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Kornbluth, Sally A.

Overview:

Our lab studies the regulation of complex cellular processes, including cell cycle progression and programmed cell death (apoptosis). These tightly orchestrated processes are critical for appropriate cell proliferation and cell death, and when they go awry can result in cancer and degenerative disorders. Within these larger fields, we have focused on understanding the cellular mechanisms that prevent the onset of mitosis prior to the completion of DNA replication, the processes that prevent cell division when the mitotic spindle is disrupted, the signaling pathways that prevent apoptotic cell death in cancer cells and the mechanisms that link cell metabolism to cell death and survival.


In our quest to answer these important cell biological and biochemical questions, we are varied in our use of experimental systems.   Traditionally, we have used cell-free extracts prepared from eggs of the frog Xenopus laevis which can recapitulate cell cycle events and apoptotic processes in vitro. For the study of cell cycle events, extracts are prepared which can undergo multiple rounds of DNA replication and mitosis in vitro. Progression through the cell cycle can be monitored by microscopic observation of nuclear morphology and by biochemically assaying the activity of serine/threonine kinases which control cell cycle transitions.


For the study of apoptosis, modifications in extract preparation have allowed us to produce extracts which can apoptotically fragment nuclei and can accurately reproduce the biochemical events of apoptosis, including internucleosomal DNA cleavage and activation of apoptotic proteases, the caspases.


More recently, we have focused on studying apoptosis and cell cycle progression in mammalian models, both tissue culture cells and mouse models of cancer.  In these studies, we are trying to determine the precise signaling mechanisms used by cancer cells to accelerate proliferation and evade apoptotic cell death mechanisms.   We also endeavor to subvert these mechanisms to therapeutic advantage.   We are particularly interested in links between metabolism and cell death, as high metabolic rates in cancer cells appear to suppress apoptosis to evade chemotherapy-induced cell death.


Finally, we also have several projects using the facile genetics of Drosophila melanogaster to further understand links between metabolism and cell death and also the ways in which mitochondrial dynamics are linked to apoptotic pathways.

Positions:

Jo Rae Wright University Professor

Biology
School of Medicine

Professor of Pharmacology & Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Vice Dean of Research, of School of Medicine

School of Medicine
School of Medicine

Education:

Ph.D. 1989

Ph.D. — Rockefeller University

News:

Grants:

Organization and Function of Cellular Structure

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1975
End Date
June 30, 2020

Center for Molecular & Cellular Studies of Ped Disease

Administered By
Pediatrics
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
April 11, 2003
End Date
November 30, 2017

Training Program in Developmental and Stem Cell Biology

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
May 01, 2001
End Date
October 31, 2017

Control of the apoptosome in epithelial ovarian cancer to enhance chemotherapy

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2014
End Date
August 31, 2017

Provost's Discretionary Fund

Administered By
Institutes and Provost's Academic Units
AwardedBy
Duke Endowment
Role
Principal Investigator
Start Date
July 01, 2014
End Date
June 30, 2017

Medical Scientist Training Program

Administered By
School of Medicine
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1997
End Date
June 30, 2017

Control of caspase activation in apoptosis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 21, 2007
End Date
February 28, 2017

Research Training In Neuro-Oncology

Administered By
Neurosurgery, Neuro-Oncology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1998
End Date
August 31, 2016

Cancer Biology Training Grant

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Cancer Institute
Role
Mentor
Start Date
July 01, 1993
End Date
March 31, 2016

Inhibition of Cytochrome c-induced Caspase Activation

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2003
End Date
January 31, 2016

Apoptosome Regulation in Leukemia by Protein Phosphatase 5 Hypoacetylation

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2013
End Date
October 31, 2015

Regulation of REDD1 and caspase-2 by FASN inhibition in ovarian cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2014
End Date
August 31, 2015

Integrating Population and Basic Science in Cancer Research

Administered By
Duke Cancer Institute
AwardedBy
National Institutes of Health
Role
Advisor
Start Date
September 01, 2009
End Date
August 31, 2015

Instrumentation for Quantitative Phosphoproteomics and Acetylomics

Administered By
Duke Center for Genomic and Computational Biology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
May 15, 2014
End Date
May 14, 2015

Automated detection of protein crystals in high-throughput crystallography experiments

Administered By
Duke Human Vaccine Institute
AwardedBy
North Carolina Biotechnology Center
Role
Major User
Start Date
April 01, 2014
End Date
April 30, 2015

Engineering tyrosine kinase-activated caspases for selective cancer cell killing

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2010
End Date
May 31, 2014

Metabolic Regulation of 14-3-3zeta Acetylation in Breast Cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2012
End Date
April 02, 2014

Glucose metabolism and cell death in cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Significant Contributor
Start Date
April 01, 2007
End Date
January 31, 2014

Molecular mechanisms of chemoresistance in breast cancer

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
May 01, 2010
End Date
April 30, 2012

Regulation of M phase exit

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 29, 2009
End Date
August 31, 2011

Control of Mitotic Entry by Regulators of Cdc2

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2003
End Date
June 30, 2011

Research Training In Neuro-Oncology

Administered By
Neurosurgery, Neuro-Oncology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
September 15, 2005
End Date
August 31, 2010

High-performance Computing System for Bioinformatics

Administered By
Institutes and Centers
AwardedBy
National Institutes of Health
Role
Advisor
Start Date
June 01, 2009
End Date
May 31, 2010

Apoptotic Signaling Pathways

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2000
End Date
November 30, 2009

Regulation of Apoptosis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1997
End Date
July 31, 2007

Control of The Mitotic Exit by the Xnf7 Ubiquitin Ligase

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 2004
End Date
June 30, 2007

Reaper Mediated Apoptosis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2004
End Date
June 30, 2007

Reaper-Scythe Regulated Apoptosis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2004
End Date
November 30, 2006

BCR-Abl-mediated inhibition of apoptosis

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2003
End Date
July 31, 2006

Regulation of the G2/2M Transition

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 01, 2000
End Date
January 31, 2005
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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, 2010

Publications:

Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin.

Evasion of apoptosis allows many cancers to resist chemotherapy. Apoptosis is mediated by the serial activation of caspase family proteins. These proteases are often activated upon the release of cytochrome c from the mitochondria, which is promoted by the proapoptotic Bcl-2 family protein, Bax. This function of Bax is enhanced by the MOAP-1 (modulator of apoptosis protein 1) protein in response to DNA damage. Previously, we reported that MOAP-1 is targeted for ubiquitylation and degradation by the APC/C(Cdh1) ubiquitin ligase. In this study, we identify the HECT (homologous to the E6-AP carboxyl terminus) family E3 ubiquitin ligase, UBR5, as a novel ubiquitin ligase for MOAP-1. We demonstrate that UBR5 interacts physically with MOAP-1, ubiquitylates MOAP-1 in vitro and inhibits MOAP-1 stability in cultured cells. In addition, we show that Dyrk2 kinase, a reported UBR5 interactor, cooperates with UBR5 in mediating MOAP-1 ubiquitylation. Importantly, we found that cisplatin-resistant ovarian cancer cell lines exhibit lower levels of MOAP-1 accumulation than their sensitive counterparts upon cisplatin treatment, consistent with the previously reported role of MOAP-1 in modulating cisplatin-induced apoptosis. Accordingly, UBR5 knockdown increased MOAP-1 expression, enhanced Bax activation and sensitized otherwise resistant cells to cisplatin-induced apoptosis. Furthermore, UBR5 expression was higher in ovarian cancers from cisplatin-resistant patients than from cisplatin-responsive patients. These results show that UBR5 downregulates proapoptotic MOAP-1 and suggest that UBR5 can confer cisplatin resistance in ovarian cancer. Thus UBR5 may be an attractive therapeutic target for ovarian cancer treatment.Oncogene advance online publication, 10 October 2016; doi:10.1038/onc.2016.336.

Authors
Matsuura, K; Huang, N-J; Cocce, K; Zhang, L; Kornbluth, S
MLA Citation
Matsuura, K, Huang, N-J, Cocce, K, Zhang, L, and Kornbluth, S. "Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin." Oncogene (October 10, 2016).
PMID
27721409
Source
epmc
Published In
Oncogene: Including Oncogene Reviews
Publish Date
2016
DOI
10.1038/onc.2016.336

Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint.

The spindle checkpoint prevents activation of the anaphase-promoting complex (APC/C) until all chromosomes are correctly attached to the mitotic spindle. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C by binding the APC/C activating protein CDC20 until the chromosomes are properly aligned and attached to the mitotic spindle, at which point MCC disassembly releases CDC20 to activate the APC/C. Once the APC/C is activated, it targets cyclin B and securin for degradation, and the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. We sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined the effect on the MCC and APC/C activation. Using this approach we found that 2 phosphatase inhibitors, calyculin A and okadaic acid (1 μM), caused MCC dissociation and APC/C activation leading to cyclin A and B degradation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.

Authors
Foss, KM; Robeson, AC; Kornbluth, S; Zhang, L
MLA Citation
Foss, KM, Robeson, AC, Kornbluth, S, and Zhang, L. "Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint." Cell cycle (Georgetown, Tex.) 15.2 (January 2016): 225-233.
PMID
26652909
Source
epmc
Published In
Cell Cycle
Volume
15
Issue
2
Publish Date
2016
Start Page
225
End Page
233
DOI
10.1080/15384101.2015.1121331

Vertebrate Reproduction.

Vertebrate reproduction requires a myriad of precisely orchestrated events-in particular, the maternal production of oocytes, the paternal production of sperm, successful fertilization, and initiation of early embryonic cell divisions. These processes are governed by a host of signaling pathways. Protein kinase and phosphatase signaling pathways involving Mos, CDK1, RSK, and PP2A regulate meiosis during maturation of the oocyte. Steroid signals-specifically testosterone-regulate spermatogenesis, as does signaling by G-protein-coupled hormone receptors. Finally, calcium signaling is essential for both sperm motility and fertilization. Altogether, this signaling symphony ensures the production of viable offspring, offering a chance of genetic immortality.

Authors
Kornbluth, S; Fissore, R
MLA Citation
Kornbluth, S, and Fissore, R. "Vertebrate Reproduction." Cold Spring Harbor perspectives in biology 7.10 (October 2015): a006064-.
PMID
26430215
Source
epmc
Published In
Cold Spring Harbor perspectives in biology
Volume
7
Issue
10
Publish Date
2015
Start Page
a006064
DOI
10.1101/cshperspect.a006064

Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis.

Caspase-2 is an initiator caspase involved in multiple apoptotic pathways, particularly in response to specific intracellular stressors (eg, DNA damage, ER stress). We recently reported that caspase-2 was pivotal for the induction of cell death triggered by excessive intracellular accumulation of long-chain fatty acids, a response known as lipoapoptosis. The liver is particularly susceptible to lipid-induced damage, explaining the pandemic status of non-alcoholic fatty liver disease (NAFLD). Progression from NAFLD to non-alcoholic steatohepatitis (NASH) results, in part, from hepatocyte apoptosis and consequential paracrine-mediated fibrogenesis. We evaluated the hypothesis that caspase-2 promotes NASH-related cirrhosis.Caspase-2 was localised in liver biopsies from patients with NASH. Its expression was evaluated in different mouse models of NASH, and outcomes of diet-induced NASH were compared in wild-type (WT) and caspase-2-deficient mice. Lipotoxicity was modelled in vitro using hepatocytes derived from WT and caspase-2-deficient mice.We showed that caspase-2 is integral to the pathogenesis of NASH-related cirrhosis. Caspase-2 is localised in injured hepatocytes and its expression was markedly upregulated in patients and animal models of NASH. During lipotoxic stress, caspase-2 deficiency reduced apoptosis, inhibited induction of profibrogenic hedgehog target genes in mice and blocked production of hedgehog ligands in cultured hepatocytes.These data point to a critical role for caspase-2 in lipid-induced hepatocyte apoptosis in vivo for the production of apoptosis-associated fibrogenic factors and in the progression of lipid-induced liver fibrosis. This raises the intriguing possibility that caspase-2 may be a promising therapeutic target to prevent progression to NASH.

Authors
Machado, MV; Michelotti, GA; Pereira, TDA; Boursier, J; Kruger, L; Swiderska-Syn, M; Karaca, G; Xie, G; Guy, CD; Bohinc, B; Lindblom, KR; Johnson, E; Kornbluth, S; Diehl, AM
MLA Citation
Machado, MV, Michelotti, GA, Pereira, TDA, Boursier, J, Kruger, L, Swiderska-Syn, M, Karaca, G, Xie, G, Guy, CD, Bohinc, B, Lindblom, KR, Johnson, E, Kornbluth, S, and Diehl, AM. "Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis." Gut 64.7 (July 2015): 1148-1157.
PMID
25053716
Source
epmc
Published In
Gut
Volume
64
Issue
7
Publish Date
2015
Start Page
1148
End Page
1157
DOI
10.1136/gutjnl-2014-307362

Fatty acid synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death.

Blockade of fatty acid synthase (FASN), a key enzyme involved in de novo lipogenesis, results in robust death of ovarian cancer cells. However, known FASN inhibitors have proven to be poor therapeutic agents due to their ability to induce cachexia. Therefore, we sought to identify additional targets in the pathway linking FASN inhibition and cell death whose modulation might kill ovarian cancer cells without the attendant side effects. Here, we show that the initiator caspase-2 is required for robust death of ovarian cancer cells induced by FASN inhibitors. REDD1 (also known as Rtp801 or DDIT4), a known mTOR inhibitor previously implicated in the response to FASN inhibition, is a novel caspase-2 regulator in this pathway. REDD1 induction is compromised in ovarian cancer cells that do not respond to FASN inhibition. Inhibition of FASN induced an ATF4-dependent transcriptional induction of REDD1; downregulation of REDD1 prevented orlistat-induced activation of caspase-2, as monitored by its cleavage, proteolytic activity and dimerization. Abrogation of REDD1-mediated suppression of mTOR by TSC2 RNAi protected FASN inhibitor-sensitive ovarian cancer cells (OVCA420 cells) from orlistat-induced death. Conversely, suppression of mTOR with the chemical inhibitors PP242 or rapamycin-sensitized DOV13, an ovarian cancer cell line incapable of inducing REDD1, to orlistat-induced cell death through caspase-2. These findings indicate that REDD1 positively controls caspase-2-dependent cell death of ovarian cancer cells by inhibiting mTOR, placing mTOR as a novel upstream regulator of caspase-2 and supporting the possibility of manipulating mTOR to enhance caspase-2 activation in ovarian cancer.

Authors
Yang, C-S; Matsuura, K; Huang, N-J; Robeson, AC; Huang, B; Zhang, L; Kornbluth, S
MLA Citation
Yang, C-S, Matsuura, K, Huang, N-J, Robeson, AC, Huang, B, Zhang, L, and Kornbluth, S. "Fatty acid synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death." Oncogene 34.25 (June 2015): 3264-3272.
PMID
25151963
Source
epmc
Published In
Oncogene: Including Oncogene Reviews
Volume
34
Issue
25
Publish Date
2015
Start Page
3264
End Page
3272
DOI
10.1038/onc.2014.271

Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis

Objective Caspase-2 is an initiator caspase involved in multiple apoptotic pathways, particularly in response to specific intracellular stressors (eg, DNA damage, ER stress). We recently reported that caspase-2 was pivotal for the induction of cell death triggered by excessive intracellular accumulation of long-chain fatty acids, a response known as lipoapoptosis. The liver is particularly susceptible to lipid-induced damage, explaining the pandemic status of non-alcoholic fatty liver disease (NAFLD). Progression from NAFLD to non-alcoholic steatohepatitis (NASH) results, in part, from hepatocyte apoptosis and consequential paracrine-mediated fibrogenesis. We evaluated the hypothesis that caspase-2 promotes NASH-related cirrhosis. Design Caspase-2 was localised in liver biopsies from patients with NASH. Its expression was evaluated in different mouse models of NASH, and outcomes of dietinduced NASH were compared in wild-type (WT) and caspase-2-deficient mice. Lipotoxicity was modelled in vitro using hepatocytes derived from WT and caspase-2-deficient mice. Results We showed that caspase-2 is integral to the pathogenesis of NASH-related cirrhosis. Caspase-2 is localised in injured hepatocytes and its expression was markedly upregulated in patients and animal models of NASH. During lipotoxic stress, caspase-2 deficiency reduced apoptosis, inhibited induction of profibrogenic hedgehog target genes in mice and blocked production of hedgehog ligands in cultured hepatocytes. Conclusions These data point to a critical role for caspase-2 in lipid-induced hepatocyte apoptosis in vivo for the production of apoptosis-associated fibrogenic factors and in the progression of lipid-induced liver fibrosis. This raises the intriguing possibility that caspase-2 may be a promising therapeutic target to prevent progression to NASH.

Authors
Machado, MV; Michelotti, GA; Pereira, TDA; Boursier, J; Kruger, L; Swiderska-Syn, M; Karaca, G; Xie, G; Guy, CD; Bohinc, B; Lindblom, KR; Johnson, E; Kornbluth, S; Diehl, AM
MLA Citation
Machado, MV, Michelotti, GA, Pereira, TDA, Boursier, J, Kruger, L, Swiderska-Syn, M, Karaca, G, Xie, G, Guy, CD, Bohinc, B, Lindblom, KR, Johnson, E, Kornbluth, S, and Diehl, AM. "Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis." Gut 64.7 (2015): 1148-1157.
Source
scival
Published In
Gut
Volume
64
Issue
7
Publish Date
2015
Start Page
1148
End Page
1157
DOI
10.1136/gutjnl-2014-307362

Metabolic control of Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated caspase-2 suppression by the B55β/protein phosphatase 2A (PP2A).

High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr(393)/Ser(395)) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.

Authors
Huang, B; Yang, C-S; Wojton, J; Huang, N-J; Chen, C; Soderblom, EJ; Zhang, L; Kornbluth, S
MLA Citation
Huang, B, Yang, C-S, Wojton, J, Huang, N-J, Chen, C, Soderblom, EJ, Zhang, L, and Kornbluth, S. "Metabolic control of Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated caspase-2 suppression by the B55β/protein phosphatase 2A (PP2A)." The Journal of biological chemistry 289.52 (December 2014): 35882-35890.
PMID
25378403
Source
epmc
Published In
The Journal of biological chemistry
Volume
289
Issue
52
Publish Date
2014
Start Page
35882
End Page
35890
DOI
10.1074/jbc.m114.585844

The complex interplay between metabolism and apoptosis

© 2014 Springer Science+Business Media New York. All rights are reserved.Energy balance is essential for cells to function properly and proliferate. Sufficient nutrient quantities are required for energy and the synthesis of building blocks of cellular structures such as lipid membranes, proteins, and nucleic acids. In contrast, nutrient excess can increase ER stress and reactive oxygen species, leading to cellular damage and apoptosis. In this chapter, we detail three important aspects of the interplay between metabolism and apoptosis: the mechanisms by which metabolic imbalances regulate major signaling effectors in apoptosis, how metabolism itself can be controlled by apoptotic proteins, and the major disease states affected when perturbations in metabolism modulate apoptosis.

Authors
Lindblom, K; Kornbluth, S
MLA Citation
Lindblom, K, and Kornbluth, S. "The complex interplay between metabolism and apoptosis." 9781461493020 (November 1, 2014): 189-209. (Chapter)
Source
scopus
Volume
9781461493020
Publish Date
2014
Start Page
189
End Page
209
DOI
10.1007/978-1-4614-9302-0_9

Sally Kornbluth: Nature's incredible contraptions.

Authors
Kornbluth, S; Sedwick, C
MLA Citation
Kornbluth, S, and Sedwick, C. "Sally Kornbluth: Nature's incredible contraptions." The Journal of cell biology 206.1 (July 2014): 4-5. (Interview)
PMID
25002675
Source
epmc
Published In
The Journal of Cell Biology
Volume
206
Issue
1
Publish Date
2014
Start Page
4
End Page
5
DOI
10.1083/jcb.2061pi

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin-proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. Here we report the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation. © 2014 Macmillan Publishers Limited All rights reserved.

Authors
Yang, CS; Sinenko, SA; Thomenius, MJ; Robeson, AC; Freel, CD; Horn, SR; Kornbluth, S
MLA Citation
Yang, CS, Sinenko, SA, Thomenius, MJ, Robeson, AC, Freel, CD, Horn, SR, and Kornbluth, S. "The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis." Cell Death and Differentiation 21.4 (April 1, 2014): 604-611.
Source
scopus
Published In
Cell Death & Differentiation
Volume
21
Issue
4
Publish Date
2014
Start Page
604
End Page
611
DOI
10.1038/cdd.2013.184

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis.

