You are here

Haystead, Timothy Arthur James

Overview:

My laboratory has three major focuses relevant to several human diseases including hypertension, cardiovascular disease, diabetes, autoimmunity and malaria. Our main goal is to define novel points for therapeutic intervention, thus enabling development of new drugs to treat these diseases. To achieve these ends we have adopted a multidisciplinary approach, combining the strengths of cutting edge technologies such as mass spectrometry and chemoproteomics with classical approaches such as physiological studies in isolated tissues, mouse genetics and biochemical methods. In project 1 we utilized a combination of proteomics and muscle physiology to identify ZIPK as a major regulator of Ca2+ independent regulation of smooth muscle contraction. We now believe that ZIPK may be an exquisite drug target for the development of a new generation of antihypertensive drugs. To validate this hypothesis we have generated two knock in flox p ZIPK mouse lines that will enable us to study the consequences of both targeted ZIPK deletion as well as global deletion on cardiovascular physiology as well as during mouse development. In project 2 we utilized a similar approach to define early phosphorylation events in response to activation of PKA and PKG in vivo. Several novel phosphoprotein targets were identified, including SMTNL1. SMTNL1 shows discrete expression that confines the protein to certain smooth muscles and not others, and type IIa fibers in striated muscle. Studies with the SMTNL1 null mouse shows that the protein plays a critical role in adaptive responses to exercise and pregnancy. SMTNL1 null mice appear to be already exercised adapted, particularly with respect to their cardiovascular responses. This finding suggests that cAMP/cGMP mediated signaling pathways contribute in previously unrecognized ways to smooth and striated muscle phenotype. In project 3 we utilized a chemoproteomic approach to define the molecular mechanisms of action of the quinoline antimalarial drugs, such chloroquine (CQ). CQ and the related drugs have been used for over 60 years to malaria and arthritic diseases such as lupus (SLE) and rheumatoid arthritis (RA). The molecular mechanisms underlying both the therapeutic actions of these drugs and their toxicity have yet to be fully defined. Using novel chemoproteomic technology (proteome mining) we identified two cellular targets of CQ and the related drugs, quinone reductase 2 (QR2) and ALDH. We believe that QR2 explains the therapeutic actions of these drugs, where as ALDH inhibition is the cause of their retinopathy side effect. The retinopathy side effect limits the therapeutic window of this class of drugs in the treatment of rheumatic diseases. We are currently developing a series of novel quinoline analogs as well as a QR2 null mouse model to validate both our hypothesis and to rapidly derive a preclinical drug candidate for the treatment of drug resistant malaria, SLE and RA.

Positions:

Professor of Pharmacology and Cancer Biology

Pharmacology & Cancer Biology
School of Medicine

Associate Professor in Pathology

Pathology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1988

Ph.D. — University of Dundee (Scotland)

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

Pharmacological Sciences Training Program

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Participating Faculty Member
Start Date
July 01, 1975
End Date
June 30, 2020

Interdisciplinary Training Program in Lung Disease

Administered By
Medicine, Pulmonary, Allergy, and Critical Care Medicine
AwardedBy
National Institutes of Health
Role
Preceptor
Start Date
July 01, 2009
End Date
March 31, 2020

Pharmacology Industry Internships for Ph.D. Students

Administered By
Pharmacology & Cancer Biology
AwardedBy
American Society for Pharmacology and Experimental Therapeutics
Role
Participating Faculty Member
Start Date
January 01, 2017
End Date
December 31, 2019

Discovery of novel liver host targets for malaria treatment

Administered By
Chemistry
AwardedBy
National Institutes of Health
Role
Co-Sponsor
Start Date
March 01, 2016
End Date
February 28, 2019

Development of Tethered Hsp90 Inhibitors Carrying Radiolabelled Probes to Specifically Descriminate and Kill Malignant

Administered By
Pharmacology & Cancer Biology
AwardedBy
Department of Defense
Role
Principal Investigator
Start Date
May 01, 2015
End Date
April 30, 2018

Targeting the synthetic essential kinases of breast cancers

Administered By
Molecular Genetics and Microbiology
AwardedBy
Department of Defense
Role
Co Investigator
Start Date
April 15, 2015
End Date
April 14, 2018

Oncogenic Signaling Networks

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Co Investigator
Start Date
September 30, 2012
End Date
September 29, 2017

Duke Training Grant in Nephrology

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Preceptor
Start Date
September 20, 1995
End Date
June 30, 2017

Preclinical Development of Novel HSP90 Inhibitors...

Administered By
School of Medicine
AwardedBy
National University of Singapore
Role
Principal Investigator
Start Date
September 01, 2014
End Date
August 31, 2016

The Duke Multidisciplinary Training Program in Pediatric Lung Disease

Administered By
Pediatrics, Pulmonary and Sleep Medicine
AwardedBy
National Institutes of Health
Role
Faculty Member
Start Date
September 01, 2010
End Date
August 31, 2016

Discovery and development of broad spectrum anti-flaviviral drugs

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
June 01, 2010
End Date
May 31, 2016

A Novel Method to Stop HIV Replication: Inhibition of the Human Purinome

Administered By
Pharmacology & Cancer Biology
AwardedBy
Ohio State University
Role
Principal Investigator
Start Date
June 01, 2010
End Date
May 31, 2014

Molecular mechanisms of chemoresistance in breast cancer

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

Calcium desensitization in Smooth Muscle.

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2010
End Date
June 30, 2011

Maturation of Normal & Sensitized Airway Contractility

Administered By
Pediatrics, Pulmonary and Sleep Medicine
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
April 01, 2006
End Date
February 28, 2011

Proteome Mining as a Predictive Tool of Drug Toxicity

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 23, 2005
End Date
January 31, 2010

G Protein Involvement in Oncogenesis and Metastasis

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

Pseudomonas invasion and the role of caveolin-2

Administered By
Medicine, Pulmonary, Allergy, and Critical Care Medicine
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 29, 2006
End Date
June 30, 2009

Regulation of smooth muscle-myosin phosphatase 1 kinase

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 14, 2005
End Date
December 31, 2008

Protein Phosphatase 1 Regulation by Multiple Subunits

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2000
End Date
January 31, 2006

Graduate Training in Pharmalogical Sciences

Administered By
Pharmacology & Cancer Biology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
July 01, 1975
End Date
June 30, 2005

Proteomic/Genetic Approaches to Monoamine Transporters

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Co-Mentor
Start Date
September 01, 2003
End Date
June 30, 2004

Activation of critical gene sets for spinal axon repair

Administered By
Neurobiology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
July 15, 2001
End Date
June 30, 2004
Show More

Publications:

Immunogenic cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity.

Immunogenic cell death (ICD) is a form of cell death that activates an adaptive immune response against dead-cell-associated antigens. Cancer cells killed via ICD can elicit antitumor immunity. ICD is efficiently induced by near-infrared photo-immunotherapy (NIR-PIT) that selectively kills target-cells on which antibody-photoabsorber conjugates bind and are activated by NIR light exposure. Advanced live cell microscopies showed that NIR-PIT caused rapid and irreversible damage to the cell membrane function leading to swelling and bursting, releasing intracellular components due to the influx of water into the cell. The process also induces relocation of ICD bio markers including calreticulin, Hsp70 and Hsp90 to the cell surface and the rapid release of immunogenic signals including ATP and HMGB1 followed by maturation of immature dendritic cells. Thus, NIR-PIT is a therapy that kills tumor cells by ICD, eliciting a host immune response against tumor.

Authors
Ogawa, M; Tomita, Y; Nakamura, Y; Lee, M-J; Lee, S; Tomita, S; Nagaya, T; Sato, K; Yamauchi, T; Iwai, H; Kumar, A; Haystead, T; Shroff, H; Choyke, PL; Trepel, JB; Kobayashi, H
MLA Citation
Ogawa, M, Tomita, Y, Nakamura, Y, Lee, M-J, Lee, S, Tomita, S, Nagaya, T, Sato, K, Yamauchi, T, Iwai, H, Kumar, A, Haystead, T, Shroff, H, Choyke, PL, Trepel, JB, and Kobayashi, H. "Immunogenic cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity." Oncotarget (January 2, 2017).
PMID
28060726
Source
epmc
Published In
Oncotarget
Publish Date
2017
DOI
10.18632/oncotarget.14425

Fasnall, a Selective FASN Inhibitor, Shows Potent Anti-tumor Activity in the MMTV-Neu Model of HER2(+) Breast Cancer.

Many tumors are dependent on de novo fatty acid synthesis to maintain cell growth. Fatty acid synthase (FASN) catalyzes the final synthetic step of this pathway, and its upregulation is correlated with tumor aggressiveness. The consequences and adaptive responses of acute or chronic inhibition of essential enzymes such as FASN are not fully understood. Herein we identify Fasnall, a thiophenopyrimidine selectively targeting FASN through its co-factor binding sites. Global lipidomics studies with Fasnall showed profound changes in cellular lipid profiles, sharply increasing ceramides, diacylglycerols, and unsaturated fatty acids as well as increasing exogenous palmitate uptake that is deviated more into neutral lipid formation rather than phospholipids. We also showed that the increase in ceramide levels contributes to some extent in the mediation of apoptosis. Consistent with this mechanism of action, Fasnall showed potent anti-tumor activity in the MMTV-Neu model of HER2(+) breast cancer, particularly when combined with carboplatin.

