
Publish Date
2011
Start Page
11
End Page
15
DOI
10.4161/cam.5.1.13196

G12 signaling through c-Jun NH2-terminal kinase promotes breast cancer cell invasion.

Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH(2)-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression.

Authors
Juneja, J; Cushman, I; Casey, PJ
MLA Citation
Juneja, J, Cushman, I, and Casey, PJ. "G12 signaling through c-Jun NH2-terminal kinase promotes breast cancer cell invasion." PLoS One 6.11 (2011): e26085-.
PMID
22087220
Source
pubmed
Published In
PloS one
Volume
6
Issue
11
Publish Date
2011
Start Page
e26085
DOI
10.1371/journal.pone.0026085

The Enzymology of CAAX Protein Prenylation

Many proteins involved in signal transduction and protein trafficking are posttranslationally modified by the covalent attachment of lipid groups. One form of lipid modification involves attachment of either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid lipid to a cysteine residue fourth from the C-terminus of the substrate protein. The attachment of the isoprenoid is the first step in a processing pathway that can include subsequent proteolysis of three carboxyl-terminal residues, methylation of the free carboxyl group of the resulting C-terminal prenylcysteine, and modification with additional lipid molecules. These modifications are necessary for targeting and attachment of these so-called CAAX proteins to the correct membrane as well as for the cellular function of the protein. The focus of this chapter is on the two protein prenyltransferases responsible for addition of the isoprenoid to the CAAX protein substrates. © 2011 Elsevier Inc.

Authors
Hightower, KE; Casey, PJ
MLA Citation
Hightower, KE, and Casey, PJ. "The Enzymology of CAAX Protein Prenylation." Enzymes 30 (2011): 1-11.
Source
scival
Published In
Enzymes
Volume
30
Publish Date
2011
Start Page
1
End Page
11
DOI
10.1016/B978-0-12-415922-8.00001-X

Signaling through Gz

Several studies have been carried out to pinpoint specific brain regions and developmental stages where Gz is transcribed or expressed. The limited tissue distribution of Gαz, its unusual biochemical properties, the identification of Gαz-specific effectors, and the inability of other Gαi subfamily members to substitute for Gαz in vivo support unique physiologic roles for Gz. The rate of GDP dissociation from Gαz is extremely slow as compared to that of most other G-protein β subunits, and almost completely suppressed at Mg2+ concentrations greater than 100 μM. Most receptors that couple to Gi proteins can also activate Gz if the G protein or receptors are overexpressed in cells. Although the precise roles of Gz in cellular signaling are still being established, accumulating evidence points to the involvement of this unique Gi subfamily member in several facets of cell biology. First, the temporal and spatial expression patterns of Gαz suggest a possible role in neuron growth and/or differentiation. Gz also appears to regulate platelet function through adrenergic receptor signaling. In addition, Gz participates in the physiologic regulation of insulin secretion from the pancreatic β cells. Finally, Gz may be involved in processes impacting on mood or behavior. These distinct roles further define Gz as unique among the Gi subfamily of heterotrimeric G proteins. © 2010 Elsevier Inc. All rights reserved.

Authors
Kimple, ME; Hultman, RC; Casey, PJ
MLA Citation
Kimple, ME, Hultman, RC, and Casey, PJ. "Signaling through Gz." Handbook of Cell Signaling, 2/e. December 1, 2010. 1649-1653.
Source
scopus
Volume
2
Publish Date
2010
Start Page
1649
End Page
1653
DOI
10.1016/B978-0-12-374145-5.00202-3

Amino Derivatives of Indole As Potent Inhibitors of Isoprenyleysteine Carboxyl Methyltransferase

Authors
Go, M-L; Leow, JL; Gorla, SK; Schueller, AP; Wang, M; Casey, PJ
MLA Citation
Go, M-L, Leow, JL, Gorla, SK, Schueller, AP, Wang, M, and Casey, PJ. "Amino Derivatives of Indole As Potent Inhibitors of Isoprenyleysteine Carboxyl Methyltransferase." JOURNAL OF MEDICINAL CHEMISTRY 53.19 (October 14, 2010): 6838-6850.
PMID
20809634
Source
wos-lite
Published In
Journal of Medicinal Chemistry
Volume
53
Issue
19
Publish Date
2010
Start Page
6838
End Page
6850
DOI
10.1021/jm1002843

Inhibition of isoprenylcysteine carboxylmethyltransferase induces autophagic-dependent apoptosis and impairs tumor growth.

Inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational C-terminal processing of prenylated proteins, suppresses tumor cell growth and induces cell death. Icmt inhibition by either a small molecule inhibitor termed as cysmethynil or inhibitory RNA induces marked autophagy leading to cell death. HepG2 cells were used to investigate the function of autophagy in tumor cell death. Suppression of autophagy, either pharmacologically or through knockdown of the autophagy essential proteins, Atg5 or Atg1, inhibits not only cysmethynil-induced autophagy, but also apoptosis in HepG2 cells. The dependence of cysmethynil-induced apoptosis on autophagy was further shown using autophagy-deficient mouse embryonic fibroblast (MEF) cells. Atg5(-/-) MEF cells were found to be resistant to cysmethynil-induced apoptosis, whereas wild-type MEFs showed high sensitivity to apoptosis induction. These data indicate that inhibition of Icmt can elicit cell death through two linked mechanisms, autophagy and apoptosis, and that autophagy can be an active player upstream of apoptosis in cell types capable of apoptotic cell death, such as HepG2 and MEFs. Further, treatment of mice-bearing HepG2-derived tumors with cysmethynil resulted in marked inhibition of tumor growth; analysis of tumor tissue from these mice revealed markers consistent with autophagy induction and cell growth arrest.

Authors
Wang, M; Hossain, MS; Tan, W; Coolman, B; Zhou, J; Liu, S; Casey, PJ
MLA Citation
Wang, M, Hossain, MS, Tan, W, Coolman, B, Zhou, J, Liu, S, and Casey, PJ. "Inhibition of isoprenylcysteine carboxylmethyltransferase induces autophagic-dependent apoptosis and impairs tumor growth." Oncogene 29.35 (September 2, 2010): 4959-4970.
PMID
20622895
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
29
Issue
35
Publish Date
2010
Start Page
4959
End Page
4970
DOI
10.1038/onc.2010.247

A Prenylated p47(phox)-p67(phox)-Rac1 Chimera Is a Quintessential NADPH Oxidase Activator MEMBRANE ASSOCIATION AND FUNCTIONAL CAPACITY

Authors
Mizrahi, A; Berdichevsky, Y; Casey, PJ; Pick, E
MLA Citation
Mizrahi, A, Berdichevsky, Y, Casey, PJ, and Pick, E. "A Prenylated p47(phox)-p67(phox)-Rac1 Chimera Is a Quintessential NADPH Oxidase Activator MEMBRANE ASSOCIATION AND FUNCTIONAL CAPACITY." JOURNAL OF BIOLOGICAL CHEMISTRY 285.33 (August 13, 2010): 25485-25499.
PMID
20529851
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
285
Issue
33
Publish Date
2010
Start Page
25485
End Page
25499
DOI
10.1074/jbc.M110.113779

Rap1 promotes multiple pancreatic islet cell functions and signals through mammalian target of rapamycin complex 1 to enhance proliferation.

Recent studies have implicated Epac2, a guanine-nucleotide exchange factor for the Rap subfamily of monomeric G proteins, as an important regulator of insulin secretion from pancreatic beta-cells. Although the Epac proteins were originally identified as cAMP-responsive activators of Rap1 GTPases, the role of Rap1 in beta-cell biology has not yet been defined. In this study, we examined the direct effects of Rap1 signaling on beta-cell biology. Using the Ins-1 rat insulinoma line, we demonstrate that activated Rap1A, but not related monomeric G proteins, promotes ribosomal protein S6 phosphorylation. Using isolated rat islets, we show that this signaling event is rapamycin-sensitive, indicating that it is mediated by the mammalian target of rapamycin complex 1-p70 S6 kinase pathway, a known growth regulatory pathway. This newly defined beta-cell signaling pathway acts downstream of cAMP, in parallel with the stimulation of cAMP-dependent protein kinase, to drive ribosomal protein S6 phosphorylation. Activated Rap1A promotes glucose-stimulated insulin secretion, islet cell hypertrophy, and islet cell proliferation, the latter exclusively through mammalian target of rapamycin complex 1, suggesting that Rap1 is an important regulator of beta-cell function. This newly defined signaling pathway may yield unique targets for the treatment of beta-cell dysfunction in diabetes.

Authors
Kelly, P; Bailey, CL; Fueger, PT; Newgard, CB; Casey, PJ; Kimple, ME
MLA Citation
Kelly, P, Bailey, CL, Fueger, PT, Newgard, CB, Casey, PJ, and Kimple, ME. "Rap1 promotes multiple pancreatic islet cell functions and signals through mammalian target of rapamycin complex 1 to enhance proliferation." J Biol Chem 285.21 (May 21, 2010): 15777-15785.
PMID
20339002
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
21
Publish Date
2010
Start Page
15777
End Page
15785
DOI
10.1074/jbc.M109.069112

Pharmacological targeting of the mitochondrial phosphatase PTPMT1.

The dual-specificity protein tyrosine phosphatases (PTPs) play integral roles in the regulation of cell signaling. There is a need for new tools to study these phosphatases, and the identification of inhibitors potentially affords not only new means for their study, but also possible therapeutics for the treatment of diseases caused by their dysregulation. However, the identification of selective inhibitors of the protein phosphatases has proven somewhat difficult. PTP localized to mitochondrion 1 (PTPMT1) is a recently discovered dual-specificity phosphatase that has been implicated in the regulation of insulin secretion. Screening of a commercially available small-molecule library yielded alexidine dihydrochloride, a dibiguanide compound, as an effective and selective inhibitor of PTPMT1 with an in vitro concentration that inhibits response by 50% of 1.08 microM. A related dibiguanide analog, chlorhexidine dihydrochloride, also significantly inhibited PTPMT1, albeit with lower potency, while a monobiguanide analog showed very weak inhibition. Treatment of isolated rat pancreatic islets with alexidine dihydrochloride resulted in a dose-dependent increase in insulin secretion, whereas treatment of a pancreatic beta-cell line with the drug affected the phosphorylation of mitochondrial proteins in a manner similar to genetic inhibition of PTPMT1. Furthermore, knockdown of PTPMT1 in rat islets rendered them insensitive to alexidine dihydrochloride treatment, providing evidence for mechanism-based activity of the inhibitor. Taken together, these studies establish alexidine dihydrochloride as an effective inhibitor of PTPMT1, both in vitro and in cells, and support the notion that PTPMT1 could serve as a pharmacological target in the treatment of type II diabetes.

Authors
Doughty-Shenton, D; Joseph, JD; Zhang, J; Pagliarini, DJ; Kim, Y; Lu, D; Dixon, JE; Casey, PJ
MLA Citation
Doughty-Shenton, D, Joseph, JD, Zhang, J, Pagliarini, DJ, Kim, Y, Lu, D, Dixon, JE, and Casey, PJ. "Pharmacological targeting of the mitochondrial phosphatase PTPMT1." J Pharmacol Exp Ther 333.2 (May 2010): 584-592.
Website
http://hdl.handle.net/10161/1303
PMID
20167843
Source
pubmed
Published In
The Journal of pharmacology and experimental therapeutics
Volume
333
Issue
2
Publish Date
2010
Start Page
584
End Page
592
DOI
10.1124/jpet.109.163329

Signaling through Gz

Several studies have been carried out to pinpoint specific brain regions and developmental stages where Gz is transcribed or expressed. The limited tissue distribution of Gαz, its unusual biochemical properties, the identification of Gαz-specific effectors, and the inability of other Gαi subfamily members to substitute for Gαz in vivo support unique physiologic roles for Gz. The rate of GDP dissociation from Gαz is extremely slow as compared to that of most other G-protein β subunits, and almost completely suppressed at Mg2+ concentrations greater than 100 μM. Most receptors that couple to Gi proteins can also activate Gz if the G protein or receptors are overexpressed in cells. Although the precise roles of Gz in cellular signaling are still being established, accumulating evidence points to the involvement of this unique Gi subfamily member in several facets of cell biology. First, the temporal and spatial expression patterns of Gαz suggest a possible role in neuron growth and/or differentiation. Gz also appears to regulate platelet function through adrenergic receptor signaling. In addition, Gz participates in the physiologic regulation of insulin secretion from the pancreatic β cells. Finally, Gz may be involved in processes impacting on mood or behavior. These distinct roles further define Gz as unique among the Gi subfamily of heterotrimeric G proteins. © 2010 Elsevier Inc. All rights reserved.

Authors
Kimple, ME; Hultman, RC; Casey, PJ
MLA Citation
Kimple, ME, Hultman, RC, and Casey, PJ. "Signaling through Gz." Handbook of Cell Signaling, 2/e 2 (2010): 1649-1653.
Source
scival
Published In
Handbook of Cell Signaling, 2/e
Volume
2
Publish Date
2010
Start Page
1649
End Page
1653
DOI
10.1016/B978-0-12-374145-5.00202-3

Rho GTPase activity modulates Wnt3a/beta-catenin signaling.

Wnt proteins constitute a family of secreted signaling molecules that regulate highly conserved pathways essential for development and, when aberrantly activated, drive oncogenesis in a number of human cancers. A key feature of the most widely studied Wnt signaling cascade is the stabilization of cytosolic beta-catenin, resulting in beta-catenin nuclear translocation and transcriptional activation of multiple target genes. In addition to this canonical, beta-catenin-dependent pathway, Wnt3A has also been shown to stimulate RhoA GTPase. While the importance of activated Rho to non-canonical Wnt signaling is well appreciated, the potential contribution of Wnt3A-stimulated RhoA to canonical beta-catenin-dependent transcription has not been examined and is the focus of this study. We find that activated Rho is required for Wnt3A-stimulated osteoblastic differentiation in C3H10T1/2 mesenchymal stem cells, a biological phenomenon mediated by stabilized beta-catenin. Using expression microarrays and real-time RT-PCR analysis, we show that Wnt3A-stimulated transcription of a subset of target genes is Rho-dependent, indicating that full induction of these Wnt targets requires both beta-catenin and Rho activation. Significantly, neither beta-catenin stabilization nor nuclear translocation stimulated by Wnt3A is affected by inhibition or activation of RhoA. These findings identify Rho activation as a critical element of the canonical Wnt3A-stimulated, beta-catenin-dependent transcriptional program.

Authors
Rossol-Allison, J; Stemmle, LN; Swenson-Fields, KI; Kelly, P; Fields, PE; McCall, SJ; Casey, PJ; Fields, TA
MLA Citation
Rossol-Allison, J, Stemmle, LN, Swenson-Fields, KI, Kelly, P, Fields, PE, McCall, SJ, Casey, PJ, and Fields, TA. "Rho GTPase activity modulates Wnt3a/beta-catenin signaling." Cell Signal 21.11 (November 2009): 1559-1568.
PMID
19482078
Source
pubmed
Published In
Cellular Signalling
Volume
21
Issue
11
Publish Date
2009
Start Page
1559
End Page
1568
DOI
10.1016/j.cellsig.2009.05.010

Role of isoprenylcysteine carboxylmethyltransferase-catalyzed methylation in Rho function and migration.

A number of proteins that play key roles in biological regulatory events undergo a process of post-translational modifications termed prenylation. The prenylation pathway consists of three enzymatic steps; the final processed protein is isoprenoid-modified and methylated on the C-terminal cysteine. This protein modification pathway plays a significant role in cancer biology because many oncogenic proteins undergo prenylation. Methylation of the C terminus by isoprenylcysteine carboxylmethyltransferase (Icmt) is the final step in the prenylation pathway. Cysmethynil, a specific Icmt inhibitor discovered in our laboratory, is able to inhibit Ras-mediated signaling, cell growth, and oncogenesis. We sought to examine the role of Icmt-mediated methylation on the behaviors of cancer cells associated with metastatic potential. Our results indicate that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading are also significantly impacted by cysmethynil. To examine the mechanism of Icmt-dependent migration we focused on RhoA and Rac1, prenylated proteins that are important mediators of cell migration through their control of the actin cytoskeleton. Inhibition of Icmt significantly decreases the activation of both RhoA and Rac1; an increase in Rho GDP-dissociation inhibitor (RhoGDI) binding in the absence of methylation appears to contribute to this effect. Furthermore, in the absence of Icmt activity the addition of exogenous RhoA or Rac1 is able to partially rescue directed and random migration, respectively. These findings establish a role for Icmt-mediated methylation in cell migration and advance our understanding of the biological consequences of Rho methylation.

Authors
Cushman, I; Casey, PJ
MLA Citation
Cushman, I, and Casey, PJ. "Role of isoprenylcysteine carboxylmethyltransferase-catalyzed methylation in Rho function and migration." J Biol Chem 284.41 (October 9, 2009): 27964-27973.
PMID
19651782
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
41
Publish Date
2009
Start Page
27964
End Page
27973
DOI
10.1074/jbc.M109.025296

Role of G12 proteins in oncogenesis and metastasis.

The G12 subfamily of heterotrimeric guanine nucleotide-binding proteins consists of two alpha subunits, G alpha12 and G alpha13. These proteins mediate signalling via G protein-coupled receptors and have been implicated in various physiological and pathophysiological processes. A number of direct and indirect effectors of G alpha12 and G alpha13 have been identified that mediate, or have been proposed to mediate, the diverse cellular responses accompanying activation of G12 proteins. This review describes the signalling pathways and cellular events stimulated by G12 proteins, with a particular emphasis on processes that are important in regulating cell migration and invasion, and could potentially be involved in the pathophysiology of cancer metastasis. Experimental findings directly implicating G12 proteins in the spread of metastatic disease are also summarized, indicating the importance of targeted inhibition of G12 signalling as a potential therapeutic option for locally advanced and metastatic disease.

Authors
Juneja, J; Casey, PJ
MLA Citation
Juneja, J, and Casey, PJ. "Role of G12 proteins in oncogenesis and metastasis." Br J Pharmacol 158.1 (September 2009): 32-40. (Review)
PMID
19422395
Source
pubmed
Published In
British Journal of Pharmacology
Volume
158
Issue
1
Publish Date
2009
Start Page
32
End Page
40
DOI
10.1111/j.1476-5381.2009.00180.x

Discovery of geranylgeranyltransferase-I inhibitors with novel scaffolds by the means of quantitative structure-activity relationship modeling, virtual screening, and experimental validation.

Geranylgeranylation is critical to the function of several proteins including Rho, Rap1, Rac, Cdc42, and G-protein gamma subunits. Geranylgeranyltransferase type I (GGTase-I) inhibitors (GGTIs) have therapeutic potential to treat inflammation, multiple sclerosis, atherosclerosis, and many other diseases. Following our standard workflow, we have developed and rigorously validated quantitative structure-activity relationship (QSAR) models for 48 GGTIs using variable selection k nearest neighbor (kNN), automated lazy learning (ALL), and partial least squares (PLS) methods. The QSAR models were employed for virtual screening of 9.5 million commercially available chemicals, yielding 47 diverse computational hits. Seven of these compounds with novel scaffolds and high predicted GGTase-I inhibitory activities were tested in vitro, and all were found to be bona fide and selective micromolar inhibitors. Notably, these novel hits could not be identified using traditional similarity search. These data demonstrate that rigorously developed QSAR models can serve as reliable virtual screening tools, leading to the discovery of structurally novel bioactive compounds.

Authors
Peterson, YK; Wang, XS; Casey, PJ; Tropsha, A
MLA Citation
Peterson, YK, Wang, XS, Casey, PJ, and Tropsha, A. "Discovery of geranylgeranyltransferase-I inhibitors with novel scaffolds by the means of quantitative structure-activity relationship modeling, virtual screening, and experimental validation." J Med Chem 52.14 (July 23, 2009): 4210-4220.
PMID
19537691
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
52
Issue
14
Publish Date
2009
Start Page
4210
End Page
4220
DOI
10.1021/jm8013772

Activation of Rap1 promotes prostate cancer metastasis.

Elucidating the mechanisms of prostate cancer (CaP) survival and metastasis are critical to the discovery of novel therapeutic targets. The monomeric G protein Rap1 has been implicated in cancer tumorigenesis. Rap1 signals to pathways involved in cell adhesion, migration, and survival, suggesting Rap1 may promote several processes associated with cancer cell metastasis. Examination of CaP cell lines revealed cells with a high metastatic ability exhibited increased Rap1 activity and reduced expression of the negative regulator Rap1GAP. Rap1 can be further stimulated in these cells by stromal-derived factor (SDF-1), an agonist known to regulate tumor cell metastasis and tropism to bone. Activation of Rap1 increased CaP cell migration and invasion, and inhibition of Rap1A activity via RNAi-mediated knockdown or ectopic expression of Rap1GAP markedly impaired CaP cell migration and invasion. Additional studies implicate integrins alpha4, beta3, and alphavbeta3 in the mechanism of Rap1-mediated CaP migration and invasion. Extending the effect of Rap1 activity in CaP metastasis in vivo, introduction of activated Rap1 into CaP cells dramatically enhanced the rate and incidence of CaP metastasis in a xenograft mouse model. These studies provide compelling evidence to support a role for aberrant Rap1 activation in CaP progression, and suggest that targeting Rap1 signaling could provide a means to control metastatic progression of this cancer.

Authors
Bailey, CL; Kelly, P; Casey, PJ
MLA Citation
Bailey, CL, Kelly, P, and Casey, PJ. "Activation of Rap1 promotes prostate cancer metastasis." Cancer Res 69.12 (June 15, 2009): 4962-4968.
PMID
19470770
Source
pubmed
Published In
Cancer Research
Volume
69
Issue
12
Publish Date
2009
Start Page
4962
End Page
4968
DOI
10.1158/0008-5472.CAN-08-4269

Topology of Mammalian Isoprenylcysteine Carboxyl Methyltransferase Determined in Live Cells with a Fluorescent Probe

Authors
Wright, LP; Court, H; Mor, A; Ahearn, IM; Casey, PJ; Philips, MR
MLA Citation
Wright, LP, Court, H, Mor, A, Ahearn, IM, Casey, PJ, and Philips, MR. "Topology of Mammalian Isoprenylcysteine Carboxyl Methyltransferase Determined in Live Cells with a Fluorescent Probe." MOLECULAR AND CELLULAR BIOLOGY 29.7 (April 1, 2009): 1826-1833.
PMID
19158273
Source
wos-lite
Published In
Molecular and Cellular Biology
Volume
29
Issue
7
Publish Date
2009
Start Page
1826
End Page
1833
DOI
10.1128/MCB.01719-08

A high-performance liquid chromatography method for the quantification of cysmethynil, an inhibitor of isoprenylcysteine carboxylmethyl transferase, in mouse plasma

Cysmethynil, a newly identified small molecule inhibitor of isoprenylcysteine carboxylmethyl transferase (Icmt) is involved in the post-translational modification of CaaX proteins. Cysmethynil causes cell death in many human cancer cells in vitro, and inhibits tumor growth in the xenograft mouse model in vivo. A HPLC method for the quantification of cysmethynil in mouse plasma was developed and validated. The lower limit of quantification of this method was 0.01 μg/ml. Inter- and intra-day variability ranged from 0.38-8.5% and accuracy was between 86% and 98%. This sensitive method was used to quantify cysmethynil in plasma of mice after intraperitoneal dosing for preliminary pharmacokinetic studies. © 2009 Elsevier B.V. All rights reserved.

Authors
Wang, M; Khoo, YM; Zhou, J; Casey, P; Lee, HS
MLA Citation
Wang, M, Khoo, YM, Zhou, J, Casey, P, and Lee, HS. "A high-performance liquid chromatography method for the quantification of cysmethynil, an inhibitor of isoprenylcysteine carboxylmethyl transferase, in mouse plasma." Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 877.5-6 (2009): 553-557.
PMID
19157999
Source
scival
Published In
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume
877
Issue
5-6
Publish Date
2009
Start Page
553
End Page
557
DOI
10.1016/j.jchromb.2008.12.067

Interacting targets of the farnesyl of transducin gamma-subunit

Authors
Katadae, M; Hagiwara, K; Wada, A; Ito, M; Umeda, M; Casey, PJ; Fukada, Y
MLA Citation
Katadae, M, Hagiwara, K, Wada, A, Ito, M, Umeda, M, Casey, PJ, and Fukada, Y. "Interacting targets of the farnesyl of transducin gamma-subunit." BIOCHEMISTRY 47.32 (August 12, 2008): 8424-8433.
PMID
18636747
Source
wos-lite
Published In
Biochemistry
Volume
47
Issue
32
Publish Date
2008
Start Page
8424
End Page
8433
DOI
10.1021/bi800359h

A small molecule inhibitor of isoprenylcysteine carboxymethyltransferase induces autophagic cell death in PC3 prostate cancer cells.

A number of proteins involved in cell growth control, including members of the Ras family of GTPases, are modified at their C terminus by a three-step posttranslational process termed prenylation. The enzyme isoprenylcysteine carboxylmethyl-transferase (Icmt) catalyzes the last step in this process, and genetic and pharmacological suppression of Icmt activity significantly impacts on cell growth and oncogenesis. Screening of a diverse chemical library led to the identification of a specific small molecule inhibitor of Icmt, cysmethynil, that inhibited growth factor signaling and tumorigenesis in an in vitro cancer cell model (Winter-Vann, A. M., Baron, R. A., Wong, W., dela Cruz, J., York, J. D., Gooden, D. M., Bergo, M. O., Young, S. G., Toone, E. J., and Casey, P. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4336-4341). To further evaluate the mechanisms through which this Icmt inhibitor impacts on cancer cells, we developed both in vitro and in vivo models utilizing PC3 prostate cancer cells. Treatment of these cells with cysmethynil resulted in both an accumulation of cells in the G(1) phase and cell death. Treatment of mice harboring PC3 cell-derived xenograft tumors with cysmethynil resulted in markedly reduced tumor size. Analysis of cell death pathways unexpectedly showed minimal impact of cysmethynil treatment on apoptosis; rather, drug treatment significantly enhanced autophagy and autophagic cell death. Cysmethynil-treated cells displayed reduced mammalian target of rapamycin (mTOR) signaling, providing a potential mechanism for the excessive autophagy as well as G(1) cell cycle arrest observed. These results identify a novel mechanism for the antitumor activity of Icmt inhibition. Further, the dual effects of cell death and cell cycle arrest by cysmethynil treatment strengthen the rationale for targeting Icmt in cancer chemotherapy.

Authors
Wang, M; Tan, W; Zhou, J; Leow, J; Go, M; Lee, HS; Casey, PJ
MLA Citation
Wang, M, Tan, W, Zhou, J, Leow, J, Go, M, Lee, HS, and Casey, PJ. "A small molecule inhibitor of isoprenylcysteine carboxymethyltransferase induces autophagic cell death in PC3 prostate cancer cells." J Biol Chem 283.27 (July 4, 2008): 18678-18684.
PMID
18434300
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
27
Publish Date
2008
Start Page
18678
End Page
18684
DOI
10.1074/jbc.M801855200

Effects of pharmacologic inhibition of protein geranylgeranyltransferase type I on aqueous humor outflow through the trabecular meshwork.