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin-proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. Here we report the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation.

Authors
Yang, C-S; Sinenko, SA; Thomenius, MJ; Robeson, AC; Freel, CD; Horn, SR; Kornbluth, S
MLA Citation
Yang, C-S, Sinenko, SA, Thomenius, MJ, Robeson, AC, Freel, CD, Horn, SR, and Kornbluth, S. "The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis." Cell Death Differ 21.4 (April 2014): 604-611.
PMID
24362437
Source
pubmed
Published In
Cell Death & Differentiation
Volume
21
Issue
4
Publish Date
2014
Start Page
604
End Page
611
DOI
10.1038/cdd.2013.184

Fatty acid synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

Blockade of fatty acid synthase (FASN), a key enzyme involved in de novo lipogenesis, results in robust death of ovarian cancer cells. However, known FASN inhibitors have proven to be poor therapeutic agents due to their ability to induce cachexia. Therefore, we sought to identify additional targets in the pathway linking FASN inhibition and cell death whose modulation might kill ovarian cancer cells without the attendant side effects. Here, we show that the initiator caspase-2 is required for robust death of ovarian cancer cells induced by FASN inhibitors. REDD1 (also known as Rtp801 or DDIT4), a known mTOR inhibitor previously implicated in the response to FASN inhibition, is a novel caspase-2 regulator in this pathway. REDD1 induction is compromised in ovarian cancer cells that do not respond to FASN inhibition. Inhibition of FASN induced an ATF4-dependent transcriptional induction of REDD1; downregulation of REDD1 prevented orlistat-induced activation of caspase-2, as monitored by its cleavage, proteolytic activity and dimerization. Abrogation of REDD1-mediated suppression of mTOR by TSC2 RNAi protected FASN inhibitor-sensitive ovarian cancer cells (OVCA420 cells) from orlistat-induced death. Conversely, suppression of mTOR with the chemical inhibitors PP242 or rapamycin-sensitized DOV13, an ovarian cancer cell line incapable of inducing REDD1, to orlistat-induced cell death through caspase-2. These findings indicate that REDD1 positively controls caspase-2-dependent cell death of ovarian cancer cells by inhibiting mTOR, placing mTOR as a novel upstream regulator of caspase-2 and supporting the possibility of manipulating mTOR to enhance caspase-2 activation in ovarian cancer.Oncogene advance online publication, 25 August 2014; doi:10.1038/onc.2014.271.

Authors
Yang, C-S; Matsuura, K; Huang, N-J; Robeson, AC; Huang, B; Zhang, L; Kornbluth, S
MLA Citation
Yang, C-S, Matsuura, K, Huang, N-J, Robeson, AC, Huang, B, Zhang, L, and Kornbluth, S. "Fatty acid synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death." Oncogene (2014).
Source
scival
Published In
Oncogene: Including Oncogene Reviews
Publish Date
2014
DOI
10.1038/onc.2014.271

Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11.

Ataxia telangiectasia mutant (ATM) is an S/T-Q-directed kinase that is critical for the cellular response to double-stranded breaks (DSBs) in DNA. Following DNA damage, ATM is activated and recruited by the MRN protein complex [meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 proteins] to sites of DNA damage where ATM phosphorylates multiple substrates to trigger cell-cycle arrest. In cancer cells, this regulation may be faulty, and cell division may proceed even in the presence of damaged DNA. We show here that the ribosomal s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that Rsk targets loading of MRN complex components onto DNA at DSB sites. Rsk can phosphorylate the Mre11 protein directly at S676 both in vitro and in intact cells and thereby can inhibit the binding of Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can reverse inhibition of the response to DSBs by Rsk. Collectively, these data point to Mre11 as an important locus of Rsk-mediated checkpoint inhibition acting upstream of ATM activation.

Authors
Chen, C; Zhang, L; Huang, N-J; Huang, B; Kornbluth, S
MLA Citation
Chen, C, Zhang, L, Huang, N-J, Huang, B, and Kornbluth, S. "Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11." Proceedings of the National Academy of Sciences of the United States of America 110.51 (December 2, 2013): 20605-20610.
Website
http://hdl.handle.net/10161/8387
PMID
24297933
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
51
Publish Date
2013
Start Page
20605
End Page
20610
DOI
10.1073/pnas.1306328110

Metabolomic profiling reveals a role for caspase-2 in lipoapoptosis.

The accumulation of long-chain fatty acids (LCFAs) in non-adipose tissues results in lipid-induced cytotoxicity (or lipoapoptosis). Lipoapoptosis has been proposed to play an important role in the pathogenesis of several metabolic diseases, including non-alcoholic fatty liver disease, diabetes mellitus, and cardiovascular disease. In this report, we demonstrate a novel role for caspase-2 as an initiator of lipoapoptosis. Using a metabolomics approach, we discovered that the activation of caspase-2, the initiator of apoptosis in Xenopus egg extracts, is associated with an accumulation of LCFA metabolites. Metabolic treatments that blocked the buildup of LCFAs potently inhibited caspase-2 activation, whereas adding back an LCFA in this scenario restored caspase activation. Extending these findings to mammalian cells, we show that caspase-2 was engaged and activated in response to treatment with the saturated LCFA palmitate. Down-regulation of caspase-2 significantly impaired cell death induced by saturated LCFAs, suggesting that caspase-2 plays a pivotal role in lipid-induced cytotoxicity. Together, these findings reveal a previously unknown role for caspase-2 as an initiator caspase in lipoapoptosis and suggest that caspase-2 may be an attractive therapeutic target for inhibiting pathological lipid-induced apoptosis.

Authors
Johnson, ES; Lindblom, KR; Robeson, A; Stevens, RD; Ilkayeva, OR; Newgard, CB; Kornbluth, S; Andersen, JL
MLA Citation
Johnson, ES, Lindblom, KR, Robeson, A, Stevens, RD, Ilkayeva, OR, Newgard, CB, Kornbluth, S, and Andersen, JL. "Metabolomic profiling reveals a role for caspase-2 in lipoapoptosis." J Biol Chem 288.20 (May 17, 2013): 14463-14475.
PMID
23553630
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
20
Publish Date
2013
Start Page
14463
End Page
14475
DOI
10.1074/jbc.M112.437210

A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.

Authors
Kurokawa, M; Kim, J; Geradts, J; Matsuura, K; Liu, L; Ran, X; Xia, W; Ribar, TJ; Henao, R; Dewhirst, MW; Kim, W-J; Lucas, JE; Wang, S; Spector, NL; Kornbluth, S
MLA Citation
Kurokawa, M, Kim, J, Geradts, J, Matsuura, K, Liu, L, Ran, X, Xia, W, Ribar, TJ, Henao, R, Dewhirst, MW, Kim, W-J, Lucas, JE, Wang, S, Spector, NL, and Kornbluth, S. "A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53. (Published online)" Sci Signal 6.274 (May 7, 2013): ra32-.
Website
http://hdl.handle.net/10161/8398
PMID
23652204
Source
pubmed
Published In
Science Signaling
Volume
6
Issue
274
Publish Date
2013
Start Page
ra32
DOI
10.1126/scisignal.2003741

Engineering a BCR-ABL-activated caspase for the selective elimination of leukemic cells.

Increased understanding of the precise molecular mechanisms involved in cell survival and cell death signaling pathways offers the promise of harnessing these molecules to eliminate cancer cells without damaging normal cells. Tyrosine kinase oncoproteins promote the genesis of leukemias through both increased cell proliferation and inhibition of apoptotic cell death. Although tyrosine kinase inhibitors, such as the BCR-ABL inhibitor imatinib, have demonstrated remarkable efficacy in the clinic, drug-resistant leukemias emerge in some patients because of either the acquisition of point mutations or amplification of the tyrosine kinase, resulting in a poor long-term prognosis. Here, we exploit the molecular mechanisms of caspase activation and tyrosine kinase/adaptor protein signaling to forge a unique approach for selectively killing leukemic cells through the forcible induction of apoptosis. We have engineered caspase variants that can directly be activated in response to BCR-ABL. Because we harness, rather than inhibit, the activity of leukemogenic kinases to kill transformed cells, this approach selectively eliminates leukemic cells regardless of drug-resistant mutations.

Authors
Kurokawa, M; Ito, T; Yang, C-S; Zhao, C; Macintyre, AN; Rizzieri, DA; Rathmell, JC; Deininger, MW; Reya, T; Kornbluth, S
MLA Citation
Kurokawa, M, Ito, T, Yang, C-S, Zhao, C, Macintyre, AN, Rizzieri, DA, Rathmell, JC, Deininger, MW, Reya, T, and Kornbluth, S. "Engineering a BCR-ABL-activated caspase for the selective elimination of leukemic cells." Proc Natl Acad Sci U S A 110.6 (February 5, 2013): 2300-2305.
Website
http://hdl.handle.net/10161/8388
PMID
23324740
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
110
Issue
6
Publish Date
2013
Start Page
2300
End Page
2305
DOI
10.1073/pnas.1206551110

Cellular mechanisms controlling caspase activation and function

Caspases are the primary drivers of apoptotic cell death, cleaving cellular proteins that are critical for dismantling the dying cell. Initially translated as inactive zymogenic precursors, caspases are activated in response to a variety of cell death stimuli. In addition to factors required for their direct activation (e.g., dimerizing adaptor proteins in the case of initiator caspases that lie at the apex of apoptotic signaling cascades), caspases are regulated by a variety of cellular factors in a myriad of physiological and pathological settings. For example, caspases may be modified posttranslationally (e.g., by phosphorylation or ubiquitylation) or through interaction of modulatory factors with either the zymogenic or active form of a caspase, altering its activation and/or activity. These regulatory events may inhibit or enhance enzymatic activity or may affect activity toward particular cellular substrates. Finally, there is emerging literature to suggest that caspases can participate in a variety of cellular processes unrelated to apoptotic cell death. In these settings, it is particularly important that caspases are maintained under stringent control to avoid inadvertent cell death. It is likely that continued examination of these processes will reveal new mechanisms of caspase regulation with implications well beyond control of apoptotic cell death. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

Authors
Parrish, AB; Freel, CD; Kornbluth, S
MLA Citation
Parrish, AB, Freel, CD, and Kornbluth, S. "Cellular mechanisms controlling caspase activation and function." Cold Spring Harbor Perspectives in Medicine 3.3 (2013).
Source
scival
Published In
Cold Spring Harbor perspectives in medicine
Volume
3
Issue
3
Publish Date
2013

The Tangled Circuitry of Metabolism and Apoptosis

For single-cell organisms, nutrient uptake and metabolism are central to the fundamental decision of whether to grow or divide. In metazoans, cell fate decisions are more complex: organismal homeostasis must be strictly maintained by balancing cell proliferation and death. Despite this increased complexity, cell fate within multicellular organisms is also influenced by metabolism; recent studies, triggered in part by an interest in tumor metabolism, are beginning to illuminate the mechanisms through which proliferation, death, and metabolism are intertwined. In particular, work on Bcl-2 family proteins suggests that the signaling pathways governing metabolism and apoptosis are inextricably linked. Here we review the crosstalk between these pathways, emphasizing recent work that illustrates the emerging dual nature of several core apoptotic proteins in regulating both metabolism and cell death. © 2013 Elsevier Inc.

Authors
Andersen, JL; Kornbluth, S
MLA Citation
Andersen, JL, and Kornbluth, S. "The Tangled Circuitry of Metabolism and Apoptosis." Molecular Cell 49.3 (2013): 399-410.
PMID
23395270
Source
scival
Published In
Molecular Cell
Volume
49
Issue
3
Publish Date
2013
Start Page
399
End Page
410
DOI
10.1016/j.molcel.2012.12.026

Cellular mechanisms controlling caspase activation and function

Caspases are the primary drivers of apoptotic cell death, cleaving cellular proteins that are critical for dismantling the dying cell. Initially translated as inactive zymogenic precursors, caspases are activated in response to a variety of cell death stimuli. In addition to factors required for their direct activation (e.g., dimerizing adaptor proteins in the case of initiator caspases that lie at the apex of apoptotic signaling cascades), caspases are regulated by a variety of cellular factors in a myriad of physiological and pathological settings. For example, caspases may be modified posttranslationally (e.g., by phosphorylation or ubiquitylation) or through interaction of modulatory factors with either the zymogenic or active form of a caspase, altering its activation and/or activity. These regulatory events may inhibit or enhance enzymatic activity or may affect activity toward particular cellular substrates. Finally, there is emerging literature to suggest that caspases can participate in a variety of cellular processes unrelated to apoptotic cell death. In these settings, it is particularly important that caspases are maintained under stringent control to avoid inadvertent cell death. It is likely that continued examination of these processes will reveal new mechanisms of caspase regulation with implications well beyond control of apoptotic cell death. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

Authors
Parrish, AB; Freel, CD; Kornbluth, S
MLA Citation
Parrish, AB, Freel, CD, and Kornbluth, S. "Cellular mechanisms controlling caspase activation and function." Cold Spring Harbor Perspectives in Medicine 3.1 (2013).
Source
scival
Published In
Cold Spring Harbor perspectives in medicine
Volume
3
Issue
1
Publish Date
2013

Ubiquitylation of p53 by the APC/C inhibitor Trim39.

Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.

Authors
Zhang, L; Huang, N-J; Chen, C; Tang, W; Kornbluth, S
MLA Citation
Zhang, L, Huang, N-J, Chen, C, Tang, W, and Kornbluth, S. "Ubiquitylation of p53 by the APC/C inhibitor Trim39." Proc Natl Acad Sci U S A 109.51 (December 18, 2012): 20931-20936.
Website
http://hdl.handle.net/10161/8389
PMID
23213260
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
109
Issue
51
Publish Date
2012
Start Page
20931
End Page
20936
DOI
10.1073/pnas.1212047110

Establishing a framework for improving the quality of clinical and translational research.

Authors
Califf, RM; Kornbluth, S
MLA Citation
Califf, RM, and Kornbluth, S. "Establishing a framework for improving the quality of clinical and translational research." J Clin Oncol 30.14 (May 10, 2012): 1725-1726. (Letter)
PMID
22493421
Source
pubmed
Published In
Journal of Clinical Oncology
Volume
30
Issue
14
Publish Date
2012
Start Page
1725
End Page
1726
DOI
10.1200/JCO.2011.41.4458

The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1.

Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/C(Cdh1)) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/C(Cdh1)-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid-mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.

Authors
Huang, N-J; Zhang, L; Tang, W; Chen, C; Yang, C-S; Kornbluth, S
MLA Citation
Huang, N-J, Zhang, L, Tang, W, Chen, C, Yang, C-S, and Kornbluth, S. "The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1." J Cell Biol 197.3 (April 30, 2012): 361-367.
Website
http://hdl.handle.net/10161/8382
PMID
22529100
Source
pubmed
Published In
The Journal of Cell Biology
Volume
197
Issue
3
Publish Date
2012
Start Page
361
End Page
367
DOI
10.1083/jcb.201111141

Rsk-mediated phosphorylation and 14-3-3ɛ binding of Apaf-1 suppresses cytochrome c-induced apoptosis.

Many pro-apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade, promote cell viability by impeding mitochondrial cytochrome c release and by inhibiting subsequent caspase activation. Here, we describe a mechanism for the inhibition of cytochrome c-induced caspase activation by MAPK signalling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). Recruitment of 14-3-3ɛ to phosphorylated Ser268 impedes the ability of cytochrome c to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14-3-3ɛ binding to Apaf-1 and rendered the cells insensitive to cytochrome c, suggesting a potential role for Rsk signalling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. Collectively, these results identify a novel locus of apoptosomal regulation wherein MAPK signalling promotes Rsk-catalysed Apaf-1 phosphorylation and consequent binding of 14-3-3ɛ, resulting in decreased cellular responsiveness to cytochrome c.

Authors
Kim, J; Parrish, AB; Kurokawa, M; Matsuura, K; Freel, CD; Andersen, JL; Johnson, CE; Kornbluth, S
MLA Citation
Kim, J, Parrish, AB, Kurokawa, M, Matsuura, K, Freel, CD, Andersen, JL, Johnson, CE, and Kornbluth, S. "Rsk-mediated phosphorylation and 14-3-3ɛ binding of Apaf-1 suppresses cytochrome c-induced apoptosis." EMBO J 31.5 (March 7, 2012): 1279-1292.
PMID
22246185
Source
pubmed
Published In
EMBO Journal
Volume
31
Issue
5
Publish Date
2012
Start Page
1279
End Page
1292
DOI
10.1038/emboj.2011.491

Mcl-1 rescues a glitch in the matrix

Bcl-2 family proteins are known to control cell death and influence mitochondrial function. The function of Mcl-1, an anti-apoptotic Bcl-2 protein, is now shown to depend on its subcellular localization. Mcl-1 at the mitochondrial outer membrane inhibits mitochondrial permeabilization to block apoptosis. However, a cleaved form of Mcl-1 localizes to the mitochondrial matrix and controls inner mitochondrial morphology and oxidative phosphorylation, without directly modulating apoptosis. © 2012 Macmillan Publishers Limited. All rights reserved.

Authors
Andersen, JL; Kornbluth, S
MLA Citation
Andersen, JL, and Kornbluth, S. "Mcl-1 rescues a glitch in the matrix." Nature Cell Biology 14.6 (2012): 563-565.
PMID
22643875
Source
scival
Published In
Nature Cell Biology
Volume
14
Issue
6
Publish Date
2012
Start Page
563
End Page
565
DOI
10.1038/ncb2511

Life, death, and the metabolically controlled protein acetylome

The complex signaling pathways that control cellular fate can be intimately influenced by metabolic status. Although the ability of nutrients to influence intracellular decisions has been appreciated for some time, the complex signaling mechanisms linking metabolic inputs to cell proliferation and death are not fully understood. An emerging theme in the literature is that intracellular metabolite levels can directly influence cell fate decisions through modulation of nutrient-derived protein modifications. It appears that varying the level of intracellular metabolites can alter the abundance of post-translational modifications, both by altering the availability of donor substrates and changing the activity of the nutrient-sensitive enzymes regulating these reactions. We focus here on protein acetylation, a modification that can modulate both cell proliferation and cell death in response to changes in extracellular nutrient supply. © 2012.

Authors
Johnson, ES; Kornbluth, S
MLA Citation
Johnson, ES, and Kornbluth, S. "Life, death, and the metabolically controlled protein acetylome." Current Opinion in Cell Biology (2012).
PMID
23103123
Source
scival
Published In
Current Opinion in Cell Biology
Publish Date
2012
DOI
10.1016/j.ceb.2012.10.002

Phosphatases driving mitosis: Pushing the gas and lifting the brakes

Entry into and progression through mitosis depends critically on the establishment and maintenance of protein phosphorylation. For this reason, studies on mitotic progression have focused heavily on the activation of MPF (M phase promoting factor), a cyclin-dependent kinase responsible for phosphorylating proteins that execute the dynamic events of mitosis. Recent work, however, has significantly expanded our understanding of mechanisms that allow accumulation of phosphoproteins at M phase, suggesting that mitotic entry relies not only on MPF activation but also on the inhibition of antimitotic phosphatases. It is now clear that there exists a separate, albeit equally important, signaling pathway for the inactivation of protein phosphatases at the G2/M transition. This pathway, which is governed by the kinase Greatwall is essential for both entry into and maintenance of M phase. This chapter will outline the molecular events regulating entry into mitosis, specifically highlighting the role that protein phosphorylation plays in triggering both MPF activation and the inhibition of phosphatase activity that would otherwise prevent accumulation of mitotic phosphoproteins. These intricate regulatory pathways are essential for maintaining normal cell division and preventing inappropriate cell proliferation, a central hallmark of cancer cells. © 2012 Elsevier Inc.

Authors
Johnson, ES; Kornbluth, S
MLA Citation
Johnson, ES, and Kornbluth, S. "Phosphatases driving mitosis: Pushing the gas and lifting the brakes." Progress in Molecular Biology and Translational Science 106 (2012): 327-341.
PMID
22340723
Source
scival
Published In
Progress in Molecular Biology and Translational Science
Volume
106
Publish Date
2012
Start Page
327
End Page
341
DOI
10.1016/B978-0-12-396456-4.00008-0

Life, death, and the metabolically controlled protein acetylome

The complex signaling pathways that control cellular fate can be intimately influenced by metabolic status. Although the ability of nutrients to influence intracellular decisions has been appreciated for some time, the complex signaling mechanisms linking metabolic inputs to cell proliferation and death are not fully understood. An emerging theme in the literature is that intracellular metabolite levels can directly influence cell fate decisions through modulation of nutrient-derived protein modifications. It appears that varying the level of intracellular metabolites can alter the abundance of post-translational modifications, both by altering the availability of donor substrates and changing the activity of the nutrient-sensitive enzymes regulating these reactions. We focus here on protein acetylation, a modification that can modulate both cell proliferation and cell death in response to changes in extracellular nutrient supply. © 2012 .