Authors
Alwarawrah, Y; Hughes, P; Loiselle, D; Carlson, DA; Darr, DB; Jordan, JL; Xiong, J; Hunter, LM; Dubois, LG; Thompson, JW; Kulkarni, MM; Ratcliff, AN; Kwiek, JJ; Haystead, TAJ
MLA Citation
Alwarawrah, Y, Hughes, P, Loiselle, D, Carlson, DA, Darr, DB, Jordan, JL, Xiong, J, Hunter, LM, Dubois, LG, Thompson, JW, Kulkarni, MM, Ratcliff, AN, Kwiek, JJ, and Haystead, TAJ. "Fasnall, a Selective FASN Inhibitor, Shows Potent Anti-tumor Activity in the MMTV-Neu Model of HER2(+) Breast Cancer." Cell chemical biology 23.6 (June 2016): 678-688.
PMID
27265747
Source
epmc
Published In
Cell chemical biology
Volume
23
Issue
6
Publish Date
2016
Start Page
678
End Page
688
DOI
10.1016/j.chembiol.2016.04.011

An inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating Hsp70 as a host antiviral target.

An estimated three billion people are at risk of Dengue virus (DENV) infection worldwide and there are currently no approved therapeutic interventions for DENV infection. Due to the relatively small size of the DENV genome, DENV is reliant on host factors throughout the viral life cycle. The inducible form of Heat Shock Protein 70 (Hsp70i) has been implicated as a host factor in DENV pathogenesis, however the complete role remains to be elucidated. Here we further illustrate the importance of Hsp70i in dengue virus pathogenesis and describe the antiviral activity of the allosteric small molecule inhibitor that is selective for Hsp70i, called HS-72. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained. HS-72 works to reduce DENV infection by inhibiting the entry stage of the viral life cycle, through disrupting the association of Hsp70i with the DENV receptor complex. This work highlights Hsp70i as an antiviral target and HS-72 as a potential anti-DENV therapeutic agent.

Authors
Howe, MK; Speer, BL; Hughes, PF; Loiselle, DR; Vasudevan, S; Haystead, TAJ
MLA Citation
Howe, MK, Speer, BL, Hughes, PF, Loiselle, DR, Vasudevan, S, and Haystead, TAJ. "An inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating Hsp70 as a host antiviral target." Antiviral research 130 (June 2016): 81-92.
PMID
27058774
Source
epmc
Published In
Antiviral Research
Volume
130
Publish Date
2016
Start Page
81
End Page
92
DOI
10.1016/j.antiviral.2016.03.017

c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells.

The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific "client" proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo.

Authors
Dunn, DM; Woodford, MR; Truman, AW; Jensen, SM; Schulman, J; Caza, T; Remillard, TC; Loiselle, D; Wolfgeher, D; Blagg, BSJ; Franco, L; Haystead, TA; Daturpalli, S; Mayer, MP; Trepel, JB; Morgan, RML; Prodromou, C; Kron, SJ; Panaretou, B; Stetler-Stevenson, WG; Landas, SK; Neckers, L; Bratslavsky, G; Bourboulia, D; Mollapour, M
MLA Citation
Dunn, DM, Woodford, MR, Truman, AW, Jensen, SM, Schulman, J, Caza, T, Remillard, TC, Loiselle, D, Wolfgeher, D, Blagg, BSJ, Franco, L, Haystead, TA, Daturpalli, S, Mayer, MP, Trepel, JB, Morgan, RML, Prodromou, C, Kron, SJ, Panaretou, B, Stetler-Stevenson, WG, Landas, SK, Neckers, L, Bratslavsky, G, Bourboulia, D, and Mollapour, M. "c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells." Cell reports 12.6 (August 2015): 1006-1018.
PMID
26235616
Source
epmc
Published In
Cell Reports
Volume
12
Issue
6
Publish Date
2015
Start Page
1006
End Page
1018
DOI
10.1016/j.celrep.2015.07.004

Pregnancy and Smoothelin-like Protein 1 (SMTNL1) Deletion Promote the Switching of Skeletal Muscle to a Glycolytic Phenotype in Human and Mice.

Pregnancy promotes physiological adaptations throughout the body, mediated by the female sex hormones progesterone and estrogen. Changes in the metabolic properties of skeletal muscle enable the female body to cope with the physiological challenges of pregnancy and may also be linked to the development of insulin resistance. We conducted global microarray, proteomic, and metabolic analyses to study the role of the progesterone receptor and its transcriptional regulator, smoothelin-like protein 1 (SMTNL1) in the adaptation of skeletal muscle to pregnancy. We demonstrate that pregnancy promotes fiber-type changes from an oxidative to glycolytic isoform in skeletal muscle. This phenomenon is regulated through an interaction between SMTNL1 and progesterone receptor, which alters the expression of contractile and metabolic proteins. smtnl1(-/-) mice are metabolically less efficient and show impaired glucose tolerance. Pregnancy antagonizes these effects by inducing metabolic activity and increasing glucose tolerance. Our results suggest that SMTNL1 has a role in mediating the actions of steroid hormones to promote fiber switching in skeletal muscle during pregnancy. Our findings also bear on the management of gestational diabetes that develops as a complication of pregnancy in ~4% of women.

Authors
Lontay, B; Bodoor, K; Sipos, A; Weitzel, DH; Loiselle, D; Safi, R; Zheng, D; Devente, J; Hickner, RC; McDonnell, DP; Ribar, T; Haystead, TA
MLA Citation
Lontay, B, Bodoor, K, Sipos, A, Weitzel, DH, Loiselle, D, Safi, R, Zheng, D, Devente, J, Hickner, RC, McDonnell, DP, Ribar, T, and Haystead, TA. "Pregnancy and Smoothelin-like Protein 1 (SMTNL1) Deletion Promote the Switching of Skeletal Muscle to a Glycolytic Phenotype in Human and Mice." The Journal of biological chemistry 290.29 (July 2015): 17985-17998.
PMID
26048986
Source
epmc
Published In
The Journal of biological chemistry
Volume
290
Issue
29
Publish Date
2015
Start Page
17985
End Page
17998
DOI
10.1074/jbc.m115.658120

Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70

©2014 Elsevier Ltd. All rights reserved.Summary Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.

Authors
Howe, MK; Bodoor, K; Carlson, DA; Hughes, PF; Alwarawrah, Y; Loiselle, DR; Jaeger, AM; Darr, DB; Jordan, JL; Hunter, LM; Molzberger, ET; Gobillot, TA; Thiele, DJ; Brodsky, JL; Spector, NL; Haystead, TAJ
MLA Citation
Howe, MK, Bodoor, K, Carlson, DA, Hughes, PF, Alwarawrah, Y, Loiselle, DR, Jaeger, AM, Darr, DB, Jordan, JL, Hunter, LM, Molzberger, ET, Gobillot, TA, Thiele, DJ, Brodsky, JL, Spector, NL, and Haystead, TAJ. "Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70." Chemistry and Biology 21.12 (December 18, 2014): 1648-1659.
Source
scopus
Published In
Chemistry & Biology
Volume
21
Issue
12
Publish Date
2014
Start Page
1648
End Page
1659
DOI
10.1016/j.chembiol.2014.10.016

Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70.

Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.

Authors
Howe, MK; Bodoor, K; Carlson, DA; Hughes, PF; Alwarawrah, Y; Loiselle, DR; Jaeger, AM; Darr, DB; Jordan, JL; Hunter, LM; Molzberger, ET; Gobillot, TA; Thiele, DJ; Brodsky, JL; Spector, NL; Haystead, TAJ
MLA Citation
Howe, MK, Bodoor, K, Carlson, DA, Hughes, PF, Alwarawrah, Y, Loiselle, DR, Jaeger, AM, Darr, DB, Jordan, JL, Hunter, LM, Molzberger, ET, Gobillot, TA, Thiele, DJ, Brodsky, JL, Spector, NL, and Haystead, TAJ. "Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70." Chemistry & biology 21.12 (December 11, 2014): 1648-1659.
PMID
25500222
Source
epmc
Published In
Chemistry & Biology
Volume
21
Issue
12
Publish Date
2014
Start Page
1648
End Page
1659
DOI
10.1016/j.chembiol.2014.10.016

Chikungunya virus nsP3 & nsP4 interacts with HSP-90 to promote virus replication: HSP-90 inhibitors reduce CHIKV infection and inflammation in vivo.

The global emergence of Chikungunya virus (CHIKV) infection is alarming and currently there is no licensed vaccine or antiviral treatment available to mitigate this disease. CHIKV infection typically results in high viral load with an outcome of high fever, skin rashes, muscle pain, and sequelae of prolonged arthritis, which occurs in >90% of the infected cases. In this study, using biochemical pull-downs, mass-spectrometry, and microscopic imaging techniques, we have identified novel interactions between CHIKV nsP3 or nsP4 proteins with the host stress-pathway chaperone HSP-90 protein. Indeed, silencing of HSP-90 transcripts using siRNA disrupts CHIKV replication in cultured cells. Furthermore, drugs targeting HSP-90, such as commercially available geldanamycin, as well as other specific HSP-90 inhibitor drugs that had been obtained from a purinome mining approach (HS-10 and SNX-2112) showed dramatic reduction in viral titers and reduced inflammation in a CHIKV mouse model of severe infection and musculopathy. The detailed study of the underlying molecular mechanism of these viral and host protein interactions may provide a platform to develop novel therapeutics against CHIKV infection.