PURPOSE: To determine the effects of inhibition of protein geranylgeranyltransferase type I (GGTase-I), which isoprenylates so-called CaaX proteins, including the GTP-binding proteins such as Rho GTPases and the betagamma subunits of heterotrimeric G-proteins, on aqueous humor outflow and trabecular meshwork cytoskeletal integrity. METHODS: A selective small molecular inhibitor of GGTase-I, GGTI-DU40, was tested in this study to investigate its effects on actin cytoskeletal integrity, cell adhesions, cell-cell junctions, myosin II phosphosphorylation, and membrane localization of GTP-binding proteins in trabecular meshwork (TM) cells, using immunofluorescence detection and immunoblotting analysis. The effects of GGTI-DU40 on aqueous humor outflow were determined using organ-cultured, perfused anterior segments of porcine eyes. RESULTS: In the TM cell lysates, GGTI-DU40 was confirmed to inhibit GGTase-I activity in a dose-dependent manner. TM cells treated with GGTI-DU40 displayed dose-dependent changes in cell morphology and reversible decreases in actin stress fibers, focal adhesions, and adherens junctions. Myosin light chain phosphorylation was decreased significantly, and membrane localization of isoprenylated small GTPases and Gbetagamma was impaired in drug-treated TM cells. Aqueous outflow facility was increased significantly in eyes perfused with GGTI-DU40. CONCLUSIONS: These data demonstrate that inhibition of geranylgeranyl isoprenylation of CaaX proteins in the aqueous outflow pathway increases aqueous humor outflow, possibly through altered cell adhesive interactions and actin cytoskeletal organization in cells of the outflow pathway. This study indicates that the GGTase-I enzyme is a promising molecular target for lowering increased ocular pressure in glaucoma patients.

Authors
Rao, PV; Peterson, YK; Inoue, T; Casey, PJ
MLA Citation
Rao, PV, Peterson, YK, Inoue, T, and Casey, PJ. "Effects of pharmacologic inhibition of protein geranylgeranyltransferase type I on aqueous humor outflow through the trabecular meshwork." Invest Ophthalmol Vis Sci 49.6 (June 2008): 2464-2471.
PMID
18316706
Source
pubmed
Published In
Investigative Ophthalmology and Visual Science
Volume
49
Issue
6
Publish Date
2008
Start Page
2464
End Page
2471
DOI
10.1167/iovs.07-1639

Galphaz negatively regulates insulin secretion and glucose clearance.

Relatively little is known about the in vivo functions of the alpha subunit of the heterotrimeric G protein Gz (Galphaz). Clues to one potential function recently emerged with the finding that activation of Galphaz inhibits glucose-stimulated insulin secretion in an insulinoma cell line (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., Young, K. H., Fields, T. A., and Casey, P. J. (2005) J. Biol. Chem. 280, 31708-31713). To extend this study in vivo, a Galphaz knock-out mouse model was utilized to determine whether Galphaz function plays a role in the inhibition of insulin secretion. No differences were discovered in the gross morphology of the pancreatic islets or in the islet DNA, protein, or insulin content between Galphaz-null and wild-type mice. There was also no difference between the insulin sensitivity of Galphaz-null mice and wild-type controls, as measured by insulin tolerance tests. Galphaz-null mice did, however, display increased plasma insulin concentrations and a corresponding increase in glucose clearance following intraperitoneal and oral glucose challenge as compared with wild-type controls. The increased plasma insulin observed in Galphaz-null mice is most likely a direct result of enhanced insulin secretion, since pancreatic islets isolated from Galphaz-null mice exhibited significantly higher glucose-stimulated insulin secretion than those of wild-type mice. Finally, the increased insulin secretion observed in Galphaz-null islets appears to be due to the relief of a tonic inhibition of adenylyl cyclase, as cAMP production was significantly increased in Galphaz-null islets in the absence of exogenous stimulation. These findings indicate that Galphaz may be a potential new target for therapeutics aimed at ameliorating beta-cell dysfunction in Type 2 diabetes.

Authors
Kimple, ME; Joseph, JW; Bailey, CL; Fueger, PT; Hendry, IA; Newgard, CB; Casey, PJ
MLA Citation
Kimple, ME, Joseph, JW, Bailey, CL, Fueger, PT, Hendry, IA, Newgard, CB, and Casey, PJ. "Galphaz negatively regulates insulin secretion and glucose clearance." J Biol Chem 283.8 (February 22, 2008): 4560-4567.
PMID
18096703
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
8
Publish Date
2008
Start Page
4560
End Page
4567
DOI
10.1074/jbc.M706481200

Gαz negatively regulates insulin secretion and glucose clearance

Relatively little is known about the in vivo functions of the α subunit of the heterotrimeric G protein Gz (Gαz). Clues to one potential function recently emerged with the finding that activation of Gαz inhibits glucose-stimulated insulin secretion in an insulinoma cell line (Kimple, M. E., Nixon, A. B., Kelly, P., Bailey, C. L., Young, K. H., Fields, T. A., and Casey, P. J. (2005) J. Biol. Chem. 280, 31708-31713). To extend this study in vivo, a Gαz knock-out mouse model was utilized to determine whether Gαz function plays a role in the inhibition of insulin secretion. No differences were discovered in the gross morphology of the pancreatic islets or in the islet DNA, protein, or insulin content between Gαz-null and wild-type mice. There was also no difference between the insulin sensitivity of Gαz-null mice and wild-type controls, as measured by insulin tolerance tests. Gαz-null mice did, however, display increased plasma insulin concentrations and a corresponding increase in glucose clearance following intraperitoneal and oral glucose challenge as compared with wild-type controls. The increased plasma insulin observed in Gαz-null mice is most likely a direct result of enhanced insulin secretion, since pancreatic islets isolated from Gαz-null mice exhibited significantly higher glucose-stimulated insulin secretion than those of wild-type mice. Finally, the increased insulin secretion observed in Gαz-null islets appears to be due to the relief of a tonic inhibition of adenylyl cyclase, as cAMP production was significantly increased in Gαz-null islets in the absence of exogenous stimulation. These findings indicate that Gαz may be a potential new target for therapeutics aimed at ameliorating β-cell dysfunction in Type 2 diabetes. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Kimple, ME; Joseph, JW; Bailey, CL; Fueger, PT; Hendry, IA; Newgard, CB; Casey, PJ
MLA Citation
Kimple, ME, Joseph, JW, Bailey, CL, Fueger, PT, Hendry, IA, Newgard, CB, and Casey, PJ. "Gαz negatively regulates insulin secretion and glucose clearance." Journal of Biological Chemistry 283.8 (February 22, 2008): 4560-4567.
Source
scopus
Published In
The Journal of biological chemistry
Volume
283
Issue
8
Publish Date
2008
Start Page
4560
End Page
4567
DOI
10.1074/jbc.M706481200

A global partnership in medical education between Duke University and the National University of Singapore.

Duke University and the National University of Singapore (NUS) have partnered to launch a new medical school that brings the American style of postbaccalaureate medical education to Asia. The new institution, called the Duke-NUS Graduate Medical School (GMS) and located in Singapore adjacent to the Singapore General Hospital, admitted its inaugural class of students representing citizens of seven nations in August 2007. The project represents an investment of more than $350 million from three ministries of the Singapore government, and a commitment on Duke's part to provide senior leadership and recruit faculty from Duke, from other international locales, and from within Singapore itself. Graduating students who complete the four-year Duke curriculum will receive an MD degree awarded jointly by Duke and NUS, thereby distinguishing this school from medical education in most Asian institutions that award an MBBS degree after a five-year period of study that follows directly from secondary school. The emphasis of the Duke-NUS GMS is to prepare physician-scientists for academic careers, with plans for 20% of each class to complete a combined MD/PhD degree. This article describes events leading up to this partnership and details of the relationship, including curriculum, organizational structure, milestones, and goals.

Authors
Williams, RS; Casey, PJ; Kamei, RK; Buckley, EG; Soo, KC; Merson, MH; Krishnan, RK; Dzau, VJ
MLA Citation
Williams, RS, Casey, PJ, Kamei, RK, Buckley, EG, Soo, KC, Merson, MH, Krishnan, RK, and Dzau, VJ. "A global partnership in medical education between Duke University and the National University of Singapore." Acad Med 83.2 (February 2008): 122-127.
PMID
18303355
Source
pubmed
Published In
Academic Medicine
Volume
83
Issue
2
Publish Date
2008
Start Page
122
End Page
127
DOI
10.1097/ACM.0b013e318160b8bc

beta-Catenin is a Nek2 substrate involved in centrosome separation

Authors
Bahmanyar, S; Kaplan, DD; DeLuca, JG; Jr, GTH; O'Toole, ET; Winey, M; Salmon, ED; Casey, PJ; Nelson, WJ; Barth, AIM
MLA Citation
Bahmanyar, S, Kaplan, DD, DeLuca, JG, Jr, GTH, O'Toole, ET, Winey, M, Salmon, ED, Casey, PJ, Nelson, WJ, and Barth, AIM. "beta-Catenin is a Nek2 substrate involved in centrosome separation." GENES & DEVELOPMENT 22.1 (January 1, 2008): 91-105.
PMID
18086858
Source
wos-lite
Published In
Genes & development
Volume
22
Issue
1
Publish Date
2008
Start Page
91
End Page
105
DOI
10.1101/gad.1596308

Biologic functions of the G12 subfamily of heterotrimeric g proteins: growth, migration, and metastasis.

The G12 subfamily of heterotrimeric G proteins has been the subject of intense scientific interest for more than 15 years. During this period, studies have revealed more than 20 potential G12-interacting proteins and numerous signaling axes emanating from the G12 proteins, Galpha12 and Galpha13. In addition, more recent studies have begun to illuminate the various and sundry functions that the G12 subfamily plays in biology. In this review, we summarize the diverse range of proteins that have been identified as Galpha12 and/or Galpha13 interactors and describe ongoing studies designed to dissect the biological roles of specific Galpha-effector protein interactions. Further, we describe and discuss the expanding role of G12 proteins in the biology of cells, focusing on the distinct properties of this subfamily in regulating cell proliferation, cell migration, and metastatic invasion.

Authors
Kelly, P; Casey, PJ; Meigs, TE
MLA Citation
Kelly, P, Casey, PJ, and Meigs, TE. "Biologic functions of the G12 subfamily of heterotrimeric g proteins: growth, migration, and metastasis." Biochemistry 46.23 (June 12, 2007): 6677-6687. (Review)
PMID
17503779
Source
pubmed
Published In
Biochemistry
Volume
46
Issue
23
Publish Date
2007
Start Page
6677
End Page
6687
DOI
10.1021/bi700235f

GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer

Authors
Sjogren, A-KM; Andersson, KME; Liu, M; Cutts, BA; Karlsson, C; Wahlstrom, AM; Dalin, M; Weinbaum, C; Casey, PJ; Tarkowski, A; Swolin, B; Young, SG; Bergo, MO
MLA Citation
Sjogren, A-KM, Andersson, KME, Liu, M, Cutts, BA, Karlsson, C, Wahlstrom, AM, Dalin, M, Weinbaum, C, Casey, PJ, Tarkowski, A, Swolin, B, Young, SG, and Bergo, MO. "GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer." JOURNAL OF CLINICAL INVESTIGATION 117.5 (May 2007): 1294-1304.
PMID
17476360
Source
wos-lite
Published In
Journal of Clinical Investigation
Volume
117
Issue
5
Publish Date
2007
Start Page
1294
End Page
1304
DOI
10.1172/JCI30868

Quantitative structure-activity relationship (QSAR) of indoloacetamides as inhibitors of human isoprenylcysteine carboxyl methyltransferase

Authors
Leow, J-L; Baron, R; Casey, PJ; Go, M-L
MLA Citation
Leow, J-L, Baron, R, Casey, PJ, and Go, M-L. "Quantitative structure-activity relationship (QSAR) of indoloacetamides as inhibitors of human isoprenylcysteine carboxyl methyltransferase." BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 17.4 (February 15, 2007): 1025-1032.
PMID
17157012
Source
wos-lite
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
17
Issue
4
Publish Date
2007
Start Page
1025
End Page
1032
DOI
10.1016/j.bmcl.2006.11.030

Time-dependent inhibition of isoprenylcysteine carboxyl methyltransferase by indole-based small molecules.

Isoprenylcysteine carboxyl methyltransferase (Icmt) catalyzes the methylation of the C-terminal prenylcysteine found on prenylated proteins. Numerous studies have shown that the methylation step is important for the correct localization and function of many prenylated proteins, most notably GTPases in the Ras superfamily. We recently reported identification of a small molecule derived from an indole core as a potent, cell-active inhibitor of Icmt whose potency was increased upon preincubation with the enzyme [Winter-Vann, A. M., Baron, R. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102 (12), 4336-41]. In the study presented here, we performed a kinetic characterization of this time-dependent inhibition of Icmt by 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). These analyses revealed that cysmethynil is a competitive inhibitor with respect to the isoprenylated cysteine substrate and a noncompetitive inhibitor with respect to AdoMet, the methyl donor in the reaction. The Ki of cysmethynil for Icmt, which represents the dissociation constant of the initial complex with the enzyme, was 2.39 +/- 0.02 microM, and the Ki*, which is the overall dissociation constant of the inhibitor for the final complex, was 0.14 +/- 0.01 microM. The first-order rate constant for the conversion of the initial enzyme-inhibitor complex to the final high-affinity complex was 0.87 +/- 0.06 min-1, and that for the reverse process was 0.053 +/- 0.003 min-1; the latter rate constant corresponds to a half-life for the high-affinity complex of 15 min. Structure-activity relationships of a number of closely related indole compounds revealed that the hydrophobicity of the substituent on the nitrogen of the indole core was responsible for the manifestation of time-dependent inhibition. These findings markedly enhance our understanding of the mechanism of inhibition of Icmt by this indole class of compounds and should facilitate ongoing efforts to assess the potential of targeting this enzyme in anticancer drug design.

Authors
Baron, RA; Peterson, YK; Otto, JC; Rudolph, J; Casey, PJ
MLA Citation
Baron, RA, Peterson, YK, Otto, JC, Rudolph, J, and Casey, PJ. "Time-dependent inhibition of isoprenylcysteine carboxyl methyltransferase by indole-based small molecules." Biochemistry 46.2 (January 16, 2007): 554-560.
PMID
17209565
Source
pubmed
Published In
Biochemistry
Volume
46
Issue
2
Publish Date
2007
Start Page
554
End Page
560
DOI
10.1021/bi060344n

The regulator of G protein signaling domain of axin selectively interacts with Galpha12 but not Galpha13.

Axin, a negative regulator of the Wnt signaling pathway, contains a canonical regulator of G protein signaling (RGS) core domain. Herein, we demonstrate both in vitro and in cells that this domain interacts with the alpha subunit of the heterotrimeric G protein G12 but not with the closely related Galpha13 or with several other heterotrimeric G proteins. Axin preferentially binds the activated form of Galpha12, a behavior consistent with other RGS proteins. However, unlike other RGS proteins, that of axin (axinRGS) does not affect intrinsic GTP hydrolysis by Galpha12. Despite its inability to act as a GTPase-activating protein, we demonstrate that in cells, axinRGS can compete for Galpha12 binding with the RGS domain of p115RhoGEF, a known G12-interacting protein that links G12 signaling to activation of the small G protein Rho. Moreover, ectopic expression of axinRGS specifically inhibits Galpha12-directed activation of the Rho pathway in MDA-MB 231 breast cancer cells. These findings establish that the RGS domain of axin is able to directly interact with the alpha subunit of heterotrimeric G protein G12 and provide a unique tool to interdict Galpha12-mediated signaling processes.

Authors
Stemmle, LN; Fields, TA; Casey, PJ
MLA Citation
Stemmle, LN, Fields, TA, and Casey, PJ. "The regulator of G protein signaling domain of axin selectively interacts with Galpha12 but not Galpha13." Mol Pharmacol 70.4 (October 2006): 1461-1468.
PMID
16868183
Source
pubmed
Published In
Molecular pharmacology
Volume
70
Issue
4
Publish Date
2006
Start Page
1461
End Page
1468
DOI
10.1124/mol.106.023705

A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion.

Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) in oncogenesis and tumor cell growth. However, few studies have examined G12 signaling in actual human cancers. In this study, we examined the role of G12 signaling in prostate cancer. We found that expression of the G12 proteins is significantly elevated in prostate cancer. Interestingly, expression of the activated forms of Galpha12 or Galpha13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Galpha12 or Galpha13 in these cell lines induced cell invasion through the activation of the RhoA family of G proteins. Furthermore, inhibition of G12 signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) in the PC3 and DU145 cell lines did not reduce cancer cell growth. However, inhibition of G12 signaling with p115-RGS in these cell lines blocked thrombin- and thromboxane A2-stimulated cell invasion. These observations identify the G12 family proteins as important regulators of prostate cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced prostate cancer patient mortality.

Authors
Kelly, P; Stemmle, LN; Madden, JF; Fields, TA; Daaka, Y; Casey, PJ
MLA Citation
Kelly, P, Stemmle, LN, Madden, JF, Fields, TA, Daaka, Y, and Casey, PJ. "A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion." J Biol Chem 281.36 (September 8, 2006): 26483-26490.
PMID
16787920
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
36
Publish Date
2006
Start Page
26483
End Page
26490
DOI
10.1074/jbc.M604376200

Conversion of protein farnesyltransferase to a geranylgeranyltransferase.

Posttranslational modifications are essential for the proper function of a number of proteins in the cell. One such modification, the covalent attachment of a single isoprenoid lipid (prenylation), is carried out by the CaaX prenyltransferases, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type-I (GGTase-I). Substrate proteins of these two enzymes are involved in a variety of cellular functions but are largely associated with signal transduction. These modified proteins include members of the Ras superfamily, heterotrimeric G-proteins, centromeric proteins, and a number of proteins involved in nuclear integrity. Although FTase and GGTase-I are highly homologous, they are quite selective for their substrates, particularly for their isoprenoid diphosphate substrates, FPP and GGPP, respectively. Here, we present both crystallographic and kinetic analyses of mutants designed to explore this isoprenoid specificity and demonstrate that this specificity is dependent upon two enzyme residues in the beta subunits of the enzymes, W102beta and Y365beta in FTase (T49beta and F324beta, respectively, in GGTase-I).

Authors
Terry, KL; Casey, PJ; Beese, LS
MLA Citation
Terry, KL, Casey, PJ, and Beese, LS. "Conversion of protein farnesyltransferase to a geranylgeranyltransferase." Biochemistry 45.32 (August 15, 2006): 9746-9755.
PMID
16893176
Source
pubmed
Published In
Biochemistry
Volume
45
Issue
32
Publish Date
2006
Start Page
9746
End Page
9755
DOI
10.1021/bi060295e

The G12 family of heterotrimeric G proteins promotes breast cancer invasion and metastasis.

Although the prognosis for patients with early-stage breast cancer has improved, the therapeutic options for patients with locally advanced and metastatic disease are limited. To improve the treatment of these patients, the molecular mechanisms underlying breast cancer invasion and metastasis must be understood. In this study, we report that signaling through the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) promotes breast cancer cell invasion. Moreover, we demonstrate that inhibition of G12 signaling reduces the metastatic dissemination of breast cancer cells in vivo. Finally, we demonstrate that the expression of Galpha12 is significantly up-regulated in the earliest stages of breast cancer, implying that amplification of G12 signaling may be an early event in breast cancer progression. Taken together, these observations identify the G12 family proteins as important regulators of breast cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced patient morbidity and mortality.

Authors
Kelly, P; Moeller, BJ; Juneja, J; Booden, MA; Der, CJ; Daaka, Y; Dewhirst, MW; Fields, TA; Casey, PJ
MLA Citation
Kelly, P, Moeller, BJ, Juneja, J, Booden, MA, Der, CJ, Daaka, Y, Dewhirst, MW, Fields, TA, and Casey, PJ. "The G12 family of heterotrimeric G proteins promotes breast cancer invasion and metastasis." Proc Natl Acad Sci U S A 103.21 (May 23, 2006): 8173-8178.
PMID
16705036
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
103
Issue
21
Publish Date
2006
Start Page
8173
End Page
8178
DOI
10.1073/pnas.0510254103

A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity.

Inhibiting protein prenylation is an attractive means to modulate cellular processes controlled by a variety of signaling proteins, including oncogenic proteins such as Ras and Rho GTPases. The largest class of prenylated proteins contain a so-called CaaX motif at their carboxyl termini and are subject to a maturation process initiated by the attachment of an isoprenoid lipid by either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I). Inhibitors of FTase, termed FTIs, have been the subject of intensive development in the past decade and have shown efficacy in clinical trials. Although GGTase-I inhibitors (GGTIs) have received less attention, accumulating evidence suggests GGTIs may augment therapies using FTIs and could be useful to treat a myriad of additional disease states. Here we describe the characterization of a selective, highly potent, and cell-active GGTase-I inhibitor, GGTI-DU40. Kinetic analysis revealed that inhibition by GGTI-DU40 is competitive with the protein substrate and uncompetitive with the isoprenoid substrate; the Ki for the inhibition is 0.8 nM. GGTI-DU40 is highly selective for GGTase-I both in vitro and in living cells. Studies indicate GGTI-DU40 blocks prenylation of a number of geranylgeranylated CaaX proteins. Treatment of MDA-MB-231 breast cancer cells with GGTI-DU40 inhibited thrombin-induced cell rounding via a process that involves inhibition of Rho proteins without significantly effecting parallel mobilization of calcium via Gbetagamma. These studies establish GGTI-DU40 as a prime tool for interrogating biologies associated with protein geranylgeranylation and define a novel structure for this emerging class of experimental therapeutics.

Authors
Peterson, YK; Kelly, P; Weinbaum, CA; Casey, PJ
MLA Citation
Peterson, YK, Kelly, P, Weinbaum, CA, and Casey, PJ. "A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity." J Biol Chem 281.18 (May 5, 2006): 12445-12450.
PMID
16517596
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
18
Publish Date
2006
Start Page
12445
End Page
12450
DOI
10.1074/jbc.M600168200

Isoprenylcysteine carboxylmethyltransferase as a target for development of novel cancer therapeutics

Authors
Wang, M; Casey, PJ
MLA Citation
Wang, M, and Casey, PJ. "Isoprenylcysteine carboxylmethyltransferase as a target for development of novel cancer therapeutics." DRUGS OF THE FUTURE 31.5 (May 2006): 437-445.
Source
wos-lite
Published In
Drugs of the future
Volume
31
Issue
5
Publish Date
2006
Start Page
437
End Page
445
DOI
10.1358/dof.2006.031.05.994707

Genetic and pharmacologic analyses of the role of Icmt in Ras membrane association and function

Authors
Svensson, AW; Casey, PJ; Young, SG; Bergo, MO
MLA Citation
Svensson, AW, Casey, PJ, Young, SG, and Bergo, MO. "Genetic and pharmacologic analyses of the role of Icmt in Ras membrane association and function." REGULATORS AND EFFECTORS OF SMALL GTPASES: RAS FAMILY 407 (2006): 144-159.
PMID
16757321
Source
wos-lite
Published In
Methods in Enzymology
Volume
407
Publish Date
2006
Start Page
144
End Page
159
DOI
10.1016/S0076-6879(05)07013-8

A role for G(z) in pancreatic islet beta-cell biology.

Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function.

Authors
Kimple, ME; Nixon, AB; Kelly, P; Bailey, CL; Young, KH; Fields, TA; Casey, PJ
MLA Citation
Kimple, ME, Nixon, AB, Kelly, P, Bailey, CL, Young, KH, Fields, TA, and Casey, PJ. "A role for G(z) in pancreatic islet beta-cell biology." J Biol Chem 280.36 (September 9, 2005): 31708-31713.
PMID
16157560
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
36
Publish Date
2005
Start Page
31708
End Page
31713
DOI
10.1074/jbc.M506700200

Involvement of a mitochondrial phosphatase in the regulation of ATP production and insulin secretion in pancreatic beta cells.

Reversible phosphorylation is the cell's most prevalent form of posttranslational modification, yet its role in the regulation of mitochondrial functions is poorly understood. We have discovered that a member of the dual-specific protein tyrosine phosphatase (DS-PTP) family, PTPMT1 (PTP localized to the Mitochondrion 1) resides nearly exclusively in mitochondria. PTPMT1 is targeted to the mitochondrion by an N-terminal signal sequence and is found anchored to the matrix face of the inner membrane. Knockdown of PTPMT1 expression in the pancreatic insulinoma cell line INS-1 832/13 alters the mitochondrial phosphoprotein profile and markedly enhances both ATP production and insulin secretion. These data define PTPMT1 as a potential drug target for the treatment of type II diabetes and strengthen the notion that mitochondria are an underappreciated site of signaling by reversible phosphorylation.

Authors
Pagliarini, DJ; Wiley, SE; Kimple, ME; Dixon, JR; Kelly, P; Worby, CA; Casey, PJ; Dixon, JE
MLA Citation
Pagliarini, DJ, Wiley, SE, Kimple, ME, Dixon, JR, Kelly, P, Worby, CA, Casey, PJ, and Dixon, JE. "Involvement of a mitochondrial phosphatase in the regulation of ATP production and insulin secretion in pancreatic beta cells." Mol Cell 19.2 (July 22, 2005): 197-207.
PMID
16039589
Source
pubmed
Published In
Molecular Cell
Volume
19
Issue
2
Publish Date
2005
Start Page
197
End Page
207
DOI
10.1016/j.molcel.2005.06.008

Selective uncoupling of G alpha 12 from Rho-mediated signaling.

The heterotrimeric G protein G(12) has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated alpha-subunit of G(12) has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G(12)-mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G(12) activation, we produced a series of substitution mutants in the regions of Galpha(12) predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of Galpha(12) that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several Galpha(12)-mediated signals. This mutant of Galpha(12) fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of Galpha(12) retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger beta-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of Galpha(12) that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G(12) proteins mediate diverse biological responses.

Authors
Meigs, TE; Juneja, J; DeMarco, CT; Stemmle, LN; Kaplan, DD; Casey, PJ
MLA Citation
Meigs, TE, Juneja, J, DeMarco, CT, Stemmle, LN, Kaplan, DD, and Casey, PJ. "Selective uncoupling of G alpha 12 from Rho-mediated signaling." J Biol Chem 280.18 (May 6, 2005): 18049-18055.
PMID
15746095
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
18
Publish Date
2005
Start Page
18049
End Page
18055
DOI
10.1074/jbc.M500445200

Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development.