Authors
Johnson, ES; Kornbluth, S
MLA Citation
Johnson, ES, and Kornbluth, S. "Life, death, and the metabolically controlled protein acetylome." Current Opinion in Cell Biology 24.6 (2012): 876-880.
Source
scival
Published In
Current Opinion in Cell Biology
Volume
24
Issue
6
Publish Date
2012
Start Page
876
End Page
880
DOI
10.1016/j.ceb.2012.10.002

Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death.

In most multicellular organisms, the decision to undergo programmed cell death in response to cellular damage or developmental cues is typically transmitted through mitochondria. It has been suggested that an exception is the apoptotic pathway of Drosophila melanogaster, in which the role of mitochondria remains unclear. Although IAP antagonists in Drosophila such as Reaper, Hid and Grim may induce cell death without mitochondrial membrane permeabilization, it is surprising that all three localize to mitochondria. Moreover, induction of Reaper and Hid appears to result in mitochondrial fragmentation during Drosophila cell death. Most importantly, disruption of mitochondrial fission can inhibit Reaper and Hid-induced cell death, suggesting that alterations in mitochondrial dynamics can modulate cell death in fly cells. We report here that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. Our in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or γ-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. Our findings are consistent with a growing body of work describing a role for mitochondrial fission and fusion machinery in the decision of cells to die.

Authors
Thomenius, M; Freel, CD; Horn, S; Krieser, R; Abdelwahid, E; Cannon, R; Balasundaram, S; White, K; Kornbluth, S
MLA Citation
Thomenius, M, Freel, CD, Horn, S, Krieser, R, Abdelwahid, E, Cannon, R, Balasundaram, S, White, K, and Kornbluth, S. "Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death." Cell Death Differ 18.10 (October 2011): 1640-1650.
Website
http://hdl.handle.net/10161/8381
PMID
21475305
Source
pubmed
Published In
Cell Death & Differentiation
Volume
18
Issue
10
Publish Date
2011
Start Page
1640
End Page
1650
DOI
10.1038/cdd.2011.26

A biotin switch-based proteomics approach identifies 14-3-3ζ as a target of Sirt1 in the metabolic regulation of caspase-2.

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes14-3-3ζ dissociation from caspase-2 in both egg extract and human cultured cells. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.

Authors
Andersen, JL; Thompson, JW; Lindblom, KR; Johnson, ES; Yang, C-S; Lilley, LR; Freel, CD; Moseley, MA; Kornbluth, S
MLA Citation
Andersen, JL, Thompson, JW, Lindblom, KR, Johnson, ES, Yang, C-S, Lilley, LR, Freel, CD, Moseley, MA, and Kornbluth, S. "A biotin switch-based proteomics approach identifies 14-3-3ζ as a target of Sirt1 in the metabolic regulation of caspase-2." Molecular cell 43.5 (September 2011): 834-842.
PMID
21884983
Source
epmc
Published In
Molecular Cell
Volume
43
Issue
5
Publish Date
2011
Start Page
834
End Page
842
DOI
10.1016/j.molcel.2011.07.028

Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability.

Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.

Authors
Horn, SR; Thomenius, MJ; Johnson, ES; Freel, CD; Wu, JQ; Coloff, JL; Yang, C-S; Tang, W; An, J; Ilkayeva, OR; Rathmell, JC; Newgard, CB; Kornbluth, S
MLA Citation
Horn, SR, Thomenius, MJ, Johnson, ES, Freel, CD, Wu, JQ, Coloff, JL, Yang, C-S, Tang, W, An, J, Ilkayeva, OR, Rathmell, JC, Newgard, CB, and Kornbluth, S. "Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability." Mol Biol Cell 22.8 (April 15, 2011): 1207-1216.
PMID
21325626
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
22
Issue
8
Publish Date
2011
Start Page
1207
End Page
1216
DOI
10.1091/mbc.E10-07-0567

Meeting the (N-terminal) end with acetylation

Cell-fate decisions are tightly linked to cellular energy status. In this issue, Yi et al. (2011) introduce a mechanism by which Bcl-xL lowers the threshold for apoptosis by suppressing acetyl-CoA production, which, in turn, suppresses the N-alpha-acetylation important for activation of the proapoptotic protease caspase-2. © 2011 Elsevier Inc.

Authors
Andersen, JL; Kornbluth, S
MLA Citation
Andersen, JL, and Kornbluth, S. "Meeting the (N-terminal) end with acetylation." Cell 146.4 (2011): 503-505.
PMID
21854977
Source
scival
Published In
Cell
Volume
146
Issue
4
Publish Date
2011
Start Page
503
End Page
505
DOI
10.1016/j.cell.2011.07.024

Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc.

Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome-wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc-mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII-mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death-inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non-phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.

Authors
Yang, C-S; Thomenius, MJ; Gan, EC; Tang, W; Freel, CD; Merritt, TJS; Nutt, LK; Kornbluth, S
MLA Citation
Yang, C-S, Thomenius, MJ, Gan, EC, Tang, W, Freel, CD, Merritt, TJS, Nutt, LK, and Kornbluth, S. "Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc." EMBO J 29.18 (September 15, 2010): 3196-3207.
PMID
20700104
Source
pubmed
Published In
EMBO Journal
Volume
29
Issue
18
Publish Date
2010
Start Page
3196
End Page
3207
DOI
10.1038/emboj.2010.191

Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism.

Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a "pseudosubstrate" mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.

Authors
Tang, W; Wu, JQ; Chen, C; Yang, C-S; Guo, JY; Freel, CD; Kornbluth, S
MLA Citation
Tang, W, Wu, JQ, Chen, C, Yang, C-S, Guo, JY, Freel, CD, and Kornbluth, S. "Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism." Mol Biol Cell 21.15 (August 1, 2010): 2589-2597.
Website
http://hdl.handle.net/10161/3328
PMID
20534816
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
21
Issue
15
Publish Date
2010
Start Page
2589
End Page
2597
DOI
10.1091/mbc.E09-08-0708

Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.

Authors
Johnson, CE; Freel, CD; Kornbluth, S
MLA Citation
Johnson, CE, Freel, CD, and Kornbluth, S. "Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring." Cell Death Differ 17.1 (January 2010): 170-179.
Website
http://hdl.handle.net/10161/8380
PMID
19730443
Source
pubmed
Published In
Cell Death & Differentiation
Volume
17
Issue
1
Publish Date
2010
Start Page
170
End Page
179
DOI
10.1038/cdd.2009.120

Women: Diversity among leaders is there if you look

Authors
Andrews, NC; Kornbluth, S; Stokke, D
MLA Citation
Andrews, NC, Kornbluth, S, and Stokke, D. "Women: Diversity among leaders is there if you look." Nature 463.7281 (2010): 608--.
PMID
20130628
Source
scival
Published In
Nature
Volume
463
Issue
7281
Publish Date
2010
Start Page
608-
DOI
10.1038/463608d

Regulation of the ATM-activator protein Aven by CRM1-dependent nuclear export

Aven is a regulator of apoptosis whose overexpression is associated with poor prognosis in several cancers, including childhood acute lymphoblastic leukemia and acute myeloid leukemia. We have recently shown that Aven serves as an activator and substrate of AtM, thereby modulating the DNA-damage response and G2/M cell cycle progression. Under physiological conditions, the cellular localization of Aven is mainly cytosolic, but a small fraction of the protein is present in the nucleus. Here, we show that treatment of cells with leptomycin B, an inhibitor of exportin-1/CRM (chromosomal region maintenance) 1, resulted in nuclear accumulation of Aven. Furthermore, we identified a functional LR-NES between amino acid residues 282-292 of the human Aven protein, a sequence that is evolutionary conserved among a range of vertebrate species. Disruption of this LR-NES by site-directed mutagenesis resulted in enhanced nuclear localization of Aven, but did not alter the ability of the protein to induce G2/M cell cycle arrest in interphase Xenopus laevis extracts. However, elimination of the LR-NES sequence led to a reduction in the capacity of Aven to arrest Xenopus oocytes containing intact nuclei. Our results suggest that the regulation of nucleocytoplasmatic traffic of Aven could modulate its ability to influence cell cycle progression. © 2010 Landes Bioscience.

Authors
Esmaili, AM; Johnson, EL; Thaivalappil, SS; Kuhn, HM; Kornbluth, S; Irusta, PM
MLA Citation
Esmaili, AM, Johnson, EL, Thaivalappil, SS, Kuhn, HM, Kornbluth, S, and Irusta, PM. "Regulation of the ATM-activator protein Aven by CRM1-dependent nuclear export." Cell Cycle 9.19 (2010): 3919-3926.
Source
scival
Published In
Cell Cycle
Volume
9
Issue
19
Publish Date
2010
Start Page
3919
End Page
3926
DOI
10.4161/cc.9.19.13138

Regulation of the ATM-activator protein Aven by CRM1-dependent nuclear export.

Aven is a regulator of apoptosis whose overexpression is associated with poor prognosis in several cancers, including childhood acute lymphoblastic leukemia and acute myeloid leukemia. We have recently shown that Aven serves as an activator and substrate of ATM, thereby modulating the DNA-damage response and G(2)/M cell cycle progression. Under physiological conditions, the cellular localization of Aven is mainly cytosolic, but a small fraction of the protein is present in the nucleus. Here, we show that treatment of cells with leptomycin B, an inhibitor of Exportin-1/CRM (chromosomal region maintenance) 1, resulted in nuclear accumulation of Aven. Furthermore, we identified a functional LR-NES between amino acid residues 282-292 of the human Aven protein, a sequence that is evolutionary conserved among a range of vertebrate species. Disruption of this LR-NES by site-directed mutagenesis resulted in enhanced nuclear localization of Aven, but did not alter the ability of the protein to induce G(2)/M cell cycle arrest in interphase Xenopus laevis extracts. However, elimination of the LR-NES sequence led to a reduction in the capacity of Aven to arrest Xenopus oocytes containing intact nuclei. Our results suggest that the regulation of nucleocytoplasmatic traffic of Aven could modulate its ability to influence cell cycle progression.

Authors
Esmaili, AM; Johnson, EL; Thaivalappil, SS; Kuhn, HM; Kornbluth, S; Irusta, PM
MLA Citation
Esmaili, AM, Johnson, EL, Thaivalappil, SS, Kuhn, HM, Kornbluth, S, and Irusta, PM. "Regulation of the ATM-activator protein Aven by CRM1-dependent nuclear export." Cell cycle (Georgetown, Tex.) 9.19 (2010): 3913-3920.
PMID
20935510
Source
scival
Published In
Cell Cycle
Volume
9
Issue
19
Publish Date
2010
Start Page
3913
End Page
3920

The engine driving the ship: Metabolic steering of cell proliferation and death

Metabolic activity is a crucial determinant of a cell's decision to proliferate or die. Although it is not fully understood how metabolic pathways such as glycolysis and the pentose phosphate pathway communicate to cell cycle and apoptotic effectors, it is clear that a complex network of signalling molecules is required to integrate metabolic inputs. D-type cyclins, cyclin-dependent kinases, the anaphase-promoting complex, p53, caspase 2 and B cell lymphoma 2 proteins, among others, have been shown to be regulated by metabolic crosstalk. Elucidating these pathways is of great importance, as metabolic aberrations and their downstream effects are known to contribute to the aetiology of cancer and degenerative disorders. © 2010 Macmillan Publishers Limited. All rights reserved.

Authors
Buchakjian, MR; Kornbluth, S
MLA Citation
Buchakjian, MR, and Kornbluth, S. "The engine driving the ship: Metabolic steering of cell proliferation and death." Nature Reviews Molecular Cell Biology 11.10 (2010): 715-727.
PMID
20861880
Source
scival
Published In
Nature Reviews Molecular Cell Biology
Volume
11
Issue
10
Publish Date
2010
Start Page
715
End Page
727
DOI
10.1038/nrm2972

Stalling in mitosis and releasing the apoptotic brake

Microtubule poisons induce mitotic arrest that leads to apoptotic cell death if not resolved in a timely manner, but the mechanisms that directly link this cell cycle arrest to apoptosis have been elusive. In this issue of The EMBO Journal, Clarke and colleagues show thatMcl-1, an anti-apoptotic Bcl-2 family protein, is phosphorylated by the mitotic kinase CDK1/cyclin B1. This targets the protein for degradation by anaphase-promoting complex/cyclosome (APC/C)-mediated ubiquitination, in a manner such that only prolonged arrest allows sufficient Mcl-1 phosphorylation and degradation to trigger apoptosis. Thus, the APC/C, a major effector of the spindle assembly checkpoint (SAC), not only ensures cell cycle arrest upon spindle disruption, but promotes cell death when the duration of mitotic arrest is too long. © 2010 European Molecular Biology Organization - All Rights Reserved.

Authors
Kurokawa, M; Kornbluth, S
MLA Citation
Kurokawa, M, and Kornbluth, S. "Stalling in mitosis and releasing the apoptotic brake." EMBO Journal 29.14 (2010): 2255-2257.
PMID
20648046
Source
scival
Published In
EMBO Journal
Volume
29
Issue
14
Publish Date
2010
Start Page
2255
End Page
2257
DOI
10.1038/emboj.2010.150

Metabolic Control of Oocyte Apoptosis Mediated by 14-3-3ζ-Regulated Dephosphorylation of Caspase-2 (DOI:10.1016/j.devcel.2009.04.005)

Authors
Nutt, LK; Buchakjian, MR; Gan, E; Darbandi, R; Yoon, S-Y; Wu, JQ; Miyamoto, YJ; Gibbons, JA; Andersen, JL; Freel, CD; Tang, W; He, C; Kurokawa, M; Wang, Y; Margolis, SS; Fissore, RA; Kornbluth, S
MLA Citation
Nutt, LK, Buchakjian, MR, Gan, E, Darbandi, R, Yoon, S-Y, Wu, JQ, Miyamoto, YJ, Gibbons, JA, Andersen, JL, Freel, CD, Tang, W, He, C, Kurokawa, M, Wang, Y, Margolis, SS, Fissore, RA, and Kornbluth, S. "Metabolic Control of Oocyte Apoptosis Mediated by 14-3-3ζ-Regulated Dephosphorylation of Caspase-2 (DOI:10.1016/j.devcel.2009.04.005)." Developmental Cell 18.1 (2010): 165--.
Source
scival
Published In
Developmental Cell
Volume
18
Issue
1
Publish Date
2010
Start Page
165-
DOI
10.1016/j.devcel.2010.01.005

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2.

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1-cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1-cyclin B1 activity must be overcome for apoptosis to occur.

Authors
Andersen, JL; Johnson, CE; Freel, CD; Parrish, AB; Day, JL; Buchakjian, MR; Nutt, LK; Thompson, JW; Moseley, MA; Kornbluth, S
MLA Citation
Andersen, JL, Johnson, CE, Freel, CD, Parrish, AB, Day, JL, Buchakjian, MR, Nutt, LK, Thompson, JW, Moseley, MA, and Kornbluth, S. "Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2." The EMBO journal 28.20 (October 2009): 3216-3227.
PMID
19730412
Source
epmc
Published In
EMBO Journal
Volume
28
Issue
20
Publish Date
2009
Start Page
3216
End Page
3227
DOI
10.1038/emboj.2009.253

Metabolic control of oocyte apoptosis mediated by 14-3-3zeta-regulated dephosphorylation of caspase-2.

Xenopus oocyte death is partly controlled by the apoptotic initiator caspase-2 (C2). We reported previously that oocyte nutrient depletion activates C2 upstream of mitochondrial cytochrome c release. Conversely, nutrient-replete oocytes inhibit C2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. We now show that C2 phosphorylated at S135 binds 14-3-3zeta, thus preventing C2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1 (PP1), which directly binds C2. Although C2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3zeta from C2 is controlled by metabolism and allows for C2 dephosphorylation. Accordingly, a C2 mutant unable to bind 14-3-3zeta is highly susceptible to dephosphorylation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine C2 by phosphorylation and 14-3-3 binding in mouse eggs. These findings provide an unexpected evolutionary link between 14-3-3 and metabolism in oocyte death.

Authors
Nutt, LK; Buchakjian, MR; Gan, E; Darbandi, R; Yoon, S-Y; Wu, JQ; Miyamoto, YJ; Gibbons, JA; Andersen, JL; Freel, CD; Tang, W; He, C; Kurokawa, M; Wang, Y; Margolis, SS; Fissore, RA; Kornbluth, S
MLA Citation
Nutt, LK, Buchakjian, MR, Gan, E, Darbandi, R, Yoon, S-Y, Wu, JQ, Miyamoto, YJ, Gibbons, JA, Andersen, JL, Freel, CD, Tang, W, He, C, Kurokawa, M, Wang, Y, Margolis, SS, Fissore, RA, and Kornbluth, S. "Metabolic control of oocyte apoptosis mediated by 14-3-3zeta-regulated dephosphorylation of caspase-2." Dev Cell 16.6 (June 2009): 856-866.
PMID
19531356
Source
pubmed
Published In
Developmental Cell
Volume
16
Issue
6
Publish Date
2009
Start Page
856
End Page
866
DOI
10.1016/j.devcel.2009.04.005

PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation.

Loss of cell division cycle 2 (Cdc2, also known as Cdk1) activity after cyclin B degradation is necessary, but not sufficient, for mitotic exit. Proteins phosphorylated by Cdc2 and downstream mitotic kinases must be dephosphorylated. We report here that protein phosphatase-1 (PP1) is the main catalyst of mitotic phosphoprotein dephosphorylation. Suppression of PP1 during early mitosis is maintained through dual inhibition by Cdc2 phosphorylation and the binding of inhibitor-1. Protein kinase A (PKA) phosphorylates inhibitor-1, mediating binding to PP1. As Cdc2 levels drop after cyclin B degradation, auto-dephosphorylation of PP1 at its Cdc2 phosphorylation site (Thr 320) allows partial PP1 activation. This promotes PP1-regulated dephosphorylation at the activating site of inhibitor-1 (Thr 35) followed by dissociation of the inhibitor-1-PP1 complex and then full PP1 activation to promote mitotic exit. Thus, Cdc2 both phosphorylates multiple mitotic substrates and inhibits their PP1-mediated dephosphorylation.

Authors
Wu, JQ; Guo, JY; Tang, W; Yang, C-S; Freel, CD; Chen, C; Nairn, AC; Kornbluth, S
MLA Citation
Wu, JQ, Guo, JY, Tang, W, Yang, C-S, Freel, CD, Chen, C, Nairn, AC, and Kornbluth, S. "PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation." Nat Cell Biol 11.5 (May 2009): 644-651.
PMID
19396163
Source
pubmed
Published In
Nature Cell Biology
Volume
11
Issue
5
Publish Date
2009
Start Page
644
End Page
651
DOI
10.1038/ncb1871

An essential role for the Glut1 PDZ-binding motif in growth factor regulation of Glut1 degradation and trafficking.

Cell surface localization of the Glut (glucose transporter), Glut1, is a cytokine-controlled process essential to support the metabolism and survival of haemopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. In the present study, we show that a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence through deletion or point mutation sharply decreased surface Glut1 levels and led to rapid targeting of internalized Glut1 to lysosomes for proteolysis, particularly in growth factor-deprived cells. The PDZ-domain protein, GIPC (G(alpha)-interacting protein-interacting protein, C-terminus), bound to Glut1 in part via the Glut1 C-terminal PDZ-binding motif, and we found that GIPC deficiency decreased Glut1 surface levels and glucose uptake. Unlike the Glut1 degradation observed on mutation of the Glut1 PDZ-binding domain, however, GIPC deficiency resulted in accumulation of intracellular Glut1 in a pool distinct from the recycling pathway of the TfR (transferrin receptor). Blockade of Glut1 lysosomal targeting after growth factor withdrawal also led to intracellular accumulation of Glut1, a portion of which could be rapidly restored to the cell surface after growth factor stimulation. These results indicate that the C-terminal PDZ-binding motif of Glut1 plays a key role in growth factor regulation of glucose uptake by both allowing GIPC to promote Glut1 trafficking to the cell surface and protecting intracellular Glut1 from lysosomal degradation after growth factor withdrawal, thus allowing the potential for a rapid return of intracellular Glut1 to the cell surface on restimulation.