Authors
Rathore, APS; Haystead, T; Das, PK; Merits, A; Ng, M-L; Vasudevan, SG
MLA Citation
Rathore, APS, Haystead, T, Das, PK, Merits, A, Ng, M-L, and Vasudevan, SG. "Chikungunya virus nsP3 & nsP4 interacts with HSP-90 to promote virus replication: HSP-90 inhibitors reduce CHIKV infection and inflammation in vivo." Antiviral research 103 (March 2014): 7-16.
PMID
24388965
Source
epmc
Published In
Antiviral Research
Volume
103
Publish Date
2014
Start Page
7
End Page
16
DOI
10.1016/j.antiviral.2013.12.010

The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules

Authors
Yasuda, K; Zhang, H; Loiselle, D; Haystead, T; Macara, IG; Mili, S
MLA Citation
Yasuda, K, Zhang, H, Loiselle, D, Haystead, T, Macara, IG, and Mili, S. "The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules." The Journal of Cell Biology 203.5 (December 9, 2013): 737-746.
Source
crossref
Published In
The Journal of Cell Biology
Volume
203
Issue
5
Publish Date
2013
Start Page
737
End Page
746
DOI
10.1083/jcb.201306058

Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor.

DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta, rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca(2+)-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.

Authors
Carlson, DA; Franke, AS; Weitzel, DH; Speer, BL; Hughes, PF; Hagerty, L; Fortner, CN; Veal, JM; Barta, TE; Zieba, BJ; Somlyo, AV; Sutherland, C; Deng, JT; Walsh, MP; MacDonald, JA; Haystead, TAJ
MLA Citation
Carlson, DA, Franke, AS, Weitzel, DH, Speer, BL, Hughes, PF, Hagerty, L, Fortner, CN, Veal, JM, Barta, TE, Zieba, BJ, Somlyo, AV, Sutherland, C, Deng, JT, Walsh, MP, MacDonald, JA, and Haystead, TAJ. "Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor." ACS chemical biology 8.12 (December 2013): 2715-2723.
PMID
24070067
Source
epmc
Published In
ACS Chemical Biology
Volume
8
Issue
12
Publish Date
2013
Start Page
2715
End Page
2723
DOI
10.1021/cb400407c

Abstract C86: Tethered Hsp90 inhibitors carrying optical or radioiodinated probes reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells.

Authors
Barrott, JJ; Smith, AP; Osada, T; Ramanujam, N; Zalutsky, MR; Lyerly, K; Haystead, TAJ
MLA Citation
Barrott, JJ, Smith, AP, Osada, T, Ramanujam, N, Zalutsky, MR, Lyerly, K, and Haystead, TAJ. "Abstract C86: Tethered Hsp90 inhibitors carrying optical or radioiodinated probes reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells." November 1, 2013.
Source
crossref
Published In
Molecular cancer therapeutics
Volume
12
Issue
11_Supplement
Publish Date
2013
Start Page
C86
End Page
C86
DOI
10.1158/1535-7163.TARG-13-C86

Optical and radioiodinated tethered Hsp90 inhibitors reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells.

Inhibitors of heat-shock protein 90 (Hsp90) have demonstrated an unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained, but could be influenced by ectopically expressed Hsp90 in tumors. In this work, we synthesized Hsp90 inhibitors that can carry optical or radioiodinated probes via a polyethyleneglycol tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein occurs at the plasma membrane. In mice, we observed exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors that target ectopic Hsp90 can be used to detect breast cancer malignancies through noninvasive imaging.

Authors
Barrott, JJ; Hughes, PF; Osada, T; Yang, X-Y; Hartman, ZC; Loiselle, DR; Spector, NL; Neckers, L; Rajaram, N; Hu, F; Ramanujam, N; Vaidyanathan, G; Zalutsky, MR; Lyerly, HK; Haystead, TA
MLA Citation
Barrott, JJ, Hughes, PF, Osada, T, Yang, X-Y, Hartman, ZC, Loiselle, DR, Spector, NL, Neckers, L, Rajaram, N, Hu, F, Ramanujam, N, Vaidyanathan, G, Zalutsky, MR, Lyerly, HK, and Haystead, TA. "Optical and radioiodinated tethered Hsp90 inhibitors reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells." Chem Biol 20.9 (September 19, 2013): 1187-1197.
PMID
24035283
Source
pubmed
Published In
Chemistry and Biology
Volume
20
Issue
9
Publish Date
2013
Start Page
1187
End Page
1197
DOI
10.1016/j.chembiol.2013.08.004

Hsp90, an unlikely ally in the war on cancer

Authors
Barrott, JJ; Haystead, TAJ
MLA Citation
Barrott, JJ, and Haystead, TAJ. "Hsp90, an unlikely ally in the war on cancer." FEBS JOURNAL 280.6 (March 2013): 1381-1396.
PMID
23356585
Source
wos-lite
Published In
FEBS Journal
Volume
280
Issue
6
Publish Date
2013
Start Page
1381
End Page
1396
DOI
10.1111/febs.12147

Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance.

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

Authors
LaMonte, G; Philip, N; Reardon, J; Lacsina, JR; Majoros, W; Chapman, L; Thornburg, CD; Telen, MJ; Ohler, U; Nicchitta, CV; Haystead, T; Chi, J-T
MLA Citation
LaMonte, G, Philip, N, Reardon, J, Lacsina, JR, Majoros, W, Chapman, L, Thornburg, CD, Telen, MJ, Ohler, U, Nicchitta, CV, Haystead, T, and Chi, J-T. "Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance." Cell Host Microbe 12.2 (August 16, 2012): 187-199.
PMID
22901539
Source
pubmed
Published In
Cell Host and Microbe
Volume
12
Issue
2
Publish Date
2012
Start Page
187
End Page
199
DOI
10.1016/j.chom.2012.06.007

Amplification and high-level expression of heat shock protein 90 marks aggressive phenotypes of human epidermal growth factor receptor 2 negative breast cancer.

INTRODUCTION: Although human epidermal growth factor receptor 2 (HER2) positive or estrogen receptor (ER) positive breast cancers are treated with clinically validated anti-HER2 or anti-estrogen therapies, intrinsic and acquired resistance to these therapies appears in a substantial proportion of breast cancer patients and new therapies are needed. Identification of additional molecular factors, especially those characterized by aggressive behavior and poor prognosis, could prioritize interventional opportunities to improve the diagnosis and treatment of breast cancer. METHODS: We compiled a collection of 4,010 breast tumor gene expression data derived from 23 datasets that have been posted on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. We performed a genome-scale survival analysis using Cox-regression survival analyses, and validated using Kaplan-Meier Estimates survival and Cox Proportional-Hazards Regression survival analyses. We conducted a genome-scale analysis of chromosome alteration using 481 breast cancer samples obtained from The Cancer Genome Atlas (TCGA), from which combined expression and copy number data were available. We assessed the correlation between somatic copy number alterations and gene expression using analysis of variance (ANOVA). RESULTS: Increased expression of each of the heat shock protein (HSP) 90 isoforms, as well as HSP transcriptional factor 1 (HSF1), was correlated with poor prognosis in different subtypes of breast cancer. High-level expression of HSP90AA1 and HSP90AB1, two cytoplasmic HSP90 isoforms, was driven by chromosome coding region amplifications and were independent factors that led to death from breast cancer among patients with triple-negative (TNBC) and HER2-/ER+ subtypes, respectively. Furthermore, amplification of HSF1 was correlated with higher HSP90AA1 and HSP90AB1 mRNA expression among the breast cancer cells without amplifications of these two genes. A collection of HSP90AA1, HSP90AB1 and HSF1 amplifications defined a subpopulation of breast cancer with up-regulated HSP90 gene expression, and up-regulated HSP90 expression independently elevated the risk of recurrence of TNBC and poor prognosis of HER2-/ER+ breast cancer. CONCLUSIONS: Up-regulated HSP90 mRNA expression represents a confluence of genomic vulnerability that renders HER2 negative breast cancers more aggressive, resulting in poor prognosis. Targeting breast cancer with up-regulated HSP90 may potentially improve the effectiveness of clinical intervention in this disease.

Authors
Cheng, Q; Chang, JT; Geradts, J; Neckers, LM; Haystead, T; Spector, NL; Lyerly, HK
MLA Citation
Cheng, Q, Chang, JT, Geradts, J, Neckers, LM, Haystead, T, Spector, NL, and Lyerly, HK. "Amplification and high-level expression of heat shock protein 90 marks aggressive phenotypes of human epidermal growth factor receptor 2 negative breast cancer. (Published online)" Breast Cancer Res 14.2 (April 17, 2012): R62-.
PMID
22510516
Source
pubmed
Published In
Breast Cancer Research
Volume
14
Issue
2
Publish Date
2012
Start Page
R62
DOI
10.1186/bcr3168

A highly selective Hsp90 affinity chromatography resin with a cleavable linker

Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media. © 2012 Elsevier Ltd. All rights reserved.