Protein farnesyltransferase (FTase) is an enzyme responsible for posttranslational modification of proteins carrying a carboxy-terminal CaaX motif. Farnesylation allows substrates to interact with membranes and protein targets. Using gene-targeted mice, we report that FTase is essential for embryonic development, but dispensable for adult homeostasis. Six-month-old FTase-deficient mice display delayed wound healing and maturation defects in erythroid cells. Embryonic fibroblasts lacking FTase have a flat morphology and reduced motility and proliferation rates. Ablation of FTase in two ras oncogene-dependent tumor models has no significant consequences for tumor initiation. However, elimination of FTase during tumor progression had a limited but significant inhibitory effect. These results should help to better understand the role of protein farnesylation in normal tissues and in tumor development.

Authors
Mijimolle, N; Velasco, J; Dubus, P; Guerra, C; Weinbaum, CA; Casey, PJ; Campuzano, V; Barbacid, M
MLA Citation
Mijimolle, N, Velasco, J, Dubus, P, Guerra, C, Weinbaum, CA, Casey, PJ, Campuzano, V, and Barbacid, M. "Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development." Cancer Cell 7.4 (April 2005): 313-324.
PMID
15837621
Source
pubmed
Published In
Cancer Cell
Volume
7
Issue
4
Publish Date
2005
Start Page
313
End Page
324
DOI
10.1016/j.ccr.2005.03.004

Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function.

The passage of leukocytes out of the blood circulation and into tissues is necessary for the normal inflammatory response, but it also occurs inappropriately in many pathological situations. This process is limited by the barrier presented by the junctions between adjacent endothelial cells that line blood vessels. Here we show that activation of the Rap1 GTPase in endothelial cells accelerated de novo assembly of endothelial cell-cell junctions and increased the barrier function of endothelial monolayers. In contrast, depressing Rap1 activity by expressing Rap1GAP led to disassembly of these junctions and increased their permeability. We also demonstrate that endogenous Rap1 was rapidly activated at early stages of junctional assembly, confirming the involvement of Rap1 during junctional assembly. Intriguingly, elevating Rap1 activity selectively within endothelial cells decreased leukocyte transendothelial migration, whereas inhibiting Rap1 activity by expression of Rap1GAP increased leukocyte transendothelial migration, providing physiological relevance to our hypothesis that Rap1 augments barrier function of inter-endothelial cell junctions. Furthermore, these results suggest that Rap1 may be a novel therapeutic target for clinical conditions in which an inappropriate inflammatory response leads to disease.

Authors
Wittchen, ES; Worthylake, RA; Kelly, P; Casey, PJ; Quilliam, LA; Burridge, K
MLA Citation
Wittchen, ES, Worthylake, RA, Kelly, P, Casey, PJ, Quilliam, LA, and Burridge, K. "Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function." J Biol Chem 280.12 (March 25, 2005): 11675-11682.
PMID
15661741
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
12
Publish Date
2005
Start Page
11675
End Page
11682
DOI
10.1074/jbc.M412595200

Androgen receptor activation by G(s) signaling in prostate cancer cells.

The androgen receptor (AR) is activated in prostate cancer patients undergoing androgen ablative therapy and mediates growth of androgen-insensitive prostate cancer cells, suggesting it is activated by nonandrogenic factors. We demonstrate that activated alpha subunit of heterotrimeric guanine nucleotide-binding G(s) protein activates the AR in prostate cancer cells and also synergizes with low concentration of androgen to more fully activate the AR. The G alpha(s) activates protein kinase A, which is required for the nuclear partition and activation of AR. These data suggest a role for G alpha(s) and PKA in the transactivation of AR in prostate cancer cells under the environment of reduced androgen levels.

Authors
Kasbohm, EA; Guo, R; Yowell, CW; Bagchi, G; Kelly, P; Arora, P; Casey, PJ; Daaka, Y
MLA Citation
Kasbohm, EA, Guo, R, Yowell, CW, Bagchi, G, Kelly, P, Arora, P, Casey, PJ, and Daaka, Y. "Androgen receptor activation by G(s) signaling in prostate cancer cells." J Biol Chem 280.12 (March 25, 2005): 11583-11589.
PMID
15653681
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
12
Publish Date
2005
Start Page
11583
End Page
11589
DOI
10.1074/jbc.M414423200

A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells.

Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of prenylation have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Recent genetic disruption studies have provided strong evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Here, we report the identification of a selective small-molecule inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). Cysmethynil treatment results in inhibition of cell growth in an Icmt-dependent fashion, demonstrating mechanism-based activity of the compound. Treatment of cancer cells with cysmethynil results in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment blocks anchorage-independent growth, and this effect is reversed by overexpression of Icmt. These findings provide a compelling rationale for development of Icmt inhibitors as another approach to anticancer drug development.

Authors
Winter-Vann, AM; Baron, RA; Wong, W; dela Cruz, J; York, JD; Gooden, DM; Bergo, MO; Young, SG; Toone, EJ; Casey, PJ
MLA Citation
Winter-Vann, AM, Baron, RA, Wong, W, dela Cruz, J, York, JD, Gooden, DM, Bergo, MO, Young, SG, Toone, EJ, and Casey, PJ. "A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells." Proc Natl Acad Sci U S A 102.12 (March 22, 2005): 4336-4341.
PMID
15784746
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
12
Publish Date
2005
Start Page
4336
End Page
4341
DOI
10.1073/pnas.0408107102

Pertussis-toxin-sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex.

Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.

Authors
Clancy, SM; Fowler, CE; Finley, M; Suen, KF; Arrabit, C; Berton, F; Kosaza, T; Casey, PJ; Slesinger, PA
MLA Citation
Clancy, SM, Fowler, CE, Finley, M, Suen, KF, Arrabit, C, Berton, F, Kosaza, T, Casey, PJ, and Slesinger, PA. "Pertussis-toxin-sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex." Mol Cell Neurosci 28.2 (February 2005): 375-389.
PMID
15691717
Source
pubmed
Published In
Molecular and Cellular Neuroscience
Volume
28
Issue
2
Publish Date
2005
Start Page
375
End Page
389
DOI
10.1016/j.mcn.2004.10.009

Analysis of the kinetic mechanism of recombinant human isoprenylcysteine carboxylmethyltransferase (Icmt).

BACKGROUND: Isoprenylcysteine carboxyl methyltransferase (Icmt) is the third of three enzymes that posttranslationally modify proteins that contain C-terminal CaaX motifs. The processing of CaaX proteins through this so-called prenylation pathway via a route initiated by addition of an isoprenoid lipid is required for both membrane targeting and function of the proteins. The involvement of many CaaX proteins such as Ras GTPases in oncogenesis and other aberrant proliferative disorders has led to the targeting of the enzymes involved in their processing for therapeutic development, necessitating a detailed understanding of the mechanisms of the enzymes. RESULTS: In this study, we have investigated the kinetic mechanism of recombinant human Icmt. In the reaction catalyzed by Icmt, S-adenosyl-L-methionine (AdoMet) provides the methyl group that is transferred to the second substrate, the C-terminal isoprenylated cysteine residue of a CaaX protein, thereby generating a C-terminal prenylcysteine methyl ester on the protein. To facilitate the kinetic analysis of Icmt, we synthesized a new small molecule substrate of the enzyme, biotin-S-farnesyl-L-cysteine (BFC). Initial kinetic analysis of Icmt suggested a sequential mechanism for the enzyme that was further analyzed using a dead end competitive inhibitor, S-farnesylthioacetic acid (FTA). Inhibition by FTA was competitive with respect to BFC and uncompetitive with respect to AdoMet, indicating an ordered mechanism with SAM binding first. To investigate the order of product dissociation, product inhibition studies were undertaken with S-adenosyl-L-homocysteine (AdoHcy) and the N-acetyl-S-farnesyl-L-cysteine methylester (AFCME). This analysis indicated that AdoHcy is a competitive inhibitor with respect to AdoMet, while AFCME shows a noncompetitive inhibition with respect to BFC and a mixed-type inhibition with respect to AdoMet. These studies established that AdoHcy is the final product released, and that BFC and AFCME bind to different forms of the enzyme. CONCLUSIONS: These studies establish that catalysis by human Icmt proceeds through an ordered sequential mechanism and provide a kinetic framework for analysis of specific inhibitors of this key enzyme.

Authors
Baron, RA; Casey, PJ
MLA Citation
Baron, RA, and Casey, PJ. "Analysis of the kinetic mechanism of recombinant human isoprenylcysteine carboxylmethyltransferase (Icmt). (Published online)" BMC Biochem 5 (December 29, 2004): 19-.
PMID
15625008
Source
pubmed
Published In
BMC Biochemistry
Volume
5
Publish Date
2004
Start Page
19
DOI
10.1186/1471-2091-5-19

Reciprocal signaling between the transcriptional co-factor Eya2 and specific members of the Galphai family.

As part of a program to elucidate signaling processes controlled by the heterotrimeric G protein Galphaz, a human fetal brain cDNA library was screened for proteins that specifically interact with the activated form of Galphaz. One of the most-encountered molecules in this screen was Eya2, a member of the Eyes absent family of proteins. Mammalian Eya proteins are predominantly cytosolic proteins that are known to interact with members of the Sine oculis (Six) family of homeodomain transcription factors. This interaction facilitates the translocation of Eya into the nucleus, where the Eya/Six complex regulates transcription during critical stages of embryonic development. In vitro binding studies confirmed that Galphaz interacts with Eya2 in an activation-dependent fashion; furthermore, most other members of the Galphai family including Galphai1, Galphai2, and Galphai3 were found to interact with Eya2. It is interesting that one of the most abundant Galphai proteins, Galphao, did not interact with Eya2. Coexpression of the activated forms of Galphai1, Galphai2, and Galphai3, but not Galphao, with Eya2 recruited Eya2 to the plasma membrane, prevented Eya2 translocation into the nucleus, and abrogated Eya2/Six4-mediated transcription. In addition, Eya2 impinged on G protein-mediated signaling, as evidenced by its ability to relieve Galphai2-mediated inhibition of adenylyl cyclase. These results demonstrate that the interaction between the Galphai proteins and Eya2 may impact on seemingly disparate regulatory events involving both classes of proteins.

Authors
Embry, AC; Glick, JL; Linder, ME; Casey, PJ
MLA Citation
Embry, AC, Glick, JL, Linder, ME, and Casey, PJ. "Reciprocal signaling between the transcriptional co-factor Eya2 and specific members of the Galphai family." Mol Pharmacol 66.5 (November 2004): 1325-1331.
PMID
15308761
Source
pubmed
Published In
Molecular pharmacology
Volume
66
Issue
5
Publish Date
2004
Start Page
1325
End Page
1331
DOI
10.1124/mol.104.004093

Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity.

Post-translational modifications are essential for the proper function of many proteins in the cell. The attachment of an isoprenoid lipid (a process termed prenylation) by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) is essential for the function of many signal transduction proteins involved in growth, differentiation, and oncogenesis. FTase and GGTase-I (also called the CaaX prenyltransferases) recognize protein substrates with a C-terminal tetrapeptide recognition motif called the Ca1a2X box. These enzymes possess distinct but overlapping protein substrate specificity that is determined primarily by the sequence identity of the Ca1a2X motif. To determine how the identity of the Ca1a2X motif residues and sequence upstream of this motif affect substrate binding, we have solved crystal structures of FTase and GGTase-I complexed with a total of eight cognate and cross-reactive substrate peptides, including those derived from the C termini of the oncoproteins K-Ras4B, H-Ras and TC21. These structures suggest that all peptide substrates adopt a common binding mode in the FTase and GGTase-I active site. Unexpectedly, while the X residue of the Ca1a2X motif binds in the same location for all GGTase-I substrates, the X residue of FTase substrates can bind in one of two different sites. Together, these structures outline a series of rules that govern substrate peptide selectivity; these rules were utilized to classify known protein substrates of CaaX prenyltransferases and to generate a list of hypothetical substrates within the human genome.

Authors
Reid, TS; Terry, KL; Casey, PJ; Beese, LS
MLA Citation
Reid, TS, Terry, KL, Casey, PJ, and Beese, LS. "Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity." J Mol Biol 343.2 (October 15, 2004): 417-433.
PMID
15451670
Source
pubmed
Published In
Journal of Molecular Biology
Volume
343
Issue
2
Publish Date
2004
Start Page
417
End Page
433
DOI
10.1016/j.jmb.2004.08.056

Identification of a role for beta-catenin in the establishment of a bipolar mitotic spindle.

beta-Catenin is a multifunctional protein that is known to participate in two well defined cellular processes, cell-cell adhesion and Wnt-stimulated transcriptional activation. Here we report that beta-catenin participates in a third cellular process, the establishment of a bipolar mitotic spindle. During mitosis, beta-catenin relocalizes to mitotic spindle poles and to the midbody. Furthermore, biochemical fractionation demonstrates the presence of beta-catenin in purified centrosome preparations. Reduction of cellular beta-catenin by RNA interference leads to the failure of centrosomes to fully separate, resulting in a marked increase in the frequency of monoastral mitotic spindles. Our results define a new and important function for beta-catenin in mitosis and demonstrate that beta-catenin is involved in vital biological processes beyond cell adhesion and Wnt signaling.

Authors
Kaplan, DD; Meigs, TE; Kelly, P; Casey, PJ
MLA Citation
Kaplan, DD, Meigs, TE, Kelly, P, and Casey, PJ. "Identification of a role for beta-catenin in the establishment of a bipolar mitotic spindle." J Biol Chem 279.12 (March 19, 2004): 10829-10832.
PMID
14744872
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
12
Publish Date
2004
Start Page
10829
End Page
10832
DOI
10.1074/jbc.C400035200

On the physiological importance of endoproteolysis of CAAX proteins: heart-specific RCE1 knockout mice develop a lethal cardiomyopathy.

Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by Rce1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking Rce1 in the heart, an organ where Rce1 is expressed at particularly high levels. The hearts from heart-specific Rce1 knockout mice manifested reduced levels of both the Rce1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific Rce1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, approximately 70% of the mice had died. Pathological studies revealed that the heart-specific Rce1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific Rce1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.

Authors
Bergo, MO; Lieu, HD; Gavino, BJ; Ambroziak, P; Otto, JC; Casey, PJ; Walker, QM; Young, SG
MLA Citation
Bergo, MO, Lieu, HD, Gavino, BJ, Ambroziak, P, Otto, JC, Casey, PJ, Walker, QM, and Young, SG. "On the physiological importance of endoproteolysis of CAAX proteins: heart-specific RCE1 knockout mice develop a lethal cardiomyopathy." J Biol Chem 279.6 (February 6, 2004): 4729-4736.
PMID
14625273
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
6
Publish Date
2004
Start Page
4729
End Page
4736
DOI
10.1074/jbc.M310081200

Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf.

Isoprenylcysteine carboxyl methyltransferase (Icmt) methylates the carboxyl-terminal isoprenylcysteine of CAAX proteins (e.g., Ras and Rho proteins). In the case of the Ras proteins, carboxyl methylation is important for targeting of the proteins to the plasma membrane. We hypothesized that a knockout of Icmt would reduce the ability of cells to be transformed by K-Ras. Fibroblasts harboring a floxed Icmt allele and expressing activated K-Ras (K-Ras-Icmt(flx/flx)) were treated with Cre-adenovirus, producing K-Ras-Icmt(Delta/Delta) fibroblasts. Inactivation of Icmt inhibited cell growth and K-Ras-induced oncogenic transformation, both in soft agar assays and in a nude mice model. The inactivation of Icmt did not affect growth factor-stimulated phosphorylation of Erk1/2 or Akt1. However, levels of RhoA were greatly reduced as a consequence of accelerated protein turnover. In addition, there was a large Ras/Erk1/2-dependent increase in p21(Cip1), which was probably a consequence of the reduced levels of RhoA. Deletion of p21(Cip1) restored the ability of K-Ras-Icmt(Delta/Delta) fibroblasts to grow in soft agar. The effect of inactivating Icmt was not limited to the inhibition of K-Ras-induced transformation: inactivation of Icmt blocked transformation by an oncogenic form of B-Raf (V599E). These studies identify Icmt as a potential target for reducing the growth of K-Ras- and B-Raf-induced malignancies.

Authors
Bergo, MO; Gavino, BJ; Hong, C; Beigneux, AP; McMahon, M; Casey, PJ; Young, SG
MLA Citation
Bergo, MO, Gavino, BJ, Hong, C, Beigneux, AP, McMahon, M, Casey, PJ, and Young, SG. "Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf." J Clin Invest 113.4 (February 2004): 539-550.
PMID
14966563
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
113
Issue
4
Publish Date
2004
Start Page
539
End Page
550
DOI
10.1172/JCI18829

Analysis of the molecular interaction of the farnesyl moiety of transducin through the use of a photoreactive farnesyl analogue.

Farnesylation of the gamma-subunit of the retinal G-protein, transducin (Talpha/Tbetagamma), is indispensable for light-initiated signaling in photoreceptor cells. However, the farnesyl-mediated molecular interactions important for signaling are not well understood. To explore this issue, we created a functional Tbetagamma analogue in which the farnesyl group was replaced with a (3-azidophenoxy)geranyl (POG) group, a novel farnesyl analogue with a distal photoreactive azido group. In the presence of lipid membranes and/or Talpha-GDP, UV irradiation of POG-modified Tbetagamma (POG-Tbetagamma) invariably yielded a cross-linked product Tgamma-Tbeta, reflecting a constitutive interaction of the Tgamma C-terminal lipid with Tbeta. In addition to the Tgamma-Tbeta adduct, a Tgamma-Talpha cross-link was detected in the aqueous fraction. Reconstitution of POG-Tbetagamma with Talpha and light-activated rhodopsin (Rh) in photoreceptor membranes resulted in cross-linking of Tgamma with a glycerophospholipid, indicating molecular interaction of the farnesyl group with cellular membranes. The Tgamma-phospholipid cross-link was observed only in the presence of both Talpha-GDP and Rh, and was abolished by the addition of GTPgammaS or by replacing Rh with opsin. These findings suggest a transient farnesyl-membrane interaction occurs only in a signaling state formed in a transducin-Rh ternary complex. On the other hand, UV irradiation of POG-Tbetagamma in a soluble complex with phosducin, a negative regulator of G-protein, yielded a Tgamma-phosducin adduct in addition to the Tgamma-Tbeta cross-link. These results illustrate that, rather than being a static membrane anchor, the farnesyl moiety plays an active role in the dynamics of protein-protein and protein-membrane interactions at defined steps in the signal transduction process.

Authors
Hagiwara, K; Wada, A; Katadae, M; Ito, M; Ohya, Y; Casey, PJ; Fukada, Y
MLA Citation
Hagiwara, K, Wada, A, Katadae, M, Ito, M, Ohya, Y, Casey, PJ, and Fukada, Y. "Analysis of the molecular interaction of the farnesyl moiety of transducin through the use of a photoreactive farnesyl analogue." Biochemistry 43.2 (January 20, 2004): 300-309.
PMID
14717583
Source
pubmed
Published In
Biochemistry
Volume
43
Issue
2
Publish Date
2004
Start Page
300
End Page
309
DOI
10.1021/bi0351514

Analysis of the regulation of microtubule dynamics by interaction of RGSZ1 (RGS20) with the neuronal stathmin, SCG10.

Regulators of G protein signaling (RGS proteins) are a diverse family of proteins that act to negatively regulate signaling by heterotrimeric G proteins; however, recent data have implied additional functions for RGS proteins. Previously, we employed the yeast two-hybrid system and identified the microtubule-destabilizing protein, superior cervical ganglia neural-specific 10 protein (SCG10), as a potential effector protein of RGSZ1. This article describes the expression and biochemical purification of both RGSZ1 and SCG10 and details the development of various in vitro assays to evaluate microtubule polymerization?depolymerization. Both turbidimetric and microscopy-based assays can be employed to study the impact that RGS proteins have on SCG10 function. The application of these in vitro assays may help identify a novel role for RGS proteins in regulating the cytoskeletal network.

Authors
Nixon, AB; Casey, PJ
MLA Citation
Nixon, AB, and Casey, PJ. "Analysis of the regulation of microtubule dynamics by interaction of RGSZ1 (RGS20) with the neuronal stathmin, SCG10." Methods Enzymol 390 (2004): 53-64.
PMID
15488170
Source
pubmed
Published In
Methods in Enzymology
Volume
390
Publish Date
2004
Start Page
53
End Page
64
DOI
10.1016/S0076-6879(04)90004-3

Improved loading and cleavage methods for solid-phase synthesis using chlorotrityl resins: Synthesis and testing of a library of 144 discrete chemicals as potential farnesyltransferase inhibitors

Authors
Park, JG; Langenwalter, KJ; Weinbaum, CA; Casey, PJ; Pang, YP
MLA Citation
Park, JG, Langenwalter, KJ, Weinbaum, CA, Casey, PJ, and Pang, YP. "Improved loading and cleavage methods for solid-phase synthesis using chlorotrityl resins: Synthesis and testing of a library of 144 discrete chemicals as potential farnesyltransferase inhibitors." JOURNAL OF COMBINATORIAL CHEMISTRY 6.3 (2004): 407-413.
PMID
15132601
Source
wos-lite
Published In
Journal of Combinatorial Chemistry
Volume
6
Issue
3
Publish Date
2004
Start Page
407
End Page
413
DOI
10.1021/cc0340729

Structure of mammalian protein geranylgeranyltransferase type-I.

Protein geranylgeranyltransferase type-I (GGTase-I), one of two CaaX prenyltransferases, is an essential enzyme in eukaryotes. GGTase-I catalyzes C-terminal lipidation of >100 proteins, including many GTP- binding regulatory proteins. We present the first structural information for mammalian GGTase-I, including a series of substrate and product complexes that delineate the path of the chemical reaction. These structures reveal that all protein prenyltransferases share a common reaction mechanism and identify specific residues that play a dominant role in determining prenyl group specificity. This hypothesis was confirmed by converting farnesyltransferase (15-C prenyl substrate) into GGTase-I (20-C prenyl substrate) with a single point mutation. GGTase-I discriminates against farnesyl diphosphate (FPP) at the product turnover step through the inability of a 15-C FPP to displace the 20-C prenyl-peptide product. Understanding these key features of specificity is expected to contribute to optimization of anti-cancer and anti-parasite drugs.

Authors
Taylor, JS; Reid, TS; Terry, KL; Casey, PJ; Beese, LS
MLA Citation
Taylor, JS, Reid, TS, Terry, KL, Casey, PJ, and Beese, LS. "Structure of mammalian protein geranylgeranyltransferase type-I." EMBO J 22.22 (November 17, 2003): 5963-5974.
PMID
14609943
Source
pubmed
Published In
EMBO Journal
Volume
22
Issue
22
Publish Date
2003
Start Page
5963
End Page
5974
DOI
10.1093/emboj/cdg571

Signaling Through G<inf>z</inf>

Authors
Meng, J; Casey, PJ
MLA Citation
Meng, J, and Casey, PJ. "Signaling Through G<inf>z</inf>." Handbook of Cell Signaling. November 7, 2003. 601-604.
Source
scopus
Volume
2-3
Publish Date
2003
Start Page
601
End Page
604
DOI
10.1016/B978-012124546-7/50580-5

High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors.

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.

Authors
Fiordalisi, JJ; Johnson, RL; Weinbaum, CA; Sakabe, K; Chen, Z; Casey, PJ; Cox, AD
MLA Citation
Fiordalisi, JJ, Johnson, RL, Weinbaum, CA, Sakabe, K, Chen, Z, Casey, PJ, and Cox, AD. "High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors." J Biol Chem 278.43 (October 24, 2003): 41718-41727.
PMID
12882980
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
43
Publish Date
2003
Start Page
41718
End Page
41727
DOI
10.1074/jbc.M305733200

Kinetic studies of protein farnesyltransferase mutants establish active substrate conformation.

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon farnesyl moiety from farnesyl diphosphate (FPP) to a cysteine residue near the C-terminus of a protein substrate. Several crystal structures of inactive FTase.FPP.peptide complexes indicate that K164alpha interacts with the alpha-phosphate and that H248beta and Y300beta form hydrogen bonds with the beta-phosphate of FPP [Strickland, C. L., et al. (1998) Biochemistry 37, 16601-16611]. Mutations K164Aalpha, H248Abeta, and Y300Fbeta were prepared and analyzed by single turnover kinetics and ligand binding studies. These mutations do not significantly affect the enzyme affinity for FPP but do decrease the farnesylation rate constant by 30-, 10-, and 500-fold, respectively. These mutations have little effect on the pH and magnesium dependence of the farnesylation rate constant, demonstrating that the side chains of K164alpha, Y300beta, and H248beta do not function either as general acid-base catalysts or as magnesium ligands. Mutation of H248beta and Y300beta, but not K164alpha, decreases the farnesylation rate constant using farnesyl monophosphate (FMP). These data suggest that, contrary to the conclusions derived from analysis of the static crystal structures, the transition state for farnesylation is stabilized by interactions between the alpha-phosphate of the isoprenoid substrate and the side chains of Y300beta and H248beta. These results suggest an active substrate conformation for FTase wherein the C1 carbon of the FPP substrate moves toward the zinc-bound thiolate of the protein substrate to react, resulting in a rearrangement of the diphosphate group relative to its ground state position in the binding pocket.

Authors
Pickett, JS; Bowers, KE; Hartman, HL; Fu, H-W; Embry, AC; Casey, PJ; Fierke, CA
MLA Citation
Pickett, JS, Bowers, KE, Hartman, HL, Fu, H-W, Embry, AC, Casey, PJ, and Fierke, CA. "Kinetic studies of protein farnesyltransferase mutants establish active substrate conformation." Biochemistry 42.32 (August 19, 2003): 9741-9748.
PMID
12911316
Source
pubmed
Published In
Biochemistry
Volume
42
Issue
32
Publish Date
2003
Start Page
9741
End Page
9748
DOI
10.1021/bi0346852

Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of methotrexate.

The antifolate methotrexate is one of the most successful drugs in cancer chemotherapy. Although its efficacy is widely attributed to a decrease in nucleotide biosynthesis (1), methotrexate is known to increase homocysteine (2), a compound associated with an elevated risk of heart disease, Alzheimer's disease (3), and neural tube defects (4). A potential mechanism for the detrimental effects of homocysteine is cellular hypomethylation from an increase in S-adenosylhomocysteine (5), an inhibitor of methyltransferases including isoprenylcysteine carboxyl methyltransferase (Icmt). Among the substrates of Icmt is the monomeric G protein Ras, a critical component of many signaling pathways that regulate cell growth and differentiation. Because carboxyl methylation of Ras is important for proper plasma membrane localization and function (6), we investigated the role of Icmt in the antiproliferative effect of methotrexate. After methotrexate treatment of DKOB8 cells, Ras methylation is decreased by almost 90%. This hypomethylation is accompanied by a mislocalization of Ras to the cytosol and a 4-fold decrease in the activation of p44 mitogen-activated protein kinase and Akt. Additionally, cells lacking Icmt are highly resistant to methotrexate. Whereas cells expressing wild-type levels of Icmt are inhibited by methotrexate, stable expression of myristoylated H-Ras, which does not require carboxyl methylation for membrane attachment (7), confers resistance to methotrexate. These results suggest that inhibition of Icmt is a critical component of the antiproliferative effect of methotrexate, expanding our understanding of this widely used drug and identifying Icmt as a target for drug discovery.