Authors
Wieman, HL; Horn, SR; Jacobs, SR; Altman, BJ; Kornbluth, S; Rathmell, JC
MLA Citation
Wieman, HL, Horn, SR, Jacobs, SR, Altman, BJ, Kornbluth, S, and Rathmell, JC. "An essential role for the Glut1 PDZ-binding motif in growth factor regulation of Glut1 degradation and trafficking." Biochem J 418.2 (March 1, 2009): 345-367.
PMID
19016655
Source
pubmed
Published In
The Biochemical journal
Volume
418
Issue
2
Publish Date
2009
Start Page
345
End Page
367
DOI
10.1042/BJ20081422

Protein phosphatase 2A-dependent dephosphorylation of replication protein A is required for the repair of DNA breaks induced by replication stress

Eukaryotic genomic integrity is safeguarded by cell cycle checkpoints and DNA repair pathways, collectively known as the DNA damage response, wherein replication protein A (RPA) is a key regulator playing multiple critical roles. The genotoxic insult-induced phosphorylation of the 32-kDa subunit of human RPA (RPA32), most notably the ATM/ATR-dependent phosphorylation at T21 and S33, acts to suppress DNA replication and recruit other checkpoint/repair proteins to the DNA lesions. It is not clear, however, how the DNA damage-responsive function of phosphorylated RPA is attenuated and how the replication-associated activity of the unphosphorylated form of RPA is restored when cells start to resume the normal cell cycle. We report here that in cells recovering from hydroxyurea (HU)-induced genotoxic stress, RPA32 is dephosphorylated by the serine/threonine protein phosphatase 2A (PP2A). Interference with PP2A catalytic activity causes persistent RPA32 phosphorylation and increased HU sensitivity. The PP2A catalytic subunit binds to RPA following DNA damage and can dephosphorylate RPA32 in vitro. Cells expressing a RPA32 persistent phosphorylation mimetic exhibit normal checkpoint activation and reenter the cell cycle normally after recovery but display a pronounced defect in the repair of DNA breaks. These data indicate that PP2A-mediated RPA32 dephosphorylation is required for the efficient DNA damage repair. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Authors
Feng, J; Wakeman, T; Yong, S; Wu, X; Kornbluth, S; Wang, X-F
MLA Citation
Feng, J, Wakeman, T, Yong, S, Wu, X, Kornbluth, S, and Wang, X-F. "Protein phosphatase 2A-dependent dephosphorylation of replication protein A is required for the repair of DNA breaks induced by replication stress." Molecular and Cellular Biology 29.21 (2009): 5696-5709.
PMID
19704001
Source
scival
Published In
Molecular and Cellular Biology
Volume
29
Issue
21
Publish Date
2009
Start Page
5696
End Page
5709
DOI
10.1128/MCB.00191-09

A Cut Above the Other Caspases

In this issue of Molecular Cell, Bouchier-Hayes et al. (2009) develop a novel approach to visualizing caspase-2 activation in real time, enabling resolution of several controversies surrounding the position of this enzyme in apoptotic signaling cascades. © 2009 Elsevier Inc. All rights reserved.

Authors
Andersen, JL; Kornbluth, S
MLA Citation
Andersen, JL, and Kornbluth, S. "A Cut Above the Other Caspases." Molecular Cell 35.6 (2009): 733-734.
PMID
19782021
Source
scival
Published In
Molecular Cell
Volume
35
Issue
6
Publish Date
2009
Start Page
733
End Page
734
DOI
10.1016/j.molcel.2009.09.001

Caspases and Kinases in a Death Grip

The complex process of apoptosis is orchestrated by caspases, a family of cysteine proteases with unique substrate specificities. Accumulating evidence suggests that cell death pathways are finely tuned by multiple signaling events, including direct phosphorylation of caspases, whereas kinases are often substrates of active caspases. Importantly, caspase-mediated cleavage of kinases can terminate prosurvival signaling or generate proapoptotic peptide fragments that help to execute the death program and facilitate packaging of the dying cells. Here, we review caspases as kinase substrates and kinases as caspase substrates and discuss how the balance between cell survival and cell death can be shifted through crosstalk between these two enzyme families. © 2009 Elsevier Inc. All rights reserved.

Authors
Kurokawa, M; Kornbluth, S
MLA Citation
Kurokawa, M, and Kornbluth, S. "Caspases and Kinases in a Death Grip." Cell 138.5 (2009): 838-854.
PMID
19737514
Source
scival
Published In
Cell
Volume
138
Issue
5
Publish Date
2009
Start Page
838
End Page
854
DOI
10.1016/j.cell.2009.08.021

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Authors
Galluzzi, L; Aaronson, SA; Abrams, J; Alnemri, ES; Andrews, DW; Baehrecke, EH; Bazan, NG; Blagosklonny, MV; Blomgren, K; Borner, C; Bredesen, DE; Brenner, C; Castedo, M; Cidlowski, JA; Ciechanover, A; Cohen, GM; Laurenzi, VD; Maria, RD; Deshmukh, M; Dynlacht, BD; El-Deiry, WS; Flavell, RA; Fulda, S; Garrido, C; Golstein, P; Gougeon, M-L; Green, DR; Gronemeyer, H; Hajnóczky, G; Hardwick, JM; Hengartner, MO; Ichijo, H; Jäättelä, M; Kepp, O; Kimchi, A; Klionsky, DJ; Knight, RA; Kornbluth, S et al.
MLA Citation
Galluzzi, L, Aaronson, SA, Abrams, J, Alnemri, ES, Andrews, DW, Baehrecke, EH, Bazan, NG, Blagosklonny, MV, Blomgren, K, Borner, C, Bredesen, DE, Brenner, C, Castedo, M, Cidlowski, JA, Ciechanover, A, Cohen, GM, Laurenzi, VD, Maria, RD, Deshmukh, M, Dynlacht, BD, El-Deiry, WS, Flavell, RA, Fulda, S, Garrido, C, Golstein, P, Gougeon, M-L, Green, DR, Gronemeyer, H, Hajnóczky, G, Hardwick, JM, Hengartner, MO, Ichijo, H, Jäättelä, M, Kepp, O, Kimchi, A, Klionsky, DJ, Knight, RA, and Kornbluth, S et al. "Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes." Cell Death and Differentiation 16.8 (2009): 1093-1107.
PMID
19373242
Source
scival
Published In
Cell Death & Differentiation
Volume
16
Issue
8
Publish Date
2009
Start Page
1093
End Page
1107
DOI
10.1038/cdd.2009.44

Inhibition of apoptosome formation by suppression of Hsp90beta phosphorylation in tyrosine kinase-induced leukemias.

Constitutively active tyrosine kinases promote leukemogenesis by increasing cell proliferation and inhibiting apoptosis. However, mechanisms underlying apoptotic inhibition have not been fully elucidated. In many settings, apoptosis occurs by mitochondrial cytochrome c release, which nucleates the Apaf-1/caspase-9 apoptosome. Here we report that the leukemogenic kinases, Bcr-Abl, FLT3/D835Y, and Tel-PDGFRbeta, all can inhibit apoptosome function. In cells expressing these kinases, the previously reported apoptosome inhibitor, Hsp90beta, bound strongly to Apaf-1, preventing cytochrome c-induced Apaf-1 oligomerization and caspase-9 recruitment. Hsp90beta interacted weakly with the apoptosome in untransformed cells. While Hsp90beta was phosphorylated at Ser 226/Ser 255 in untransformed cells, phosphorylation was absent in leukemic cells. Expression of mutant Hsp90beta (S226A/S255A), which mimics the hypophosphorylated form in leukemic cells, conferred resistance to cytochrome c-induced apoptosome activation in normal cells, reflecting enhanced binding of nonphosphorylatable Hsp90beta to Apaf-1. In Bcr-Abl-positive mouse bone marrow cells, nonphosphorylatable Hsp90beta expression conferred imatinib (Gleevec) resistance. These data provide an explanation for apoptosome inhibition by activated leukemogenic tyrosine kinases and suggest that alterations in Hsp90beta-apoptosome interactions may contribute to chemoresistance in leukemias.

Authors
Kurokawa, M; Zhao, C; Reya, T; Kornbluth, S
MLA Citation
Kurokawa, M, Zhao, C, Reya, T, and Kornbluth, S. "Inhibition of apoptosome formation by suppression of Hsp90beta phosphorylation in tyrosine kinase-induced leukemias." Mol Cell Biol 28.17 (September 2008): 5494-5506.
PMID
18591256
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
28
Issue
17
Publish Date
2008
Start Page
5494
End Page
5506
DOI
10.1128/MCB.00265-08

Cdc2 and Mos regulate Emi2 stability to promote the meiosis I-meiosis II transition.

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.

Authors
Tang, W; Wu, JQ; Guo, Y; Hansen, DV; Perry, JA; Freel, CD; Nutt, L; Jackson, PK; Kornbluth, S
MLA Citation
Tang, W, Wu, JQ, Guo, Y, Hansen, DV, Perry, JA, Freel, CD, Nutt, L, Jackson, PK, and Kornbluth, S. "Cdc2 and Mos regulate Emi2 stability to promote the meiosis I-meiosis II transition." Mol Biol Cell 19.8 (August 2008): 3536-3543.
PMID
18550795
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
19
Issue
8
Publish Date
2008
Start Page
3536
End Page
3543
DOI
10.1091/mbc.E08-04-0417

Aven-dependent activation of ATM following DNA damage.

BACKGROUND: In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cell's capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. RESULTS: In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpressed in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM after DNA damage is enhanced by ATM-mediated Aven phosphorylation. CONCLUSIONS: These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal.

Authors
Guo, JY; Yamada, A; Kajino, T; Wu, JQ; Tang, W; Freel, CD; Feng, J; Chau, BN; Wang, MZ; Margolis, SS; Yoo, HY; Wang, X-F; Dunphy, WG; Irusta, PM; Hardwick, JM; Kornbluth, S
MLA Citation
Guo, JY, Yamada, A, Kajino, T, Wu, JQ, Tang, W, Freel, CD, Feng, J, Chau, BN, Wang, MZ, Margolis, SS, Yoo, HY, Wang, X-F, Dunphy, WG, Irusta, PM, Hardwick, JM, and Kornbluth, S. "Aven-dependent activation of ATM following DNA damage." Curr Biol 18.13 (July 8, 2008): 933-942.
PMID
18571408
Source
pubmed
Published In
Current Biology
Volume
18
Issue
13
Publish Date
2008
Start Page
933
End Page
942
DOI
10.1016/j.cub.2008.05.045

Mitochondrial localization of Reaper to promote inhibitors of apoptosis protein degradation conferred by GH3 domain-lipid interactions.

Morphological hallmarks of apoptosis result from activation of the caspase family of cysteine proteases, which are opposed by a pro-survival family of inhibitors of apoptosis proteins (IAPs). In Drosophila, disruption of IAP function by Reaper, HID, and Grim (RHG) proteins is sufficient to induce cell death. RHG proteins have been reported to localize to mitochondria, which, in the case of both Reaper and Grim proteins, is mediated by an amphipathic helical domain known as the GH3. Through direct binding, Reaper can bring the Drosophila IAP (DIAP1) to mitochondria, concomitantly promoting IAP auto-ubiquitination and destruction. Whether this localization is sufficient to induce DIAP1 auto-ubiquitination has not been reported. In this study we characterize the interaction between Reaper and the mitochondria using both Xenopus and Drosophila systems. We find that Reaper concentrates on the outer surface of mitochondria in a nonperipheral manner largely mediated by GH3-lipid interactions. Importantly, we show that mitochondrial targeting of DIAP1 alone is not sufficient for degradation and requires Reaper binding. Conversely, Reaper able to bind IAPs, but lacking a mitochondrial targeting GH3 domain (DeltaGH3 Reaper), can induce DIAP1 turnover only if DIAP1 is otherwise targeted to membranes. Surprisingly, targeting DIAP1 to the endoplasmic reticulum instead of mitochondria is partially effective in allowing DeltaGH3 Reaper to promote DIAP1 degradation, suggesting that co-localization of DIAP and Reaper at a membrane surface is critical for the induction of DIAP degradation. Collectively, these data provide a specific function for the GH3 domain in conferring protein-lipid interactions, demonstrate that both Reaper binding and mitochondrial localization are required for accelerated IAP degradation, and suggest that membrane localization per se contributes to DIAP1 auto-ubiquitination and degradation.

Authors
Freel, CD; Richardson, DA; Thomenius, MJ; Gan, EC; Horn, SR; Olson, MR; Kornbluth, S
MLA Citation
Freel, CD, Richardson, DA, Thomenius, MJ, Gan, EC, Horn, SR, Olson, MR, and Kornbluth, S. "Mitochondrial localization of Reaper to promote inhibitors of apoptosis protein degradation conferred by GH3 domain-lipid interactions." J Biol Chem 283.1 (January 4, 2008): 367-379.
PMID
17998202
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
1
Publish Date
2008
Start Page
367
End Page
379
DOI
10.1074/jbc.M708931200

Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era

Vertebrate eggs are arrested at the metaphase stage of meiosis II. Only upon fertilization will the metaphase-II-arrested eggs exit meiosis II and enter interphase. In 1971, Masui and Markert injected egg extracts into a two-cell-stage embryo and found that the injected blastomere arrested at the next mitosis. On the basis of these observations, they proposed the existence of an activity present in the eggs that is responsible for meiosis-II arrest and can induce mitotic arrest, and named this activity cytostatic factor (CSF). Although the existence of CSF was hypothesized more than 35 years ago, its precise identity remained unclear until recently. The discovery of the Mos-MAPK pathway and characterization of the anaphase-promoting complex/cyclosome (APC/C) as a central regulator of M-phase exit provided the framework for a molecular understanding of CSF. These pathways have now been linked by the discovery and characterization of the protein Emi2, a meiotic APC/C inhibitor, the activity and stability of which are controlled by the Mos-MAPK pathway. Continued investigation into the mechanism of action and mode of regulation of Emi2 promises to shed light not only on CSF function, but also on the general principles of APC/C regulation and the control of protein function by MAPK pathways.

Authors
Wu, JQ; Kornbluth, S
MLA Citation
Wu, JQ, and Kornbluth, S. "Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era." Journal of Cell Science 121.21 (2008): 3509-3514.
Website
http://hdl.handle.net/10161/8390
PMID
18946022
Source
scival
Published In
Journal of cell science
Volume
121
Issue
21
Publish Date
2008
Start Page
3509
End Page
3514
DOI
10.1242/jcs.036855

Caspase Cleavage Is Not for Everyone

During apoptosis, caspases cleave cellular substrates to break down and package the apoptotic cell for removal. Reporting in Cell, Mahrus et al. (2008) and Dix et al. (2008) use new approaches that identify hundreds of previously unrecognized caspase substrates, many of which appear to produce polypeptide fragments with potentially new functional activities. © 2008 Elsevier Inc. All rights reserved.

Authors
Johnson, CE; Kornbluth, S
MLA Citation
Johnson, CE, and Kornbluth, S. "Caspase Cleavage Is Not for Everyone." Cell 134.5 (2008): 720-721.
PMID
18775303
Source
scival
Published In
Cell
Volume
134
Issue
5
Publish Date
2008
Start Page
720
End Page
721
DOI
10.1016/j.cell.2008.08.019

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantificationmethods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was ∼20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV ≤27%). The MS-based method strongly correlated with the immunoquantification method (r2≥0.87) and marker activities (r2≥0.88), including testosterone 6β-hydroxylation (CYP3A), midazolam 1′-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliablemeans of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Authors
Wang, MZ; Wu, JQ; Dennison, JB; Bridges, AS; Hall, SD; Kornbluth, S; Tidwell, RR; Smith, PC; Voyksner, RD; Paine, MF; Hall, JE
MLA Citation
Wang, MZ, Wu, JQ, Dennison, JB, Bridges, AS, Hall, SD, Kornbluth, S, Tidwell, RR, Smith, PC, Voyksner, RD, Paine, MF, and Hall, JE. "A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes." Proteomics 8.20 (2008): 4186-4196.
PMID
18792928
Source
scival
Published In
Proteomics
Volume
8
Issue
20
Publish Date
2008
Start Page
4186
End Page
4196
DOI
10.1002/pmic.200800144

Not-So-Pseudo a Substrate: Acm1-Mediated Inhibition of the APC

In a recent issue of Molecular Cell, Enquist-Newman et al. (2008) demonstrate that Acm1 is ubiquitinated by APCCdc20. By contrast, the high-affinity interaction between Acm1 and APCCdh1 renders it a poor substrate, but a specific inhibitor, of the APCCdh1 complex. © 2008 Elsevier Inc. All rights reserved.

Authors
Wu, JQ; Kornbluth, S
MLA Citation
Wu, JQ, and Kornbluth, S. "Not-So-Pseudo a Substrate: Acm1-Mediated Inhibition of the APC." Molecular Cell 30.5 (2008): 543-544.
PMID
18538651
Source
scival
Published In
Molecular Cell
Volume
30
Issue
5
Publish Date
2008
Start Page
543
End Page
544
DOI
10.1016/j.molcel.2008.05.011

Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues.

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.

Authors
Johnson, CE; Huang, YY; Parrish, AB; Smith, MI; Vaughn, AE; Zhang, Q; Wright, KM; Van Dyke, T; Wechsler-Reya, RJ; Kornbluth, S; Deshmukh, M
MLA Citation
Johnson, CE, Huang, YY, Parrish, AB, Smith, MI, Vaughn, AE, Zhang, Q, Wright, KM, Van Dyke, T, Wechsler-Reya, RJ, Kornbluth, S, and Deshmukh, M. "Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues." Proc Natl Acad Sci U S A 104.52 (December 26, 2007): 20820-20825.
Website
http://hdl.handle.net/10161/8392
PMID
18093951
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
52
Publish Date
2007
Start Page
20820
End Page
20825
DOI
10.1073/pnas.0709101105

Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A.

Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.

Authors
Wu, JQ; Hansen, DV; Guo, Y; Wang, MZ; Tang, W; Freel, CD; Tung, JJ; Jackson, PK; Kornbluth, S
MLA Citation
Wu, JQ, Hansen, DV, Guo, Y, Wang, MZ, Tang, W, Freel, CD, Tung, JJ, Jackson, PK, and Kornbluth, S. "Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A." Proc Natl Acad Sci U S A 104.42 (October 16, 2007): 16564-16569.
Website
http://hdl.handle.net/10161/8391
PMID
17881560
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
42
Publish Date
2007
Start Page
16564
End Page
16569
DOI
10.1073/pnas.0707537104

Filling a GAP(DH) in caspase-independent cell death.

Mitochondrial outer-membrane permeabilization can lead to cell death even without activation of caspases. In this issue of Cell, Colell et al. (2007) identify the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase as a potent inhibitor of caspase-independent cell death that may allow metabolically active cells to survive mitochondrial insult.

Authors
Rathmell, JC; Kornbluth, S
MLA Citation
Rathmell, JC, and Kornbluth, S. "Filling a GAP(DH) in caspase-independent cell death." Cell 129.5 (June 1, 2007): 861-863.
PMID
17540167
Source
pubmed
Published In
Cell
Volume
129
Issue
5
Publish Date
2007
Start Page
861
End Page
863
DOI
10.1016/j.cell.2007.05.030

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest.

BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.

Authors
Wu, Q; Guo, Y; Yamada, A; Perry, JA; Wang, MZ; Araki, M; Freel, CD; Tung, JJ; Tang, W; Margolis, SS; Jackson, PK; Yamano, H; Asano, M; Kornbluth, S
MLA Citation
Wu, Q, Guo, Y, Yamada, A, Perry, JA, Wang, MZ, Araki, M, Freel, CD, Tung, JJ, Tang, W, Margolis, SS, Jackson, PK, Yamano, H, Asano, M, and Kornbluth, S. "A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest." Curr Biol 17.3 (February 6, 2007): 213-224.
PMID
17276914
Source
pubmed
Published In
Current Biology
Volume
17
Issue
3
Publish Date
2007
Start Page
213
End Page
224
DOI
10.1016/j.cub.2006.12.045

Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug pafuramidine

CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent Km and Vmax values ranged from 0.6 to 2.4 μM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl) formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid ω-hydroxylation rates (r2 = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.