Authors
Hughes, PF; Barrott, JJ; Carlson, DA; Loiselle, DR; Speer, BL; Bodoor, K; Rund, LA; Haystead, TAJ
MLA Citation
Hughes, PF, Barrott, JJ, Carlson, DA, Loiselle, DR, Speer, BL, Bodoor, K, Rund, LA, and Haystead, TAJ. "A highly selective Hsp90 affinity chromatography resin with a cleavable linker." Bioorganic and Medicinal Chemistry 20.10 (2012): 3298-3305.
PMID
22520629
Source
scival
Published In
Bioorganic & Medicinal Chemistry
Volume
20
Issue
10
Publish Date
2012
Start Page
3298
End Page
3305
DOI
10.1016/j.bmc.2012.03.043

Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP

Protein kinase inhibitors have emerged as indispensable tools for the elucidation of the biological functions of specific signal transduction pathways and as promising candidates for molecular-targeted therapy. However, because many protein kinase inhibitors are ATP-competitive inhibitors targeting the catalytic site of specific protein kinases, the large number of protein kinases that are encoded within eukaryotic genomes and the existence of many other cellular proteins that bind ATP result in the prospect of off-target effects for many of these compounds. Many of the potential off-target effects remain unrecognized because protein kinase inhibitors are often developed and tested primarily on the basis of in vitro assays using purified components. To overcome this limitation, we describe a systematic approach to characterize ATP-competitive protein kinase inhibitors employing ATP-sepharose to capture the purine-binding proteome from cell extracts. Protein kinase inhibitors can be used in competition experiments to prevent binding of specific cellular proteins to ATP-sepharose or to elute bound proteins from ATP-sepharose. Collectively, these strategies can enable validation of interactions between a specific protein kinase and an inhibitor in complex mixtures and can yield the identification of inhibitor targets. © 2012 Springer Science+Business Media, LLC.

Authors
Duncan, JS; Haystead, TAJ; Litchfield, DW
MLA Citation
Duncan, JS, Haystead, TAJ, and Litchfield, DW. "Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP." Methods in Molecular Biology 795 (2012): 119-134.
PMID
21960219
Source
scival
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
795
Publish Date
2012
Start Page
119
End Page
134
DOI
10.1007/978-1-61779-337-0_8

Smoothelin-like 1 protein is a bifunctional regulator of the progesterone receptor during pregnancy.

During pregnancy, uterine smooth muscle (USM) coordinately adapts its contractile phenotype in order to accommodate the developing fetus and then prepare for delivery. Herein we show that SMTNL1 plays a major role in pregnancy to promote adaptive responses in USM and that this process is specifically mediated through interactions of SMTNL1 with the steroid hormone receptor PR-B. In vitro and in vivo SMTNL1 selectively binds PR and not other steroid hormone receptors. The physiological relationship between the two proteins was also established in global gene expression and transcriptional reporter studies in pregnant smtnl1(-/-) mice and by RNA interference in progesterone-sensitive cell lines. We show that the contraction-associated and progestin-sensitive genes (oxytocin receptor, connexin 43, and cyclooxygenase-2) and prolactins are down-regulated in pregnant smtnl1(-/-) mice. We suggest that SMTNL1 is a bifunctional co-regulator of PR-B signaling and thus provides a molecular mechanism whereby PR-B is targeted to alter gene expression patterns within USM cells to coordinately promote alterations in USM function during pregnancy.

Authors
Bodoor, K; Lontay, B; Safi, R; Weitzel, DH; Loiselle, D; Wei, Z; Lengyel, S; McDonnell, DP; Haystead, TA
MLA Citation
Bodoor, K, Lontay, B, Safi, R, Weitzel, DH, Loiselle, D, Wei, Z, Lengyel, S, McDonnell, DP, and Haystead, TA. "Smoothelin-like 1 protein is a bifunctional regulator of the progesterone receptor during pregnancy." J Biol Chem 286.36 (September 9, 2011): 31839-31851.
PMID
21771785
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
36
Publish Date
2011
Start Page
31839
End Page
31851
DOI
10.1074/jbc.M111.270397

Angiotensin II type 1A receptors in vascular smooth muscle cells do not influence aortic remodeling in hypertension.

Vascular injury and remodeling are common pathological sequelae of hypertension. Previous studies have suggested that the renin-angiotensin system acting through the type 1 angiotensin II (AT(1)) receptor promotes vascular pathology in hypertension. To study the role of AT(1) receptors in this process, we generated mice with cell-specific deletion of AT(1) receptors in vascular smooth muscle cells using Cre/Loxp technology. We crossed the SM22α-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Agtr1a gene (Agtr1a (flox)), encoding the major murine AT(1) receptor isoform (AT(1A)). In SM22α-Cre(+)Agtr1a (flox/flox) (SMKO) mice, AT(1A) receptors were efficiently deleted from vascular smooth muscle cells in larger vessels but not from resistance vessels such as preglomerular arterioles. Thus, vasoconstrictor responses to angiotensin II were preserved in SMKO mice. To induce hypertensive vascular remodeling, mice were continuously infused with angiotensin II for 4 weeks. During infusion of angiotensin II, blood pressures increased significantly and to a similar extent in SMKO and control mice. In control mice, there was evidence of vascular oxidative stress indicated by enhanced nitrated tyrosine residues in segments of aorta; this was significantly attenuated in SMKO mice. Despite these differences in oxidative stress, the extent of aortic medial expansion induced by angiotensin II infusion was virtually identical in both groups. Thus, vascular AT(1A) receptors promote oxidative stress in the aortic wall but are not required for remodeling in angiotensin II-dependent hypertension.

Authors
Sparks, MA; Parsons, KK; Stegbauer, J; Gurley, SB; Vivekanandan-Giri, A; Fortner, CN; Snouwaert, J; Raasch, EW; Griffiths, RC; Haystead, TAJ; Le, TH; Pennathur, S; Koller, B; Coffman, TM
MLA Citation
Sparks, MA, Parsons, KK, Stegbauer, J, Gurley, SB, Vivekanandan-Giri, A, Fortner, CN, Snouwaert, J, Raasch, EW, Griffiths, RC, Haystead, TAJ, Le, TH, Pennathur, S, Koller, B, and Coffman, TM. "Angiotensin II type 1A receptors in vascular smooth muscle cells do not influence aortic remodeling in hypertension." Hypertension 57.3 (March 2011): 577-585.
PMID
21242463
Source
pubmed
Published In
Hypertension
Volume
57
Issue
3
Publish Date
2011
Start Page
577
End Page
585
DOI
10.1161/HYPERTENSIONAHA.110.165274

Efficient detection of RNA-protein interactions using tethered RNAs

The diverse localization of transcripts in cells suggests that there are many specific RNA-protein interactions that have yet to be identified. Progress has been limited, however, by the lack of a robust method to detect and isolate the RNA-binding proteins. Here we describe the use of an RNA aptamer, scaffolded to a tRNA, to create an affinity matrix that efficiently pulls down transcript-specific RNA-binding proteins from cell lysates. The addition of the tRNA scaffold to a Streptavidin aptamer (tRSA) increased binding efficiency by ∼10-fold. The tRSA system with an attached G-quartet sequence also could efficiently and specifically capture endogenous Fragile X Mental Retardation Protein (FMRP), which recognizes this RNA sequence. An alternative method, using biotinylated RNA, captured FMRP less efficiently than did our tRSA method. Finally we demonstrate the identification of novel RNA-binding proteins that interact with intron2 or 3'-UTR of the polarity protein Crumbs3 transcript. Proteins captured by these RNA sequences attached to the tRNA scaffold were identified by mass spectrometry. GFP-tagged versions of these proteins also showed specific interaction with either the Crb3 intron2 or 3'-UTR. Our tRSA technique should find wide application in mapping the RNA-protein interactome. © 2010 The Author(s).

Authors
Iioka, H; Loiselle, D; Haystead, TA; MacAra, IG
MLA Citation
Iioka, H, Loiselle, D, Haystead, TA, and MacAra, IG. "Efficient detection of RNA-protein interactions using tethered RNAs." Nucleic Acids Research 39.8 (2011): e53-.
PMID
21300640
Source
scival
Published In
Nucleic Acids Research
Volume
39
Issue
8
Publish Date
2011
Start Page
e53
DOI
10.1093/nar/gkq1316

Phosphorylation-dependent control of ZIPK nuclear import is species specific

ZIPK (zipper-interacting protein kinase) is a Ca2+-independent protein kinase that promotes myosin phosphorylation in both smooth muscle and non-muscle cells. A recent report attempted to clarify a debate over the subcellular localization of ZIPK in non-muscle cells (Shoval et. al. (2007) Plos Genetics. 3: 1884-1883). A species-specific loss of a key phosphorylation site (T299) in murine (mouse and rat) ZIPK seems to direct it to the nucleus, while the presence of the T299 site in human ZIPK correlates with cytoplasmic localization. T299 is immediately adjacent to a putative nuclear localization sequence (NLS) and may mask its function when phosphorylated, therefore explaining the species-specific dichotomy of intracellular localization. However, despite the murine ZIPK (mZIPK) lacking the T299 residue that is critical for controlling human ZIPK (hZIPK) subcellular localization, mutational analysis showed that this NLS control locus is nonfunctional in the murine context. A constitutively active Rho promoted the cytoplasmic retention of a human ZIPK mutant that would otherwise localize to the nucleus. Endogenous hZIPK showed sensitivity to the nuclear export inhibitor leptomycin B, suggesting a continuous shuttling between cytoplasm and nucleus that is dependent upon T299 dephosphorylation. Thus, the C-terminal domain of human and murine ZIPK demonstrated quite divergent nuclear import and export functionality. We conclude that in the case of ZIPK, studies between the species may not be directly comparable to each other given the gross differences in intracellular localization and movement. © 2010 Elsevier Inc.