Authors
Winter-Vann, AM; Kamen, BA; Bergo, MO; Young, SG; Melnyk, S; James, SJ; Casey, PJ
MLA Citation
Winter-Vann, AM, Kamen, BA, Bergo, MO, Young, SG, Melnyk, S, James, SJ, and Casey, PJ. "Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of methotrexate." Proc Natl Acad Sci U S A 100.11 (May 27, 2003): 6529-6534.
PMID
12750467
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
11
Publish Date
2003
Start Page
6529
End Page
6534
DOI
10.1073/pnas.1135239100

Overview of the Alliance for Cellular Signaling.

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

Authors
Gilman, AG; Simon, MI; Bourne, HR; Harris, BA; Long, R; Ross, EM; Stull, JT; Taussig, R; Bourne, HR; Arkin, AP; Cobb, MH; Cyster, JG; Devreotes, PN; Ferrell, JE; Fruman, D; Gold, M; Weiss, A; Stull, JT; Berridge, MJ; Cantley, LC; Catterall, WA; Coughlin, SR; Olson, EN; Smith, TF; Brugge, JS; Botstein, D; Dixon, JE; Hunter, T; Lefkowitz, RJ; Pawson, AJ; Sternberg, PW; Varmus, H; Subramaniam, S; Sinkovits, RS; Li, J; Mock, D; Ning, Y; Saunders, B; Sternweis, PC; Hilgemann, D; Scheuermann, RH et al.
MLA Citation
Gilman, AG, Simon, MI, Bourne, HR, Harris, BA, Long, R, Ross, EM, Stull, JT, Taussig, R, Bourne, HR, Arkin, AP, Cobb, MH, Cyster, JG, Devreotes, PN, Ferrell, JE, Fruman, D, Gold, M, Weiss, A, Stull, JT, Berridge, MJ, Cantley, LC, Catterall, WA, Coughlin, SR, Olson, EN, Smith, TF, Brugge, JS, Botstein, D, Dixon, JE, Hunter, T, Lefkowitz, RJ, Pawson, AJ, Sternberg, PW, Varmus, H, Subramaniam, S, Sinkovits, RS, Li, J, Mock, D, Ning, Y, Saunders, B, Sternweis, PC, Hilgemann, D, and Scheuermann, RH et al. "Overview of the Alliance for Cellular Signaling." Nature 420.6916 (December 12, 2002): 703-706.
PMID
12478301
Source
pubmed
Published In
Nature
Volume
420
Issue
6916
Publish Date
2002
Start Page
703
End Page
706
DOI
10.1038/nature01304

Activation of Gz attenuates Rap1-mediated differentiation of PC12 cells.

We previously identified a specific activation-dependent interaction between the alpha subunit of the heterotrimeric G protein, G(z), and a regulator of Rap1 signaling, Rap1GAP (Meng, J., Glick, J. L., Polakis, P., and Casey, P. J. (1999) J. Biol. Chem. 274, 36663-36669). We now demonstrate that activated forms of Galpha(z) are able to recruit Rap1GAP from a cytosolic location to the membrane. Using PC12 cells as a model for neuronal differentiation, the influence of G(z) activation on Rap1-mediated cell differentiation was examined. Introduction of constitutively-activated Galpha(z) into PC12 cells markedly attenuated the differentiation process of these cells induced by a cAMP analogue. Treatment of PC12 cells expressing wild type Galpha(z) with a specific agonist to the alpha(2A)-adrenergic receptor also attenuated cAMP-induced PC12 cell differentiation, demonstrating that receptor-mediated activation of G(z) was also effective in this regard. Furthermore, activation of G(z) decreased the ability of the cAMP analogue to trigger both Rap1 and extracellular-regulated kinase (ERK) activation. Differentiation of PC12 cells induced by nerve growth factor (NGF) is also thought to be a Rap1-mediated process, and G(z) activation was found to attenuate this process as well. Rap1 activation, ERK phosphorylation, and PC12 cell differentation induced by NGF treatment were all significantly attenuated by either transfection of constitutively activated Galpha(z) or receptor-mediated G(z) activation. Based on these findings, a model is proposed in which activation of G(z) results in recruitment of Rap1GAP to the membrane where it can effectively down-regulate Rap1 signaling. The implications of these findings in regard to a possible role for G(z) in neuronal development are discussed.

Authors
Meng, J; Casey, PJ
MLA Citation
Meng, J, and Casey, PJ. "Activation of Gz attenuates Rap1-mediated differentiation of PC12 cells." J Biol Chem 277.45 (November 8, 2002): 43417-43424.
PMID
12198116
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
45
Publish Date
2002
Start Page
43417
End Page
43424
DOI
10.1074/jbc.M204074200

Stereospecificity and kinetic mechanism of human prenylcysteine lyase, an unusual thioether oxidase.

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus. The cellular abundance of prenylated proteins, as well as the stability of the thioether bond, poses a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that degrades a variety of prenylcysteines. Prenylcysteine lyase is a FAD-dependent thioether oxidase that produces free cysteine, an isoprenoid aldehyde, and hydrogen peroxide as products of the reaction. Here we report initial studies of the kinetic mechanism and stereospecificity of this unusual enzyme. We utilized product and dead end inhibitors of prenylcysteine lyase to probe the kinetic mechanism of the multistep reaction. The results with these inhibitors, together with those of other experiments, suggest that the reaction catalyzed by prenylcysteine lyase proceeds through a sequential mechanism. The reaction catalyzed by the enzyme is stereospecific, in that the pro-S hydride of the farnesylcysteine is transferred to FAD to initiate the reaction. With (2R,1'S)-[1'-(2)H(1)]farnesylcysteine as a substrate, a primary deuterium isotope effect of 2 was observed on the steady state rate. However, the absence of an isotope effect on an observed pre-steady-state burst of hydrogen peroxide formation implicates a partially rate-determining proton transfer after a relatively fast C-H (C-D) bond cleavage step. Furthermore, no pre-steady-state burst of cysteine was observed. The finding that the rate of cysteine formation was within 2-fold of the steady-state k(cat) value indicates that cysteine production is one of the primary rate-limiting steps in the reaction. These results provide substantial new information on the catalytic mechanism of prenylcysteine lyase.

Authors
Digits, JA; Pyun, H-J; Coates, RM; Casey, PJ
MLA Citation
Digits, JA, Pyun, H-J, Coates, RM, and Casey, PJ. "Stereospecificity and kinetic mechanism of human prenylcysteine lyase, an unusual thioether oxidase." J Biol Chem 277.43 (October 25, 2002): 41086-41093.
PMID
12186880
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
43
Publish Date
2002
Start Page
41086
End Page
41093
DOI
10.1074/jbc.M208069200

Prenylcysteine lyase deficiency in mice results in the accumulation of farnesylcysteine and geranylgeranylcysteine in brain and liver.

In in vitro experiments, prenylcysteine lyase (Pcly) cleaves the thioether bond of prenylcysteines to yield free cysteine and the aldehyde of the isoprenoid lipid. However, the importance of this enzyme has not yet been fully defined at the biochemical or physiologic level. In this study, we show that Pcly is expressed at high levels in mouse liver, kidney, heart, and brain. To test whether Pcly deficiency would cause prenylcysteines to accumulate in tissues and result in pathologic consequences, we produced Pcly-deficient cell lines and Pcly-deficient mice (Pcly-/-). Pcly activity levels were markedly reduced in Pcly-/- cells and tissues. Pcly-/- fibroblasts were more sensitive than wild-type fibroblasts to growth inhibition when prenylcysteines were added to the cell culture medium. To determine if the reduced Pcly enzyme activity levels led to an accumulation of prenylcysteines within cells, mass spectrometry was used to measure farnesylcysteine and geranylgeranylcysteine levels in the tissues of Pcly-/- mice and wild-type controls. These studies revealed a striking accumulation of both farnesylcysteine and geranylgeranylcysteine in the brain and liver of Pcly-/- mice. This accumulation did not appear to be accompanied by significant pathologic consequences. Pcly-/- mice were healthy and fertile, and surveys of more than 30 tissues did not uncover any abnormalities. We conclude that prenylcysteine lyase does play a physiologic role in cleaving prenylcysteines in mammals, but the absence of this activity does not lead to major pathologic consequences.

Authors
Beigneux, A; Withycombe, SK; Digits, JA; Tschantz, WR; Weinbaum, CA; Griffey, SM; Bergo, M; Casey, PJ; Young, SG
MLA Citation
Beigneux, A, Withycombe, SK, Digits, JA, Tschantz, WR, Weinbaum, CA, Griffey, SM, Bergo, M, Casey, PJ, and Young, SG. "Prenylcysteine lyase deficiency in mice results in the accumulation of farnesylcysteine and geranylgeranylcysteine in brain and liver." J Biol Chem 277.41 (October 11, 2002): 38358-38363.
PMID
12151402
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
41
Publish Date
2002
Start Page
38358
End Page
38363
DOI
10.1074/jbc.M205183200

Reaction path of protein farnesyltransferase at atomic resolution.

Protein farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction proteins, including members of the Ras superfamily. The farnesylation of Ras oncoproteins, which are associated with 30% of human cancers, is essential for their transforming activity. FTase inhibitors are currently in clinical trials for the treatment of cancer. Here we present a complete series of structures representing the major steps along the reaction coordinate of this enzyme. From these observations can be deduced the determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes. A structural model for the transition state consistent with previous mechanistic studies was also constructed. The processive nature of the reaction suggests the structural basis for the successive addition of two prenyl groups to Rab proteins by the homologous enzyme geranylgeranyltransferase type-II. Finally, known FTase inhibitors seem to differ in their mechanism of inhibiting the enzyme.

Authors
Long, SB; Casey, PJ; Beese, LS
MLA Citation
Long, SB, Casey, PJ, and Beese, LS. "Reaction path of protein farnesyltransferase at atomic resolution." Nature 419.6907 (October 10, 2002): 645-650.
PMID
12374986
Source
pubmed
Published In
Nature
Volume
419
Issue
6907
Publish Date
2002
Start Page
645
End Page
650
DOI
10.1038/nature00986

Galpha12 and Galpha13 negatively regulate the adhesive functions of cadherin.

Cadherins function to promote adhesion between adjacent cells and play critical roles in such cellular processes as development, tissue maintenance, and tumor suppression. We previously demonstrated that heterotrimeric G proteins of the G12 subfamily comprised of Galpha12 and Galpha13 interact with the cytoplasmic domain of cadherins and cause the release of the transcriptional activator beta-catenin (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). Because of the importance of beta-catenin in cadherin-mediated cell-cell adhesion, we examined whether G12 subfamily proteins could also regulate cadherin function. The introduction of mutationally activated G12 proteins into K562 cells expressing E-cadherin blocked cadherin-mediated cell adhesion in steady-state assays. Also, in breast cancer cells, the introduction of activated G12 proteins blocked E-cadherin function in a fast aggregation assay. Aggregation mediated by a mutant cadherin that lacks G12 binding ability was not affected by activated G12 proteins, indicating a requirement for direct G12-cadherin interaction. Furthermore, in wound-filling assays in which ectopic expression of E-cadherin inhibits cell migration, the expression of activated G12 proteins reversed the inhibition via a mechanism that was independent of G12-mediated Rho activation. These results validate the G12-cadherin interaction as a potentially important event in cell biology and suggest novel roles for G12 proteins in the regulation of cadherin-mediated developmental events and in the loss of cadherin function that is characteristic of metastatic tumor progression.

Authors
Meigs, TE; Fedor-Chaiken, M; Kaplan, DD; Brackenbury, R; Casey, PJ
MLA Citation
Meigs, TE, Fedor-Chaiken, M, Kaplan, DD, Brackenbury, R, and Casey, PJ. "Galpha12 and Galpha13 negatively regulate the adhesive functions of cadherin." J Biol Chem 277.27 (July 5, 2002): 24594-24600.
PMID
11976333
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
27
Publish Date
2002
Start Page
24594
End Page
24600
DOI
10.1074/jbc.M201984200

Gα12 and Gα13 negatively regulate the adhesive functions of cadherin

Cadherins function to promote adhesion between adjacent cells and play critical roles in such cellular processes as development, tissue maintenance, and tumor suppression. We previously demonstrated that heterotrimeric G proteins of the G12 subfamily comprised of Gα12 and Gα13 interact with the cytoplasmic domain of cadherins and cause the release of the transcriptional activator β-catenin (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). Because of the importance of β-catenin in cadherin-mediated cell-cell adhesion, we examined whether G12 subfamily proteins could also regulate cadherin function. The introduction of mutationally activated G12 proteins into K562 cells expressing E-cadherin blocked cadherin-mediated cell adhesion in steady-state assays. Also, in breast cancer cells, the introduction of activated G12 proteins blocked E-cadherin function in a fast aggregation assay. Aggregation mediated by a mutant cadherin that lacks G12 binding ability was not affected by activated G12 proteins, indicating a requirement for direct G12-cadherin interaction. Furthermore, in wound-filling assays in which ectopic expression of E-cadherin inhibits cell migration, the expression of activated G12 proteins reversed the inhibition via a mechanism that was independent of G12-mediated Rho activation. These results validate the G12-cadherin interaction as a potentially important event in cell biology and suggest novel roles for G12 proteins in the regulation of cadherin-mediated developmental events and in the loss of cadherin function that is characteristic of metastatic tumor progression.

Authors
Meigs, TE; Fedor-Chaiken, M; Kaplan, DD; Brackenbury, R; Casey, PJ
MLA Citation
Meigs, TE, Fedor-Chaiken, M, Kaplan, DD, Brackenbury, R, and Casey, PJ. "Gα12 and Gα13 negatively regulate the adhesive functions of cadherin." Journal of Biological Chemistry 277.27 (July 5, 2002): 24594-24600.
Source
scopus
Published In
The Journal of biological chemistry
Volume
277
Issue
27
Publish Date
2002
Start Page
24594
End Page
24600
DOI
10.1074/jbc.M201984200

The interaction of RGSZ1 with SCG10 attenuates the ability of SCG10 to promote microtubule disassembly.

RGS proteins (regulators of G protein signaling) are a diverse family of proteins that act to negatively regulate signaling by heterotrimeric G proteins. Initially characterized as GTPase-activating proteins for Galpha subunits, recent data have implied additional functions for RGS proteins. We previously identified an RGS protein (termed RGSZ1) whose expression is quite specific to neuronal tissue (Glick, J. L., Meigs, T. E., Miron, A., and Casey, P. J. (1998) J. Biol. Chem. 273, 26008-26013). In a continuing effort to understand the role of RGSZ1 in cellular signaling, the yeast two-hybrid system was employed to identify potential effector proteins of RGSZ1. The microtubule-destabilizing protein SCG10 (superior cervical ganglia, neural specific 10) was found to directly interact with RGSZ1 in the yeast system, and this interaction was further verified using direct binding assays. Treatment of PC12 cells with nerve growth factor resulted in Golgi-specific distribution of SCG10. A green fluorescent protein-tagged variant of RGSZ1 translocated to the Golgi complex upon treatment of PC12 cells with nerve growth factor, providing evidence that RGSZ1 and SCG10 interact in cells as well as in vitro. Analysis of in vitro microtubule polymerization/depolymerization showed that binding of RGSZ1 to SCG10 effectively blocked the ability of SCG10 to induce microtubule disassembly as determined by both turbidimetric and microscopy-based assays. These results identify a novel connection between RGS proteins and the cytoskeletal network that points to a broader role than previously envisioned for RGS proteins in regulating biological processes.

Authors
Nixon, AB; Grenningloh, G; Casey, PJ
MLA Citation
Nixon, AB, Grenningloh, G, and Casey, PJ. "The interaction of RGSZ1 with SCG10 attenuates the ability of SCG10 to promote microtubule disassembly." J Biol Chem 277.20 (May 17, 2002): 18127-18133.
PMID
11882662
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
20
Publish Date
2002
Start Page
18127
End Page
18133
DOI
10.1074/jbc.M201065200

Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation.

After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications-endoproteolytic release of the last three amino acids of the protein by the protease Rce1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting Rce1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional Rce1 allele (Rce1(flox)) and produced Rce1(flox/flox) fibroblasts. Cre-mediated excision of Rce1 (thereby producing Rce1(Delta/Delta) fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. Rce1(Delta/Delta) fibroblasts grew more slowly than Rce1(flox/flox) fibroblasts. The excision of Rce1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of Rce1 from a Rce1(flox/flox) skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.

Authors
Bergo, MO; Ambroziak, P; Gregory, C; George, A; Otto, JC; Kim, E; Nagase, H; Casey, PJ; Balmain, A; Young, SG
MLA Citation
Bergo, MO, Ambroziak, P, Gregory, C, George, A, Otto, JC, Kim, E, Nagase, H, Casey, PJ, Balmain, A, and Young, SG. "Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation." Mol Cell Biol 22.1 (January 2002): 171-181.
PMID
11739732
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
22
Issue
1
Publish Date
2002
Start Page
171
End Page
181

Prenylation of CaaX-type proteins: Basic principles through clinical applications

Post-translational modification by attachment of lipid moieties is critical for the biological activity of many membrane-associated proteins. This process, termed lipidation, serves to direct and anchor specific proteins to the cell membrane and can also play a role in important protein-protein interactions. A wide variety of lipids can be attached to proteins, including saturated acyl groups such as myristoyl and palmitoyl chains, glycosylphosphatidylinositol (GPI) moieties, and the 15- and 20-carbon isoprenoid groups famesyl and geranylgeranyl, respectively. Some lipidated proteins have only a single modification, whereas many others may have multiple, in some cases variable, modifications, in which each permuation confers a unique chemical property and distinct functional characteristic to the parent protein. This chapter deals primarily with the biochemistry and biology of protein prenylation, focusing on those proteins that contain a so-called CaaX motif at their C-terminus. The C-terminal processing of CaaX proteins is initiated by addition of either a farnesyl or geranylgeranyl isoprenoid to the conserved cysteine of the CaaX motif. Farnesylation of Ras proteins in particular has attracted a great deal of attention and is the primary reason why the enzyme responsible, termed protein farnesyltransferase (FTase), has been targeted for development of inhibitors that are currently being evaluated in clinical trials as anticancer agents. © 2002.

Authors
Hurwitz, HI; Casey, PJ
MLA Citation
Hurwitz, HI, and Casey, PJ. "Prenylation of CaaX-type proteins: Basic principles through clinical applications." Current Topics in Membranes 52 (2002): 531-550.
Source
scival
Published In
Current Topis in Membranes
Volume
52
Publish Date
2002
Start Page
531
End Page
550

Lysine(164)alpha of protein farnesyltransferase is important for both CaaX substrate binding and catalysis.

Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys(164) of the alpha subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys(164)alpha is involved in formation of the enzyme (E).farnesyl diphosphate (FPP).peptide ternary complex. Furthermore, mutation of Lys(164)alpha resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys(164)alpha, as well as the positioning of the charge, are important for overall catalysis by FTase.

Authors
Hightower, KE; De, S; Weinbaum, C; Spence, RA; Casey, PJ
MLA Citation
Hightower, KE, De, S, Weinbaum, C, Spence, RA, and Casey, PJ. "Lysine(164)alpha of protein farnesyltransferase is important for both CaaX substrate binding and catalysis." Biochem J 360.Pt 3 (December 15, 2001): 625-631.
PMID
11736652
Source
pubmed
Published In
The Biochemical journal
Volume
360
Issue
Pt 3
Publish Date
2001
Start Page
625
End Page
631

Distinct regions of the cadherin cytoplasmic domain are essential for functional interaction with Galpha 12 and beta-catenin.

Heterotrimeric G proteins of the G(12) subfamily mediate cellular signals leading to events such as cytoskeletal rearrangements, cell proliferation, and oncogenic transformation. Several recent studies have revealed direct effector proteins through which G(12) subfamily members may transmit signals leading to various cellular responses. Our laboratory recently demonstrated that Galpha(12) and Galpha(13) specifically interact with the cytoplasmic domains of several members of the cadherin family of cell adhesion molecules (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 519-524). This interaction causes beta-catenin to release from cadherin and relocalize to the cytoplasm and nucleus, where it participates in transcriptional activation. Here we report that two distinct regions of the epithelial cadherin (E-cadherin) tail are required for interaction with beta-catenin and Galpha(12), respectively. Deletion of an acidic, 19-amino acid region of E-cadherin abolishes its ability to bind beta-catenin in vitro, to inhibit beta-catenin-mediated transactivation, or to stabilize beta-catenin; causes subcellular mislocalization of beta-catenin; and disrupts cadherin-mediated cell adhesion. On the other hand, deletion of a distinct 11-amino acid region of E-cadherin dramatically attenuates interaction with Galpha(12); furthermore, Galpha(12) is ineffective in stimulating beta-catenin release from an E-cadherin cytoplasmic domain lacking this putative Galpha(12)-binding region. These findings indicate that Galpha(12) and beta-catenin do not compete for the same binding site on cadherin and provide molecular targets for selectively disrupting the interaction of these proteins with cadherin.

Authors
Kaplan, DD; Meigs, TE; Casey, PJ
MLA Citation
Kaplan, DD, Meigs, TE, and Casey, PJ. "Distinct regions of the cadherin cytoplasmic domain are essential for functional interaction with Galpha 12 and beta-catenin." J Biol Chem 276.47 (November 23, 2001): 44037-44043.
PMID
11546809
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
47
Publish Date
2001
Start Page
44037
End Page
44043
DOI
10.1074/jbc.M106121200

Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10).

Heterotrimeric G proteins are involved in the transduction of hormonal and sensory signals across plasma membranes of eukaryotic cells. Hence, they are a critical point of control for a variety of agents that modulate cellular function. Activation of these proteins is dependent on GTP binding to their alpha (Galpha) subunits. Regulators of G protein signaling (RGS) bind specifically to activated Galpha proteins, potentiating the intrinsic GTPase activity of the Galpha proteins and thus expediting the termination of Galpha signaling. Although there are several points in most G protein controlled signaling pathways that are affected by reversible covalent modification, little evidence has been shown addressing whether or not the functions of RGS proteins are themselves regulated by such modifications. We report in this study the acute functional regulation of RGS10 thru the specific and inducible phosphorylation of RGS10 protein at serine 168 by cAMP-dependent kinase A. This phosphorylation nullifies the RGS10 activity at the plasma membrane, which controls the G protein-dependent activation of the inwardly rectifying potassium channel. Surprisingly, the phosphorylation-mediated attenuation of RGS10 activity was not manifested in an alteration of its ability to accelerate GTPase activity of Galpha. Rather, the phosphorylation event correlates with translocation of RGS10 from the plasma membrane and cytosol into the nucleus.

Authors
Burgon, PG; Lee, WL; Nixon, AB; Peralta, EG; Casey, PJ
MLA Citation
Burgon, PG, Lee, WL, Nixon, AB, Peralta, EG, and Casey, PJ. "Phosphorylation and nuclear translocation of a regulator of G protein signaling (RGS10)." J Biol Chem 276.35 (August 31, 2001): 32828-32834.
PMID
11443111
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
35
Publish Date
2001
Start Page
32828
End Page
32834
DOI
10.1074/jbc.M100960200

Isoprenylcysteine carboxyl methyltransferase deficiency in mice.

After isoprenylation, Ras and other CAAX proteins undergo endoproteolytic processing by Rce1 and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). We reported previously that Rce1-deficient mice died during late gestation or soon after birth. We hypothesized that Icmt deficiency might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of other methyltransferase activities, we generated Icmt-deficient mice. Contrary to our expectation, Icmt deficiency caused a more severe phenotype than Rce1 deficiency, with virtually all of the knockout embryos (Icmt-/-) dying by mid-gestation. An analysis of chimeric mice produced from Icmt-/- embryonic stem cells showed that the Icmt-/- cells retained the capacity to contribute to some tissues (e.g. skeletal muscle) but not to others (e.g. brain). Lysates from Icmt-/- embryos lacked the ability to methylate either recombinant K-Ras or small molecule substrates (e.g. N-acetyl-S-geranylgeranyl-l-cysteine). In addition, Icmt-/- cells lacked the ability to methylate Rab proteins. Thus, Icmt appears to be the only enzyme participating in the carboxyl methylation of isoprenylated proteins.

Authors
Bergo, MO; Leung, GK; Ambroziak, P; Otto, JC; Casey, PJ; Gomes, AQ; Seabra, MC; Young, SG
MLA Citation
Bergo, MO, Leung, GK, Ambroziak, P, Otto, JC, Casey, PJ, Gomes, AQ, Seabra, MC, and Young, SG. "Isoprenylcysteine carboxyl methyltransferase deficiency in mice." J Biol Chem 276.8 (February 23, 2001): 5841-5845.
PMID
11121396
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
8
Publish Date
2001
Start Page
5841
End Page
5845
DOI
10.1074/jbc.C000831200

Non-peptidic, non-prenylic inhibitors of the prenyl protein-specific protease Rce1.

Several compounds designed as bisubstrate analogues of protein farnesyltransferase inhibited the prenyl protein-specific protease Rce1, qualifying them as lead structures for a novel class of non-peptidic, non-prenylic inhibitors of this protease.

Authors
Schlitzer, M; Winter-Vann, A; Casey, PJ
MLA Citation
Schlitzer, M, Winter-Vann, A, and Casey, PJ. "Non-peptidic, non-prenylic inhibitors of the prenyl protein-specific protease Rce1." Bioorg Med Chem Lett 11.3 (February 12, 2001): 425-427.
PMID
11212127
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
11
Issue
3
Publish Date
2001
Start Page
425
End Page
427

Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase.

Protein farnesyltransferase catalyzes the modification of protein substrates containing specific carboxyl-terminal Ca(1)a(2)X motifs with a 15-carbon farnesyl group. The thioether linkage is formed between the cysteine of the Ca(1)a(2)X motif and C1 of the farnesyl group. Protein substrate specificity is essential to the function of the enzyme and has been exploited to find enzyme-specific inhibitors for antitumor therapies. In this work, we investigate the thiol substrate specificity of protein farnesyltransferase by demonstrating that a variety of nonpeptidic thiol compounds, including glutathione and dithiothreitol, are substrates. However, the binding energy of these thiols is decreased 4-6 kcal/mol compared to a peptide derived from the carboxyl terminus of H-Ras. Furthermore, for these thiol substrates, both the farnesylation rate constant and the apparent magnesium affinity decrease significantly. Surprisingly, no correlation is observed between the pH-independent log(k(max)) and the thiol pK(a); model nucleophilic reactions of thiols display a Brønsted correlation of approximately 0.4. These data demonstrate that zinc-sulfur coordination is a primary criterion for classification as a FTase substrate, but other interactions between the peptide and the FTase.isoprenoid complex provide significant enhancement of binding and catalysis. Finally, these results suggest that the mechanism of FTase provides in vivo selectivity for the farnesylation of protein substrates even in the presence of high concentrations of intracellular thiols.