Authors
Wang, MZ; Wu, JQ; Bridges, AS; Zeldin, DC; Kornbluth, S; Tidwell, RR; Hall, JE; Paine, MF
MLA Citation
Wang, MZ, Wu, JQ, Bridges, AS, Zeldin, DC, Kornbluth, S, Tidwell, RR, Hall, JE, and Paine, MF. "Human enteric microsomal CYP4F enzymes O-demethylate the antiparasitic prodrug pafuramidine." Drug Metabolism and Disposition 35.11 (2007): 2067-2075.
PMID
17709372
Source
scival
Published In
Drug metabolism and disposition: the biological fate of chemicals
Volume
35
Issue
11
Publish Date
2007
Start Page
2067
End Page
2075
DOI
10.1124/dmd.107.016428

Cdc25 and Wee1: Analogous opposites?

Movement through the cell cycle is controlled by the temporally and spatially ordered activation of cyclin-dependent kinases paired with their respective cyclin binding partners. Cell cycle events occur in a stepwise fashion and are monitored by molecular surveillance systems to ensure that each cell cycle process is appropriately completed before subsequent events are initiated. Cells prevent entry into mitosis while DNA replication is ongoing, or if DNA is damaged, via checkpoint mechanisms that inhibit the activators and activate the inhibitors of mitosis, Cdc25 and Wee1, respectively. Once DNA replication has been faithfully completed, Cdc2/Cyclin B is swiftly activated for a timely transition from interphase into mitosis. This sharp transition is propagated through both positive and negative feedback loops that impinge upon Cdc25 and Wee1 to ensure that Cdc2/Cyclin B is fully activated. Recent reports from a number of laboratories have revealed a remarkably complex network of kinases and phosphatases that coordinately control Cdc25 and Wee1, thereby precisely regulating the transition into mitosis. Although not all factors that inhibit Cdc25 have been shown to activate Wee1 and vice versa, a number of regulatory modules are clearly shared in common. Thus, studies on either the Cdc25 or Wee1-regulatory arm of the mitotic control pathway should continue to shed light on how both arms are coordinated to smoothly regulate mitotic entry. © 2007 Perry and Kornbluth; licensee BioMed Central Ltd.

Authors
Perry, JA; Kornbluth, S
MLA Citation
Perry, JA, and Kornbluth, S. "Cdc25 and Wee1: Analogous opposites?." Cell Division 2 (2007).
Website
http://hdl.handle.net/10161/8383
Source
scival
Published In
Cell Division
Volume
2
Publish Date
2007
DOI
10.1186/1747-1028-2-12

Mcm10 and And-1/CTF4 recruit DNA polymerase α to chromatin for initiation of DNA replication

The MCM2-7 helicase complex is loaded on DNA replication origins during the G1 phase of the cell cycle to license the origins for replication in S phase. How the initiator primase-polymerase complex, DNA polymerase α (pol α), is brought to the origins is still unclear. We show that And-1/Ctf4 (Chromosome transmission fidelity 4) interacts with Mcm10, which associates with MCM2-7, and with the p180 subunit of DNA pol α. And-1 is essential for DNA synthesis and the stability of p180 in mammalian cells. In Xenopus egg extracts And-1 is loaded on the chromatin after Mcm10, concurrently with DNA pol α, and is required for efficient DNA synthesis. Mcm10 is required for chromatin loading of And-1 and an antibody that disrupts the Mcm10-And-1 interaction interferes with the loading of And-1 and of pol α, inhibiting DNA synthesis. And-1/Ctf4 is therefore a new replication initiation factor that brings together the MCM2-7 helicase and the DNA pol α-primase complex, analogous to the linker between helicase and primase or helicase and polymerase that is seen in the bacterial replication machinery. The discovery also adds to the connection between replication initiation and sister chromatid cohesion. © 2007 by Cold Spring Harbor Laboratory Press.

Authors
Zhu, W; Ukomadu, C; Jha, S; Senga, T; Dhar, SK; Wohlschlegel, JA; Nutt, LK; Kornbluth, S; Dutta, A
MLA Citation
Zhu, W, Ukomadu, C, Jha, S, Senga, T, Dhar, SK, Wohlschlegel, JA, Nutt, LK, Kornbluth, S, and Dutta, A. "Mcm10 and And-1/CTF4 recruit DNA polymerase α to chromatin for initiation of DNA replication." Genes and Development 21.18 (2007): 2288-2299.
Website
http://hdl.handle.net/10161/8385
PMID
17761813
Source
scival
Published In
Genes & development
Volume
21
Issue
18
Publish Date
2007
Start Page
2288
End Page
2299
DOI
10.1101/gad.1585607

HLA-B-associated transcript 3 (Bat3)/Scythe is essential for p300-mediated acetylation of p53

In response to DNA damage, p53 undergoes post-translational modifications (including acetylation) that are critical for its transcriptional activity. However, the mechanism by which p53 acetylation is regulated is still unclear. Here, we describe an essential role for HLA-B-associated transcript 3 (Bat3)/Scythe in controlling the acetylation of p53 required for DNA damage responses. Depletion of Bat3 from human and mouse cells markedly impairs p53-mediated transactivation of its target genes Puma and p21. Although DNA damage-induced phosphorylation, stabilization, and nuclear accumulation of p53 are not significantly affected by Bat3 depletion, p53 acetylation is almost completely abolished. Bat3 forms a complex with p300, and an increased amount of Bat3 enhances the recruitment of p53 to p300 and facilitates subsequent p53 acetylation. In contrast, Bat3-depleted cells show reduced p53-p300 complex formation and decreased p53 acetylation. Furthermore, consistent with our in vitro findings, thymocytes from Bat3-deficient mice exhibit reduced induction of puma and p21, and are resistant to DNA damage-induced apoptosis in vivo. Our data indicate that Bat3 is a novel and essential regulator of p53-mediated responses to genotoxic stress, and that Bat3 controls DNA damage-induced acetylation of p53. © 2007 by Cold Spring Harbor Laboratory Press.

Authors
Sasaki, T; Gan, EC; Wakeham, A; Kornbluth, S; Mak, TW; Okada, H
MLA Citation
Sasaki, T, Gan, EC, Wakeham, A, Kornbluth, S, Mak, TW, and Okada, H. "HLA-B-associated transcript 3 (Bat3)/Scythe is essential for p300-mediated acetylation of p53." Genes and Development 21.7 (2007): 848-861.
Website
http://hdl.handle.net/10161/8384
PMID
17403783
Source
scival
Published In
Genes & development
Volume
21
Issue
7
Publish Date
2007
Start Page
848
End Page
861
DOI
10.1101/gad.1534107

Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis.

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. We show here that DNA-responsive checkpoints also activate PP2A/B56delta phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Our data suggest that creation of a 14-3-3 "sink," consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56delta as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis.

Authors
Margolis, SS; Perry, JA; Forester, CM; Nutt, LK; Guo, Y; Jardim, MJ; Thomenius, MJ; Freel, CD; Darbandi, R; Ahn, J-H; Arroyo, JD; Wang, X-F; Shenolikar, S; Nairn, AC; Dunphy, WG; Hahn, WC; Virshup, DM; Kornbluth, S
MLA Citation
Margolis, SS, Perry, JA, Forester, CM, Nutt, LK, Guo, Y, Jardim, MJ, Thomenius, MJ, Freel, CD, Darbandi, R, Ahn, J-H, Arroyo, JD, Wang, X-F, Shenolikar, S, Nairn, AC, Dunphy, WG, Hahn, WC, Virshup, DM, and Kornbluth, S. "Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis." Cell 127.4 (November 17, 2006): 759-773.
PMID
17110335
Source
pubmed
Published In
Cell
Volume
127
Issue
4
Publish Date
2006
Start Page
759
End Page
773
DOI
10.1016/j.cell.2006.10.035

A role for PP1 in the Cdc2/Cyclin B-mediated positive feedback activation of Cdc25.

The Cdc25 phosphatase promotes entry into mitosis through the removal of inhibitory phosphorylations on the Cdc2 subunit of the Cdc2/CyclinB complex. During interphase, or after DNA damage, Cdc25 is suppressed by phosphorylation at Ser287 (Xenopus numbering; Ser216 of human Cdc25C) and subsequent binding of the small acidic protein, 14-3-3. As reported recently, at the time of mitotic entry, 14-3-3 protein is removed from Cdc25 and S287 is dephosphorylated by protein phosphatase 1 (PP1). After the initial activation of Cdc25 and consequent derepression of Cdc2/CyclinB, Cdc25 is further activated through a Cdc2-catalyzed positive feedback loop. Although the existence of such a loop has been appreciated for some time, the molecular mechanism for this activation has not been described. We report here that phosphorylation of S285 by Cdc2 greatly enhances recruitment of PP1 to Cdc25, thereby accelerating S287 dephosphorylation and mitotic entry. Moreover, we show that two other previously reported sites of Cdc2-catalyzed phosphorylation on Cdc25 are required for maximal biological activity of Cdc25, but they do not contribute to PP1 regulation and do not act solely through controlling S287 phosphorylation. Therefore, multiple mechanisms, including enhanced recruitment of PP1, are used to promote full activation of Cdc25 at the time of mitotic entry.

Authors
Margolis, SS; Perry, JA; Weitzel, DH; Freel, CD; Yoshida, M; Haystead, TA; Kornbluth, S
MLA Citation
Margolis, SS, Perry, JA, Weitzel, DH, Freel, CD, Yoshida, M, Haystead, TA, and Kornbluth, S. "A role for PP1 in the Cdc2/Cyclin B-mediated positive feedback activation of Cdc25." Mol Biol Cell 17.4 (April 2006): 1779-1789.
PMID
16467385
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
17
Issue
4
Publish Date
2006
Start Page
1779
End Page
1789
DOI
10.1091/mbc.E05-08-0751

Enhanced sensitivity to cytochrome c-induced apoptosis mediated by PHAPI in breast cancer cells.

Apoptotic signaling defects both promote tumorigenesis and confound chemotherapy. Typically, chemotherapeutics stimulate cytochrome c release to the cytoplasm, thereby activating the apoptosome. Although cancer cells can be refractory to cytochrome c release, many malignant cells also exhibit defects in cytochrome c-induced apoptosome activation, further promoting chemotherapeutic resistance. We have found that breast cancer cells display an unusual sensitivity to cytochrome c-induced apoptosis when compared with their normal counterparts. This sensitivity, not observed in other cancers, resulted from enhanced recruitment of caspase-9 to the Apaf-1 caspase recruitment domain. Augmented caspase activation was mediated by PHAPI, which is overexpressed in breast cancers. Furthermore, cytochrome c microinjection into mammary epithelial cells preferentially killed malignant cells, suggesting that this phenomenon might be exploited for chemotherapeutic purposes.

Authors
Schafer, ZT; Parrish, AB; Wright, KM; Margolis, SS; Marks, JR; Deshmukh, M; Kornbluth, S
MLA Citation
Schafer, ZT, Parrish, AB, Wright, KM, Margolis, SS, Marks, JR, Deshmukh, M, and Kornbluth, S. "Enhanced sensitivity to cytochrome c-induced apoptosis mediated by PHAPI in breast cancer cells." Cancer Res 66.4 (February 15, 2006): 2210-2218.
PMID
16489023
Source
pubmed
Published In
Cancer Research
Volume
66
Issue
4
Publish Date
2006
Start Page
2210
End Page
2218
DOI
10.1158/0008-5472.CAN-05-3923

Analysis of the cell cycle using Xenopus egg extracts.

In this unit, Xenopus eggs are isolated from hormonally primed female frogs, and then the extract is treated with cyclohexamide so it remains in interphase of the cell cycle. In the presence of sperm chromatin and ATP, membrane vesicles in the extract fuse to assemble nuclei, making the extract suitable for studies of DNA replication and nuclear transport.

Authors
Kornbluth, S; Yang, J; Powers, M
MLA Citation
Kornbluth, S, Yang, J, and Powers, M. "Analysis of the cell cycle using Xenopus egg extracts." Curr Protoc Cell Biol Chapter 11 (January 2006): Unit-11.11.
PMID
18228475
Source
pubmed
Published In
Current Protocols in Cell Biology
Volume
Chapter 11
Publish Date
2006
Start Page
Unit
End Page
11.11
DOI
10.1002/0471143030.cb1111s29

Multifunctional reaper: Sixty-five amino acids of fury

Authors
Thomenius, M; Kornbluth, S
MLA Citation
Thomenius, M, and Kornbluth, S. "Multifunctional reaper: Sixty-five amino acids of fury." Cell Death and Differentiation 13.8 (2006): 1305-1309.
PMID
16710365
Source
scival
Published In
Cell Death & Differentiation
Volume
13
Issue
8
Publish Date
2006
Start Page
1305
End Page
1309
DOI
10.1038/sj.cdd.4401954

The Apoptosome: Physiological, Developmental, and Pathological Modes of Regulation

Apoptosis, a form of programmed cell death, is executed by a family of zymogenic proteases known as caspases, which cleave an array of intracellular substrates in the dying cell. Many proapoptotic stimuli trigger cytochrome c release from mitochondria, promoting the formation of a complex between Apaf-1 and caspase-9 in a caspase-activating structure known as the apoptosome. In this review, we describe knockout and knockin studies of apoptosome components, elegant structural and biochemical experiments, and analyses of the apoptosome in various cancers and other disease states, all of which have provided new insight into this critical locus of apoptotic control. © 2006 Elsevier Inc. All rights reserved.

Authors
Schafer, ZT; Kornbluth, S
MLA Citation
Schafer, ZT, and Kornbluth, S. "The Apoptosome: Physiological, Developmental, and Pathological Modes of Regulation." Developmental Cell 10.5 (2006): 549-561.
PMID
16678772
Source
scival
Published In
Developmental Cell
Volume
10
Issue
5
Publish Date
2006
Start Page
549
End Page
561
DOI
10.1016/j.devcel.2006.04.008

Direct ribosomal binding by a cellular inhibitor of translation

During apoptosis and under conditions of cellular stress, several signaling pathways promote inhibition of cap-dependent translation while allowing continued translation of specific messenger RNAs encoding regulatory and stress-response proteins. We report here that the apoptotic regulator Reaper inhibits protein synthesis by binding directly to the 40S ribosomal subunit. This interaction does not affect either ribosomal association of initiation factors or formation of 43S or 48S complexes. Rather, it interferes with late initiation events upstream of 60S subunit joining, apparently modulating start-codon recognition during scanning. CrPV IRES-driven translation, involving direct ribosomal recruitment to the start site, is relatively insensitive to Reaper. Thus, Reaper is the first known cellular ribosomal binding factor with the potential to allow selective translation of mRNAs initiating at alternative start codons or from certain IRES elements. This function of Reaper may modulate gene expression programs to affect cell fate. © 2006 Nature Publishing Group.

Authors
Colón-Ramos, DA; Shenvi, CL; Weitzel, DH; Gan, EC; Matts, R; Cate, J; Kornbluth, S
MLA Citation
Colón-Ramos, DA, Shenvi, CL, Weitzel, DH, Gan, EC, Matts, R, Cate, J, and Kornbluth, S. "Direct ribosomal binding by a cellular inhibitor of translation." Nature Structural and Molecular Biology 13.2 (2006): 103-111.
PMID
16429152
Source
scival
Published In
Nature Structural & Molecular Biology
Volume
13
Issue
2
Publish Date
2006
Start Page
103
End Page
111
DOI
10.1038/nsmb1052

Study of apoptosis in vitro using the Xenopus egg extract reconstitution system.

It was first shown by Newmeyer and colleagues in the 1990s that the molecular events of apoptosis could be reconstituted in vitro using Xenopus egg extracts. When the egg extract is allowed to incubate at room temperature for an extended time, the biochemical events of apoptosis are activated spontaneously. The features of apoptosis in the Xenopus reconstitution system mimic those that occur in mammalian cells: Cytochrome c is released from the mitochondria, caspases are activated, cellular substrates are cleaved, and added nuclei are fragmented. Moreover, these apoptotic events can be inhibited by addition of antiapoptotic proteins such as Bcl-2 and conventional caspase inhibitors (e.g., ZVAD-fmk). The mitochondria, which are triggered to release cytochrome c by as-yet-unknown signals in the extract, are essential for induction of the spontaneous apoptotic program in these extracts. However, one can study the apoptotic events that occur downstream of mitochondrial cytochrome c release by preparing extracts devoid of membrane components (including mitochondria). When purified cytochrome c is added to such cytosolic extracts, biochemical markers of apoptosis (e.g., activation of caspases) can be monitored. The egg extract therefore offers a tractable system for studying either separately or together the events of apoptosis occurring upstream and downstream of the mitochondria.

Authors
Deming, P; Kornbluth, S
MLA Citation
Deming, P, and Kornbluth, S. "Study of apoptosis in vitro using the Xenopus egg extract reconstitution system." Methods in molecular biology (Clifton, N.J.) 322 (2006): 379-393.
PMID
16739738
Source
scival
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
322
Publish Date
2006
Start Page
379
End Page
393

Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2.

Vertebrate female reproduction is limited by the oocyte stockpiles acquired during embryonic development. These are gradually depleted over the organism's lifetime through the process of apoptosis. The timer that triggers this cell death is yet to be identified. We used the Xenopus egg/oocyte system to examine the hypothesis that nutrient stores can regulate oocyte viability. We show that pentose-phosphate-pathway generation of NADPH is critical for oocyte survival and that the target of this regulation is caspase-2, previously shown to be required for oocyte death in mice. Pentose-phosphate-pathway-mediated inhibition of cell death was due to the inhibitory phosphorylation of caspase-2 by calcium/calmodulin-dependent protein kinase II (CaMKII). These data suggest that exhaustion of oocyte nutrients, resulting in an inability to generate NADPH, may contribute to ooctye apoptosis. These data also provide unexpected links between oocyte metabolism, CaMKII, and caspase-2.

Authors
Nutt, LK; Margolis, SS; Jensen, M; Herman, CE; Dunphy, WG; Rathmell, JC; Kornbluth, S
MLA Citation
Nutt, LK, Margolis, SS, Jensen, M, Herman, CE, Dunphy, WG, Rathmell, JC, and Kornbluth, S. "Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2." Cell 123.1 (October 7, 2005): 89-103.
PMID
16213215
Source
pubmed
Published In
Cell
Volume
123
Issue
1
Publish Date
2005
Start Page
89
End Page
103
DOI
10.1016/j.cell.2005.07.032

Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

Authors
Casaletto, JB; Nutt, LK; Wu, Q; Moore, JD; Etkin, LD; Jackson, PK; Hunt, T; Kornbluth, S
MLA Citation
Casaletto, JB, Nutt, LK, Wu, Q, Moore, JD, Etkin, LD, Jackson, PK, Hunt, T, and Kornbluth, S. "Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase." J Cell Biol 169.1 (April 11, 2005): 61-71.
Website
http://hdl.handle.net/10161/8379
PMID
15824132
Source
pubmed
Published In
The Journal of Cell Biology
Volume
169
Issue
1
Publish Date
2005
Start Page
61
End Page
71
DOI
10.1083/jcb.200411056

Apoptosis in Drosophila: Neither fish nor fowl (nor man, nor worm)

Studies in a wide variety of organisms have produced a general model for the induction of apoptosis in which multiple signaling pathways lead ultimately to activation of the caspase family of proteases. Once activated, these enzymes cleave key cellular substrates to promote the orderly dismantling of dying cells. A broad similarity exists in the cell death pathways operating in different organisms and there is a clear evolutionary conservation of apoptotic regulators such as caspases, Bcl-2 family members, inhibitor of apoptosis (IAP) proteins, IAP antagonists and caspase activators. Despite this, studies in Caenorhabditis elegans, Drosophila and vertebrates have revealed some apparent differences both in the way apoptosis is regulated and in the way individual molecules contribute to the propagation of the death signal. For example, whereas cytochrome c released from mitochondria clearly promotes caspase activation in vertebrates, there is no documented role for cytochrome c in C. elegans apoptosis and its role in Drosophila is highly controversial. In addition, the apoptotic potency of IAP antagonists appears to be greater in Drosophila than in vertebrates, indicating that IAPs may be of different relative importance in different organisms. Thus, although Drosophila, worms and humans share a host of apoptotic regulators, the way in which they function may not be identical.