Authors
Weitzel, DH; Chambers, J; Haystead, TAJ
MLA Citation
Weitzel, DH, Chambers, J, and Haystead, TAJ. "Phosphorylation-dependent control of ZIPK nuclear import is species specific." Cellular Signalling 23.1 (2011): 297-303.
PMID
20854903
Source
scival
Published In
Cellular Signalling
Volume
23
Issue
1
Publish Date
2011
Start Page
297
End Page
303
DOI
10.1016/j.cellsig.2010.09.016

Application of chemoproteomics to drug discovery: Identification of a clinical candidate targeting Hsp90

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies. © 2010 Elsevier Ltd.

Authors
Fadden, P; Huang, KH; Veal, JM; Steed, PM; Barabasz, AF; Foley, B; Hu, M; Partridge, JM; Rice, J; Scott, A; Dubois, LG; Freed, TA; Silinski, MAR; Barta, TE; Hughes, PF; Ommen, A; Ma, W; Smith, ED; Spangenberg, AW; Eaves, J; Hanson, GJ; Hinkley, L; Jenks, M; Lewis, M; Otto, J; Pronk, GJ; Verleysen, K; Haystead, TA; Hall, SE
MLA Citation
Fadden, P, Huang, KH, Veal, JM, Steed, PM, Barabasz, AF, Foley, B, Hu, M, Partridge, JM, Rice, J, Scott, A, Dubois, LG, Freed, TA, Silinski, MAR, Barta, TE, Hughes, PF, Ommen, A, Ma, W, Smith, ED, Spangenberg, AW, Eaves, J, Hanson, GJ, Hinkley, L, Jenks, M, Lewis, M, Otto, J, Pronk, GJ, Verleysen, K, Haystead, TA, and Hall, SE. "Application of chemoproteomics to drug discovery: Identification of a clinical candidate targeting Hsp90." Chemistry and Biology 17.7 (2010): 686-694.
PMID
20659681
Source
scival
Published In
Chemistry & Biology
Volume
17
Issue
7
Publish Date
2010
Start Page
686
End Page
694
DOI
10.1016/j.chembiol.2010.04.015

Smoothelin-like 1 protein regulates myosin phosphatase-targeting subunit 1 expression during sexual development and pregnancy

Pregnancy coordinately alters the contractile properties of both vascular and uterine smooth muscles reducing systemic blood pressure and maintaining uterine relaxation. The precise molecular mechanisms underlying these pregnancy-induced adaptations have yet to be fully defined but are likely to involve changes in the expression of proteins regulating myosin phosphorylation. Here we show that smoothelin like protein 1 (SMTNL1) is a key factor governing sexual development and pregnancy induced adaptations in smooth and striated muscle. A primary target gene of SMTNL1 in these muscles is myosin phosphatase-targeting subunit 1 (MYPT1). Deletion of SMTNL1 increases expression of MYPT1 30-40-fold in neonates and during development expression of both SMTNL1 and MYPT1 increases over 20-fold. Pregnancy also regulates SMTNL1 and MYPT1 expression, and deletion SMTNL1 greatly exaggerates expression of MYPT1 in vascular smooth muscle, producing a profound reduction in force development in response to phenylephrine as well as sensitizing the muscle to acetylcholine. We also show that MYPT1 is expressed in Type2a muscle fibers in mice and humans and its expression is regulated during pregnancy, suggesting unrecognized roles in mediating skeletal muscle plasticity in both species. Our findings define a new conserved pathway in which sexual development and pregnancy mediate smooth and striated muscle adaptations through SMTNL1 and MYPT1. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Lontay, B; Bodoor, K; Weitzel, DH; Loiselle, D; Fortner, C; Lengyel, S; Zheng, D; Devente, J; Hickner, R; Haystead, TAJ
MLA Citation
Lontay, B, Bodoor, K, Weitzel, DH, Loiselle, D, Fortner, C, Lengyel, S, Zheng, D, Devente, J, Hickner, R, and Haystead, TAJ. "Smoothelin-like 1 protein regulates myosin phosphatase-targeting subunit 1 expression during sexual development and pregnancy." Journal of Biological Chemistry 285.38 (2010): 29357-29366.
PMID
20634291
Source
scival
Published In
The Journal of biological chemistry
Volume
285
Issue
38
Publish Date
2010
Start Page
29357
End Page
29366
DOI
10.1074/jbc.M110.143966

Rapid characterization of in vivo phosphorylation sites and the protein kinases and phosphatases that regulate them by affinity capture

This chapter discusses methodologies for analysis of the phosphoproteome, and describes how affinity captures using naturally occurring small molecules can be utilized to define protein kinases and phosphatases regulating phosphorylation events in vivo. In this light, it offers alternatives to strategies driven purely by molecular biology for defining phosphoregulation of signaling pathways in vivo. Protein phosphorylation is a reversible and widespread mechanism for regulating protein function, so the phosphoproteome is of great interest. There are several advanced mass spectrometric techniques available to the investigator that enable detailed characterization of phosphoproteomes. These techniques typically use cultured cells, and include phosphopeptide capture approaches, precursor ion scanning, isotope-coded affinity (ICAT), and stable isotope labeling with amino acids in cell culture (SILAC). However, the majority of these techniques have several limitations that result in missing important regulatory sites of phosphorylation. This study describes a procedure that has the considerable advantage over purely MS-based approaches to phosphopeptide analysis in that it is highly quantitative and comprehensive. Finally, it deals with rapid protein kinase and phosphatase identification by small molecule affinity capture and high throughput mass spectrometry. © 2010 Elsevier Inc. All rights reserved.

Authors
Haystead, TAJ
MLA Citation
Haystead, TAJ. "Rapid characterization of in vivo phosphorylation sites and the protein kinases and phosphatases that regulate them by affinity capture." Handbook of Cell Signaling, 2/e 2 (2010): 1247-1251.
Source
scival
Published In
Handbook of Cell Signaling, 2/e
Volume
2
Publish Date
2010
Start Page
1247
End Page
1251
DOI
10.1016/B978-0-12-374145-5.00154-6

Tyrosine phosphorylation of the human glutathione S-transferase P1 by epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR) gene amplification, mutations, and/or aberrant activation are frequent abnormalities in malignant gliomas and other human cancers and have been associated with an aggressive clinical course and a poor therapeutic outcome. Elevated glutathione S-transferase P1 (GSTP1), a major drug-metabolizing and stress response signaling protein, is also associated with drug resistance and poor clinical outcome in gliomas and other cancers. Here, we provide evidence that GSTP1 is a downstream EGFR target and that EGFR binds to and phosphorylates tyrosine residues in the GSTP1 protein in vitro and in vivo. Mass spectrometry and mutagenesis analyses in a cell-free system and in gliomas cells identified Tyr-7 and Tyr-198 as major EGFR-specific phospho-acceptor residues in the GSTP1 protein. The phosphorylation increased GSTP1 enzymatic activity significantly, and computer-based modeling showed a corresponding increase in electronegativity of the GSTP1 active site. In human glioma and breast cancer cells, epidermal growth factor stimulation rapidly increased GSTP1 tyrosine phosphorylation and decreased cisplatin sensitivity. Lapatinib, a clinically active EGFR inhibitor, significantly reversed the epidermal growth factor-induced cisplatin resistance. These data define phosphorylation and activation of GSTP1 by EGFR as a novel, heretofore unrecognized component of the EGFR signaling network and a novel mechanism of tumor drug resistance, particularly in tumors with elevated GSTP1 and/or activated EGFR.

Authors
Okamura, T; Singh, S; Buolamwini, J; Haystead, T; Friedman, H; Bigner, D; Ali-Osman, F
MLA Citation
Okamura, T, Singh, S, Buolamwini, J, Haystead, T, Friedman, H, Bigner, D, and Ali-Osman, F. "Tyrosine phosphorylation of the human glutathione S-transferase P1 by epidermal growth factor receptor." J Biol Chem 284.25 (June 19, 2009): 16979-16989.
PMID
19254954
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
25
Publish Date
2009
Start Page
16979
End Page
16989
DOI
10.1074/jbc.M808153200

The role of the calponin homology domain of smoothelin-like 1 (SMTNL1) in myosin phosphatase inhibition and smooth muscle contraction

In this study, we provide further insight into the contribution of the smoothelin-like 1 (SMTNL1) calponin homology (CH)-domain on myosin light chain phosphatase (SMPP-1M) activity and smooth muscle contraction. SMTNL1 protein was shown to have inhibitory effects on SMPP-1M activity but not on myosin light chain kinase (MLCK) activity. Treatment of β-escin permeabilized rabbit, ileal smooth muscle with SMTNL1 had no effect on the time required to reach half-maximal force (t1/2) during stimulation with pCa6.3 solution. The addition of recombinant SMTNL1 protein to permeabilized, smooth muscle strips caused a significant decrease in contractile force. While the calponin homology (CH)-domain was essential for maximal SMTNL1-associated relaxation, it alone did not cause significant changes in force. SMTNL1 was poorly dephosphorylated by PP-1C in the presence of the myosin targeting subunit (MYPT1), suggesting that phosphorylated SMTNL1 does not possess "substrate trapping" properties. Moreover, while full-length SMTNL1 could suppress SMPP-1M activity toward LC20 in vitro, truncated SMTNL1 lacking the CH-domain was ineffective. In summary, our findings suggest an important role for the CH-domain in mediating the effects of SMTNL1 on smooth muscle contraction. © Springer Science+Business Media, LLC. 2009.