Authors
Hightower, KE; Casey, PJ; Fierke, CA
MLA Citation
Hightower, KE, Casey, PJ, and Fierke, CA. "Farnesylation of nonpeptidic thiol compounds by protein farnesyltransferase." Biochemistry 40.4 (January 30, 2001): 1002-1010.
PMID
11170422
Source
pubmed
Published In
Biochemistry
Volume
40
Issue
4
Publish Date
2001
Start Page
1002
End Page
1010

Lysosomal prenylcysteine lyase is a FAD-dependent thioether oxidase.

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached via a thioether bond to a cysteine residue at or near their C terminus. As prenylated proteins comprise up to 2% of the total protein in eukaryotic cells, and the thioether bond is a stable modification, their degradation raises a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that cleaves prenylcysteines to cysteine and an unidentified isoprenoid product. Here we show that the isoprenoid product of prenylcysteine lyase is the C-1 aldehyde of the isoprenoid moiety (farnesal in the case of C-15). The enzyme requires molecular oxygen as a cosubstrate and utilizes a noncovalently bound flavin cofactor in an NAD(P)H-independent manner. Additionally, a stoichiometric amount of hydrogen peroxide is produced during the reaction. These surprising findings indicate that prenylcysteine lyase utilizes a novel oxidative mechanism to cleave thioether bonds and provide insight into the unique role this enzyme plays in the cellular metabolism of prenylcysteines.

Authors
Tschantz, WR; Digits, JA; Pyun, HJ; Coates, RM; Casey, PJ
MLA Citation
Tschantz, WR, Digits, JA, Pyun, HJ, Coates, RM, and Casey, PJ. "Lysosomal prenylcysteine lyase is a FAD-dependent thioether oxidase." J Biol Chem 276.4 (January 26, 2001): 2321-2324.
PMID
11078725
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
4
Publish Date
2001
Start Page
2321
End Page
2324
DOI
10.1074/jbc.C000616200

Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release.

The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis. In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12. Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins. Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins. Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation. These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation.

Authors
Meigs, TE; Fields, TA; McKee, DD; Casey, PJ
MLA Citation
Meigs, TE, Fields, TA, McKee, DD, and Casey, PJ. "Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release." Proc Natl Acad Sci U S A 98.2 (January 16, 2001): 519-524.
PMID
11136230
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
2
Publish Date
2001
Start Page
519
End Page
524
DOI
10.1073/pnas.021350998

1 Mechanism of catalysis by protein farnesyltransferase

Authors
Spence, RA; Casey, PJ
MLA Citation
Spence, RA, and Casey, PJ. "1 Mechanism of catalysis by protein farnesyltransferase." Enzymes 21.C (2001): 1-18.
Source
scival
Published In
Enzymes
Volume
21
Issue
C
Publish Date
2001
Start Page
1
End Page
18
DOI
10.1016/S1874-6047(01)80014-7

The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules.

After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.

Authors
Chen, Z; Otto, JC; Bergo, MO; Young, SG; Casey, PJ
MLA Citation
Chen, Z, Otto, JC, Bergo, MO, Young, SG, and Casey, PJ. "The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules." J Biol Chem 275.52 (December 29, 2000): 41251-41257.
PMID
11007785
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
52
Publish Date
2000
Start Page
41251
End Page
41257
DOI
10.1074/jbc.M006687200

Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition.

Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.

Authors
Spence, RA; Hightower, KE; Terry, KL; Beese, LS; Fierke, CA; Casey, PJ
MLA Citation
Spence, RA, Hightower, KE, Terry, KL, Beese, LS, Fierke, CA, and Casey, PJ. "Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition." Biochemistry 39.45 (November 14, 2000): 13651-13659.
PMID
11076503
Source
pubmed
Published In
Biochemistry
Volume
39
Issue
45
Publish Date
2000
Start Page
13651
End Page
13659

Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.

After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/-). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.

Authors
Bergo, MO; Leung, GK; Ambroziak, P; Otto, JC; Casey, PJ; Young, SG
MLA Citation
Bergo, MO, Leung, GK, Ambroziak, P, Otto, JC, Casey, PJ, and Young, SG. "Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells." J Biol Chem 275.23 (June 9, 2000): 17605-17610.
PMID
10747846
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
23
Publish Date
2000
Start Page
17605
End Page
17610
DOI
10.1074/jbc.C000079200

The basis for K-Ras4B binding specificity to protein farnesyltransferase revealed by 2 A resolution ternary complex structures.

BACKGROUND: The protein farnesyltransferase (FTase) catalyzes addition of the hydrophobic farnesyl isoprenoid to a cysteine residue fourth from the C terminus of several protein acceptors that are essential for cellular signal transduction such as Ras and Rho. This addition is necessary for the biological function of the modified proteins. The majority of Ras-related human cancers are associated with oncogenic variants of K-RasB, which is the highest affinity natural substrate of FTase. Inhibition of FTase causes regression of Ras-mediated tumors in animal models. RESULTS: We present four ternary complexes of rat FTase co-crystallized with farnesyl diphosphate analogs and K-Ras4B peptide substrates. The Ca(1)a(2)X portion of the peptide substrate binds in an extended conformation in the hydrophobic cavity of FTase and coordinates the active site zinc ion. These complexes offer the first view of the polybasic region of the K-Ras4B peptide substrate, which confers the major enhancement of affinity of this substrate. The polybasic region forms a type I beta turn and binds along the rim of the hydrophobic cavity. Removal of the catalytically essential zinc ion results in a dramatically different peptide conformation in which the Ca(1)a(2)X motif adopts a beta turn. A manganese ion binds to the diphosphate mimic of the farnesyl diphosphate analog. CONCLUSIONS: These ternary complexes provide new insight into the molecular basis of peptide substrate specificity, and further define the roles of zinc and magnesium in the prenyltransferase reaction. Zinc is essential for productive Ca(1)a(2)X peptide binding, suggesting that the beta-turn conformation identified in previous nuclear magnetic resonance (NMR) studies reflects a state in which the cysteine is not coordinated to the zinc ion. The structural information presented here should facilitate structure-based design and optimization of inhibitors of Ca(1)a(2)X protein prenyltransferases.

Authors
Long, SB; Casey, PJ; Beese, LS
MLA Citation
Long, SB, Casey, PJ, and Beese, LS. "The basis for K-Ras4B binding specificity to protein farnesyltransferase revealed by 2 A resolution ternary complex structures." Structure 8.2 (February 15, 2000): 209-222.
PMID
10673434
Source
pubmed
Published In
Structure
Volume
8
Issue
2
Publish Date
2000
Start Page
209
End Page
222

Functional interaction between Galpha(z) and Rap1GAP suggests a novel form of cellular cross-talk.

G(z) is a member of the G(i) family of trimeric G proteins whose primary role in cell physiology is still unknown. In an ongoing effort to elucidate the cellular functions of G(z), the yeast two-hybrid system was employed to identify proteins that specifically interact with a mutationally activated form of Galpha(z). One of the molecules uncovered in this screen was Rap1GAP, a previously identified protein that specifically stimulates GTP hydrolytic activity of the monomeric G protein Rap1 and thus is believed to function as a down-regulator of Rap1 signaling. Like G(z), the precise role of Rap1 in cell physiology is poorly understood. Biochemical analysis using purified recombinant proteins revealed that the physical interaction between Galpha(z) and Rap1GAP blocks the ability of RGSs (regulators of G protein signaling) to stimulate GTP hydrolysis of the alpha subunit, and also attenuates the ability of activated Galpha(z) to inhibit adenylyl cyclase. Structure-function analyses indicate that the first 74 amino-terminal residues of Rap1GAP, a region distinct from the catalytic core domain responsible for the GAP activity toward Rap1, is required for this interaction. Co-precipitation assays revealed that Galpha(z), Rap1GAP, and Rap1 can form a stable complex. These data suggest that Rap1GAP acts as a signal integrator to somehow coordinate and/or integrate G(z) signaling and Rap1 signaling in cells.

Authors
Meng, J; Glick, JL; Polakis, P; Casey, PJ
MLA Citation
Meng, J, Glick, JL, Polakis, P, and Casey, PJ. "Functional interaction between Galpha(z) and Rap1GAP suggests a novel form of cellular cross-talk." J Biol Chem 274.51 (December 17, 1999): 36663-36669.
PMID
10593970
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
51
Publish Date
1999
Start Page
36663
End Page
36669

Cloning, expression, and cellular localization of a human prenylcysteine lyase.

Prenylated proteins contain either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus. These proteins constitute up to 2% of total cellular protein in eukaryotic cells. The degradation of prenylated proteins raises a metabolic challenge to the cell, because the thioether bond of the modified cysteine is quite stable. We recently identified and isolated an enzyme termed prenylcysteine lyase that cleaves the prenylcysteine to free cysteine and an isoprenoid product (Zhang, L., Tschantz, W. R., and Casey, P. J. (1997) J. Biol. Chem. 272, 23354-23359). To facilitate the molecular characterization of this enzyme, its cloning was undertaken. Overlapping cDNA clones encoding the complete coding sequence of this enzyme were obtained from a human cDNA library. The open reading frame of the gene encoding prenylcysteine lyase is 1515 base pairs and has a nearly ubiquitous expression pattern with a message size of 6 kilobase pairs. Recombinant prenylcysteine lyase was produced in a baculovirus-Sf9 expression system. Analysis of both the recombinant and native enzyme revealed that the enzyme is glycosylated and contains a signal peptide that is cleaved during processing. Additionally, the subcellular localization of this enzyme was determined to be lysosomal. These findings strengthen the notion that prenylcysteine lyase plays an important role in the final step in the degradation of prenylated proteins and will allow further physiological and biochemical characterization of this enzyme.

Authors
Tschantz, WR; Zhang, L; Casey, PJ
MLA Citation
Tschantz, WR, Zhang, L, and Casey, PJ. "Cloning, expression, and cellular localization of a human prenylcysteine lyase." J Biol Chem 274.50 (December 10, 1999): 35802-35808.
PMID
10585463
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
50
Publish Date
1999
Start Page
35802
End Page
35808

Cloning and characterization of a mammalian prenyl protein-specific protease.

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Ggamma1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.

Authors
Otto, JC; Kim, E; Young, SG; Casey, PJ
MLA Citation
Otto, JC, Kim, E, Young, SG, and Casey, PJ. "Cloning and characterization of a mammalian prenyl protein-specific protease." J Biol Chem 274.13 (March 26, 1999): 8379-8382.
PMID
10085068
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
13
Publish Date
1999
Start Page
8379
End Page
8382

Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells

Authors
Kim, E; Ambroziak, P; Otto, JC; Taylor, B; Ashby, M; Shannon, K; Casey, PJ; Young, SG
MLA Citation
Kim, E, Ambroziak, P, Otto, JC, Taylor, B, Ashby, M, Shannon, K, Casey, PJ, and Young, SG. "Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells." JOURNAL OF BIOLOGICAL CHEMISTRY 274.13 (March 26, 1999): 8383-8390.
PMID
10085069
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
274
Issue
13
Publish Date
1999
Start Page
8383
End Page
8390
DOI
10.1074/jbc.274.13.8383

Enzymology and biology of CaaX protein prenylation.

Protein prenylation refers to a type of lipid modification in which either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins. The majority of prenylated proteins belong to a group termed "CaaX proteins" that are defined by a specific C-terminal motif that directs their modification by this process. The ménage of CaaX-type prenylated proteins encompasses a wide variety of molecules that are found primarily at the cytoplasmic face of cellular membranes. These include nuclear lamins, Ras and a multitude of GTP-binding proteins (G proteins), several protein kinases and phosphatases, as well as other important proteins. A tremendous number of cellular signaling processes and regulatory events are under the control of CaaX prenyl proteins. While the attached isoprenoid lipids, in general, support the membrane association of the modified proteins, some proteins also clearly participate directly in protein-protein interactions. This chapter will emphasize 1) the biochemistry of the two enzymes termed farnesyltransferase and geranylgeranyltransferase type I, responsible for CaaX protein prenylation, and 2) biological roles for these modifications. Throughout, we will attempt to highlight the significance of prenylation in specific cellular events. The critical importance of this class of lipid modifications is attested to by the emergence of farnesyltransferase as a target for the development of anti-cancer therapeutics.

Authors
Fu, HW; Casey, PJ
MLA Citation
Fu, HW, and Casey, PJ. "Enzymology and biology of CaaX protein prenylation." Recent Prog Horm Res 54 (1999): 315-342. (Review)
PMID
10548882
Source
pubmed
Published In
Recent progress in hormone research
Volume
54
Publish Date
1999
Start Page
315
End Page
342

H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate.

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the alkylation of a cysteine residue of protein substrates with a 15 carbon farnesyl group. We have developed fluorescence assays to directly measure the affinity of the enzyme for peptide and protein (Ras) substrates. A peptide corresponding to the carboxyl terminus of H-Ras binds to FTase in the microM range (KD = 4 microM) at physiological pH; however, the peptide affinity is enhanced approximately 70-fold in a ternary complex with an enzyme-bound farnesyl diphosphate (FPP) analogue, indicating that the two substrates bind synergistically. The pH dependence of substrate binding was also investigated, and two ionizations were observed: for the ternary complex, the pKa values are 8.1, reflecting ionization of the thiol of the free peptide, and 6.4. The pH dependence of the ligand-metal charge-transfer band in the optical absorption spectra of a Co2+-substituted FTase ternary complex suggests that a metal-coordinated thiol ionizes with a pKa of 6.3. These data indicate that metal coordination of the peptide sulfur with the zinc ion in FTase lowers the pKa of the thiol resulting in formation of a bound thiolate at physiological pH.

Authors
Hightower, KE; Huang, CC; Casey, PJ; Fierke, CA
MLA Citation
Hightower, KE, Huang, CC, Casey, PJ, and Fierke, CA. "H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate." Biochemistry 37.44 (November 3, 1998): 15555-15562.
PMID
9799520
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
44
Publish Date
1998
Start Page
15555
End Page
15562
DOI
10.1021/bi981525v

RGSZ1, a Gz-selective regulator of G protein signaling whose action is sensitive to the phosphorylation state of Gzalpha.

Regulators of G protein signaling (RGS) are a family of proteins that attenuate the activity of the trimeric G proteins. RGS proteins act as GTPase-activating proteins (GAPs) for the alpha subunits of several trimeric G proteins, much like the GAPs that regulate the activity of monomeric G proteins such as Ras. RGS proteins have been cloned from many eukaryotes, and those whose biochemical activity has been characterized regulate the members of the Gi family of G proteins; some forms can also act on Gq proteins. In an ongoing effort to elucidate the role of Gzalpha in cell signaling, the yeast two-hybrid system was employed to identify proteins that could interact with a mutationally activated form of Gzalpha. A novel RGS, termed RGSZ1, was identified that is most closely related to two existing RGS proteins termed RetRGS1 and GAIP. Northern blot analysis revealed that expression of RGSZ1 was limited to brain, and expression was particularly high in the caudate nucleus. Biochemical characterization of recombinant RGSZ1 protein revealed that RGSZ1 was indeed a GAP and, most significantly, showed a marked preference for Gzalpha over other members of the Gialpha family. Phosphorylation of Gzalpha by protein kinase C, an event known to occur in cells and that was previously shown to influence alpha-betagamma interactions of Gz, rendered the G protein much less susceptible to RGSZ1 action.

Authors
Glick, JL; Meigs, TE; Miron, A; Casey, PJ
MLA Citation
Glick, JL, Meigs, TE, Miron, A, and Casey, PJ. "RGSZ1, a Gz-selective regulator of G protein signaling whose action is sensitive to the phosphorylation state of Gzalpha." J Biol Chem 273.40 (October 2, 1998): 26008-26013.
PMID
9748279
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
40
Publish Date
1998
Start Page
26008
End Page
26013

Cocrystal structure of protein farnesyltransferase complexed with a farnesyl diphosphate substrate.

Protein farnesyltransferase (FTase) catalyzes the transfer of the hydrophobic farnesyl group from farnesyl diphosphate (FPP) to cellular proteins such as Ras at a cysteine residue near their carboxy-terminus. This process is necessary for the subcellular localization of these proteins to the plasma membrane and is required for the transforming activity of oncogenic variants of Ras, making FTase a prime target for anticancer therapeutics. The high-resolution crystal structure of rat FTase was recently determined, and we present here the X-ray crystal structure of the first complex of FTase with a FPP substrate bound at the active site. The isoprenoid moiety of FPP binds in an extended conformation in a hydrophobic cavity of the beta subunit of the FTase enzyme, and the diphosphate moiety binds to a positively charged cleft at the top of this cavity near the subunit interface. The observed location of the FPP molecule is consistent with mutagenesis data. This binary complex of FTase with FPP leads us to suggest a "molecular ruler" hypothesis for isoprenoid substrate specificity, where the depth of the hydrophobic binding cavity acts as a ruler discriminating between isoprenoids of differing lengths. Although other length isoprenoids may bind in the cavity, only the 15-carbon farnesyl moiety binds with its C1 atom in register with a catalytic zinc ion as required for efficient transfer to the Ras substrate.

Authors
Long, SB; Casey, PJ; Beese, LS
MLA Citation
Long, SB, Casey, PJ, and Beese, LS. "Cocrystal structure of protein farnesyltransferase complexed with a farnesyl diphosphate substrate." Biochemistry 37.27 (July 7, 1998): 9612-9618.
PMID
9657673
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
27
Publish Date
1998
Start Page
9612
End Page
9618
DOI
10.1021/bi980708e

Kinetic analysis of zinc ligand mutants of mammalian protein farnesyltransferase.

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that catalyzes the prenylation of several proteins that are important in cellular regulatory events. A specific residue of FTase, Cys299 in the beta subunit previously identified as essential for zinc binding and catalysis, had been tentatively assigned as one of the zinc ligands. This assignment was subsequently confirmed in the X-ray structure of FTase, which also identified two additional residues, Asp297 and His362 in the beta subunit, as the remaining protein-derived metal ligands. To more fully explore the role of zinc in the catalytic mechanism of FTase, site-directed mutagenesis was performed on these two zinc ligands. Although the abilities of all the mutants to bind the farnesyl diphosphate substrate were similar to that of the wild-type enzyme, all the mutants displayed markedly reduced enzymatic activities and zinc affinities. Steady-state and pre-steady-state kinetic analyses of the residual activities indicated that the rate-limiting step changed from product release in the wild-type enzyme to the chemical step of product formation for three of the mutant enzymes. Additionally, single-turnover experiments indicated that the greatest effect of alteration of zinc ligands for all the mutants was on the product formation step, this being reduced 10(3)-10(5)-fold in the mutant forms compared to the wild-type enzyme. These results confirm a critical involvement of the zinc in catalysis by FTase and support a model in which the metal ion is directly involved in the chemical step of the enzymatic reaction.

Authors
Fu, HW; Beese, LS; Casey, PJ
MLA Citation
Fu, HW, Beese, LS, and Casey, PJ. "Kinetic analysis of zinc ligand mutants of mammalian protein farnesyltransferase." Biochemistry 37.13 (March 31, 1998): 4465-4472.
PMID
9521766
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
13
Publish Date
1998
Start Page
4465
End Page
4472
DOI
10.1021/bi972511c

Prenylation-dependent association of Ki-Ras with microtubules. Evidence for a role in subcellular trafficking.

We recently identified a prenyl peptide-binding protein in microsomal membranes from bovine brain (Thissen, J. A., and Casey, P. J. (1993) J. Biol. Chem. 268, 13780-13783). Through a variety of approaches, this binding protein has been identified as the cytoskeletal protein tubulin. Prenyl peptides bind to purified tubulin with a Kd of 40 nM and also bind to tubulin polymerized into microtubules. Microtubule affinity chromatography of extracts from cells in which the prenyl protein pool was metabolically labeled revealed that prenyl proteins bound to the immobilized microtubules; one, a 24-kDa protein, was tentatively identified as a GTP-binding protein. Of several prenylated GTP-binding proteins tested, including Ki-Ras4B, Ha-Ras, RhoB, RhoA, and Rap1B, only Ki-Ras was found to bind significantly to microtubules, and this was in a prenylation-dependent fashion. A potential significance of the interaction of Ki-Ras4B with microtubules was indicated from analysis of the localization of newly synthesized Ki-Ras4B and Ha-Ras, each tagged with green fluorescence protein (GFP). Treatment of NIH-3T3 cells expressing GFP-Ki-Ras with Taxol (paclitaxel) resulted in accumulation of the expressed protein in intracellular locations, whereas in control cells the protein was correctly targeted to the plasma membrane. Importantly, such treatment with paclitaxel did not affect the cellular localization of expressed GFP-Ha-Ras. These results indicate that an intact microtubule network may be directly involved in Ki-Ras processing and/or targeting and provide direct evidence for a physiological distinction between Ki-Ras and Ha-Ras in cells. Additionally, the finding that paclitaxel treatment of cells disrupts Ki-Ras trafficking suggests an additional mechanism for the anti-proliferative effects of this drug.

Authors
Thissen, JA; Gross, JM; Subramanian, K; Meyer, T; Casey, PJ
MLA Citation
Thissen, JA, Gross, JM, Subramanian, K, Meyer, T, and Casey, PJ. "Prenylation-dependent association of Ki-Ras with microtubules. Evidence for a role in subcellular trafficking." J Biol Chem 272.48 (November 28, 1997): 30362-30370.
PMID
9374526
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
48
Publish Date
1997
Start Page
30362
End Page
30370

Can prenylcysteines be exploited as ligands for mammalian multidrug-resistance transporters?

The overexpression of specific transport proteins in the membrane of many cancer cells renders these cells resistant to many therapeutic drugs. Some lipid-modified cysteine compounds inhibit one drug-transporting protein, indicating the potential of developing such compounds as therapeutic agents.

Authors
Hurwitz, LM; Casey, PJ
MLA Citation
Hurwitz, LM, and Casey, PJ. "Can prenylcysteines be exploited as ligands for mammalian multidrug-resistance transporters?." Chem Biol 4.10 (October 1997): 711-715. (Review)
PMID
9375248
Source
pubmed
Published In
Chemistry & Biology
Volume
4
Issue
10
Publish Date
1997
Start Page
711
End Page
715

Isolation and characterization of a prenylcysteine lyase from bovine brain.

Prenylated proteins contain one of two isoprenoid lipids, either the 15-carbon farnesyl or the 20-carbon geranylgeranyl, covalently attached to cysteine residues at or near their C terminus. The cellular abundance of prenylated proteins, which can comprise up to 2% of total cellular protein, raises the question of how cells dispose of prenylcysteines produced during the normal turnover of prenylated proteins. We have identified and characterized a novel enzyme, which we term prenylcysteine lyase, that is capable of cleaving the thioether bond of prenylcysteines. The enzyme was isolated from bovine brain membranes and exhibits an apparent molecular mass of 63 kDa. The enzyme did not require NADPH as cofactor for prenylcysteine degradation, thus distinguishing it from cytochrome P450- and flavin-containing monooxygenases that catalyze S-oxidation of thioethers. Purified prenylcysteine lyase shows similar kinetics in utilization of both farnesylcysteine and geranylgeranylcysteine as substrates, although Vmax is 2-fold higher with the former compound. Interaction of prenylcysteine substrates with the enzyme requires that they possess a free amino group; N-acetylated prenylcysteines and prenyl peptides are not substrates. These findings suggest that prenylcysteine lyase is a specific enzyme involved in prenylcysteine metabolism in mammalian cells, most likely comprising the final step in the degradation of prenylated proteins.

Authors
Zhang, L; Tschantz, WR; Casey, PJ
MLA Citation
Zhang, L, Tschantz, WR, and Casey, PJ. "Isolation and characterization of a prenylcysteine lyase from bovine brain." J Biol Chem 272.37 (September 12, 1997): 23354-23359.
PMID
9287348
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
37
Publish Date
1997
Start Page
23354
End Page
23359

Analysis of the C-terminal proteolysis of prenylated proteins

Authors
Otto, JC; Casey, PJ
MLA Citation
Otto, JC, and Casey, PJ. "Analysis of the C-terminal proteolysis of prenylated proteins." FASEB JOURNAL 11.9 (July 31, 1997): A1088-A1088.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1088
End Page
A1088

Substrate binding is required for release of product from mammalian protein farnesyltransferase

Authors
Tschantz, WR; Furfine, ES; Casey, PJ
MLA Citation
Tschantz, WR, Furfine, ES, and Casey, PJ. "Substrate binding is required for release of product from mammalian protein farnesyltransferase." FASEB JOURNAL 11.9 (July 31, 1997): A1087-A1087.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1087
End Page
A1087

Structure-function analysis of residues important in zinc binding and activity in protein farnesyltransferase

Authors
Fu, HW; Casey, PJ
MLA Citation
Fu, HW, and Casey, PJ. "Structure-function analysis of residues important in zinc binding and activity in protein farnesyltransferase." FASEB JOURNAL 11.9 (July 31, 1997): A964-A964.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A964
End Page
A964

Functions of metals in protein farnesyltransferase

Authors
Huang, CC; Casey, PJ; Fierke, CA
MLA Citation
Huang, CC, Casey, PJ, and Fierke, CA. "Functions of metals in protein farnesyltransferase." FASEB JOURNAL 11.9 (July 31, 1997): A1308-A1308.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1308
End Page
A1308

Farnesyltransferase inhibitors alter the prenylation and growth-stimulating function of RhoB

Authors
Lebowitz, PF; Casey, PJ; Prendergast, GC; Thissen, JA
MLA Citation
Lebowitz, PF, Casey, PJ, Prendergast, GC, and Thissen, JA. "Farnesyltransferase inhibitors alter the prenylation and growth-stimulating function of RhoB." JOURNAL OF BIOLOGICAL CHEMISTRY 272.25 (June 20, 1997): 15591-15594.
PMID
9188444
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
272
Issue
25
Publish Date
1997
Start Page
15591
End Page
15594
DOI
10.1074/jbc.272.25.15591

Substrate binding is required for release of product from mammalian protein farnesyltransferase.