Authors
Kornbluth, S; White, K
MLA Citation
Kornbluth, S, and White, K. "Apoptosis in Drosophila: Neither fish nor fowl (nor man, nor worm)." Journal of Cell Science 118.9 (2005): 1779-1787.
Website
http://hdl.handle.net/10161/8399
PMID
15860727
Source
scival
Published In
Journal of Cell Science
Volume
118
Issue
9
Publish Date
2005
Start Page
1779
End Page
1787
DOI
10.1242/jcs.02377

DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organsims.

Authors
Yamada, A; Duffy, B; Perry, JA; Kornbluth, S
MLA Citation
Yamada, A, Duffy, B, Perry, JA, and Kornbluth, S. "DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7." Journal of Cell Biology 167.5 (2004): 841-849.
PMID
15583029
Source
scival
Published In
Journal of Cell Biology
Volume
167
Issue
5
Publish Date
2004
Start Page
841
End Page
849
DOI
10.1083/jcb.200406048

Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release

Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.

Authors
Deming, PB; Schafer, ZT; Tashker, JS; Potts, MB; Deshmukh, M; Kornbluth, S
MLA Citation
Deming, PB, Schafer, ZT, Tashker, JS, Potts, MB, Deshmukh, M, and Kornbluth, S. "Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release." Molecular and Cellular Biology 24.23 (2004): 10289-10299.
PMID
15542838
Source
scival
Published In
Molecular and Cellular Biology
Volume
24
Issue
23
Publish Date
2004
Start Page
10289
End Page
10299
DOI
10.1128/MCB.24.23.10289-10299.2004

A dimerized coiled-coil domain and an adjoining part of geminin interact with two sites on Cdt1 for replication inhibition

Geminin is a cellular protein that associates with Cdt1 and inhibits Mcm2-7 loading during S phase. It prevents multiple cycles of replication per cell cycle and prevents episome replication. It also directly inhibits the HoxA11 transcription factor. Here we report that geminin forms a parallel coiled-coil homodimer with atypical residues in the dimer interface. Point mutations that disrupt the dimerization abolish interaction with Cdt1 and inhibition of replication. An array of glutamic acid residues on the coiled-coil domain surface interacts with positive charges in the middle of Cdt1. An adjoining region interacts independently with the N-terminal 100 residues of Cdt1. Both interactions are essential for replication inhibition. The negative residues on the coiled-coil domain and a different part of geminin are also required for interaction with HoxA11. Therefore a rigid cylinder with negative surface charges is a critical component of a bipartite interaction interface between geminin and its cellular targets.

Authors
Saxena, S; Yuan, P; Dhar, SK; Senga, T; Takeda, D; Robinson, H; Kornbluth, S; Swaminathan, K; Dutta, A
MLA Citation
Saxena, S, Yuan, P, Dhar, SK, Senga, T, Takeda, D, Robinson, H, Kornbluth, S, Swaminathan, K, and Dutta, A. "A dimerized coiled-coil domain and an adjoining part of geminin interact with two sites on Cdt1 for replication inhibition." Molecular Cell 15.2 (2004): 245-258.
PMID
15260975
Source
scival
Published In
Molecular Cell
Volume
15
Issue
2
Publish Date
2004
Start Page
245
End Page
258
DOI
10.1016/j.molcel.2004.06.045

When the checkpoints have gone: Insights into Cdc25 functional activation

DNA-responsive checkpoints operate at the G2/M transition to prevent premature mitosis in the presence of incompletely replicated or damaged DNA. These pathways prevent mitotic entry, at least in part, by suppressing Cdc25, the phosphatase that activates Cdc2/Cyclin B. To gain insight into how checkpoint signaling controls Cdc25 function, we have carefully examined the individual steps in Cdc25 activation. We found that removal of the regulatory protein, 14-3-3, that binds to phosphorylated Cdc25 during interphase is one of the early steps in mitotic activation. Moreover, our studies unexpectedly implicated the phosphatase PP1 and the G1/S kinase Cdk2 in the process of Cdc25 activation. Here we integrate our findings and those of others to propose a model for Cdc25 activation in an effort to provide insight into novel loci of DNA-responsive checkpoint control of mitotic entry.

Authors
Margolis, SS; Kornbluth, S
MLA Citation
Margolis, SS, and Kornbluth, S. "When the checkpoints have gone: Insights into Cdc25 functional activation." Cell Cycle 3.4 (2004): 425-428.
PMID
15020839
Source
scival
Published In
Cell Cycle
Volume
3
Issue
4
Publish Date
2004
Start Page
425
End Page
428

PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation.

It has been known for over a decade that inhibition of protein phosphatase 1 (PP1) activity prevents entry into M phase, but the relevant substrate has not been identified. We report here that PP1 is required for dephosphorylation of the Cdc2-directed phosphatase Cdc25 at Ser287 (of Xenopus Cdc25; Ser216 of human Cdc25C), a site that suppresses Cdc25 during interphase. Moreover, PP1 recognizes Cdc25 directly by interacting with a PP1-binding motif in the Cdc25 N-terminus. We have also found that 14-3-3 binding to phospho-Ser287 protects Cdc25 from premature dephosphorylation. Upon entry into M phase, 14-3-3 removal from Cdc25 precedes Ser287 dephosphorylation, suggesting the existence of a phosphatase- independent pathway for 14-3-3 removal from Cdc25. We show here that this dissociation of 14-3-3 from Cdc25 requires the activity of the cyclin-dependent kinase Cdk2, providing a molecular explanation for the previously reported requirement for Cdk2 in promoting mitotic entry. Collectively, our data clarify several steps important for Cdc25 activation and provide new insight into the role of PP1 in Cdc2 activation and mitotic entry.

Authors
Margolis, SS; Walsh, S; Weiser, DC; Yoshida, M; Shenolikar, S; Kornbluth, S
MLA Citation
Margolis, SS, Walsh, S, Weiser, DC, Yoshida, M, Shenolikar, S, and Kornbluth, S. "PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation." EMBO J 22.21 (November 3, 2003): 5734-5745.
PMID
14592972
Source
pubmed
Published In
EMBO Journal
Volume
22
Issue
21
Publish Date
2003
Start Page
5734
End Page
5745
DOI
10.1093/emboj/cdg545

Inhibition of translation and induction of apoptosis by Bunyaviral nonstructural proteins bearing sequence similarity to reaper.

Members of the California serogroup of bunyaviruses (family Bunyaviridae) are the leading cause of pediatric viral encephalitis in North America. Significant cell death is observed as part of the infection pathology. We now report that a Bunyaviral nonstructural protein termed NSs shows sequence similarity to Reaper, a proapoptotic protein from Drosophila. Although NSs proteins lack the Reaper N-terminal motif critical for IAP inhibition, they do retain other functions of Reaper that map to conserved C-terminal regions. Like Reaper, NSs proteins induce mitochondrial cytochrome c release and caspase activation in cell-free extracts and promote neuronal apoptosis and mortality in a mouse model. Independent of caspase activation, Bunyavirus NSs proteins also share with Reaper the ability to directly inhibit cellular protein translation. We have found that the shared capacity to inhibit translation and induce apoptosis resides in common sequence motifs present in both Reaper and NSs proteins. Data presented here suggest that NSs induce apoptosis through a mechanism similar to that used by Reaper, as both proteins bind to an apoptotic regulator called Scythe and can relieve Scythe inhibition of Hsp70. Thus, bunyavirus NSs proteins have multiple Reaper-like functions that likely contribute to viral pathogenesis by promoting cell death and/or inhibiting cellular translation.

Authors
Colón-Ramos, DA; Irusta, PM; Gan, EC; Olson, MR; Song, J; Morimoto, RI; Elliott, RM; Lombard, M; Hollingsworth, R; Hardwick, JM; Smith, GK; Kornbluth, S
MLA Citation
Colón-Ramos, DA, Irusta, PM, Gan, EC, Olson, MR, Song, J, Morimoto, RI, Elliott, RM, Lombard, M, Hollingsworth, R, Hardwick, JM, Smith, GK, and Kornbluth, S. "Inhibition of translation and induction of apoptosis by Bunyaviral nonstructural proteins bearing sequence similarity to reaper." Mol Biol Cell 14.10 (October 2003): 4162-4172.
PMID
14517326
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
14
Issue
10
Publish Date
2003
Start Page
4162
End Page
4172
DOI
10.1091/mbc.E03-03-0139

Cell division, growth and death: Cell growth: Live and let die

Authors
Kornbluth, S; Pines, J
MLA Citation
Kornbluth, S, and Pines, J. "Cell division, growth and death: Cell growth: Live and let die." Current Opinion in Cell Biology 15.6 (2003): 645-647.
PMID
14644187
Source
scival
Published In
Current Opinion in Cell Biology
Volume
15
Issue
6
Publish Date
2003
Start Page
645
End Page
647
DOI
10.1016/j.ceb.2003.10.017

A GH3-like Domain in Reaper Is Required for Mitochondrial Localization and Induction of IAP Degradation

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. We report here that this domain, although required for IAP destabilization, is not sufficient. Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain was deficient in these properties, these defects could be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells.

Authors
Olson, MR; Holley, CL; Gan, EC; Colón-Ramos, DA; Kaplan, B; Kornbluth, S
MLA Citation
Olson, MR, Holley, CL, Gan, EC, Colón-Ramos, DA, Kaplan, B, and Kornbluth, S. "A GH3-like Domain in Reaper Is Required for Mitochondrial Localization and Induction of IAP Degradation." Journal of Biological Chemistry 278.45 (2003): 44758-44768.
PMID
12917412
Source
scival
Published In
Journal of Biological Chemistry
Volume
278
Issue
45
Publish Date
2003
Start Page
44758
End Page
44768
DOI
10.1074/jbc.M308055200

Reaper is regulated by IAP-mediated ubiquitination

In most cases, apoptotic cell death culminates in the activation of the caspase family of cysteine proteases, leading to the orderly dismantling and elimination of the cell. The IAPs (inhibitors of apoptosis) comprise a family of proteins that oppose caspases and thus act to raise the apoptotic threshold. Disruption of IAP-mediated caspase inhibition has been shown to be an important activity for pro-apoptotic proteins in Drosophila (Reaper, HID, and Grim) and in mammalian cells (Smael DIABLO and Omi/HtrA2). In addition, in the case of the fly, these proteins are able to stimulate the ubiquitination and degradation of IAPs by a mechanism involving the ubiquitin ligase activity of the IAP itself. In this report, we show that the Drosophila RHG proteins (Reaper, HID, and Grim) are themselves substrates for IAP-mediated ubiquitination. This ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, we also show that regulation of Reaper by ubiquitination is a significant factor in determining its biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance.

Authors
Olson, MR; Holley, CL; Yoo, SJ; Huh, JR; Hay, BA; Kornbluth, S
MLA Citation
Olson, MR, Holley, CL, Yoo, SJ, Huh, JR, Hay, BA, and Kornbluth, S. "Reaper is regulated by IAP-mediated ubiquitination." Journal of Biological Chemistry 278.6 (2003): 4028-4034.
PMID
12446669
Source
scival
Published In
Journal of Biological Chemistry
Volume
278
Issue
6
Publish Date
2003
Start Page
4028
End Page
4034
DOI
10.1074/jbc.M209734200

Phosphorylation of the cyclin B1 cytoplasmic retention sequence by mitogen-activated protein kinase and Plx

The cyclin B1/Cdc2 complex regulates many of the dramatic cellular rearrangements observed at mitosis. Although predominantly cytoplasmic during interphase, this kinase complex translocates precipitously to the nucleus at the G2-M transition. The interphase cytoplasmic location of cyclin B1/Cdc2 reflects continuous, albeit slow, nuclear import and much more rapid nuclear export. In contrast, the sudden nuclear accumulation of the complex before entry into mitosis reflects a marked increase in the import rate, with a concomitant inhibition of cyclin B1 nuclear export. These dynamic changes in cyclin B1/Cdc2 localization are regulated by phosphorylation of four serines within a region of cyclin B1 known as the cytoplasmic retention sequence (CRS). Phosphorylation of all four serines is required for rapid nuclear entry, whereas phosphorylation of only the last in the series (Ser 113) is required to prevent nuclear export by CRM1. As these residues represent key loci of regulation, it is important to identify the kinases acting on these sites. Here we report that Xenopus cyclin B1 is regulated by both Erk and Plx kinases, and that Cdc2, counter to previous speculation, is not required for CRS phosphorylation. Phosphorylation of the first two of the CRS serines (Ser 94 and Ser 96) is catalyzed by Erk in the Xenopus system. Although it was previously reported that Ser 113 is a Plx substrate, we were unable to observe phosphorylation of this residue in isolation by purified Plx. Rather, in contrast to previously published data, we have found that the penultimate CRS serine (Ser 101) is a Plx substrate. Collectively, these data demonstrate a new role for Erk in mitotic regulation, identify the Ser 101-directed kinase, and provide a picture of cyclin B1/Cdc2 regulation by the combinatorial action of distinct kinases.

Authors
Walsh, S; Margolis, SS; Kornbluth, S
MLA Citation
Walsh, S, Margolis, SS, and Kornbluth, S. "Phosphorylation of the cyclin B1 cytoplasmic retention sequence by mitogen-activated protein kinase and Plx." Molecular Cancer Research 1.4 (2003): 280-289.
PMID
12612056
Source
scival
Published In
Molecular Cancer Research
Volume
1
Issue
4
Publish Date
2003
Start Page
280
End Page
289

Xnf7 and the regulation of Xenopus cyclin B2

Authors
Casaletto, JB; Moore, JD; Etkin, LD; Kornbluth, SA
MLA Citation
Casaletto, JB, Moore, JD, Etkin, LD, and Kornbluth, SA. "Xnf7 and the regulation of Xenopus cyclin B2." November 2002.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
13
Publish Date
2002
Start Page
300A
End Page
300A

Post-cytochrome C protection from apoptosis conferred by a MAPK pathway in Xenopus egg extracts.

In response to many different apoptotic stimuli, cytochrome c is released from the intermembrane space of the mitochondria into the cytoplasm, where it serves as a cofactor in the activation of procaspase 9. Inhibition of this process can occur either by preventing cytochrome c release or by blocking caspase activation or activity. Experiments involving in vitro reconstitution of apoptosis in cell-free extracts of Xenopus laevis eggs have suggested that extracts arrested in interphase are susceptible to an endogenous apoptotic program leading to caspase activation, whereas extracts arrested in meiotic metaphase are not. We report here that Mos/MEK/MAPK pathways active in M phase-arrested eggs are responsible for rendering them refractory to apoptosis. Interestingly, M phase-arrested extracts are competent to release cytochrome c, yet still do not activate caspases. Concomitantly, we have also demonstrated that recombinant Mos, MEK, and ERK are sufficient to block cytochrome c-dependent caspase activation in purified Xenopus cytosol, which lacks both transcription and translation. These data indicate that the MAP kinase pathway can target and inhibit post-cytochrome c release apoptotic events in the absence of new mRNA/protein synthesis and that this biochemical pathway is responsible for the apoptotic inhibition observed in meiotic X. laevis egg extracts.

Authors
Tashker, JS; Olson, M; Kornbluth, S
MLA Citation
Tashker, JS, Olson, M, and Kornbluth, S. "Post-cytochrome C protection from apoptosis conferred by a MAPK pathway in Xenopus egg extracts." Mol Biol Cell 13.2 (February 2002): 393-401.
PMID
11854398
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
13
Issue
2
Publish Date
2002
Start Page
393
End Page
401
DOI
10.1091/mbc.01-06-0291

Identification of the nuclear localization signal in Xenopus cyclin E and analysis of its role in replication and mitosis

Cyclin-dependent kinase (Cdk)2/cyclin E is imported into nuclei assembled in Xenopus egg extracts by a pathway that requires importin-α and -β. Here, we identify a basic nuclear localization sequence (NLS) in the N-terminus of Xenopus cyclin E. Mutation of the NLS eliminated nuclear accumulation of both cyclin E and Cdk2, and such versions of cyclin E were unable to trigger DNA replication. Addition of a heterologous NLS from SV40 large T antigen restored both nuclear targeting of Cdk2/cyclin E and DNA replication. We present evidence indicating that Cdk2/cyclin E complexes must become highly concentrated within nuclei to support replication and find that cyclin A can trigger replication at much lower intranuclear concentrations. We confirmed that depletion of endogenous cyclin E increases the concentration of cyclin B necessary to promote entry into mitosis. In contrast to its inability to promote DNA replication, cyclin E lacking its NLS was able to cooperate with cyclin B in promoting mitotic entry.

Authors
Moore, JD; Kornbluth, S; Hunt, T
MLA Citation
Moore, JD, Kornbluth, S, and Hunt, T. "Identification of the nuclear localization signal in Xenopus cyclin E and analysis of its role in replication and mitosis." Molecular Biology of the Cell 13.12 (2002): 4388-4400.
PMID
12475960
Source
scival
Published In
Molecular Biology of the Cell
Volume
13
Issue
12
Publish Date
2002
Start Page
4388
End Page
4400
DOI
10.1091/mbc.E02-07-0449

Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition

Inhibitors of apoptosis (IAPs) inhibit caspases, thereby preventing proteolysis of apoptotic substrates. IAPs occlude the active sites of caspases to which they are bound-3 and can function as ubiquitin ligases. IAPs are also reported to ubiquitinate themselves and caspases. Several proteins induce apoptosis, at least in part, by binding and inhibiting IAPs. Among these are the Drosophila melanogaster proteins Reaper (Rpr), Grim, and HID, and the mammalian proteins Smac/Diablo and Omi/HtrA2, all of which share a conserved amino-terminal IAP-binding motif. We report here that Rpr not only inhibits IAP function, but also greatly decreases IAP abundance. This decrease in IAP levels results from a combination of increased IAP degradation and a previously unrecognized ability of Rpr to repress total protein translation. Rpr-stimulated IAP degradation required both IAP ubiquitin ligase activity and an unblocked Rpr N terminus. In contrast, Rpr lacking a free N terminus still inhibited protein translation. As the abundance of short-lived proteins are severely affected after translational inhibition, the coordinated dampening of protein synthesis and the ubiquitin-mediated destruction of IAPs can effectively reduce IAP levels to lower the threshold for apoptosis.

Authors
Holley, CL; Olson, MR; Colón-Ramos, DA; Kornbluth, S
MLA Citation
Holley, CL, Olson, MR, Colón-Ramos, DA, and Kornbluth, S. "Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition." Nature Cell Biology 4.6 (2002): 439-444.
PMID
12021770
Source
scival
Published In
Nature Cell Biology
Volume
4
Issue
6
Publish Date
2002
Start Page
439
End Page
444
DOI
10.1038/ncb798

Apoptotic regulation by the Crk adapter protein mediated by interactions with Wee1 and Crm1/exportin

The adapter protein Crk contains an SH2 domain and two SH3 domains. Through binding of particular ligands to the SH2 domain and the N-terminal SH3 domain, Crk has been implicated in a number of signaling processes, including regulation of cell growth, cell motility, and apoptosis. We report here that the C-terminal SH3 domain, never shown to bind any specific signaling molecules, contains a binding site for the nuclear export factor Crm1. We find that a mutant Crk protein, deficient in Crm1 binding, promotes apoptosis. Moreover, this nuclear export sequence mutant [NES(-) Crk] interacts strongly, through its SH2 domain, with the nuclear tyrosine kinase, Wee1. Collectively, these data suggest that a nuclear population of Crk bound to Wee1 promotes apoptotic death of mammalian cells.

Authors
Smith, JJ; Richardson, DA; Kopf, J; Yoshida, M; Hollingsworth, RE; Kornbluth, S
MLA Citation
Smith, JJ, Richardson, DA, Kopf, J, Yoshida, M, Hollingsworth, RE, and Kornbluth, S. "Apoptotic regulation by the Crk adapter protein mediated by interactions with Wee1 and Crm1/exportin." Molecular and Cellular Biology 22.5 (2002): 1412-1423.
PMID
11839808
Source
scival
Published In
Molecular and Cellular Biology
Volume
22
Issue
5
Publish Date
2002
Start Page
1412
End Page
1423
DOI
10.1128/MCB.22.5.1412-1423.2002

Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper.

Protein folding mediated by the Hsp70 family of molecular chaperones requires both ATP and the co-chaperone Hdj-1. BAG-1 was recently identified as a bcl-2-interacting, anti-apoptotic protein that binds to the ATPase domain of Hsp70 and prevents the release of the substrate. While this suggested that cells had the potential to modulate Hsp70-mediated protein folding, physiological regulators of BAG-1 have yet to be identified. We report here that the apoptotic regulator Scythe, originally isolated through binding to the potent apoptotic inducer Reaper, shares limited sequence identity with BAG-1 and inhibits Hsp70- mediated protein refolding. Scythe-mediated inhibition of Hsp70 is reversed by Reaper, providing evidence for the regulated reversible inhibition of chaperone activity. As Scythe functions downstream of Reaper in apoptotic induction, these findings suggest that Scythe/Reaper may signal apoptosis, in part through regulating the folding and activity of apoptotic signaling molecules.