Authors
Borman, MA; Freed, TA; Haystead, TAJ; MacDonald, JA
MLA Citation
Borman, MA, Freed, TA, Haystead, TAJ, and MacDonald, JA. "The role of the calponin homology domain of smoothelin-like 1 (SMTNL1) in myosin phosphatase inhibition and smooth muscle contraction." Molecular and Cellular Biochemistry 327.1-2 (2009): 93-100.
PMID
19219534
Source
scival
Published In
Molecular and Cellular Biochemistry
Volume
327
Issue
1-2
Publish Date
2009
Start Page
93
End Page
100
DOI
10.1007/s11010-009-0047-z

An unbiased evaluation of CK2 inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets.

Recently protein kinases have emerged as some of the most promising drug targets; and therefore, pharmaceutical strategies have been developed to inhibit kinases in the treatment of a variety of diseases. CK2 is a serine/threonine-protein kinase that has been implicated in a number of cellular processes, including maintenance of cell viability, protection of cells from apoptosis, and tumorigenesis. Elevated CK2 activity has been established in a number of cancers where it was shown to promote tumorigenesis via the regulation of the activity of various oncogenes and tumor suppressor proteins. Consequently the development of CK2 inhibitors has been ongoing in preclinical studies, resulting in the generation of a number of CK2-directed compounds. In the present study, an unbiased evaluation of CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz), and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) was carried out to elucidate the mechanism of action as well as inhibitor specificity of these compounds. Utilizing a chemoproteomics approach in conjunction with inhibitor-resistant mutant studies, CK2alpha and CK2alpha' were identified as bona fide targets of TBB, TBBz, and DMAT in cells. However, inhibitor-specific cellular effects were observed indicating that the structurally related compounds had unique biological properties, suggesting differences in inhibitor specificity. Rescue experiments utilizing inhibitor-resistant CK2 mutants were unable to rescue the apoptosis associated with TBBz and DMAT treatment, suggesting the inhibitors had off-target effects. Exploitation of an unbiased chemoproteomics approach revealed a number of putative off-target inhibitor interactions, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme quinone reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors as well as drug specificity that will assist in the development of more specific next generation CK2 inhibitors.

Authors
Duncan, JS; Gyenis, L; Lenehan, J; Bretner, M; Graves, LM; Haystead, TA; Litchfield, DW
MLA Citation
Duncan, JS, Gyenis, L, Lenehan, J, Bretner, M, Graves, LM, Haystead, TA, and Litchfield, DW. "An unbiased evaluation of CK2 inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets." Mol Cell Proteomics 7.6 (June 2008): 1077-1088.
PMID
18258654
Source
pubmed
Published In
Molecular & cellular proteomics : MCP
Volume
7
Issue
6
Publish Date
2008
Start Page
1077
End Page
1088
DOI
10.1074/mcp.M700559-MCP200

Human cytidine triphosphate synthetase 1 interacting proteins.

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.

Authors
Higgins, MJ; Loiselle, D; Haystead, TA; Graves, LM
MLA Citation
Higgins, MJ, Loiselle, D, Haystead, TA, and Graves, LM. "Human cytidine triphosphate synthetase 1 interacting proteins." Nucleosides Nucleotides Nucleic Acids 27.6 (June 2008): 850-857.
PMID
18600551
Source
pubmed
Published In
Nucleosides, Nucleotides & Nucleic Acids
Volume
27
Issue
6
Publish Date
2008
Start Page
850
End Page
857
DOI
10.1080/15257770802146502

Deletion of the protein kinase A/protein kinase G target SMTNL1 promotes an exercise-adapted phenotype in vascular smooth muscle.

In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1(-/-) mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to alpha-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1(-/-) mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1(+/+) mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.

Authors
Wooldridge, AA; Fortner, CN; Lontay, B; Akimoto, T; Neppl, RL; Facemire, C; Datto, MB; Kwon, A; McCook, E; Li, P; Wang, S; Thresher, RJ; Miller, SE; Perriard, J-C; Gavin, TP; Hickner, RC; Coffman, TM; Somlyo, AV; Yan, Z; Haystead, TAJ
MLA Citation
Wooldridge, AA, Fortner, CN, Lontay, B, Akimoto, T, Neppl, RL, Facemire, C, Datto, MB, Kwon, A, McCook, E, Li, P, Wang, S, Thresher, RJ, Miller, SE, Perriard, J-C, Gavin, TP, Hickner, RC, Coffman, TM, Somlyo, AV, Yan, Z, and Haystead, TAJ. "Deletion of the protein kinase A/protein kinase G target SMTNL1 promotes an exercise-adapted phenotype in vascular smooth muscle." J Biol Chem 283.17 (April 25, 2008): 11850-11859.
PMID
18310078
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
17
Publish Date
2008
Start Page
11850
End Page
11859
DOI
10.1074/jbc.M708628200

Murine CD7 shares antigenic cross-reactivity with HSP-60.

Human (h) CD7 is a 40 kDa single chain Ig superfamily molecule that is expressed on thymocytes, a major subunit of peripheral T cells, and most natural killer cells. Ligands for hCD7 include the epithelial cell-produced molecule, K-12, and galectin. Mice deficient in CD7 have been shown to be resistant to LPS-induced endotoxic shock syndromes. However, monoclonal antibodies (MAb) to mouse (m) CD7 have yet to be produced, nor is the distribution of mCD7 protein in mice known. We have raised a panel of three rat MAbs to mCD7 by immunizing rats with recombinant mCD7 protein. However, using Western blot and immunoprecipitation of tissue extracts from mouse thymus, spleen, liver, brain, lymph node and skin, these anti-mouse CD7 MAbs bound only to murine heat shock protein 60 (HSP-60) present both in wild-type (CD7+/+) and CD7-deficient (CD7-/-) mice. Epitope mapping of the sites on HSP-60 and recombinant mCD7 recognized by mCD7 MAbs demonstrated non-homologous amino acid sequence epitopes recognized by anti-CD7 MAbs on both proteins. These data demonstrated molecular mimicry of mCD7 with HSP-60, and leave open the question of surface expression of mCD7.

Authors
Howard, BA; Sempowski, GD; Scearce, RM; Liao, H-X; Lee, DM; Lam, GK; Chen, H; Fadden, P; Haystead, T; Haynes, BF
MLA Citation
Howard, BA, Sempowski, GD, Scearce, RM, Liao, H-X, Lee, DM, Lam, GK, Chen, H, Fadden, P, Haystead, T, and Haynes, BF. "Murine CD7 shares antigenic cross-reactivity with HSP-60." Hybridoma (Larchmt) 27.2 (April 2008): 81-89.
PMID
18642672
Source
pubmed
Published In
Hybridoma
Volume
27
Issue
2
Publish Date
2008
Start Page
81
End Page
89
DOI
10.1089/hyb.2007.0553

Protein kinases of malaria parasites: an update

Protein kinases (PKs) play crucial roles in the control of proliferation and differentiation in eukaryotic cells. Research on protein phosphorylation has expanded tremendously in the past few years, in part as a consequence of the realization that PKs represent attractive drug targets in a variety of diseases. Activity in Plasmodium PK research has followed this trend, and several reports on various aspects of this subject were delivered at the Molecular Approaches to Malaria 2008 meeting (MAM2008), a sharp increase from the previous meeting. Here, the authors of most of these communications join to propose an integrated update of the development of the rapidly expanding field of Plasmodium kinomics. © 2008 Elsevier Ltd. All rights reserved.

Authors
Doerig, C; Billker, O; Haystead, T; Sharma, P; Tobin, AB; Waters, NC
MLA Citation
Doerig, C, Billker, O, Haystead, T, Sharma, P, Tobin, AB, and Waters, NC. "Protein kinases of malaria parasites: an update." Trends in Parasitology 24.12 (2008): 570-577.
PMID
18845480
Source
scival
Published In
Trends in Parasitology
Volume
24
Issue
12
Publish Date
2008
Start Page
570
End Page
577
DOI
10.1016/j.pt.2008.08.007

Proteomic identification of the cerebral cavernous malformation signaling complex.

Cerebral cavernous malformations (CCM) are sporadic or inherited vascular lesions of the central nervous system characterized by dilated, thin-walled, leaky vessels. Linkage studies have mapped autosomal dominant mutations to three loci: ccm1 (KRIT1), ccm2 (OSM), and ccm3 (PDCD10). All three proteins appear to be scaffolds or adaptor proteins, as no enzymatic function can be attributed to them. Our previous results demonstrated that OSM is a scaffold for the assembly of the GTPase Rac and the MAPK kinase kinase MEKK3, for the hyperosmotic stress-dependent activation of p38 MAPK. Herein, we show that the three CCM proteins are members of a larger signaling complex. To define this complex, epitope-tagged wild type OSM or OSM harboring the mutation of F217-->A, which renders the OSM phosphotyrosine binding (PTB) domain unable to bind KRIT1, were stably introduced into RAW264.7 mouse macrophages. FLAG-OSM or FLAG-OSMF217A and the associated complex members were purified by immunoprecipitation using anti-FLAG antibody. OSM binding partners were identified by gel-based methods combined with electrospray ionization-MS or by multidimensional protein identification technology (MudPIT). Previously identified proteins that associate with OSM including KRIT1, MEKK3, Rac, and the KRIT1-binding protein ICAP-1 were found in the immunoprecipitates. In addition, we show for the first time that PDCD10 binds to OSM and is found in cellular CCM complexes. Other prominent proteins that bound the CCM complex include EF1A1, RIN2, and tubulin, with each interaction disrupted with the OSMF217A mutant protein. We further show that PDCD10 binds phosphatidylinositol di- and triphosphates and OSM binds phosphatidylinositol monophosphates. The findings define the targeting of the CCM complex to membranes and to proteins regulating trafficking and the cytoskeleton.