Protein farnesyltransferase (FTase) catalyzes the modification by a farnesyl lipid of Ras and several other key proteins involved in cellular regulation. Previous studies on this important enzyme have indicated that product dissociation is the rate-limiting step in catalysis. A detailed examination of this has now been performed, and the results provide surprising insights into the mechanism of the enzyme. Examination of the binding of a farnesylated peptide product to free enzyme revealed a binding affinity of approximately 1 microM. However, analysis of the product release step under single turnover conditions led to the surprising observation that the peptide product did not dissociate from the enzyme unless additional substrate was provided. Once additional substrate was provided, the enzyme released the farnesylated peptide product with rates comparable with that of overall catalysis by FTase. Additionally, stable FTase-farnesylated product complexes were formed using Ras proteins as substrates, and these complexes also require additional substrate for product release. These data have major implications in both our understanding of overall mechanism of this enzyme and in design of inhibitors against this therapeutic target.

Authors
Tschantz, WR; Furfine, ES; Casey, PJ
MLA Citation
Tschantz, WR, Furfine, ES, and Casey, PJ. "Substrate binding is required for release of product from mammalian protein farnesyltransferase." J Biol Chem 272.15 (April 11, 1997): 9989-9993.
PMID
9092540
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
15
Publish Date
1997
Start Page
9989
End Page
9993

Crystal structure of protein farnesyltransferase at 2.25 angstrom resolution.

Protein farnesyltransferase (FTase) catalyzes the carboxyl-terminal lipidation of Ras and several other cellular signal transduction proteins. The essential nature of this modification for proper function of these proteins has led to the emergence of FTase as a target for the development of new anticancer therapy. Inhibition of this enzyme suppresses the transformed phenotype in cultured cells and causes tumor regression in animal models. The crystal structure of heterodimeric mammalian FTase was determined at 2.25 angstrom resolution. The structure shows a combination of two unusual domains: a crescent-shaped seven-helical hairpin domain and an alpha-alpha barrel domain. The active site is formed by two clefts that intersect at a bound zinc ion. One cleft contains a nine-residue peptide that may mimic the binding of the Ras substrate; the other cleft is lined with highly conserved aromatic residues appropriate for binding the farnesyl isoprenoid with required specificity.

Authors
Park, HW; Boduluri, SR; Moomaw, JF; Casey, PJ; Beese, LS
MLA Citation
Park, HW, Boduluri, SR, Moomaw, JF, Casey, PJ, and Beese, LS. "Crystal structure of protein farnesyltransferase at 2.25 angstrom resolution." Science 275.5307 (March 21, 1997): 1800-1804.
PMID
9065406
Source
pubmed
Published In
Science
Volume
275
Issue
5307
Publish Date
1997
Start Page
1800
End Page
1804

Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins.

Pertussis toxin (PTX) has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in signalling. This toxin catalyses the ADP-ribosylation of specific G-protein alpha subunits of the Gi family, and this modification prevents the occurrence of the receptor-G-protein interaction. This review focuses on the biochemical properties and signalling of those G-proteins historically classified as 'PTX-resistant' due to the inability of the toxin to influence signalling through them. These G-proteins include members of the Gq and G12 families and one Gi family member, i.e. Gz. Signalling pathways controlled by these G-proteins are well characterized only for Gq family members, which activate specific isoforms of phospholipase C, resulting in increases in intracellular calcium and activation of protein kinase C (PKC), among other responses. While members of the G12 family have been implicated in processes that regulate cell growth, and Gz has been shown to inhibit adenylate cyclase, the specific downstream targets to these G-proteins in vivo have not been clearly established. Since two of these proteins, G12 alpha and Gz alpha, are excellent substrates for PKC, there is the potential for cross-talk between their signalling and Gq-dependent processes leading to activation of PKC. In tissues that express these G-proteins, a number of guanine-nucleotide-dependent, PTX-resistant, signalling pathways have been defined for which the G-protein involved has not been identified. This review summarizes these pathways and discusses the evidence both for the participation of specific PTX-resistant G-proteins in them and for the regulation of these processes by PKC.

Authors
Fields, TA; Casey, PJ
MLA Citation
Fields, TA, and Casey, PJ. "Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins." Biochem J 321 ( Pt 3) (February 1, 1997): 561-571. (Review)
PMID
9032437
Source
pubmed
Published In
The Biochemical journal
Volume
321 ( Pt 3)
Publish Date
1997
Start Page
561
End Page
571

Evidence for a catalytic role of zinc in protein farnesyltransferase. Spectroscopy of Co2+-farnesyltransferase indicates metal coordination of the substrate thiolate.

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that catalyzes the addition of a farnesyl isoprenoid to a conserved cysteine in peptide or protein substrates. We have substituted the essential Zn2+ in FTase with Co2+ to investigate the function of the metal polyhedron using optical absorption spectroscopy. The catalytic activity of FTase is unchanged by the substitution of cobalt for zinc. The absorption spectrum of Co2+-FTase displays a thiolate-Co2+ charge transfer band (epsilon320 = 1030 M(-1) cm(-1)) consistent with the coordination of one cysteine side chain and also ligand field bands (epsilon560 = 140 M(-1) cm(-1)) indicative of a pentacoordinate or distorted tetrahedral metal geometry. Most importantly, the ligand-metal charge transfer band displays an increased intensity (epsilon320 = 1830 M(-1) cm(-1)) in the ternary complex of FTase x isoprenoid x peptide substrate indicative of the formation of a second Co2+-thiolate bond as cobalt coordinates the thiolate of the peptide substrate. A similar increase in the ligand-metal charge transfer band in a product complex indicates that the sulfur atom of the farnesylated peptide also coordinates the metal. Transient kinetics demonstrate that thiolate-cobalt metal coordination also occurs in an active FTase x FPP x peptide substrate complex and that the rate constant for the chemical step is 17 s(-1). These data provide evidence that the zinc ion plays an important catalytic role in FTase, most likely by activation of the cysteine thiol of the protein substrate for nucleophilic attack on the isoprenoid.

Authors
Huang, CC; Casey, PJ; Fierke, CA
MLA Citation
Huang, CC, Casey, PJ, and Fierke, CA. "Evidence for a catalytic role of zinc in protein farnesyltransferase. Spectroscopy of Co2+-farnesyltransferase indicates metal coordination of the substrate thiolate." J Biol Chem 272.1 (January 3, 1997): 20-23.
PMID
8995218
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
1
Publish Date
1997
Start Page
20
End Page
23

Functions of metals in protein farnesyltransferase

Mammalian protein farnesyltransferase (FTase) is a zinc metalloenzyme that catalyzes the addition of a farnesyl isoprenoid to a conserved cysteine in peptide or protein substrates. In addition to zinc, FTase also requires mM concentrations of magnesium for optimal reactivity. In order to further understand the catalytic roles of these two metals in l,'Tase, we have used metal-substituted enzymes in combination with transient kinetic experiments to directly measure the catalytic step. Metal coordination of the peptide thiot is indicated by observation of a metal-sulfur charge transfer band in the absorption spectrum of cobalt-substituted FrI'ase ternary complexes. Additionally. the rate constant of product formation of (admium-substituted FTase is similar to that ofzinc-FTase. Using the zinc-enzyme with two peptide substrates, GCVLS and CVIM, derived from H-Ras and KRas, respectively, we have found that product can be formed in the absence of magnesium although the rate constant is decreased. In addition, the ('VIM substrate has a decreased requirement for magnesium. The combination of detailed kinetic and thermodynamic analysis with metal substitution experiments provides insights into the mechanism of protein prenyl transferases. (supported by NItt GM 40602).

Authors
Huang, CC; Casey, PJ; Fierke, CA
MLA Citation
Huang, CC, Casey, PJ, and Fierke, CA. "Functions of metals in protein farnesyltransferase." FASEB Journal 11.9 (1997): A1307-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1307

Substrate binding is required for release of product from mammalian protein farnesyltransferase

Protein farnesyltransferase (FTase) catalyzes the modification by a farnesyl lipid of Ras and several other key proteins involved in cellular regulation. Previous studies on this important enzyme have indicated that product dissociation is the rate-limiting step in catalysis. A detailed examination of this has now been performed, and the results provide surprising insights into the mechanism of the enzyme. Examination of the binding of a farnesylated peptide product to free enzyme revealed a binding affinity of 1 #M. However, analysis of the product release step under single turnover conditions led to the surprising observation that the peptide product did not dissociate from the enzyme unless additional substrate was provided. Once additional substrate was provided, the enzyme released the farnesylated peptide product with rates comparable to the that of the overall catalysis by FTase. Additionally, stable FTase:farnesylated product complexes were formed using Ras proteins as substrates, and these complexes also require additional substrate for product release. These data have major implications in both our understanding of the overall mechanism of this enzyme and in design of inhibitors against this therapeutic target.

Authors
Tschantz, WR; Furfine, ES; Casey, PJ
MLA Citation
Tschantz, WR, Furfine, ES, and Casey, PJ. "Substrate binding is required for release of product from mammalian protein farnesyltransferase." FASEB Journal 11.9 (1997): A1086-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1086

Analysis of the C-terminal proteolysis of prenylated proteins

A variety of proteins, including many GTP-binding proteins, are modified by an isoprenoid lipid which is attached at a C-terminal motif. This motif consists of the tetrapeptide CaaX, where 6C5 is the cysteine on which prenylation occurs, 6a6 are commonly aliphatic residues, and 6X5 can be one of several amino acids. Following prenylation of the CaaX motif, the prenylated protein undergoes two additional processing steps. First, a specific protease cleaves the -aaX tripeptide from the C-terminus of the protein. After this proteolytic cleavage, the now exposed cagboxyl group of the prenylated cysteine residue is methylated by a specific carboxylmethyltransferase. Using purified, recombinant prenylated proteins and a recombinant yeast carboxytmethyltransferase, we have developed an assay by which we can observe the proteolysis of prenylated proteins in vitro. Using this assay, we have characterized the specific prenyl protein protease activity using a variety of prenytated protein substrates, and have eliminated non-specific proteolysis events that occur in prenyl peptide-based assays.

Authors
Otto, JC; Casey, PJ
MLA Citation
Otto, JC, and Casey, PJ. "Analysis of the C-terminal proteolysis of prenylated proteins." FASEB Journal 11.9 (1997): A1087-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1087

Influence of metal ions on substrate binding and catalytic activity of mammalian protein geranylgeranyltransferase type-I.

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group from the prenyl donor geranylgeranyl diphosphate (GGPP) to the cysteine residue of substrate proteins containing a C-terminal CaaX-motif (a sequence motif of proteins consisting of an invariant Cys residue fourth from the C-terminus). The GGTase-I heterodimer contains one atom of zinc, and this metal is required for enzyme activity. In this regard, GGTase-I is similar to the related enzyme protein farnesyltransferase (FTase); the latter enzyme also requires Mg2+ for activity. The current studies were undertaken in an attempt to explore further the role of bivalent metal ions in the activity of GGTase-I. Surprisingly, we found that GGTase-I and FTase have different metal requirements. Specifically, in marked contrast to FTase, GGTase-I does not require Mg2+ for activity. Direct binding assays, including a novel fluorescence-based technique, were employed to obtain quantitative information on the interaction of substrates with GGTase-I. Using these assays, we demonstrate that the Zn2+ in GGTase-I is required for peptide, but not for isoprenoid, substrate binding. Moreover, binding of GGPP protects GGTase-I from inactivation by zinc-chelating reagents; this protective effect is not seen with binding of peptide substrates. Metal substitution studies show that the Zn2+ in GGTase-I can be replaced by Cd2+, and that the Cd form of GGTase-I has altered specificity with regard to utilization of both peptide and isoprenoid substrates. The significance of these findings in relation to proposed mechanisms for the GGTase-I reaction is discussed.

Authors
Zhang, FL; Casey, PJ
MLA Citation
Zhang, FL, and Casey, PJ. "Influence of metal ions on substrate binding and catalytic activity of mammalian protein geranylgeranyltransferase type-I." Biochem J 320 ( Pt 3) (December 15, 1996): 925-932.
PMID
9003382
Source
pubmed
Published In
The Biochemical journal
Volume
320 ( Pt 3)
Publish Date
1996
Start Page
925
End Page
932

Kinetics of protein farnesyltransferase: sigmoidal vs hyperbolic behavior as a function of assay conditions.

Protein farnesyl transferase (FTase) catalyzes the addition of a farnesyl isoprenoid to a conserved cysteine residue in Ras and several other key proteins involved in cell regulation. An assay technique commonly used to measure FTase activity involves vacuum filtration. This assay, which traps precipitated, radiolabeled prenylated proteins on a glass fiber filter for analysis by scintillation counting, was designed to be fast and accurate. In the case of FTase, substrate saturation curves generated by this assay technique using Ras as a substrate often show a lag at low Ras concentrations, resulting in curves with sigmoidal character. We have found that the sigmoidal behavior is due to the use of the filter binding assay and not to any inherent property of FTase. Specifically, the glass fiber filters do not adequately trap precipitated Ras proteins, especially at low concentrations. Addition of cytosol from either bovine brain or liver, or of purified tubulin to the FTase assay mixture prior to the precipitation step, results in the apparent formation of stable complexes of farnesylated Ras protein that can then be optimally trapped on the glass fiber filter. This appears to be at least in part due to the ability of tubulin to bind the prenyl protein reaction product. The ability to obtain accurate kinetics for the FTase using the standard filter binding assay should greatly enhance its use to accurately assess the properties of FTase inhibitors.

Authors
Thissen, JA; Casey, PJ
MLA Citation
Thissen, JA, and Casey, PJ. "Kinetics of protein farnesyltransferase: sigmoidal vs hyperbolic behavior as a function of assay conditions." Anal Biochem 243.1 (December 1, 1996): 80-85.
PMID
8954528
Source
pubmed
Published In
Analytical Biochemistry
Volume
243
Issue
1
Publish Date
1996
Start Page
80
End Page
85
DOI
10.1006/abio.1996.0484

Identification of a cysteine residue essential for activity of protein farnesyltransferase. Cys299 is exposed only upon removal of zinc from the enzyme.

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [3H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys299, that is in the beta subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys299 in the beta subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.

Authors
Fu, HW; Moomaw, JF; Moomaw, CR; Casey, PJ
MLA Citation
Fu, HW, Moomaw, JF, Moomaw, CR, and Casey, PJ. "Identification of a cysteine residue essential for activity of protein farnesyltransferase. Cys299 is exposed only upon removal of zinc from the enzyme." J Biol Chem 271.45 (November 8, 1996): 28541-28548.
PMID
8910483
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
45
Publish Date
1996
Start Page
28541
End Page
28548

RCS10 is a selective activator of G alpha(i) GTPase activity

Authors
Hunt, TW; Fields, TA; Casey, PJ; Peralta, EG
MLA Citation
Hunt, TW, Fields, TA, Casey, PJ, and Peralta, EG. "RCS10 is a selective activator of G alpha(i) GTPase activity." NATURE 383.6596 (September 12, 1996): 175-177.
PMID
8774883
Source
wos-lite
Published In
Nature
Volume
383
Issue
6596
Publish Date
1996
Start Page
175
End Page
177
DOI
10.1038/383175a0

The role of prenylation in G-protein assembly and function.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are vital components of numerous signal transduction pathways, including sensory and hormonal response systems. G-proteins transduce signals from heptahelical transmembrane receptors to downstream effectors. The localization of a G-protein to the plasma membrane, as well as its interaction with the appropriate receptor and effector, are essential for its function. In addition, the association of a G-protein's subunits to form its trimer is required for interaction with its receptor. The G-protein gamma subunits (G gamma) are subject to a set of carboxyl-terminal processing events that include prenylation of a cysteine, proteolysis, and methylation. Recent advances which elucidate the contributions that the post-translational modifications of the G gamma subunit have on the assembly, membrane association, and function of the G-protein trimer reveal that these modifications are required for important protein-protein, in addition to membrane-protein, interactions.

Authors
Higgins, JB; Casey, PJ
MLA Citation
Higgins, JB, and Casey, PJ. "The role of prenylation in G-protein assembly and function." Cell Signal 8.6 (September 1996): 433-437. (Review)
PMID
8958445
Source
pubmed
Published In
Cellular Signalling
Volume
8
Issue
6
Publish Date
1996
Start Page
433
End Page
437

Substitution of cadmium for zinc in farnesyl:protein transferase alters its substrate specificity

Authors
Zhang, FL; Fu, HW; Casey, PJ; Bishop, WR
MLA Citation
Zhang, FL, Fu, HW, Casey, PJ, and Bishop, WR. "Substitution of cadmium for zinc in farnesyl:protein transferase alters its substrate specificity." BIOCHEMISTRY 35.25 (June 25, 1996): 8166-8171.
PMID
8679569
Source
wos-lite
Published In
Biochemistry
Volume
35
Issue
25
Publish Date
1996
Start Page
8166
End Page
8171
DOI
10.1021/bi960574+

Prenylation of the hepatitis delta virus large antigen: A new target for farnesyltransferase inhibitors.

Authors
Otto, JC; Hayward, KN; Casey, PJ
MLA Citation
Otto, JC, Hayward, KN, and Casey, PJ. "Prenylation of the hepatitis delta virus large antigen: A new target for farnesyltransferase inhibitors." FASEB JOURNAL 10.6 (April 30, 1996): 2893-2893.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
6
Publish Date
1996
Start Page
2893
End Page
2893

Protein prenyltransferases.

Authors
Casey, PJ; Seabra, MC
MLA Citation
Casey, PJ, and Seabra, MC. "Protein prenyltransferases." J Biol Chem 271.10 (March 8, 1996): 5289-5292. (Review)
PMID
8621375
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
10
Publish Date
1996
Start Page
5289
End Page
5292

The hepatitis delta virus large antigen is farnesylated both in vitro and in animal cells.

The hepatitis delta virus large antigen (lHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20-carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S-transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate (apparent Km = 0.8 microM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. As prenylation of lHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection.

Authors
Otto, JC; Casey, PJ
MLA Citation
Otto, JC, and Casey, PJ. "The hepatitis delta virus large antigen is farnesylated both in vitro and in animal cells." J Biol Chem 271.9 (March 1, 1996): 4569-4572.
PMID
8617711
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
9
Publish Date
1996
Start Page
4569
End Page
4572

Arachidonate and related unsaturated fatty acids selectively inactivate the guanine nucleotide-binding regulatory protein, Gz.

Gz is a member of the family of trimeric guanine nucleotide-binding regulatory proteins (G proteins), which plays a crucial role in signaling across cell membranes. The expression of Gz is predominately confined to neuronal cells and platelets, suggesting an involvement in a neuroendocrine process. Although the signaling pathway in which Gz participates is not yet known, it has been linked to inhibition of adenylyl cyclase. We have found that arachidonate and related unsaturated fatty acids suppress guanine nucleotide binding to the alpha subunit of Gz. This inhibition of nucleotide binding by cis-unsaturated fatty acids is specific for Gz alpha; other G protein alpha subunits are relatively insensitive to these lipids. The IC50 for inhibition by the lipids closely corresponds to their critical micellar concentrations, suggesting that the interaction of the lipid micelle with Gzalpha is the primary event leading to inhibition. The presence of the acidic group of the fatty acid is critical for inhibition, as no effect is observed with the corresponding fatty alcohol. While arachidonic acid produces near-complete inhibition of both GDP and guanosine 5-(3-O-thio)triphosphate binding by Gzalpha, release of GDP from the protein was unaffected. Furthermore, the rate of inactivation of Gzalpha by arachidonate is essentially identical to the rate of GDP release from the protein, indicating that GDP release is required for inactivation. These observations indicate that the mechanism of inactivation of Gzalpha by unsaturated fatty acids is through an interaction of an acidic lipid micelle with the nucleotide-free form of the protein. Although the physiologic significance of this finding is unclear, similar effects of unsaturated fatty acids on other proteins involved in cell signaling indicate potential roles for these lipids in signal modulation. Additionally, the ability of arachidonate to inactivate this adenylyl cyclase-inhibitory G protein provides a molecular mechanism for previous findings that treatment of platelets with arachidonate results in elevated cAMP levels.

Authors
Glick, J; Santoyo, G; Casey, PJ
MLA Citation
Glick, J, Santoyo, G, and Casey, PJ. "Arachidonate and related unsaturated fatty acids selectively inactivate the guanine nucleotide-binding regulatory protein, Gz." J Biol Chem 271.6 (February 9, 1996): 2949-2954.
PMID
8621685
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
6
Publish Date
1996
Start Page
2949
End Page
2954

Protein prenylation: molecular mechanisms and functional consequences.

Prenylation is a class of lipid modification involving covalent addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. Known prenylated proteins include fungal mating factors, nuclear lamins, Ras and Ras-related GTP-binding proteins (G proteins), the subunits of trimeric G proteins, protein kinases, and at least one viral protein. Prenylation promotes membrane interactions of most of these proteins, which is not surprising given the hydrophobicity of the lipids involved. In addition, however, prenylation appears to play a major role in several protein-protein interactions involving these species. The emphasis in this review is on the enzymology of prenyl protein processing and the functional significance of prenylation in cellular events. Several other recent reviews provide more detailed coverage of aspects of prenylation that receive limited attention here owing to length restrictions (1-4).

Authors
Zhang, FL; Casey, PJ
MLA Citation
Zhang, FL, and Casey, PJ. "Protein prenylation: molecular mechanisms and functional consequences." Annu Rev Biochem 65 (1996): 241-269. (Review)
PMID
8811180
Source
pubmed
Published In
Annual Review of Biochemistry
Volume
65
Publish Date
1996
Start Page
241
End Page
269
DOI
10.1146/annurev.bi.65.070196.001325

[8] Assays for G protein βγ subunit activity

Authors
Higgins, JB; Casey, PJ
MLA Citation
Higgins, JB, and Casey, PJ. "[8] Assays for G protein βγ subunit activity." Methods in Neurosciences 29.C (1996): 90-100.
Source
scival
Published In
Methods in Neurosciences
Volume
29
Issue
C
Publish Date
1996
Start Page
90
End Page
100
DOI
10.1016/S1043-9471(96)80044-9

Prenylation of the hepatitis delta virus large antigen: a new target for farnesyltransferase inhibitors

The hepatitis delta virus large antigen (IHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. The prenylation of IHDAg was examined in vitro using a fusion protein between glutathione-S-transferase and the C-terminal 117 amino acids of IHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase-I was not detected. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic IHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of IHDAg expressed in COS cells was not observed. Thus, IHDAg is exclusively a substrate for FTase. As prenylation of IHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection. We are currently examining the effects of farnesyltransferase inhibitors on hepatitis delta viral assembly events in a human hepatoma cell line.

Authors
Otto, JÇ; Hayward, KN; Casey, PJ
MLA Citation
Otto, JÇ, Hayward, KN, and Casey, PJ. "Prenylation of the hepatitis delta virus large antigen: a new target for farnesyltransferase inhibitors." FASEB Journal 10.6 (1996): A1501-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
6
Publish Date
1996
Start Page
A1501

EVIDENCE THAT DIRECT BINDING OF G(BETA-GAMMA) TO THE GIRK1 G-PROTEIN-GATED INWARDLY RECTIFYING K+ CHANNEL IS IMPORTANT FOR CHANNEL ACTIVATION

Authors
HUANG, CL; SLESINGER, PA; CASEY, PJ; JAN, YN; JAN, LY
MLA Citation
HUANG, CL, SLESINGER, PA, CASEY, PJ, JAN, YN, and JAN, LY. "EVIDENCE THAT DIRECT BINDING OF G(BETA-GAMMA) TO THE GIRK1 G-PROTEIN-GATED INWARDLY RECTIFYING K+ CHANNEL IS IMPORTANT FOR CHANNEL ACTIVATION." NEURON 15.5 (November 1995): 1133-1143.
PMID
7576656
Source
wos-lite
Published In
Neuron
Volume
15
Issue
5
Publish Date
1995
Start Page
1133
End Page
1143
DOI
10.1016/0896-6273(95)90101-9

Characterization of prenylcysteines that interact with P-glycoprotein and inhibit drug transport in tumor cells.

Prenylcysteine methyl esters that represent the C-terminal structures of prenylated proteins demonstrate specific substrate-like interactions with P-glycoprotein (Zhang, L., Sachs, C. W., Fine, R. L., and Casey, P. J. (1994) J. Biol. Chem. 269, 15973-15976). The simplicity of these compounds provides a unique system for probing the structural specificity of P-glycoprotein substrates. We have further assessed the structural elements of prenylcysteines involved in the interaction with P-glycoprotein. Carboxyl group methylation, a modification in many prenylated proteins, plays an essential role of blocking the negative charge at the free carboxylate. Substitution of the methyl ester with a methyl amide or simple amide does not change the ability of the molecule to stimulate P-glycoprotein ATPase activity, but substitution with a glycine is not tolerated unless the carboxyl group of glycine is methylated. The presence of a nitrogen atom, which is found in many P-glycoprotein substrates and modifiers, is also essential for prenylcysteines to interact with P-glycoprotein. The structure at the nitrogen atom can, however, influence the type of interaction. Acetylation of the free amino group of prenylcysteine/results in a significant loss in the ability of prenylcysteines to stimulate P-glycoprotein ATPase activity. Instead, certain acetylated prenylcysteines behave as inhibitors of this activity. In studies using MDR1-transfected human breast cancer cells, the acetylated prenylcysteine analogs inhibit P-glycoprotein-mediated drug transport and enhance the steady-state accumulation of [3H]vinblastine, [3H]colchicine, and [3H]taxol. These inhibitors do not, however, affect drug accumulation in parental cells. These studies provide a novel approach for designing P-glycoprotein inhibitors that could prove effective in reversing the phenotype of multidrug resistance in tumor cells.

Authors
Zhang, L; Sachs, CW; Fu, HW; Fine, RL; Casey, PJ
MLA Citation
Zhang, L, Sachs, CW, Fu, HW, Fine, RL, and Casey, PJ. "Characterization of prenylcysteines that interact with P-glycoprotein and inhibit drug transport in tumor cells." J Biol Chem 270.39 (September 29, 1995): 22859-22865.
PMID
7559420
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
39
Publish Date
1995
Start Page
22859
End Page
22865

Phosphorylation of Gz alpha by protein kinase C blocks interaction with the beta gamma complex.

Gz alpha is a G protein alpha subunit with biochemical properties that distinguish it from other members of the G protein alpha subunit family. One such property is its ability to be stoichiometrically phosphorylated by protein kinase C (PKC), both in vitro and in intact cells. The site of this phosphorylation has been mapped to a region near the N terminus of Gz alpha, but no functional significance of the modification has been established. To investigate this question, we have developed a baculovirus/Sf9 cell expression system to produce Gz alpha. The protein purified from Sf9 cells is functional as assessed by its ability both to bind guanine nucleotide in a Mg(2+)-sensitive fashion and to serve as a substrate for phosphorylation by PKC. Furthermore, addition of the G protein beta gamma complex purified from bovine brain inhibits phosphorylation of Gz alpha in a dose-dependent manner. Conversely, phosphorylation of Gz alpha inhibits its ability to interact with beta gamma subunits. These results establish a functional consequence for PKC-catalyzed phosphorylation of Gz alpha and suggest a mechanism for regulation of signaling through Gz by preventing reassociation of its subunits.