Authors
Thress, K; Song, J; Morimoto, RI; Kornbluth, S
MLA Citation
Thress, K, Song, J, Morimoto, RI, and Kornbluth, S. "Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper." EMBO J 20.5 (March 1, 2001): 1033-1041.
PMID
11230127
Source
pubmed
Published In
EMBO Journal
Volume
20
Issue
5
Publish Date
2001
Start Page
1033
End Page
1041
DOI
10.1093/emboj/20.5.1033

Mitochondria in apoptosis and human disease.

Apoptosis is a process of cell suicide whereby individual cells are destroyed while preserving the integrity and architecture of surrounding tissue. This targeted cell destruction is critical both in physiological contexts as well as pathological states. It seems increasingly evident that mitochondria play an important role in the regulation of programmed cell death via release of proapoptotic agents and/or disruption of cellular energy metabolism. The mechanisms of mitochondrial involvement are beginning to be elucidated, and may involve the participation of bcl-2 family members, reactive oxygen species, and caspases. As part of a central mechanism of amplification of the apoptotic signal, mitochondria may be an appropriate target for therapeutic agents designed to modulate apoptosis. This review focuses on recent advances in understanding mitochondrial mechanisms in apoptosis and the involvement of these pathways in human disease.

Authors
Olson, M; Kornbluth, S
MLA Citation
Olson, M, and Kornbluth, S. "Mitochondria in apoptosis and human disease." Curr Mol Med 1.1 (March 2001): 91-122.
PMID
11899246
Source
pubmed
Published In
Current molecular medicine
Volume
1
Issue
1
Publish Date
2001
Start Page
91
End Page
122

Combinatorial control of cyclin B1 nuclear trafficking through phosphorylation at multiple sites.

Entry into mitosis is regulated by the Cdc2 kinase complexed to B-type cyclins. We and others recently reported that cyclin B1/Cdc2 complexes, which appear to be constitutively cytoplasmic during interphase, actually shuttle continually into and out of the nucleus, with the rate of nuclear export exceeding the import rate (). At the time of entry into mitosis, the import rate is increased, whereas the export rate is decreased, leading to rapid nuclear accumulation of Cdc2/cyclin B1. Although it has recently been reported that phosphorylation of 4 serines within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at G(2)/M, the role that individual phosphorylation sites play in this process has not been examined (, ). We report here that phosphorylation of a single serine residue (Ser(113) of Xenopus cyclin B1) abrogates nuclear export of cyclin B1. This serine lies directly within the cyclin B1 nuclear export sequence and, when phosphorylated, prevents binding of the nuclear export factor, CRM1. In contrast, analysis of phosphorylation site mutants suggests that coordinate phosphorylation of all 4 serines (94, 96, 101, and 113) is required for the accelerated nuclear import of cyclin B1/Cdc2 characteristic of G(2)/M. Additionally, binding of cyclin B1 to importin-beta, the factor known to be responsible for the slow interphase nuclear entry of cyclin B1, appears to be unaffected by the phosphorylation state of cyclin B. These data suggest that a distinct import factor must be recruited to enhance nuclear entry of Cdc2/cyclin B1 at the G(2)/M transition.

Authors
Yang, J; Song, H; Walsh, S; Bardes, ES; Kornbluth, S
MLA Citation
Yang, J, Song, H, Walsh, S, Bardes, ES, and Kornbluth, S. "Combinatorial control of cyclin B1 nuclear trafficking through phosphorylation at multiple sites." J Biol Chem 276.5 (February 2, 2001): 3604-3609.
PMID
11060306
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
5
Publish Date
2001
Start Page
3604
End Page
3609
DOI
10.1074/jbc.M008151200

Analysis of apoptosis using Xenopus egg extracts.

In the presence of a subcellular fraction enriched for mitochondria, after prolonged incubation the Xenopus egg extract can mimic biochemical aspects of apoptosis such as caspase activation, and DNA fragmentation. This unit describes preparation of an apoptotic extract from a crude interphase extract and an extract fractionated into latent and execution phases. An apoptotic extract can also be reconstituted from a fractionated interphase extract and purified mitochondria. Protocols are also included for monitoring apoptotic progression in the extract either by following activation of apoptotic proteases (caspases) or by assessing translocation of cytochrome c from the mitochondria to the cytosol.

Authors
Kornbluth, S; Evans, EK
MLA Citation
Kornbluth, S, and Evans, EK. "Analysis of apoptosis using Xenopus egg extracts." Current protocols in cell biology / editorial board, Juan S. Bonifacino .. [et al.] Chapter 11 (2001): Unit-11.12.
PMID
18228303
Source
scival
Published In
Current Protocols in Cell Biology
Volume
Chapter 11
Publish Date
2001
Start Page
Unit
End Page
11.12
DOI
10.1002/0471143030.cb1112s09

Preparation and use of interphase Xenopus egg extracts.

In this unit, Xenopus eggs are isolated from hormonally primed female frogs, and then the extract is treated with cyclohexamide so it remains in interphase of the cell cycle. In the presence of sperm chromatin and ATP, membrane vesicles in the extract fuse to assemble nuclei, making the extract suitable for studies of DNA replication and nuclear transport.

Authors
Powers, M; Evans, EK; Yang, J; Kornbluth, S
MLA Citation
Powers, M, Evans, EK, Yang, J, and Kornbluth, S. "Preparation and use of interphase Xenopus egg extracts." Current protocols in cell biology / editorial board, Juan S. Bonifacino .. [et al.] Chapter 11 (2001): Unit-11.10.
PMID
18228302
Source
scival
Published In
Current Protocols in Cell Biology
Volume
Chapter 11
Publish Date
2001
Start Page
Unit
End Page
11.10
DOI
10.1002/0471143030.cb1110s09

Wee1-regulated apoptosis mediated by the crk adaptor protein in Xenopus egg extracts.

Many of the biochemical reactions of apoptotic cell death, including mitochondrial cytochrome c release and caspase activation, can be reconstituted in cell-free extracts derived from Xenopus eggs. In addition, because caspase activation does not occur until the egg extract has been incubated for several hours on the bench, upstream signaling processes occurring before full apoptosis are rendered accessible to biochemical manipulation. We reported previously that the adaptor protein Crk is required for apoptotic signaling in egg extracts (Evans, E.K., W. Lu, S.L. Strum, B.J. Mayer, and S. Kornbluth. 1997. EMBO (Eur. Mol. Biol. Organ.) J. 16:230-241). Moreover, we demonstrated that removal of Crk Src homology (SH)2 or SH3 interactors from the extracts prevented apoptosis. We now report the finding that the relevant Crk SH2-interacting protein, important for apoptotic signaling in the extract, is the well-known cell cycle regulator, Wee1. We have demonstrated a specific interaction between tyrosine-phosphorylated Wee1 and the Crk SH2 domain and have shown that recombinant Wee1 can restore apoptosis to an extract depleted of SH2 interactors. Moreover, exogenous Wee1 accelerated apoptosis in egg extracts, and this acceleration was largely dependent on the presence of endogenous Crk protein. As other Cdk inhibitors, such as roscovitine and Myt1, did not act like Wee1 to accelerate apoptosis, we propose that Wee1-Crk complexes signal in a novel apoptotic pathway, which may be unrelated to Wee1's role as a cell cycle regulator.

Authors
Smith, JJ; Evans, EK; Murakami, M; Moyer, MB; Moseley, MA; Vande Woude, G; Kornbluth, S
MLA Citation
Smith, JJ, Evans, EK, Murakami, M, Moyer, MB, Moseley, MA, Vande Woude, G, and Kornbluth, S. "Wee1-regulated apoptosis mediated by the crk adaptor protein in Xenopus egg extracts." J Cell Biol 151.7 (December 25, 2000): 1391-1400.
Website
http://hdl.handle.net/10161/8378
PMID
11134069
Source
pubmed
Published In
The Journal of Cell Biology
Volume
151
Issue
7
Publish Date
2000
Start Page
1391
End Page
1400

Requirement of the prolyl isomerase Pin1 for the replication checkpoint.

The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication.

Authors
Winkler, KE; Swenson, KI; Kornbluth, S; Means, AR
MLA Citation
Winkler, KE, Swenson, KI, Kornbluth, S, and Means, AR. "Requirement of the prolyl isomerase Pin1 for the replication checkpoint." Science 287.5458 (March 3, 2000): 1644-1647.
PMID
10698738
Source
pubmed
Published In
Science
Volume
287
Issue
5458
Publish Date
2000
Start Page
1644
End Page
1647

A family of ubiquitin-like proteins binds the ATPase domain of Hsp70-like Stch

We have isolated two human ubiquitin-like (UbL) proteins that bind to a short peptide within the ATPase domain of the Hsp70-like Stch protein. Chap1 is a duplicated homologue of the yeast Dsk2 gene that is required for transit through the G2/M phase of the cell cycle and expression of the human full-length cDNA restored viability and suppressed the G2/M arrest phenotype of dsk2Δ rad23Δ Saccharomyces cerevisiae mutants. Chap2 is a homologue for Xenopus scythe which is an essential component of reaper-induced apoptosis in egg extracts. While the N-terminal UbL domains were not essential for Stch binding, Chap1/Dsk2 contains a Sti1-like repeat sequence that is required for binding to Stch and is also conserved in the Hsp70 binding proteins, Hip and p60/Sti1/Hop. These findings extend the association between Hsp70 members and genes encoding UbL sequences and suggest a broader role for the Hsp70-like ATPase family in regulating cell cycle and cell death events. Copyright (C) 2000 Federation of European Biochemical Societies.

Authors
Kaye, FJ; Modi, S; Ivanovska, I; Koonin, EV; Thress, K; Kubo, A; Kornbluth, S; Rose, MD
MLA Citation
Kaye, FJ, Modi, S, Ivanovska, I, Koonin, EV, Thress, K, Kubo, A, Kornbluth, S, and Rose, MD. "A family of ubiquitin-like proteins binds the ATPase domain of Hsp70-like Stch." FEBS Letters 467.2-3 (2000): 348-355.
PMID
10675567
Source
scival
Published In
FEBS Letters
Volume
467
Issue
2-3
Publish Date
2000
Start Page
348
End Page
355
DOI
10.1016/S0014-5793(00)01135-2

All aboard the cyclin train: subcellular trafficking of cyclins and their CDK partners.

Progression through the cell cycle is governed by the periodic activation and inactivation of cyclin-dependent kinase complexes (CDK-cyclins). Although the enzymatic activity of these complexes is regulated tightly, it has recently been demonstrated that an additional facet of cell-cycle control involves the modulation of CDK-cyclin subcellular localization. Recent discoveries include the identification of nuclear transport factors responsible for ferrying some of the CDK-cyclins in and out of the nucleus, the demonstration that phosphorylation can regulate these transport processes and the establishment of potential links between cell-cycle checkpoints and the control of CDK-cyclin subcellular localization.

Authors
Yang, J; Kornbluth, S
MLA Citation
Yang, J, and Kornbluth, S. "All aboard the cyclin train: subcellular trafficking of cyclins and their CDK partners." Trends Cell Biol 9.6 (June 1999): 207-210. (Review)
PMID
10354564
Source
pubmed
Published In
Trends in Cell Biology
Volume
9
Issue
6
Publish Date
1999
Start Page
207
End Page
210

Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import.

Cdc2-cyclin B1 in the G2-arrested Xenopus oocyte is held inactive by phosphorylation of Cdc2 at two negative regulatory sites, Thr14 and Tyr15. Upon treatment with progesterone, these sites are dephosphorylated by the dual specificity phosphatase, Cdc25, leading to Cdc2-cyclin B1 activation. Whereas maintenance of the G2 arrest depends upon preventing Cdc25-induced Cdc2 dephosphorylation, the mechanisms responsible for keeping Cdc25 in check in these cells have not yet been described. Here we report that Cdc25 in the G2-arrested oocyte is bound to 14-3-3 proteins and that progesterone treatment abrogates this binding. We demonstrate that Cdc25, apparently statically localized in the cytoplasm, is actually capable of shuttling in and out of the oocyte nucleus. Binding of 14-3-3 protein markedly reduces the nuclear import rate of Cdc25, allowing nuclear export mediated by a nuclear export sequence present in the N-terminus of Cdc25 to predominate. If 14-3-3 binding to Cdc25 is prevented while nuclear export is inhibited, the coordinate nuclear accumulation of Cdc25 and Cdc2-cyclin B1 facilitates their mutual activation, thereby promoting oocyte maturation.

Authors
Yang, J; Winkler, K; Yoshida, M; Kornbluth, S
MLA Citation
Yang, J, Winkler, K, Yoshida, M, and Kornbluth, S. "Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import." EMBO J 18.8 (April 15, 1999): 2174-2183.
PMID
10205171
Source
pubmed
Published In
EMBO Journal
Volume
18
Issue
8
Publish Date
1999
Start Page
2174
End Page
2183
DOI
10.1093/emboj/18.8.2174

Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

Authors
Moore, JD; Yang, J; Truant, R; Kornbluth, S
MLA Citation
Moore, JD, Yang, J, Truant, R, and Kornbluth, S. "Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1." J Cell Biol 144.2 (January 25, 1999): 213-224.
Website
http://hdl.handle.net/10161/8377
PMID
9922449
Source
pubmed
Published In
The Journal of Cell Biology
Volume
144
Issue
2
Publish Date
1999
Start Page
213
End Page
224

Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. We recently reported that Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. We now show that Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper which is detectably homologous to Grim and Hid is dispensable for Scythe binding.

Authors
Thress, K; Evans, EK; Kornbluth, S
MLA Citation
Thress, K, Evans, EK, and Kornbluth, S. "Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity." EMBO Journal 18.20 (1999): 5486-5493.
PMID
10523293
Source
scival
Published In
EMBO Journal
Volume
18
Issue
20
Publish Date
1999
Start Page
5486
End Page
5493
DOI
10.1093/emboj/18.20.5486

Mitochondria at the crossroad of apoptotic cell death

In the past few years, it has become widely appreciated that apoptotic cell death generally involves activation of a family of proteases, the caspases, which undermine the integrity of the cell by cleavage of critical intracellular substrates. Caspases, which arc synthesized as inactive zymogens, are themselves caspase substrates and this cleavage leads to their activation. Hence, the potential exists for cascades of caspases leading to cell death. However, it has been recently recognized that another, perhaps more prominent route to caspase activation, involves the mitochondria. Upon receipt of apoptotic stimuli, either externally or internally generated, cells initiate signaling pathways which converge upon the mitochondria to promote release of cytochrome C to the cytoplasm; cytochrome c, thus released, acts as a potent cofactor in caspase activation. Even cell surface 'death receptors' such as Fas, which can trigger direct caspase activation (and potentially a caspase cascade), appear to utilize mitochondria as part of an amplification mechanism; it has been recently demonstrated that activated caspases can cleave key substrates to trigger mitochondrial release of cytochrome c, thereby inducing further caspase activation and amplifying the apoptotic signal. Therefore, mitochondria play a central role in apoptotic cell death, serving as a repository for cytochrome c.

Authors
Thress, K; Kornbluth, S; Smith, JJ
MLA Citation
Thress, K, Kornbluth, S, and Smith, JJ. "Mitochondria at the crossroad of apoptotic cell death." Journal of Bioenergetics and Biomembranes 31.4 (1999): 321-326.
PMID
10665522
Source
scival
Published In
Journal of Bioenergetics and Biomembranes
Volume
31
Issue
4
Publish Date
1999
Start Page
321
End Page
326
DOI
10.1023/A:1005471701441

The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1.

The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man. Here we demonstrate that depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B. We show biochemically that this phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1. Although Pin1 could interact with Plx1 during interphase and mitosis, only the phosphorylated, mitotically active form of Cdc25 was able to bind Pin1, an event we have recapitulated using in vitro phosphorylated Cdc25. Taken together, these data suggest that Pin1 may modulate cell cycle control through interaction with Cdc25 and its activator, Plx1.

Authors
Crenshaw, DG; Yang, J; Means, AR; Kornbluth, S
MLA Citation
Crenshaw, DG, Yang, J, Means, AR, and Kornbluth, S. "The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1." EMBO J 17.5 (August 10, 1998): 1315-1327.
PMID
9482729
Source
pubmed
Published In
EMBO Journal
Volume
17
Issue
5
Publish Date
1998
Start Page
1315
End Page
1327
DOI
10.1093/emboj/17.5.1315

Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.

Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.

Authors
Yang, J; Bardes, ES; Moore, JD; Brennan, J; Powers, MA; Kornbluth, S
MLA Citation
Yang, J, Bardes, ES, Moore, JD, Brennan, J, Powers, MA, and Kornbluth, S. "Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1." Genes Dev 12.14 (July 15, 1998): 2131-2143.
Website
http://hdl.handle.net/10161/8386
PMID
9679058
Source
pubmed
Published In
Genes & development
Volume
12
Issue
14
Publish Date
1998
Start Page
2131
End Page
2143

Scythe: A novel reaper-binding apoptotic regulator

Reaper is a central regulator of apoptosis in Drosophila melanogaster. With no obvious catalytic activity or homology to other known apoptotic regulators, reaper's mechanism of action has been obscure. We recently reported that recombinant Drosophila reaper protein induced rapid mitochondrial cytochrome c release, caspase activation and apoptotic nuclear fragmentation in extracts of Xenopus eggs. We now report the purification of a 150 kDa reaper-interacting protein from Xenopus egg extracts, which we have named Scythe. Scythe is highly conserved among vertebrates and contains a ubiquitin-like domain near its N-terminus. Immunodepletion of Scythe from extracts completely prevented reaper-induced apoptosis without affecting apoptosis triggered by activated caspases. Moreover, a truncated variant of Scythe lacking the N-terminal domain induced apoptosis even in the absence of reaper. These data suggest that Scythe is a novel apoptotic regulator that is an essential component in the pathway of reaper-induced apoptosis.

Authors
Thress, K; Henzel, W; Shillinglaw, W; Kornbluth, S
MLA Citation
Thress, K, Henzel, W, Shillinglaw, W, and Kornbluth, S. "Scythe: A novel reaper-binding apoptotic regulator." EMBO Journal 17.21 (1998): 6135-6143.
PMID
9799223
Source
scival
Published In
EMBO Journal
Volume
17
Issue
21
Publish Date
1998
Start Page
6135
End Page
6143
DOI
10.1093/emboj/17.21.6135

Regulation of apoptosis in Xenopus egg extracts

Although it is clear that caspase activation is an integral part of the apoptotic program, it is likely that caspases participate in the dismantling of the cell late in the apoptotic process. The upstream signaling events leading to caspase activation are only beginning to be elucidated. Although our data and that of others suggest that phosphotyrosine-mediated events participate in transmission of the apoptotic signal, it is not yet clear how these events are linked to caspase activation. Moreover, it remains to be determined whether tyrosine kinase pathways impinge upon apoptotic signaling in all tissues or are specific for certain cell types. The identification of crk as a key mediator of apoptotic signal transmission in the Xenopus egg extracts provides an excellent starting point to identify and characterize proteins which function in the induction of apoptosis. In particular, proteins interacting with the crk SH2 and crk SH3 domains are likely to be involved either upstream or downstream of crk in apoptotic signaling. Furthermore, the ability of bcr/abl to inhibit apoptosis in the egg extract should greatly facilitate the identification of abl substrates important for apoptotic inhibition.