Authors
Hilder, TL; Malone, MH; Bencharit, S; Colicelli, J; Haystead, TA; Johnson, GL; Wu, CC
MLA Citation
Hilder, TL, Malone, MH, Bencharit, S, Colicelli, J, Haystead, TA, Johnson, GL, and Wu, CC. "Proteomic identification of the cerebral cavernous malformation signaling complex." J Proteome Res 6.11 (November 2007): 4343-4355.
PMID
17900104
Source
pubmed
Published In
Journal of Proteome Research
Volume
6
Issue
11
Publish Date
2007
Start Page
4343
End Page
4355
DOI
10.1021/pr0704276

Characterization of a UBC13 kinase in Plasmodium falciparum.

Protein kinases are generally recognized as attractive drug targets to treat a variety of human diseases. Recent analysis of the Plasmodium falciparum kinome identified several kinases that are entirely unique to Plasmodium species. The specific functions and targets of most of these enzymes remain largely unknown. Here, we have identified a P. falciparum kinase (PfPK9/PF13_0085 ORF) that does not cluster with any of the typical eukaryotic protein kinases. PfPK9 protein expression was induced approximately 18 h after red blood cell infection, and was mainly localized to the parasitophorous vacuolar membrane as well as the cytosol. Recombinant PfPK9 autophosphorylated in vitro and specifically phosphorylated the exogenous substrate histone H1, indicating that it is catalytically active. Phosphopeptide mapping studies showed that autophosphorylation occurred at three residues: T082, T265, and T269. We identified a P. falciparum homolog of the E2 ubiquitin-conjugating enzyme 13 (UBC13) as an endogenous substrate for PfPK9. PfPK9 phosphorylated UBC13 at S106, a highly conserved residue among eukaryotic E2s, and suppressed its ubiquitin-conjugating activity. Our findings not only describe a previously uncharacterized Plasmodium kinase and its likely in vivo target, but also suggest that modulation of UBC13 activity by phosphorylation may be a common regulatory mechanism in eukaryotes.

Authors
Philip, N; Haystead, TA
MLA Citation
Philip, N, and Haystead, TA. "Characterization of a UBC13 kinase in Plasmodium falciparum." Proc Natl Acad Sci U S A 104.19 (May 8, 2007): 7845-7850.
PMID
17452636
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
19
Publish Date
2007
Start Page
7845
End Page
7850
DOI
10.1073/pnas.0611601104

A conserved family of enzymes that phosphorylate inositol hexakisphosphate.

Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.

Authors
Mulugu, S; Bai, W; Fridy, PC; Bastidas, RJ; Otto, JC; Dollins, DE; Haystead, TA; Ribeiro, AA; York, JD
MLA Citation
Mulugu, S, Bai, W, Fridy, PC, Bastidas, RJ, Otto, JC, Dollins, DE, Haystead, TA, Ribeiro, AA, and York, JD. "A conserved family of enzymes that phosphorylate inositol hexakisphosphate." Science 316.5821 (April 6, 2007): 106-109.
PMID
17412958
Source
pubmed
Published In
Science
Volume
316
Issue
5821
Publish Date
2007
Start Page
106
End Page
109
DOI
10.1126/science.1139099

Dual kinase-mediated regulation of PITK by CaMKII and GSK3.

Phosphatase Interactor Targeting K protein (PITK) was previously identified as a novel PP1 targeting subunit implicated in modulating the phosphorylation of the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) [Kwiek NC, Thacker DF, Datto MB, Megosh HB, Haystead TA. Cell Signal 18 (10) (2006) 1769.]. Through the phosphorylation of PITK at S1013 and S1017 (residues that flank or reside within a PP1C-binding motif), the binding of the PP1 catalytic subunit to PITK, and subsequently the activity of the holoenzyme, are discretely controlled. Herein, we demonstrate that PITK phosphorylation at S1013 and S1017 also dictates the subcellular localization of the holoenzyme. Whereas both wildtype-and an S1013,1017D-PITK mutant displayed a speckled nuclear localization, a constitutively dephosphorylated form of PITK (S1013,1017A-PITK) resulted in a diffuse localization throughout the cell including the cytoplasm. Additionally, through the use of unbiased proteomics techniques, we provide evidence for a dual kinase-mediated regulation of the PITK holoenzyme whereby PITK phosphorylation at S1017 is catalyzed by calcium/calmodulin-dependent kinase II-delta (CaMKIIdelta), promoting the subsequent phosphorylation of S1013 by glycogen synthase kinase-3 (GSK3) in vitro. Taken together, our findings provide further insight into the regulation of PITK, PP1, and hnRNP K by reversible phosphorylation.

Authors
Kwiek, NC; Thacker, DF; Haystead, TAJ
MLA Citation
Kwiek, NC, Thacker, DF, and Haystead, TAJ. "Dual kinase-mediated regulation of PITK by CaMKII and GSK3." Cell Signal 19.3 (March 2007): 593-599.
PMID
17023142
Source
pubmed
Published In
Cellular Signalling
Volume
19
Issue
3
Publish Date
2007
Start Page
593
End Page
599
DOI
10.1016/j.cellsig.2006.08.009

ROCK1 phosphorylates and activates zipper-interacting protein kinase.

Zipper-interacting protein kinase (ZIPK) regulates Ca(2+)-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.

Authors
Hagerty, L; Weitzel, DH; Chambers, J; Fortner, CN; Brush, MH; Loiselle, D; Hosoya, H; Haystead, TAJ
MLA Citation
Hagerty, L, Weitzel, DH, Chambers, J, Fortner, CN, Brush, MH, Loiselle, D, Hosoya, H, and Haystead, TAJ. "ROCK1 phosphorylates and activates zipper-interacting protein kinase." J Biol Chem 282.7 (February 16, 2007): 4884-4893.
PMID
17158456
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
7
Publish Date
2007
Start Page
4884
End Page
4893
DOI
10.1074/jbc.M609990200

Clostridium taeniosporum spore ribbon-like appendage structure, composition and genes.

Clostridium taeniosporum spores have about 12 large, flat, ribbon-like appendages attached through a common trunk at one spore pole to a previously unknown surface layer outside the coat that is proposed to be called the 'encasement'. Appendages are about 4.5 microm long, 0.5 microm wide and 30 nm thick and taper at the attachment end into a semicircle that is twisted relative to the flat ribbon. Individual fibrils, about 45 nm in length with spherical heads and long thin tails, form a hair-like nap, visible along the appendage edge. Four appendage proteins have been detected: a paralogous pair of 29 kDa (designated P29a and P29b), a glycoprotein of about 37 kDa (designated GP85) and an orthologue of the Bacillus spore morphogenetic protein SpoVM. The P29 proteins consist of duplicated regions and each region includes a domain of unknown function 11. The GP85 glycoprotein contains a collagen-like region. The genes for P29a and b, GP85 and possibly related proteins are closely linked on two small chromosome fragments. Putative sigma(K)-dependent promoters upstream of the P29a and b genes indicate that they likely are expressed late in the mother cell, consistent with their deposition into the layer external to the coat.

Authors
Walker, JR; Gnanam, AJ; Blinkova, AL; Hermandson, MJ; Karymov, MA; Lyubchenko, YL; Graves, PR; Haystead, TA; Linse, KD
MLA Citation
Walker, JR, Gnanam, AJ, Blinkova, AL, Hermandson, MJ, Karymov, MA, Lyubchenko, YL, Graves, PR, Haystead, TA, and Linse, KD. "Clostridium taeniosporum spore ribbon-like appendage structure, composition and genes." Mol Microbiol 63.3 (February 2007): 629-643.
PMID
17302797
Source
pubmed
Published In
Molecular Microbiology
Volume
63
Issue
3
Publish Date
2007
Start Page
629
End Page
643
DOI
10.1111/j.1365-2958.2006.05494.x

Staurosporine inhibition of zipper-interacting protein kinase contractile effects in gastrointestinal smooth muscle.

Zipper-interacting protein kinase (ZIPK) is a serine-threonine kinase that has been implicated in Ca2+-independent myosin II phosphorylation and contractile force generation in vascular smooth muscle. However, relatively little is known about the contribution of this kinase to gastrointestinal smooth muscle contraction. The addition of a recombinant version of ZIPK that lacked the leucine zipper domain to permeabilized ileal strips evoked a Ca2+-independent contraction and resulted in myosin regulatory light chain diphosphorylation at Ser19 and Thr18. Neither Ca2+-independent force development nor myosin regulatory light chain phosphorylation was elicited by the addition of kinase-dead ZIPK to the ileal strips. The sensitivity of ZIPK-induced contraction to various kinase inhibitors was similar to the in vitro sensitivity of purified ZIPK to these inhibitors. Staurosporine was the most effective ZIPK inhibitor, with a Ki value calculated to be 2.6 +/- 0.3 micromol/L. Through the use of specific kinase inhibitors, we determined that Rho-associated protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C) do not mitigate ZIPK-induced contraction in ileum. Our findings support a role for ZIPK in Ca2+-independent contractile force generation in gastrointestinal smooth muscle.