Authors
Fields, TA; Casey, PJ
MLA Citation
Fields, TA, and Casey, PJ. "Phosphorylation of Gz alpha by protein kinase C blocks interaction with the beta gamma complex." J Biol Chem 270.39 (September 29, 1995): 23119-23125.
PMID
7559455
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
39
Publish Date
1995
Start Page
23119
End Page
23125

PROTEIN FARNESYLTRANSFERASE - KINETICS OF FARNESYL PYROPHOSPHATE BINDING AND PRODUCT RELEASE

Authors
FURFINE, ES; LEBAN, JJ; LANDAVAZO, A; MOOMAW, JF; CASEY, PJ
MLA Citation
FURFINE, ES, LEBAN, JJ, LANDAVAZO, A, MOOMAW, JF, and CASEY, PJ. "PROTEIN FARNESYLTRANSFERASE - KINETICS OF FARNESYL PYROPHOSPHATE BINDING AND PRODUCT RELEASE." BIOCHEMISTRY 34.20 (May 23, 1995): 6857-6862.
PMID
7756316
Source
wos-lite
Published In
Biochemistry
Volume
34
Issue
20
Publish Date
1995
Start Page
6857
End Page
6862
DOI
10.1021/bi00020a032

Protein lipidation in cell signaling.

The ability of cells to communicate with and respond to their external environment is critical for their continued existence. A universal feature of this communication is that the external signal must in some way penetrate the lipid bilayer surrounding the cell. In most cases of such signal acquisition, the signaling entity itself does not directly enter the cell but rather transmits its information to specific proteins present on the surface of the cell membrane. These proteins then communicate with additional proteins associated with the intracellular face of the membrane. Membrane localization and function of many of these proteins are dependent on their covalent modification by specific lipids, and it is the processes involved that form the focus of this article.

Authors
Casey, PJ
MLA Citation
Casey, PJ. "Protein lipidation in cell signaling." Science 268.5208 (April 14, 1995): 221-225. (Review)
PMID
7716512
Source
pubmed
Published In
Science
Volume
268
Issue
5208
Publish Date
1995
Start Page
221
End Page
225

Mechanisms of protein prenylation and role in G protein function.

Authors
Casey, PJ
MLA Citation
Casey, PJ. "Mechanisms of protein prenylation and role in G protein function." Biochem Soc Trans 23.1 (February 1995): 161-166. (Review)
PMID
7758720
Source
pubmed
Published In
Biochemical Society transactions
Volume
23
Issue
1
Publish Date
1995
Start Page
161
End Page
166

Isolation of protein prenyltransferases from bovine brain and baculovirus expression system.

Authors
Moomaw, JF; Zhang, FL; Casey, PJ
MLA Citation
Moomaw, JF, Zhang, FL, and Casey, PJ. "Isolation of protein prenyltransferases from bovine brain and baculovirus expression system." Methods Enzymol 250 (1995): 12-21.
PMID
7651144
Source
pubmed
Published In
Methods in Enzymology
Volume
250
Publish Date
1995
Start Page
12
End Page
21

Prenylated peptides in identification of specific binding proteins.

Authors
Thissen, JA; Barrett, MG; Casey, PJ
MLA Citation
Thissen, JA, Barrett, MG, and Casey, PJ. "Prenylated peptides in identification of specific binding proteins." Methods Enzymol 250 (1995): 158-168.
PMID
7651148
Source
pubmed
Published In
Methods in Enzymology
Volume
250
Publish Date
1995
Start Page
158
End Page
168

Lipid modifications of protein: Preface

Authors
Casey, PJ; Buss, JE
MLA Citation
Casey, PJ, and Buss, JE. "Lipid modifications of protein: Preface." Methods in Enzymology 250 (1995): xvii-xviii.
Source
scival
Published In
Methods in Enzymology
Volume
250
Publish Date
1995
Start Page
xvii
End Page
xviii
DOI
10.1016/0076-6879(95)50055-3

Properties and kinetic mechanism of recombinant mammalian protein geranylgeranyltransferase type I.

Protein geranylgeranyltransferase type I (GGTase I) catalyzes the prenylation of a number of proteins that play important roles in cellular signaling. The recent cDNA cloning of this enzyme (Zhang, F. L., Diehl, R. E., Kohl, N. E., Gibbs, J. B., Giros, B., Casey, P. J., and Omer, C. A. (1994) J. Biol. Chem. 269, 3175-3180) has allowed us to develop an expression system for obtaining large quantities of the enzyme. Co-infection of insect cells with recombinant baculoviruses encoding the two subunits of the enzyme results in GGTase I accumulation within the cells to levels of > 20% of cytosolic protein. The recombinant enzyme could be readily purified by ion-exchange chromatography and is shown to possess the activity and specificity of the enzyme obtained from mammalian tissues. Production of the recombinant enzyme allowed us to confirm its identity as a zinc metalloenzyme by direct identification of the metal using atomic absorption spectroscopy. We also identify two substrate analogs that are competitive inhibitors of GGTase I. One is a novel isoprenoid analog, 3-aza-GGPP, which inhibits the enzyme with a Ki of 15 nM. The second inhibitor is the tetrapeptide Cys-Val-Phe-Leu, which exhibits a Ki of 50 nM. The use of these inhibitors, coupled with a steady-state kinetic analysis of the enzyme, reveals that the reaction catalyzed by GGTase I proceeds through a random ordered sequential mechanism.

Authors
Zhang, FL; Moomaw, JF; Casey, PJ
MLA Citation
Zhang, FL, Moomaw, JF, and Casey, PJ. "Properties and kinetic mechanism of recombinant mammalian protein geranylgeranyltransferase type I." J Biol Chem 269.38 (September 23, 1994): 23465-23470.
PMID
8089111
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
38
Publish Date
1994
Start Page
23465
End Page
23470

A G-PROTEIN BETA-GAMMA-SUBUNIT-RESPONSIVE PHOSPHOINOSITIDE 3-KINASE ACTIVITY IN HUMAN PLATELET CYTOSOL

Authors
THOMASON, PA; JAMES, SR; CASEY, PJ; DOWNES, CP
MLA Citation
THOMASON, PA, JAMES, SR, CASEY, PJ, and DOWNES, CP. "A G-PROTEIN BETA-GAMMA-SUBUNIT-RESPONSIVE PHOSPHOINOSITIDE 3-KINASE ACTIVITY IN HUMAN PLATELET CYTOSOL." JOURNAL OF BIOLOGICAL CHEMISTRY 269.24 (June 17, 1994): 16525-16528.
PMID
8206965
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
269
Issue
24
Publish Date
1994
Start Page
16525
End Page
16528

Interaction of prenylcysteine methyl esters with the multidrug resistance transporter.

The multidrug resistance transporter is an integral membrane protein, termed P-glycoprotein, which can function as an ATP-dependent drug efflux pump to reduce intracellular drug accumulation in treated cells. The physiologic function of this protein in normal cells, however, is not completely understood. We report here that prenylcysteine methyl esters, which represent the C-terminal structures of prenylated proteins, both stimulate the transporter's intrinsic ATPase activity and compete for drug binding. The structural elements of prenylcysteine methyl esters involved in their interaction with P-glycoprotein include the isoprenoid moiety, the carboxyl methyl group, and the free amino group. These findings indicate that these molecules are potential physiologic ligands of the transporter. Furthermore, as the structures of the active prenylcysteines are distinct from the known substrates of P-glycoprotein, this information may facilitate design of novel inhibitors of the transporter.

Authors
Zhang, L; Sachs, CW; Fine, RL; Casey, PJ
MLA Citation
Zhang, L, Sachs, CW, Fine, RL, and Casey, PJ. "Interaction of prenylcysteine methyl esters with the multidrug resistance transporter." J Biol Chem 269.23 (June 10, 1994): 15973-15976.
PMID
7911465
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
23
Publish Date
1994
Start Page
15973
End Page
15976

Subtype-specific binding of azidoanilido-GTP by purified G protein alpha subunits.

Azidoanilido-GTP (AA-GTP), a hydrolysis-resistant, photoreactive GTP analog, is becoming an increasingly popular tool for identifying activation of specific G proteins by receptors within native plasma membranes. Despite the use of AA-GTP as an affinity probe, surprisingly little is known regarding the ability of various G protein alpha subunits to bind this analog. To directly address this issue, we compared the ability of four purified G protein alpha subunits (Go, Gi2, Gs, and Gz) to bind AA-GTP with their ability to bind GTP gamma S, a GTP analog commonly used to characterize the GTP-binding properties of G proteins. All four G alpha subunits tested bound AA-GTP in a manner distinct from their binding of GTP gamma S. One of these proteins, Gs alpha, required millimolar levels of free Mg2+ for significant binding of AA-GTP, while Go alpha and Gi alpha 2 displayed peak AA-GTP binding at approximately 100 microM free Mg2+. The fourth G alpha subunit, Gz, bound AA-GTP very poorly relative to GTP gamma S regardless of the magnesium concentration. These results indicate that individual G protein alpha subunits differ markedly in their ability to bind AA-GTP. Use of AA-GTP to identify specific G protein-receptor interactions must therefore take into account the varied abilities of G alpha subunits to bind this analog.

Authors
Fields, TA; Linder, ME; Casey, PJ
MLA Citation
Fields, TA, Linder, ME, and Casey, PJ. "Subtype-specific binding of azidoanilido-GTP by purified G protein alpha subunits." Biochemistry 33.22 (June 7, 1994): 6877-6883.
PMID
8204622
Source
pubmed
Published In
Biochemistry
Volume
33
Issue
22
Publish Date
1994
Start Page
6877
End Page
6883

Lipid modifications of G proteins.

Covalent attachment of lipids is a near-universal mechanism through which eukaryotic cells direct and, in some cases, control membrane localization of G proteins. Studies conducted over the past year have substantially advanced our understanding of both the molecular mechanisms and the functional consequences of these modifications. Of particular note are the processes of palmitoylation of the alpha-subunits of heterotrimeric G proteins, and prenylation of members of the Ras superfamily of monomeric G proteins, where recent findings point to unexpected roles for lipid modifications in signaling through these proteins.

Authors
Casey, PJ
MLA Citation
Casey, PJ. "Lipid modifications of G proteins." Curr Opin Cell Biol 6.2 (April 1994): 219-225. (Review)
PMID
8024813
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
6
Issue
2
Publish Date
1994
Start Page
219
End Page
225

In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex.

The gamma subunits of the heterotrimeric G proteins (G gamma) are subject to carboxyl-terminal processing. This processing involves prenylation of a cysteine residue initially 4 amino acids from the carboxyl terminus, endoproteolytic truncation of the 3 terminal amino acids, and methylation of the now carboxyl-terminal prenylcysteine residue. The significance of each of these modifications in the ultimate properties of G proteins is not yet clear. We have developed in vitro systems for the production of the three processing intermediates (unmodified, prenylated, and truncated-prenylated) for two G gamma subunits, one which is subject to farnesylation (G gamma 1) and one which is geranylgeranylated (G gamma 2). Assessment of the functional consequences of the processing of G gamma was found to require reconstitution of the polypeptides with a G protein beta subunit (G beta). The ability of recombinant G beta, produced in Sf9 cells, to assemble into stable beta gamma complexes (G beta gamma) with each of the G gamma processing intermediates was assessed. Both prenylated and unprenylated G gamma subunits formed stable complexes with G beta, but surprisingly, neither of the truncated-prenylated G gamma subunits were competent for this assembly. The G beta gamma complexes which were formed were examined for their ability to interact with a G protein alpha subunit (G alpha). Only those G beta gamma complexes containing a prenylated G gamma subunit were functional in this assay. These data indicate that: 1) prenylation of G gamma is not required for G beta gamma assembly; 2) assembly of the G beta gamma complex occurs prior to the proteolytic processing of G gamma; and 3) G beta gamma complexes require prenylated G gamma for interaction with G alpha.

Authors
Higgins, JB; Casey, PJ
MLA Citation
Higgins, JB, and Casey, PJ. "In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex." J Biol Chem 269.12 (March 25, 1994): 9067-9073.
PMID
8132644
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
12
Publish Date
1994
Start Page
9067
End Page
9073

cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I.

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48-kilodalton alpha subunit and a 43-kilodalton beta subunit. Peptides isolated from the alpha subunit of GGTase-I were shown to be identical with the alpha subunit of a related enzyme, protein farnesyltransferase. Overlapping cDNA clones containing the complete coding sequence for the beta subunit of GGTase-I were obtained from rat and human cDNA libraries. The cDNA clones from both species each predicted a protein of 377 amino acids with molecular masses of 42.4 kilodaltons (human) and 42.5 kilodaltons (rat). Amino acid sequence comparison suggests that the protein encoded by the Saccharomyces cerevisiae gene CDC43 is the yeast counterpart of the mammalian GGTase-I beta subunit. Co-expression of the GGTase-I beta subunit cDNA together with the alpha subunit of protein farnesyltransferase in Escherichia coli produced recombinant GGTase-I with electrophoretic and enzymatic properties indistinguishable from native GGTase-I.

Authors
Zhang, FL; Diehl, RE; Kohl, NE; Gibbs, JB; Giros, B; Casey, PJ; Omer, CA
MLA Citation
Zhang, FL, Diehl, RE, Kohl, NE, Gibbs, JB, Giros, B, Casey, PJ, and Omer, CA. "cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I." J Biol Chem 269.5 (February 4, 1994): 3175-3180.
PMID
8106351
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
5
Publish Date
1994
Start Page
3175
End Page
3180

Prenylation and G protein signaling.

Authors
Casey, PJ; Moomaw, JF; Zhang, FL; Higgins, YB; Thissen, JA
MLA Citation
Casey, PJ, Moomaw, JF, Zhang, FL, Higgins, YB, and Thissen, JA. "Prenylation and G protein signaling." Recent Prog Horm Res 49 (1994): 215-238. (Review)
PMID
8146425
Source
pubmed
Published In
Recent progress in hormone research
Volume
49
Publish Date
1994
Start Page
215
End Page
238

Microsomal membranes contain a high affinity binding site for prenylated peptides.

Prenylation and subsequent processing of many proteins involved in cellular signaling serves to direct and/or anchor these proteins to specific membranes in the cell. One major class of prenylated proteins contains the so-called CAAX motif; such proteins contain a cysteine residue fourth from the COOH terminus. After addition of the isoprenoid to this cysteine residue by specific cytosolic protein prenyltransferases, the proteins are subject to further processing by enzymes associated with microsomal membranes. This suggests that newly prenylated proteins are initially directed to a microsomal membrane compartment for completion of their processing. Using a radiolabeled, prenylated peptide as a ligand, we have identified a specific, high affinity binding site or receptor on microsomal membranes. This receptor is both protease- and heat-sensitive and exhibits saturable binding of the prenylated peptide with a KD of 30 nM. Competition analysis indicates that both geranylgeranylated and farnesylated, but not myristoylated, peptides bind to this receptor. A fully processed prenylated protein also does not compete, indicating a role for the three terminal residues of the prenylated peptide in receptor recognition. This receptor may serve to direct newly prenylated proteins to a microsomal compartment for completion of processing prior to trafficking to their final destination in the cell.

Authors
Thissen, JA; Casey, PJ
MLA Citation
Thissen, JA, and Casey, PJ. "Microsomal membranes contain a high affinity binding site for prenylated peptides." J Biol Chem 268.19 (July 5, 1993): 13780-13783.
PMID
8314746
Source
pubmed
Published In
The Journal of biological chemistry
Volume
268
Issue
19
Publish Date
1993
Start Page
13780
End Page
13783

HIGH-LEVEL EXPRESSION OF MAMMALIAN PROTEIN FARNESYLTRANSFERASE IN A BACULOVIRUS SYSTEM - THE PURIFIED PROTEIN CONTAINS ZINC

Authors
CHEN, WJ; MOOMAW, JF; OVERTON, L; KOST, TA; CASEY, PJ
MLA Citation
CHEN, WJ, MOOMAW, JF, OVERTON, L, KOST, TA, and CASEY, PJ. "HIGH-LEVEL EXPRESSION OF MAMMALIAN PROTEIN FARNESYLTRANSFERASE IN A BACULOVIRUS SYSTEM - THE PURIFIED PROTEIN CONTAINS ZINC." JOURNAL OF BIOLOGICAL CHEMISTRY 268.13 (May 5, 1993): 9675-9680.
PMID
8486655
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
268
Issue
13
Publish Date
1993
Start Page
9675
End Page
9680

Biochemistry of protein prenylation.

Authors
Casey, PJ
MLA Citation
Casey, PJ. "Biochemistry of protein prenylation." J Lipid Res 33.12 (December 1992): 1731-1740. (Review)
PMID
1479283
Source
pubmed
Published In
Journal of lipid research
Volume
33
Issue
12
Publish Date
1992
Start Page
1731
End Page
1740

FARNESYLATION OF YDJ1P IS REQUIRED FOR FUNCTION AT ELEVATED GROWTH TEMPERATURES IN SACCHAROMYCES-CEREVISIAE

Authors
CAPLAN, AJ; TSAI, J; CASEY, PJ; DOUGLAS, MG
MLA Citation
CAPLAN, AJ, TSAI, J, CASEY, PJ, and DOUGLAS, MG. "FARNESYLATION OF YDJ1P IS REQUIRED FOR FUNCTION AT ELEVATED GROWTH TEMPERATURES IN SACCHAROMYCES-CEREVISIAE." JOURNAL OF BIOLOGICAL CHEMISTRY 267.26 (September 15, 1992): 18890-18895.
PMID
1527016
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
267
Issue
26
Publish Date
1992
Start Page
18890
End Page
18895

Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors.

The rate and extent of the agonist-dependent phosphorylation of beta 2-adrenergic receptors and rhodopsin by beta-adrenergic receptor kinase (beta ARK) are markedly enhanced on addition of G protein beta gamma subunits. With a model peptide substrate it was demonstrated that direct activation of the kinase could not account for this effect. G protein beta gamma subunits were shown to interact directly with the COOH-terminal region of beta ARK, and formation of this beta ARK-beta gamma complex resulted in receptor-facilitated membrane localization of the enzyme. The beta gamma subunits of transducin were less effective at both enhancing the rate of receptor phosphorylation and binding to the COOH-terminus of beta ARK, suggesting that the enzyme preferentially binds specific beta gamma complexes. The beta gamma-mediated membrane localization of beta ARK serves to intimately link receptor activation to beta ARK-mediated desensitization.

Authors
Pitcher, JA; Inglese, J; Higgins, JB; Arriza, JL; Casey, PJ; Kim, C; Benovic, JL; Kwatra, MM; Caron, MG; Lefkowitz, RJ
MLA Citation
Pitcher, JA, Inglese, J, Higgins, JB, Arriza, JL, Casey, PJ, Kim, C, Benovic, JL, Kwatra, MM, Caron, MG, and Lefkowitz, RJ. "Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors." Science 257.5074 (August 28, 1992): 1264-1267.
PMID
1325672
Source
pubmed
Published In
Science
Volume
257
Issue
5074
Publish Date
1992
Start Page
1264
End Page
1267

Mammalian protein geranylgeranyltransferase. Subunit composition and metal requirements.

An enzyme capable of specifically modifying, with a geranylgeranyl isoprenoid, candidate proteins containing a consensus prenylation sequence ending in leucine has been purified from bovine brain. This protein geranylgeranyltransferase (PGGT), isolated using affinity chromatography on an immobilized peptide column, contains two subunits with molecular masses of 48 and 43 kDa, designated alpha and beta, respectively. An antiserum raised to the alpha subunit of the related enzyme, protein farnesyltransferase (PFT), also recognizes this chromatographically identical alpha-subunit of the PGGT by immunoblot analysis. The PGGT and PFT enzymes from bovine brain are shown to be dependent on both Mg2+ and Zn2+ for optimal activity. Demonstration of the Zn2+ dependence of the enzymes requires prolonged incubation or purification in the presence of a chelating agent; we therefore propose that these enzymes be placed into the category of metalloenzymes. Under optimal assay conditions, these enzymes show high specificity toward their prenyl diphosphate substrates, with only a weak competition observed with farnesyl diphosphate in the PGGT reaction or geranylgeranyl diphosphate in the PFT reaction. The two enzymes are differentially sensitive to several detergents tested to determine suitable ones for product stabilization in the reactions. These results confirm previous predictions on the subunit structure of the PGGT and provide an avenue to initiating a molecular analysis of the geranylgeranyl modification of many mammalian proteins.

Authors
Moomaw, JF; Casey, PJ
MLA Citation
Moomaw, JF, and Casey, PJ. "Mammalian protein geranylgeranyltransferase. Subunit composition and metal requirements." J Biol Chem 267.24 (August 25, 1992): 17438-17443.
PMID
1512274
Source
pubmed
Published In
The Journal of biological chemistry
Volume
267
Issue
24
Publish Date
1992
Start Page
17438
End Page
17443

Rac1, a low-molecular-mass GTP-binding-protein with high intrinsic GTPase activity and distinct biochemical properties.

Rac1, a member of the family of low-molecular-mass GTP-binding proteins, functions in phagocytic leukocytes as a component necessary for activation of the respiratory burst. To characterize the biochemical properties of rac1, the protein was expressed as a fusion protein in Escherichia coli and purified to greater than 99% homogeneity by affinity chromatography. Rac1 protein bound maximally bound and hydrolyzed GTP under low free-Mg2+ concentrations. Under those conditions, (45 nm free Mg2+), purified rac1 exhibited a steady-state GTPase activity of 18 nmol.min-1.mg protein-1 (turnover number approximately 0.39 min-1 at 37 degrees C), which is 40-fold higher than H-ras. The high intrinsic GTPase activity of rac1 under low free Mg2+ was mainly due to an increased kcat, the rate constant for hydrolysis of bound GTP, which was 0.29 min-1 for rac1 vs 0.007 min-1 for H-ras (at 20 degrees C). Rac1 also released bound GDP faster than H-ras (koff.GDP = 1.02 min-1 for rac1 vs 0.33 min-1 for H-ras at 20 degrees C). In contrast, rac1 released bound guanosine 5'-[gamma-thio]triphosphate (GTP[S]) at a slower rate than H-ras (koff.GTP[S] approximately 0.04 min-1 for rac1 vs 0.31 min-1 for H-ras at 20 degrees C). Rac1 was a very good substrate for in vitro geranylgeranylation (C20) but not for farnesylation (C15), whereas the converse is true for H-ras. Surprisingly, rac1 was a very poor substrate for in vitro ADP-ribosylation by the C3 component of Clostridium botulinum toxin compared to rhoA. As a further characterization of rac1, a mutant was made in which the Thr115 was replaced by asparagine. This protein (referred to as [Thr115----Asn]rac1) contains the consensus amino acids of all four GTP-binding domains of H-ras. The koff.GDP of [Thr115----Asn]rac1 was reduced to that of H-ras, but [Thr115----Asn]rac1 exhibited essentially identical kcat (0.13 min-1 at 20 degrees C) and koff-GTP[S] (0.03 min-1 at 20 degrees C) values as the wild-type protein. Thus, the region(s) in rac1 which control the dissociation of GTP[S] (and presumably GTP) do not entirely coincide with those controlling GDP dissociation. Biochemical analysis of [Thr115----Asn]rac1 also suggests that the region responsible for the increased kcat of rac1 is not within the consensus amino acids of the four guanine-nucleotide-binding domains.

Authors
Ménard, L; Tomhave, E; Casey, PJ; Uhing, RJ; Snyderman, R; Didsbury, JR
MLA Citation
Ménard, L, Tomhave, E, Casey, PJ, Uhing, RJ, Snyderman, R, and Didsbury, JR. "Rac1, a low-molecular-mass GTP-binding-protein with high intrinsic GTPase activity and distinct biochemical properties." Eur J Biochem 206.2 (June 1, 1992): 537-546.
PMID
1597193
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
206
Issue
2
Publish Date
1992
Start Page
537
End Page
546

EVIDENCE OF A ROLE FOR HETEROTRIMERIC GTP-BINDING PROTEINS IN ENDOSOME FUSION

Authors
COLOMBO, MI; MAYORGA, LS; CASEY, PJ; STAHL, PD
MLA Citation
COLOMBO, MI, MAYORGA, LS, CASEY, PJ, and STAHL, PD. "EVIDENCE OF A ROLE FOR HETEROTRIMERIC GTP-BINDING PROTEINS IN ENDOSOME FUSION." SCIENCE 255.5052 (March 27, 1992): 1695-1697.
PMID
1348148
Source
wos-lite
Published In
Science
Volume
255
Issue
5052
Publish Date
1992
Start Page
1695
End Page
1697
DOI
10.1126/science.1348148

Visual differences

Authors
Casey, P
MLA Citation
Casey, P. "Visual differences." Nature 359.6397 (1992): 671-672.
PMID
1436026
Source
scival
Published In
Nature
Volume
359
Issue
6397
Publish Date
1992
Start Page
671
End Page
672
DOI
10.1038/359671a0

Enzymatic modification of proteins with a geranylgeranyl isoprenoid.

The prenylation of several proteins involved in oncogenesis and signal transduction plays an essential role in regulating their biological activities. Two distinct isoprenoids are known to be involved in this modification, the 15-carbon farnesyl and 20-carbon geranylgeranyl groups. Thus far, identified farnesylated proteins contain methionine or serine at the COOH terminus, while those modified by geranylgeranyl end in leucine. This report describes the characterization of an enzyme activity that transfers the geranylgeranyl group to candidate proteins. The enzyme, termed a "protein geranylgeranyltransferase," exhibits a marked preference for substrate proteins that contain leucine at the COOH terminus. In fact, the enzyme will efficiently modify a normally farnesylated protein, Ha-ras, if its COOH-terminal amino acid is switched from serine to leucine. Additional studies characterize this enzyme and suggest that it is responsible for the geranylgeranyl modification of a number of GTP-binding proteins (or their subunits) that contain a consensus prenylation sequence ending in leucine.