Authors
Evans, EK; Kornbluth, S
MLA Citation
Evans, EK, and Kornbluth, S. "Regulation of apoptosis in Xenopus egg extracts." Advances in Enzyme Regulation 38.1 (1998): 265-280.
PMID
9762358
Source
scival
Published In
Advances in Enzyme Regulation
Volume
38
Issue
1
Publish Date
1998
Start Page
265
End Page
280
DOI
10.1016/S0065-2571(97)00020-4

Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7

A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

Authors
Sterner, JM; Dew-Knight, S; Musahl, C; Kornbluth, S; Horowitz, JM
MLA Citation
Sterner, JM, Dew-Knight, S, Musahl, C, Kornbluth, S, and Horowitz, JM. "Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7." Molecular and Cellular Biology 18.5 (1998): 2748-2757.
PMID
9566894
Source
scival
Published In
Molecular and Cellular Biology
Volume
18
Issue
5
Publish Date
1998
Start Page
2748
End Page
2757

Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c

Apoptosis often involves the release of cytochrome c from mitochondria, leading to caspase activation. However, in apoptosis mediated by CD95 (Fas/APO-1), caspase-8 (FLICE/MACH/Mch5) is immediately activated and, in principle, could process other caspases directly. To investigate whether caspase-8 could also act through mitochondria, we added active caspase-8 to a Xenopus cell-free system requiring these organelles. Caspase-8 rapidly promoted the apoptotic program, culminating in fragmentation of chromatin and the nuclear membrane. In extracts devoid of mitochondria, caspase-8 produced DNA degradation, but left nuclear membranes intact. Thus, mitochondria were required for complete engagement of the apoptotic machinery. In the absence of mitochondria, high concentrations of caspase-8 were required to activate downstream caspases. However, when mitochondria were present, the effects of low concentrations of caspase-8 were vastly amplified through cytochrome c- dependent caspase activation. Caspase-8 promoted cytochrome c release indirectly, by cleaving at least one cytosolic substrate. Bcl-2 blocked apoptosis only at the lowest caspase-8 concentrations, potentially explaining why CD95-induced apoptosis can often evade inhibition by Bcl-2.

Authors
Kuwana, T; Smith, JJ; Muzio, M; Dixit, V; Newmeyer, DD; Kornbluth, S
MLA Citation
Kuwana, T, Smith, JJ, Muzio, M, Dixit, V, Newmeyer, DD, and Kornbluth, S. "Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c." Journal of Biological Chemistry 273.26 (1998): 16589-16594.
PMID
9632731
Source
scival
Published In
The Journal of biological chemistry
Volume
273
Issue
26
Publish Date
1998
Start Page
16589
End Page
16594
DOI
10.1074/jbc.273.26.16589

Regulation of apoptotic signaling pathways in cell-free extracts

ApoptOBis is a program of cellular suicide which rids multicellular otanisms of damaged or extraneous cells. In order to elucidate the cellular signaling pathways which contribute to apoptotic cell death, we are utilizing an extract of Xenopus eggs which can recapitulate the apoptotic process in vitro. Using these extracts, we have found that the SH2/SH3-containlng adaptor protein, crk, is required for in vitro apoptosls. Moreover, proteins interacting with both the SH2 and SH3 domains are critical for transmitting the apoptotic signal. We are currently purifying proteins interacting with these domains. We have also found that the reaper protein of Drosophila Melanogaster, critical for apoptosis in that organism, can induce rapid apoptosis in extracts. Using reaper as a bait in affinity Chromatography experiments, we have purified a reaper-Interacting protein from egg extracts. Characterization of this protein will be discussed.

Authors
Kornbluth, S
MLA Citation
Kornbluth, S. "Regulation of apoptotic signaling pathways in cell-free extracts." FASEB Journal 12.8 (1998): A1330-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
12
Issue
8
Publish Date
1998
Start Page
A1330

Crk is required for apoptosis in Xenopus egg extracts

Apoptosis is essential for the development and homeostasis of multicellular organisms. Recently, a cell-free extract prepared from Xenopus eggs was shown to recapitulate intracellular apoptotic pathways in vitro. While many stimuli have been shown to trigger apoptosis in a variety of cell types, the intracellular signaling pathways involved in apoptosis remain largely unknown. Here we show that addition of a recombinant protein containing the phosphotyrosine binding (SH2) domain from the adaptor protein crk, but not those derived from a panel of other signaling proteins, can prevent apoptosis in the Xenopus egg extract system. Furthermore, immunodepletion of endogenous crk protein from the egg extracts, or addition of anti-crk antisera to these extracts, prevents apoptosis. The ability to undergo apoptosis can be restored to these extracts by addition of recombinant crk protein. These results directly demonstrate that crk participates in apoptotic signaling.

Authors
Evans, EK; Lu, W; Strum, SL; Mayer, BJ; Kornbluth, S
MLA Citation
Evans, EK, Lu, W, Strum, SL, Mayer, BJ, and Kornbluth, S. "Crk is required for apoptosis in Xenopus egg extracts." EMBO Journal 16.2 (1997): 230-241.
PMID
9029144
Source
scival
Published In
EMBO Journal
Volume
16
Issue
2
Publish Date
1997
Start Page
230
End Page
241
DOI
10.1093/emboj/16.2.230

Reaper-induced apoptosis in a vertebrate system

The reaper protein of Drosophila melanogaster has been shown to be a central regulator of apoptosis in that organism. However, it has not been shown to function in any vertebrate nor have the cellular components required for its action been defined. In this report we show that reaper can induce rapid apoptosis in vitro using an apoptotic reconstitution system derived from Xenopus eggs. Moreover, we show that a subcellular fraction enriched in mitochondria is required for this process and that reaper, acting in conjunction with cytosolic factors, can trigger mitochondrial cytochrome c release. Bcl-2 antagonizes these effects, but high levels of reaper can overcome the Bcl-2 block. These results demonstrate that reaper can function in a vertebrate context, suggesting that reaper-responsive factors are conserved elements of the apoptotic program.

Authors
Evans, EK; Kuwana, T; Strum, SL; Smith, JJ; Newmeyer, DD; Kornbluth, S
MLA Citation
Evans, EK, Kuwana, T, Strum, SL, Smith, JJ, Newmeyer, DD, and Kornbluth, S. "Reaper-induced apoptosis in a vertebrate system." EMBO Journal 16.24 (1997): 7372-7381.
PMID
9405366
Source
scival
Published In
EMBO Journal
Volume
16
Issue
24
Publish Date
1997
Start Page
7372
End Page
7381
DOI
10.1093/emboj/16.24.7372

Apoptosis in Xenopus egg extracts

Authors
Kornbluth, S
MLA Citation
Kornbluth, S. "Apoptosis in Xenopus egg extracts." Methods in Enzymology 283 (1997): 600-614.
PMID
9251051
Source
scival
Published In
Methods in Enzymology
Volume
283
Publish Date
1997
Start Page
600
End Page
614
DOI
10.1016/S0076-6879(97)83047-9

Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control.

Cyclins and cyclin-dependent kinases (Cdks) are universal regulators of cell cycle progression in eukaryotic cells. Cdk activity is controlled by phosphorylation at three conserved sites, and many of the enzymes that act on these sites have now been identified. Although the biochemistry of Cdk phosphorylation is relatively well understood, the regulatory roles of such phosphorylation are, in many cases, obscure. Recent studies have uncovered new and unexpected potential roles, and prompted re-examination of previously assumed roles, of Cdk phosphorylation.

Authors
Lew, DJ; Kornbluth, S
MLA Citation
Lew, DJ, and Kornbluth, S. "Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control." Curr Opin Cell Biol 8.6 (December 1996): 795-804. (Review)
PMID
8939679
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
8
Issue
6
Publish Date
1996
Start Page
795
End Page
804

Cyclin-binding motifs are essential for the function of p21(CIP1)

The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin- binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin- Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.

Authors
Chen, J; Saha, P; Kornbluth, S; Dynlacht, BD; Dutta, A
MLA Citation
Chen, J, Saha, P, Kornbluth, S, Dynlacht, BD, and Dutta, A. "Cyclin-binding motifs are essential for the function of p21(CIP1)." Molecular and Cellular Biology 16.9 (1996): 4673-4682.
PMID
8756624
Source
scival
Published In
Molecular and Cellular Biology
Volume
16
Issue
9
Publish Date
1996
Start Page
4673
End Page
4682

Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14

The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.

Authors
Kornbluth, S; Sebastian, B; Hunter, T; Newport, J
MLA Citation
Kornbluth, S, Sebastian, B, Hunter, T, and Newport, J. "Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14." Molecular Biology of the Cell 5.3 (1994): 273-282.
PMID
8049520
Source
scival
Published In
Molecular Biology of the Cell
Volume
5
Issue
3
Publish Date
1994
Start Page
273
End Page
282

Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP-bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34(cdc2) on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.

Authors
Kornbluth, S; Dasso, M; Newport, J
MLA Citation
Kornbluth, S, Dasso, M, and Newport, J. "Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression." Journal of Cell Biology 125.4 (1994): 705-719.
PMID
8188741
Source
scival
Published In
The Journal of Cell Biology
Volume
125
Issue
4
Publish Date
1994
Start Page
705
End Page
719
DOI
10.1083/jcb.125.4.705

RCC1, a regulator of mitosis, is essential for DNA replication

Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.

Authors
Dasso, M; Nishitani, H; Kornbluth, S; Nishimoto, T; Newport, JW
MLA Citation
Dasso, M, Nishitani, H, Kornbluth, S, Nishimoto, T, and Newport, JW. "RCC1, a regulator of mitosis, is essential for DNA replication." Molecular and Cellular Biology 12.8 (1992): 3337-3345.
PMID
1630449
Source
scival
Published In
Molecular and Cellular Biology
Volume
12
Issue
8
Publish Date
1992
Start Page
3337
End Page
3345

DNA replication and progression through the cell cycle.

Somatic cells possess control mechanisms which monitor DNA replication and assure that it is complete before mitosis is initiated. We have been investigating these mechanisms in Xenopus egg extracts. Using in vitro cycling extracts, which spontaneously alternate between interphase and mitosis, we found that the onset of mitosis is inhibited by the presence of unreplicated DNA, demonstrating that the completion of DNA replication and the initiation of mitosis are coupled in these extracts. As in somatic cells, this coupling is sensitive to caffeine and to okadaic acid. In Xenopus extracts unreplicated DNA increases the tyrosine phosphorylation of p34cdc2, thereby maintaining MPF (mitosis-promoting factor) in an inactive state and preventing the onset of mitosis. The block to mitosis in the presence of unreplicated DNA can be reversed by the addition of bacterially expressed cdc25 protein. The extent of MPF activation by cdc25 protein under these conditions depends on the number of nuclei present. We have developed an assay to examine the rate of tyrosine phosphorylation on p34cdc2. It is increased by unreplicated DNA, in a manner consistent with unreplicated DNA up-regulating the kinase that phosphorylates p34cdc2. We have begun to examine how unreplicated DNA generates the signal that inhibits MPF activation by testing the ability of naked single- and double-stranded DNA templates to inhibit mitosis, and by investigating the role of RCC1, a chromatin-associated protein required for the coupling of DNA replication and mitosis.

Authors
Dasso, M; Smythe, C; Milarski, K; Kornbluth, S; Newport, JW
MLA Citation
Dasso, M, Smythe, C, Milarski, K, Kornbluth, S, and Newport, JW. "DNA replication and progression through the cell cycle." Ciba Foundation symposium 170 (1992): 161-180.
PMID
1483344
Source
scival
Published In
Ciba Foundation symposium
Volume
170
Publish Date
1992
Start Page
161
End Page
180

In vitro cell cycle arrest induced by using artificial DNA templates

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylaling p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.

Authors
Kornbluth, S; Smythe, C; Newport, JW
MLA Citation
Kornbluth, S, Smythe, C, and Newport, JW. "In vitro cell cycle arrest induced by using artificial DNA templates." Molecular and Cellular Biology 12.7 (1992): 3216-3223.
PMID
1320197
Source
scival
Published In
Molecular and Cellular Biology
Volume
12
Issue
7
Publish Date
1992
Start Page
3216
End Page
3223

Characterization of the murine BEK fibroblast growth factor (FGF) receptor: Activation by three members, of the FGF family and requirement for heparin

The bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinases with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands.

Authors
Mansukhani, A; Dell'era, P; Moscatelli, D; Kornbluth, S; Hanafusa, H; Basilico, C
MLA Citation
Mansukhani, A, Dell'era, P, Moscatelli, D, Kornbluth, S, Hanafusa, H, and Basilico, C. "Characterization of the murine BEK fibroblast growth factor (FGF) receptor: Activation by three members, of the FGF family and requirement for heparin." Proceedings of the National Academy of Sciences of the United States of America 89.8 (1992): 3305-3309.
Website
http://hdl.handle.net/10161/8400
PMID
1373495
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
89
Issue
8
Publish Date
1992
Start Page
3305
End Page
3309

Association of p62c-yes with polyomavirus middle T-antigen mutants correlates with transforming ability

A number of mutants of polyomavirus middle T antigen (MTag) were constructed into replication-competent avian retroviruses. To assess the ability of these MTag variants to transform and to associate with the avian p60c-src and p62c-yes proto-oncogenc products, we used these viruses to infect chicken embryo ibroblasts. We found that the ability of individual mutant MTags to associate with p62c-src correlated well with the ability of these mutants to transform, as has been previously shown for the association of MTag with p60c-src. All transformation-competent mutant MTags retained the ability to complex with p62c-yes. Two transformation-defective mutants, RX67 and RX68, which could weakly associate with p60c-src, were unable to associate with p62c-yes. dl1015, a transformation-defective mutant which could associate with p60c-src and with a phosphati-dylinositol kinase activity, was also able to associate with p62c-yes. Therefore, some as yet unmeasured biochemical property is defective in this mutant.

Authors
Kornbluth, S; Cheng, SH; Markland, W; Fukui, Y; Hanafusa, H
MLA Citation
Kornbluth, S, Cheng, SH, Markland, W, Fukui, Y, and Hanafusa, H. "Association of p62c-yes with polyomavirus middle T-antigen mutants correlates with transforming ability." Journal of Virology 64.4 (1990): 1584-1589.
PMID
1690822
Source
scival
Published In
Journal of virology
Volume
64
Issue
4
Publish Date
1990
Start Page
1584
End Page
1589

Phosphatidylinositol kinase type I activity associates with various oncogene products

We have assayed immunoprecipitates of several oncogene products from retrovirally infected chicken embryo fibroblasts (CEF) for phosphatidylinositol (PI) kinase activity. Immunoprecipitates of P68(gag-ros), P130(gag-fps), P47(gag-crk), polyoma middle T (mT)-p60(c-src) complex, and mT-p62(c-yes) complex exhibited PI kinase activity when assayed without detergents. This activity was sensitive to the nonionic detergent, Triton X-100, and the product was indistinguishable from phosphatidylinositol-3-phosphate, the product of kinase type I. Immunoprecipitates of p21(Ha-ras) protein did not contain any PI kinase type I activity. It has been suggested that an 81 kD protein phosphorylated in in vitro kinase assays of immunoprecipitates from mT-transformed rodent cells is responsible for the PI kinase type I activity seen in these immunoprecipitates. We have detected a chicken homologue of this 81 kD protein in immunoprecipitates of lysates from mT-transformed CEF. However, the chicken 81 kD protein sedimented more quickly than the PI kinase activity in sucrose gradients. In addition, the 81 kD protein was not detectable in protein kinase assays of immunoprecipitates of P68(gag-ros) or P130(gag-fps). These results suggest that the 81 kD protein may not be the PI kinase.

Authors
Fukui, Y; Kornbluth, S; Jong, S-M; Wang, L-H; Hanafusa, H
MLA Citation
Fukui, Y, Kornbluth, S, Jong, S-M, Wang, L-H, and Hanafusa, H. "Phosphatidylinositol kinase type I activity associates with various oncogene products." Oncogene Research 4.4 (1989): 283-292.
PMID
2549489
Source
scival
Published In
Oncogene Research
Volume
4
Issue
4
Publish Date
1989
Start Page
283
End Page
292

Novel tyrosine kinase identified by phosphotyrosine antibody screening of cDNA libraries

Authors
Kornbluth, S; Paulson, KE; Hanafusa, H
MLA Citation
Kornbluth, S, Paulson, KE, and Hanafusa, H. "Novel tyrosine kinase identified by phosphotyrosine antibody screening of cDNA libraries." Molecular and Cellular Biology 8.12 (1988): 5541-5544.
PMID
2468999
Source
scival
Published In
Molecular and Cellular Biology
Volume
8
Issue
12
Publish Date
1988
Start Page
5541
End Page
5544

Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region

Cellular src protein, p60c-src, is phosphorylated on tyrosine 527 in chicken embryo fibroblasts, and this phosphorylation is implicated in suppressing the protein-tyrosine kinase activity and transforming potential of p60c-src. To determine whether tyrosine 527 phosphorylation is dependent on p60c-src kinase activity, the ATP-binding site of chicken p60c-src was destroyed by substitution of lysine 295 with methionine. The resultant protein, p60c-src(M295), expressed either in chicken cells or in yeast, lacked detectable kinase activity. Nevertheless, tyrosine and serine phosphorylation of p60c-src(M295) overproduced in chicken cells were indistinguishable from that of authentic p60c-src. By contrast, p60c-src(M295) was not phosphorylated on tyrosine in yeast. These results suggest that a protein kinase present in chicken cells but not in yeast phosphorylates tyrosine 527 in trans, and are consistent with the possibility that this kinase is distinct from p60c-src. © 1987.

Authors
Jove, R; Kornbluth, S; Hanafusa, H
MLA Citation
Jove, R, Kornbluth, S, and Hanafusa, H. "Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region." Cell 50.6 (1987): 937-943.
PMID
2441875
Source
scival
Published In
Cell
Volume
50
Issue
6
Publish Date
1987
Start Page
937
End Page
943

Characterization of avian and viral p60src proteins expressed in yeast.

Avian and viral p60src proteins were expressed from a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. Both the viral and cellular src proteins produced in yeast cells were myristoylated at their amino termini, as is the case for src proteins expressed in chicken embryo fibroblasts. The viral src protein produced in yeast autophosphorylated at tyrosine-416 in vivo and had approximately the same level of in vitro kinase activity as p60v-src expressed in Rous sarcoma virus-transformed cells. Unlike p60c-src expressed in chicken cells, which is phosphorylated on tyrosine in vivo almost exclusively at tyrosine-527, p60c-src expressed in yeast was phosphorylated 2.5-3 times more at tyrosine-416 than at tyrosine-527. The specific activity of the p60c-src produced in yeast was 2.5-5.0 times higher than that of p60c-src overexpressed from a retroviral vector in chicken cells, implicating the altered state of in vivo phosphorylation in modulation of the in vitro kinase activity. The expression of p60v-src substantially slowed down the growth of the yeast cells, suggesting that phosphorylation of yeast proteins essential for cell growth may have interfered with their proper functioning.

Authors
Kornbluth, S; Jove, R; Hanafusa, H
MLA Citation
Kornbluth, S, Jove, R, and Hanafusa, H. "Characterization of avian and viral p60src proteins expressed in yeast." Proceedings of the National Academy of Sciences of the United States of America 84.13 (1987): 4455-4459.
PMID
2440023
Source
scival
Published In
Proceedings of the National Academy of Sciences of the United States of America
Volume
84
Issue
13
Publish Date
1987
Start Page
4455
End Page
4459

Association of the polyomavirus middle-T antigen with c-yes protein

Expression of the middle-T antigen of polyomavirus is sufficient to induce transformation of fibroblasts in culture and tumour formation in whole animals. Middle-T can form a complex with the cellular src gene product (p60(c-src)) and can be phosphorylated by p60(c-src) in vitro. Studies using middle-T mutants have suggested that the association of middle-T with p60(c-src) may be necessary but not sufficient for transformation. Therefore, we addressed the possibility that middle-T could interact with other tyrosine protein kinases structurally related to p60(c-src). Using antibody raised against a fusion protein between β-galactosidase and amino-terminal sequences of p90(gag-yes) from Y73 virus (anti-yes antibody), we have found that middle-T can associate with and be phosphorylated by the c-yes proto-oncogene product, a protein of relative molecular mass (M(r)) 62,000 (62K). This raises the possibility that the middle-T-p62(c-yes) complex contributes to transformation by polyomavirus.

Authors
Kornbluth, S; Sudol, M; Hanafusa, H
MLA Citation
Kornbluth, S, Sudol, M, and Hanafusa, H. "Association of the polyomavirus middle-T antigen with c-yes protein." Nature 325.6100 (1987): 171-173.
PMID
3027584
Source
scival
Published In
Nature
Volume
325
Issue
6100
Publish Date
1987
Start Page
171
End Page
173

Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector

Authors
Kornbluth, S; Cross, FR; Harbison, M; Hanafusa, H
MLA Citation
Kornbluth, S, Cross, FR, Harbison, M, and Hanafusa, H. "Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector." Molecular and Cellular Biology 6.5 (1986): 1545-1551.
PMID
3023895
Source
scival
Published In
Molecular and Cellular Biology
Volume
6
Issue
5
Publish Date
1986
Start Page
1545
End Page
1551
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