Authors
Casey, PJ; Thissen, JA; Moomaw, JF
MLA Citation
Casey, PJ, Thissen, JA, and Moomaw, JF. "Enzymatic modification of proteins with a geranylgeranyl isoprenoid." Proc Natl Acad Sci U S A 88.19 (October 1, 1991): 8631-8635.
PMID
1924324
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
88
Issue
19
Publish Date
1991
Start Page
8631
End Page
8635

PROTEIN FARNESYLTRANSFERASE AND GERANYLGERANYLTRANSFERASE SHARE A COMMON ALPHA-SUBUNIT

Authors
SEABRA, MC; REISS, Y; CASEY, PJ; BROWN, MS; GOLDSTEIN, JL
MLA Citation
SEABRA, MC, REISS, Y, CASEY, PJ, BROWN, MS, and GOLDSTEIN, JL. "PROTEIN FARNESYLTRANSFERASE AND GERANYLGERANYLTRANSFERASE SHARE A COMMON ALPHA-SUBUNIT." CELL 65.3 (May 3, 1991): 429-434.
PMID
2018975
Source
wos-lite
Published In
Cell
Volume
65
Issue
3
Publish Date
1991
Start Page
429
End Page
434
DOI
10.1016/0092-8674(91)90460-G

The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

Authors
Buss, JE; Quilliam, LA; Kato, K; Casey, PJ; Solski, PA; Wong, G; Clark, R; McCormick, F; Bokoch, GM; Der, CJ
MLA Citation
Buss, JE, Quilliam, LA, Kato, K, Casey, PJ, Solski, PA, Wong, G, Clark, R, McCormick, F, Bokoch, GM, and Der, CJ. "The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein." Molecular and Cellular Biology 11.3 (1991): 1523-1530.
PMID
1899909
Source
scival
Published In
Molecular and Cellular Biology
Volume
11
Issue
3
Publish Date
1991
Start Page
1523
End Page
1530

Phosphorylation of Gz in human platelets: Selectivity and site of modification

We have demonstrated previously that the G protein α subunit GZα (or GXα) in human platelets is subject to phosphorylation by agents that activate protein kinase C, including phorbol 12-myristate 13-acetate, thrombin, and the thromboxane A2 analog U46619. We examine here the site and selectivity of phosphorylation both in vitro using recombinant G protein α subunits and in situ using permeabilized and intact platelets. Protein kinase C catalyzes the rapid and nearly stoichiometric phosphorylation of recombinant GZα, with the modification occurring preferentially for the GDP-bound form of the subunit. Under the same conditions, phosphorylation of recombinant Giα1, Giα2, Giα3, Gsα-S, Gsα-L, and Goα1 was minimal. Phosphorylation of both rGZα, and platelet GZα occurs at a serine residue near the amino terminus. This conclusion is supported by phosphoamino acid analysis and the incorporation of radiolabel from [γ-32P]ATP into the amino-terminal CNBr peptide (residues 2-53 of the encoded protein). One of the antisera used in this study (6354, directed toward residues 24-33) recognizes only the nonphosphorylated form of GZα, providing strong evidence that Ser25 or Ser27 is the site of phosphorylation. Results obtained with 6354 also suggest that phorbol ester-promoted phosphorylation of GZα approaches 1 mol of phosphate per mol of subunit in permeabilized platelets.

Authors
Lounsbury, KM; Casey, PJ; Brass, LF; Manning, DR
MLA Citation
Lounsbury, KM, Casey, PJ, Brass, LF, and Manning, DR. "Phosphorylation of Gz in human platelets: Selectivity and site of modification." Journal of Biological Chemistry 266.32 (1991): 22051-22056.
PMID
1939224
Source
scival
Published In
Journal of Biological Chemistry
Volume
266
Issue
32
Publish Date
1991
Start Page
22051
End Page
22056

Assay of G-protein βγ-subunit complex by catalytic support of ADP-ribosylation of G(oα)

Authors
Casey, PJ; Pang, I-H; Gilman, AG
MLA Citation
Casey, PJ, Pang, I-H, and Gilman, AG. "Assay of G-protein βγ-subunit complex by catalytic support of ADP-ribosylation of G(oα)." Methods in Enzymology 195 (1991): 315-321.
PMID
1903494
Source
scival
Published In
Methods in Enzymology
Volume
195
Publish Date
1991
Start Page
315
End Page
321

Structural characterization of prenyl groups attached to proteins

Certain mammalian proteins are modified at a carboxyl-terminal cysteine by a thioether-linked prenyl group, the 15-carbon farnesyl or the 20-carbon geranylgeranyl moiety. Here, we describe analytical methods to determine the presence of a prenyl modification, the structure of the prenyl group, and the lipid: protein molar ratio for candidate proteins or their proteolytic fragments. Methods for the synthesis of prenyl standards are also presented. Methyl iodide or Raney nickel treatment is used to release the prenyl group from the protein for further analysis. When the prenyl group has been radiolabeled biosynthetically, two analytical techniques are available. Methyl iodide-released material, primarily the prenyl alcohol, can be cochromatographed with known standards using reverse-phase high-performance liquid chromatography to provide indirect structural data. Raney nickel-released material, primarily the unsubstituted hydrocarbon, can be analyzed by radiometric gas chromatography, which offers more precise structural information. However, unequivocal determination of the structure and quantitation of the mass of the released prenyl compound requires the use of gas chromatography-coupled mass spectrometry. © 1990 Academic Press, Inc. All rights reserved.

Authors
Farnsworth, CC; Casey, PJ; Howald, WN; Glomset, JA; Gelb, MH
MLA Citation
Farnsworth, CC, Casey, PJ, Howald, WN, Glomset, JA, and Gelb, MH. "Structural characterization of prenyl groups attached to proteins." Methods 1.3 (1990): 231-240.
Source
scival
Published In
Methods
Volume
1
Issue
3
Publish Date
1990
Start Page
231
End Page
240

Novel localization of a G protein, Gz-α, in neurons of brain and retina

Recently, a cDNA coding for a novel G protein α-subunit, Gz-α, was isolated from a human retinal cDNA library and shown by Northern blot analysis to be expressed at high levels in neural tissues. We have prepared affinity-purified antibodies specifically directed against synthetic Gz-α peptides and employed immunohistochemical methods to map the localization of Gz-α in human, bovine, and murine retina and brain. By light microscopy, Gz-α was localized to the cytoplasm of neurons, with predominant reactivity in ganglion cells of the retina, Purkinje cells of the cerebellum, and most neurons of the hippocampus and cerebral cortex. Reactivity was confined to perikaryon, dendrites, and a very short segment of proximal axons, except for the retinal ganglion cells, in which the axons in the nerve fiber layer showed intense Gz-α immunoreactivity proximal to the lamina cribrosa. Pre-embedding immunoelectron microscopy demonstrated the presence of focal Gz-α immunoreactivity on the nuclear membranes, endoplasmic reticulum, and plasma membranes of Purkinje cell perikarya and in association with microtubules in their proximal dendrites. Subcellular fractionation studies confirmed the association of Gz-α with plasma and intracellular membranes. The localization of Gz-α and its unique amino acid sequence suggest that it may have a specialized function in neural tissues.

Authors
Hinton, DR; Blanks, JC; Fong, HKW; Casey, PJ; Hildebrandt, E; Simons, MI
MLA Citation
Hinton, DR, Blanks, JC, Fong, HKW, Casey, PJ, Hildebrandt, E, and Simons, MI. "Novel localization of a G protein, Gz-α, in neurons of brain and retina." Journal of Neuroscience 10.8 (1990): 2763-2770.
PMID
2117645
Source
scival
Published In
Journal of Neuroscience
Volume
10
Issue
8
Publish Date
1990
Start Page
2763
End Page
2770

G protein γ subunits contain a 20-carbon isoprenoid

A small subset of cellular proteins are covalently modified by the addition of isoprenoid groups. These include p21ras, fungal mating factors, and nuclear lamins, which are isoprenylated at carboxyl-terminal cysteine residues with a 15-carbon farnesyl group. The similarity of the carboxyl-terminal sequences of these proteins with the α and γ subunits of signal-transducing guanine nucleotide-binding regulatory proteins (G proteins) prompted examination of isoprenylation of G protein subunits. PC-12 cells were incubated with the isoprenoid precursor [3H]mevalonolactone. The β and γ subunits were isolated by specific association with an affinity column of immobilized α subunits. The γ subunits were radiolabeled, and the tritiated lipid released from them by treatment with methyl iodide comigrated chromatographically with the 20-carbon isoprenoid geranylgeraniol. Label was not detected in G protein α or β subunits. Isoprenylation of γ subunits by the geranylgeranyl group is presumed to contribute to the association of G proteins with membranes.

Authors
Mumby, SM; Casey, PJ; Gilman, AG; Gutowski, S; Sternweis, PC
MLA Citation
Mumby, SM, Casey, PJ, Gilman, AG, Gutowski, S, and Sternweis, PC. "G protein γ subunits contain a 20-carbon isoprenoid." Proceedings of the National Academy of Sciences of the United States of America 87.15 (1990): 5873-5877.
PMID
2116011
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
87
Issue
15
Publish Date
1990
Start Page
5873
End Page
5877
DOI
10.1073/pnas.87.15.5873

Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides

We report the identification, purification, and characterization of a farnesyl:protein transferase that transfers the farnesyl moiety from farnesyl pyrophosphate to a cysteine in p21ras proteins. The enzyme was purified ∼60,000-fold from rat brain cytosol through use of a chromatography step based on the enzyme's ability to bind to a hexapeptide containing the consensus sequence (Cys-AAX) for farnesylation. The purified enzyme migrated on gel filtration chromatography with an apparent molecular weight of 70,000-100,000. High resolution SDS-polyacrylamide gels showed two closely spaced ∼50 kd protein bands in the final preparation. The enzyme was inhibited competitively by peptides as short as 4 residues that contained the Cys-AAX motif. These peptides acted as alternative substrates that competed with p21H-ras for farnesylation. Effective peptides included the COOH-terminal sequences of all known p21ras proteins as well as those of lamin A and B.

Authors
Reiss, Y; Goldstein, JL; Seabra, MC; Casey, PJ; Brown, MS
MLA Citation
Reiss, Y, Goldstein, JL, Seabra, MC, Casey, PJ, and Brown, MS. "Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides." Cell 62.1 (1990): 81-88.
PMID
2194674
Source
scival
Published In
Cell
Volume
62
Issue
1
Publish Date
1990
Start Page
81
End Page
88

Gz, a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for Gzα, a newly apprecitated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the βγ subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gzα (rGzα) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGzα has several properties that distinguish it from other G protein α subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min-1), which is reduced >20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gzα is extremely slow. The hydrolysis rate (kcat) for rGzα at 30 °C is 0.05 min-1, or 200-fold slower than that determined for other G protein α subunits. rGzα can interact with bovine brain βγ but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.

Authors
Casey, PJ; Fong, HKW; Simon, MI; Gilman, AG
MLA Citation
Casey, PJ, Fong, HKW, Simon, MI, and Gilman, AG. "Gz, a guanine nucleotide-binding protein with unique biochemical properties." Journal of Biological Chemistry 265.4 (1990): 2383-2390.
PMID
2105321
Source
scival
Published In
The Journal of biological chemistry
Volume
265
Issue
4
Publish Date
1990
Start Page
2383
End Page
2390

G protein βγ subunits from bovine brain and retina: Equivalent catalytic support of ADP-ribosylation of α subunits by pertussis toxin but differential interactions with G

We have examined the ability of the βγ subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein α subunits. Substoichiometric amounts of the βγ complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the α subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective α and βγ subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain βγ does not require Mg2+. Although the βγ subunit complexes purified from bovine brain G proteins and the βγ complex of Gt support equally the ADP-ribosylation of a subunits by PT, there is a marked difference in their abilities to interact with Gsα. The enhancement of deactivation of fluoride-activated Gsα requires 25-fold more βγ from Gt than from brain G proteins to produce a similar response. This difference in potency of βγ complexes from the two sources was also observed in the ability of βγ to produce an increase in the activity of recombinant Gsα produced in Escherichia coli. © 1989 American Chemical Society.

Authors
Casey, PJ; Graziano, MP; Gilman, AG
MLA Citation
Casey, PJ, Graziano, MP, and Gilman, AG. "G protein βγ subunits from bovine brain and retina: Equivalent catalytic support of ADP-ribosylation of α subunits by pertussis toxin but differential interactions with G." Biochemistry 28.2 (1989): 611-616.
PMID
2496748
Source
scival
Published In
Biochemistry
Volume
28
Issue
2
Publish Date
1989
Start Page
611
End Page
616

G proteins control diverse pathways of transmembrane signaling

Hormones, neurotransmitters, and autacoids interact with specific receptors and thereby trigger a series of molecular events that ultimately produce their biological effects. These receptors, localized in the plasma membrane, carry binding sites for ligands as diverse as peptides (e.g., glucagon, neuropeptides), lipids (e.g., prostaglandins), nucleosides and nucleotides (e.g., adenosine), and amines (e.g., catecholamines, serotonin). These receptors do not interact directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger); rather, they control one or several target systems via the activation of an intermediary guanine nucleotide-binding regulatory protein or G protein. G proteins serve as signal transducers, linking extracellulary oriented receptors to membrane-bound effectors. Traffic in these pathways is regulated by a GTP (on)-GDP (off) switch, which is regulated by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. These approaches, complemented in particular by electrophysiological experiments, have also identified several effectors that are regulated by G proteins. We can safely assume that current lists of G proteins and the functions that they control are incomplete.

Authors
Freissmuth, M; Casey, PJ; Gilman, AG
MLA Citation
Freissmuth, M, Casey, PJ, and Gilman, AG. "G proteins control diverse pathways of transmembrane signaling." FASEB Journal 3.10 (1989): 2125-2139.
PMID
2546847
Source
scival
Published In
FASEB Journal
Volume
3
Issue
10
Publish Date
1989
Start Page
2125
End Page
2139

p21ras is modified by a farnesyl isoprenoid

Association of oncogenic ras proteins with cellular membranes appears to be a crucial step in transformation. ras is synthesized as a cytosolic precursor, which is processed to a mature form that localizes to the plasma membrane. This processing involves, in part, a conserved sequence, Cys-Ali-Ali-Xaa (in which Ali is an amino acid with an aliphatic side chain and Xaa is any amino acid), at the COOH terminus of ras proteins. Yeast a-factor mating hormone precursor also possesses a COOH-terminal Cys-Ali-Ali-Xaa sequence. However, while the COOH-terminal cysteine has been implicated as a site of palmitoylation of ras proteins, in mature a-type mating factor this residue is modified by an isoprenoid, a farnesyl moiety. We asked whether the Cys-Ali-Ali-Xaa sequence signaled different modifications for the yeast peptides (farnesylation) than for ras proteins (palmitoylation) or whether ras proteins were similar to the mating factors and contained a previously undiscovered isoprenoid. We report here that the processing of ras proteins involves addition of a farnesyl moiety, apparently at the COOH-terminal cysteine analogous to the cysteine modified in the yeast peptides, and that farnesylation may be important for membrane association and transforming activity of ras proteins.

Authors
Casey, PJ; Solski, PA; Der, CJ; Buss, JE
MLA Citation
Casey, PJ, Solski, PA, Der, CJ, and Buss, JE. "p21ras is modified by a farnesyl isoprenoid." Proceedings of the National Academy of Sciences of the United States of America 86.21 (1989): 8323-8327.
PMID
2682646
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
86
Issue
21
Publish Date
1989
Start Page
8323
End Page
8327

Role of G proteins in transmembrane signaling.

Authors
Casey, PJ; Graziano, MP; Freissmuth, M; Gilman, AG
MLA Citation
Casey, PJ, Graziano, MP, Freissmuth, M, and Gilman, AG. "Role of G proteins in transmembrane signaling." January 1988.
PMID
3151167
Source
epmc
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
53 Pt 1
Publish Date
1988
Start Page
203
End Page
208
DOI
10.1101/sqb.1988.053.01.026

Role of G proteins in transmembrane signaling

Authors
Casey, PJ; Graziano, MP; Friessmuth, M; Gilman, AG
MLA Citation
Casey, PJ, Graziano, MP, Friessmuth, M, and Gilman, AG. "Role of G proteins in transmembrane signaling." Cold Spring Harbor Symposia on Quantitative Biology 53.1 (1988): 203-208.
Source
scival
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
53
Issue
1
Publish Date
1988
Start Page
203
End Page
208

G protein involvement in receptor-effector coupling

The recent plethora of information on G proteins has suggested that they play a major role in signaling processes. The use of bacterial toxins, GTP analogs, specific antisera, and oligonucleotide probes has revealed that members or close relatives of the family exist in such primitive organisms as Saccharomyces and Dictyostelium, speaking to a high degree of conservation of these proteins during evolution. The tools of the molecular biologist and structural biochemist will soon allow a more detailed examination of the crucial protein-ligand and protein-protein interactions required for G protein action. The alliance of all of these disciplines will facilitate investigation of G protein-linked signaling processes and of possible pathological disturbances therein.

Authors
Casey, PJ; Gilman, AG
MLA Citation
Casey, PJ, and Gilman, AG. "G protein involvement in receptor-effector coupling." Journal of Biological Chemistry 263.6 (1988): 2577-2580.
PMID
2830256
Source
scival
Published In
Journal of Biological Chemistry
Volume
263
Issue
6
Publish Date
1988
Start Page
2577
End Page
2580

Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins.

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the alpha subunits of Gs (stimulatory) (alpha 45 and alpha 52), a 41-kDa subunit of Gi (inhibitory) (alpha 41), a 40-kDa protein (alpha 40), and the 36-kDa beta subunit. No protein that comigrated with the alpha subunit of Go (unknown function) (alpha 39) was detected. In cells grown in the presence of [3H]myristic acid, alpha 41 and alpha 40 contained 3H label, while the beta subunit did not. Chemical analysis of lipids attached covalently to purified alpha 41 and alpha 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein beta and gamma subunits and of Gt (transducin) subunits (alpha, beta, and gamma) failed to reveal fatty acids. The fatty acid associated with alpha 41, alpha 40, and alpha 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.

Authors
Buss, JE; Mumby, SM; Casey, PJ; Gilman, AG; Sefton, BM
MLA Citation
Buss, JE, Mumby, SM, Casey, PJ, Gilman, AG, and Sefton, BM. "Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins." Proceedings of the National Academy of Sciences of the United States of America 84.21 (1987): 7493-7497.
PMID
3118369
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
84
Issue
21
Publish Date
1987
Start Page
7493
End Page
7497

Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate.

Adenylosuccinate lyase from rat skeletal muscle was purified to apparent homogeneity by a combination of ion-exchange chromatography and affinity chromatography on agarose containing covalently bound adenylophosphonopropionate. The purified enzyme is stable when stored in 20% glycerol at -70 degrees C, and can be thawed and re-frozen with minimal loss of activity. Adenylosuccinate lyase has a specific activity of 11 mumol/min per mg of protein at 25 degrees C. Its subunit Mr is 52,000, by SDS/polyacrylamide-gel electrophoresis, and its apparent native Mr is approx. 200,000, by gel filtration. The purified enzyme has Km values for adenylosuccinate and 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) of 1.5 microM and approximately 1 microM respectively, in Hepes/KOH buffer, pH 7.4. Several monoanions and dianions activate the enzyme at low concentration; several of these inhibit the enzyme at high concentrations. Fluoro analogues of adenylosuccinate and SAICAR were synthesized by using highly purified adenylosuccinate synthase and SAICAR synthase respectively, and erythro-beta-fluoroaspartate in place of aspartate. Both analogues are competitive inhibitors of adenylosuccinate lyase in both of the reactions catalysed by the enzyme, with Ki values well below the Km values for the two substrates.

Authors
Casey, PJ; Lowenstein, JM
MLA Citation
Casey, PJ, and Lowenstein, JM. "Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate." Biochemical Journal 246.2 (1987): 263-269.
PMID
3689310
Source
scival
Published In
Biochemical Journal
Volume
246
Issue
2
Publish Date
1987
Start Page
263
End Page
269

Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase.

Complementary DNAs that encode two forms of the alpha subunit (Gs alpha) of the guanine nucleotide-binding protein responsible for stimulation of adenylate cyclase (Gs) have been inserted into plasmid vectors for expression in Escherichia coli. Following transformation of either of these plasmids into E. coli K38, Gs alpha accumulates to 0.4-0.8 mg/liter (approximately 0.1% of total protein), as judged by immunoblot analysis with specific antisera. Based on deduced amino acid sequence, the two cDNAs should encode proteins with molecular weights of 44,500 and 46,000, respectively (Robishaw, J.D., Smigel, M. D., and Gilman, A. G. (1986) J. Biol. Chem. 261, 9587-9590). Expression of these cDNAs in E. coli yields proteins that co-migrate on sodium dodecyl sulfate-polyacrylamide gels with the Gs alpha subunits from S49 lymphoma cell membranes, with apparent molecular weights of 45,000 and 52,000, respectively. Low levels of activity are detected in the 100,000 X g supernatant after lysis and fractionation of E. coli expressing either form of Gs alpha. Partial purification of Gs alpha from E. coli lysates yields preparations in which significant and stable activity can be assayed. Both forms of Gs alpha migrate through sucrose gradients as soluble, monodisperse species in the absence of detergent. As expressed in E. coli, both forms of Gs alpha can reconstitute isoproterenol-, guanine nucleotide-, and fluoride-stimulated adenylate cyclase activity in S49 cyc-cell membranes to approximately the same degree and can be ADP-ribosylated with [32P]NAD+ and cholera toxin. However, based on the specific activity of purified rabbit liver Gs, only 1-2% of the Gs alpha expressed in E. coli appears to be active. Incubation of partially purified fractions of recombinant Gs alpha with guanosine 5'-(3-O-thio)triphosphate and resolved beta gamma subunits isolated from purified bovine brain G proteins results in a 7-10-fold increase in Gs activity. Incubation of bovine brain beta gamma with recombinant Gs alpha also leads to a dramatic increase in observed levels of cholera toxin-catalyzed [32P]ADP-ribosylation.

Authors
Graziano, MP; Casey, PJ; Gilman, AG
MLA Citation
Graziano, MP, Casey, PJ, and Gilman, AG. "Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase." Journal of Biological Chemistry 262.23 (1987): 11375-11381.
PMID
3112160
Source
scival
Published In
Journal of Biological Chemistry
Volume
262
Issue
23
Publish Date
1987
Start Page
11375
End Page
11381

Inhibition of adenylosuccinate lyase by L-alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-aicor)

L-Alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR) has been synthesized enzymatically using 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase in conjunction with 5-aminoimidazole-4-carboxylic acid ribonucleotide and L-2-amino-3-(N-hydroxy-N-nitrosoamino)propionic acid (alanosine). The product was characterized by chromatography, ultraviolet spectrum and NMR spectrum at 300 MHz. Alanosyl-AICOR was not a substrate of adenylosuccinate lyase from rat skeletal muscle, but it was an apparent competitive inhibitor in both of the reactions catalyzed by the enzyme. The KI values for alanosyl-AICOR were ∼ 1.5 and 1.3 μM in the SAICAR and adenylosuccinate cleavage reactions respectively. These KI values were essentially the same as the Km values for the two substrates of adenylosuccinate lyase. They compare with an accumulation of 70 μM alanosyl-AICOR in leukemic nodules of mice treated with alanosine [A. K. Tyagi and D. Cooney, Cancer Res. 40, 4390 (1980)]. Thus, inhibition of adenylosuccinate lyase may account for much of the inhibitory effect exerted by alanosyl-AICOR in vivo. We confirmed the previous observation that alanosyl-AICOR is an inhibitor of adenylosuccinate synthetase. © 1987.

Authors
Casey, PJ; Lowenstein, JM
MLA Citation
Casey, PJ, and Lowenstein, JM. "Inhibition of adenylosuccinate lyase by L-alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-aicor)." Biochemical Pharmacology 36.5 (1987): 705-709.
PMID
3827951
Source
scival
Published In
Biochemical Pharmacology
Volume
36
Issue
5
Publish Date
1987
Start Page
705
End Page
709

Metabolism of threo-β-fluoroaspartate by H4 cells. Inhibition of adenylosuccinate lyase by fluoro analogs of its substrates

DL-threo-β-Fluoroaspartate is a substrate for the two enzymes in de novo purine biosynthesis that use aspartate, namely 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase and adenylosuccinate synthetase. With both enzymes, V(max) with threo-β-fluoroaspartate is about 50% of that observed with aspartate. The products of the two enzyme reactions, threo-β-fluoro-SAICAR and threo-β-fluoroadenylosuccinate, are inhibitors of adenylosuccinate lyase purified from rat skeletal muscle. In 20 mM phosphate buffer, pH 7.4, the K(I) values for threo-β-fluoro-SAICAR are 5 and 3 μM and for threo-β-fluoroadenylosuccinate are 3 and 1 μM, in the SAICAR and adenylosuccinate cleavage reactions, respectively. In 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, the K(I) values for threo-β-fluoro-SAICAR are ~0.14 and 0.03 μM and for threo-β-fluoroadenylosuccinate are ~0.05 and 0.015 μM, in the same two reactions, respectively. These K(I) values are one-half to one-hundredth of the K(m) values for SAICAR and adenylosuccinate, the two substrates of adenylosuccinate lyase. After an 8-h incubation with 45 μM threo-β-fluoroaspartate, H4 cells contain 200-300 μM threo-β-fluoro-SAICAR and 60-90 μM threo-β-fluoroadenylosuccinate. These concentrations of fluoro analogs are sufficient to substantially inhibit adenylosuccinate lyase and hence the de novo synthesis of purines in H4 cells.

Authors
Casey, PJ; Abeles, RH; Lowenstein, JM
MLA Citation
Casey, PJ, Abeles, RH, and Lowenstein, JM. "Metabolism of threo-β-fluoroaspartate by H4 cells. Inhibition of adenylosuccinate lyase by fluoro analogs of its substrates." Journal of Biological Chemistry 261.29 (1986): 13637-13642.
PMID
3759987
Source
scival
Published In
Journal of Biological Chemistry
Volume
261
Issue
29
Publish Date
1986
Start Page
13637
End Page
13642

Purification and characterization of adenylosuccinate lyase from rat skeletal muscle

Authors
Casey, PJ; Lowenstein, JM
MLA Citation
Casey, PJ, and Lowenstein, JM. "Purification and characterization of adenylosuccinate lyase from rat skeletal muscle." Federation Proceedings 44.5 (1985): No.-6944.
Source
scival
Published In
Federation Proceedings
Volume
44
Issue
5
Publish Date
1985
Start Page
No.
End Page
6944

Post-prenylation-processing enzymes as new targets in oncogenesis.

RAS and many other oncogenic proteins undergo a complex series of post-translational modifications that are initiated by the addition of an isoprenoid lipid through a process known as prenylation. Following prenylation, these proteins usually undergo endoproteolytic processing by the RCE1 protease and then carboxyl methylation by a unique methyltransferase known as isoprenylcysteine carboxyl methyltransferase (ICMT). Although inhibitors that have been designed to target the prenylation step are now in advanced-stage clinical trials, their utility and efficacy seem to be limited. Recent findings, however, indicate that the inhibition of these post-prenylation-processing steps--particularly that of ICMT-catalysed methylation--might provide a better approach to the control of cancer-cell proliferation.

Authors
Winter-Vann, AM; Casey, PJ
MLA Citation
Winter-Vann, AM, and Casey, PJ. "Post-prenylation-processing enzymes as new targets in oncogenesis." Nat Rev Cancer 5.5: 405-412. (Review)
PMID
15864282
Source
pubmed
Published In
Nature Reviews Cancer
Volume
5
Issue
5
Start Page
405
End Page
412
DOI
10.1038/nrc1612
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