Authors
Borman, MA; MacDonald, JA; Haystead, TAJ
MLA Citation
Borman, MA, MacDonald, JA, and Haystead, TAJ. "Staurosporine inhibition of zipper-interacting protein kinase contractile effects in gastrointestinal smooth muscle." Biochem Cell Biol 85.1 (February 2007): 111-120.
PMID
17464351
Source
pubmed
Published In
Biochemistry and Cell Biology
Volume
85
Issue
1
Publish Date
2007
Start Page
111
End Page
120
DOI
10.1139/o06-209

Synthesis and use of the protein phosphatase affinity matrices microcystin-sepharose and microcystin-biotin-sepharose.

Microcystin-based affinity matrices have been utilized to demonstrate the association of signaling proteins with protein phosphatases and for the purification of low-abundance microcystin-sensitive protein phosphatases. Here, we describe the procedure for the synthesis and use of microcystin-Sepharose and microcystin-biotin-Sepharose.

Authors
Moorhead, GBG; Haystead, TAJ; MacKintosh, C
MLA Citation
Moorhead, GBG, Haystead, TAJ, and MacKintosh, C. "Synthesis and use of the protein phosphatase affinity matrices microcystin-sepharose and microcystin-biotin-sepharose." Methods Mol Biol 365 (2007): 39-45.
PMID
17200552
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
365
Publish Date
2007
Start Page
39
End Page
45
DOI
10.1385/1-59745-267-X:39

Real-time in vivo proteomic identification of novel kinase substrates in smooth muscle.

Relaxation of smooth muscle can occur through agonists (such as nitric oxide) that activate guanylyl cyclase and stimulate the production of cGMP, activating its target, cGMP-dependent protein kinase (PKG). This kinase can raise the Ca2+ threshold for contraction, thus causing Ca2+ desensitization, but the mechanism for this event is not completely understood. Ca2+ sensitization/desensitization pathways are essential for maintenance of normal smooth muscle tone, and abnormalities in these pathways have been shown to be key components in the pathogenesis of diseases such as hypertension and asthma in humans. Our laboratory has devised a proteomic method to specifically address the question of what proteins are early phosphorylation targets in calcium desensitization. Using ileum smooth muscle, we metabolically labeled the muscle with (32P)-orthophosphate, permeabilized the muscle, established constant calcium concentrations, and stimulated with 8-bromo-cGMP, which activates PKG. Proteins whose phosphorylation state changed in response to cGMP at constant levels of calcium were separated with two-dimensional gel electrophoresis, identified by autoradiography, and sequenced with nanospray mass spectrometry. Using this technique, we identified a previously uncharacterized PKG phosphoprotein, which we have termed CHASM (Calponin Homology Smooth Muscle protein). Using physiological muscle bath contraction studies, we have validated CHASM as a component of calcium desensitization pathways in smooth muscle.

Authors
Wooldridge, AA; Haystead, TA
MLA Citation
Wooldridge, AA, and Haystead, TA. "Real-time in vivo proteomic identification of novel kinase substrates in smooth muscle." Methods Mol Biol 357 (2007): 235-252.
PMID
17172692
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
357
Publish Date
2007
Start Page
235
End Page
252
DOI
10.1385/1-59745-214-9:235

PITK, a PP1 targeting subunit that modulates the phosphorylation of the transcriptional regulator hnRNP K.

Protein phosphatase-1 (PP1), through interactions with substrate targeting subunits, plays critical roles in the regulation of numerous cellular processes. Herein, we describe a newly identified regulatory subunit (PITK; Phosphatase Interactor Targeting K protein) that specifically targets the catalytic subunit of PP1 to nuclear foci to selectively bind and dephosphorylate the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) at a regulatory S284 site. Additionally, PITK is phosphorylated in vivo at S1013 and S1017, residues that flank or reside within the PP1C-binding motif, and this phosphorylation negatively regulates the binding of the phosphatase to PITK. A mutant variant, S1013,1017A-PITK, when expressed in intact cells, exhibited an increase in native PP1 binding and elicited a more profound dephosphorylation of hnRNPK at S284. A global analysis of transcription by Affymetrix microarray revealed that the expression of PITK resulted in the altered expression of 47 genes, including a marked induction of MEK5 (>14-fold, p<0.007). Additionally, the effects of PITK and S1013,1017A-PITK on transcription could be modulated by the co-expression of hnRNP K. Taken together, our findings provide a putative mechanism by which transcriptional activity of hnRNP K can be discretely controlled through the regulation of PP1 activity.

Authors
Kwiek, NC; Thacker, DF; Datto, MB; Megosh, HB; Haystead, TAJ
MLA Citation
Kwiek, NC, Thacker, DF, Datto, MB, Megosh, HB, and Haystead, TAJ. "PITK, a PP1 targeting subunit that modulates the phosphorylation of the transcriptional regulator hnRNP K." Cell Signal 18.10 (October 2006): 1769-1778.
PMID
16564677
Source
pubmed
Published In
Cellular Signalling
Volume
18
Issue
10
Publish Date
2006
Start Page
1769
End Page
1778
DOI
10.1016/j.cellsig.2006.01.019

Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice.

The carboxypeptidase ACE2 is a homologue of angiotensin-converting enzyme (ACE). To clarify the physiological roles of ACE2, we generated mice with targeted disruption of the Ace2 gene. ACE2-deficient mice were viable, fertile, and lacked any gross structural abnormalities. We found normal cardiac dimensions and function in ACE2-deficient animals with mixed or inbred genetic backgrounds. On the C57BL/6 background, ACE2 deficiency was associated with a modest increase in blood pressure, whereas the absence of ACE2 had no effect on baseline blood pressures in 129/SvEv mice. After acute Ang II infusion, plasma concentrations of Ang II increased almost 3-fold higher in ACE2-deficient mice than in controls. In a model of Ang II-dependent hypertension, blood pressures were substantially higher in the ACE2-deficient mice than in WT. Severe hypertension in ACE2-deficient mice was associated with exaggerated accumulation of Ang II in the kidney, as determined by MALDI-TOF mass spectrometry. Although the absence of functional ACE2 causes enhanced susceptibility to Ang II-induced hypertension, we found no evidence for a role of ACE2 in the regulation of cardiac structure or function. Our data suggest that ACE2 is a functional component of the renin-angiotensin system, metabolizing Ang II and thereby contributing to regulation of blood pressure.

Authors
Gurley, SB; Allred, A; Le, TH; Griffiths, R; Mao, L; Philip, N; Haystead, TA; Donoghue, M; Breitbart, RE; Acton, SL; Rockman, HA; Coffman, TM
MLA Citation
Gurley, SB, Allred, A, Le, TH, Griffiths, R, Mao, L, Philip, N, Haystead, TA, Donoghue, M, Breitbart, RE, Acton, SL, Rockman, HA, and Coffman, TM. "Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice." J Clin Invest 116.8 (August 2006): 2218-2225.
PMID
16878172
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
116
Issue
8
Publish Date
2006
Start Page
2218
End Page
2225
DOI
10.1172/JCI16980

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

Delineating signal transduction pathways in smooth muscle through focused proteomics.

This review will outline examples of the authors' focused proteomics approaches to studying signal transduction pathways in smooth muscle. By focusing the use of traditional proteomics techniques with hypothesis-driven selection methods, this approach efficiently addresses the identification of novel elements in a signal transduction pathway of interest. However, focused proteomics serves only as a starting point in the investigation of novel signaling proteins. While focused proteomics studies can suggest the involvement and general biochemical function of a protein in a signaling pathway, these findings must be further investigated and validated. Through the integrated use of focused proteomics with complementary approaches such as genetics, biochemistry and cell physiology, a complete and detailed mechanism of signal transduction can be determined.

Authors
Hagerty, L; Haystead, TAJ
MLA Citation
Hagerty, L, and Haystead, TAJ. "Delineating signal transduction pathways in smooth muscle through focused proteomics." Expert Rev Proteomics 3.1 (February 2006): 75-85. (Review)
PMID
16445352
Source
pubmed
Published In
Expert review of proteomics
Volume
3
Issue
1
Publish Date
2006
Start Page
75
End Page
85
DOI
10.1586/14789450.3.1.75

The purinome, a complex mix of drug and toxicity targets.

Much attention has focused on the development of protein kinases as drug targets to treat a variety of human diseases including diabetes, cancer, hypertension and arthritis. To date, Gleevec is one example of a drug targeting protein that has successfully treated human cancer. Several other protein kinase inhibitors are in clinical development. However, protein kinases are in fact part of a larger collection of some 2000 distinct proteins expressed by the genome that like the protein kinases also bind purines (the purinome), either to be utilized as substrates or as co-factors in the form of NAD, NADP and co-enzyme A. The solution structures of many representative gene family members within the purinome show these proteins bind purines in a similar orientations to that observed in all protein kinases. Several non-protein kinase purine utilizing proteins are established drug targets such as HMG CoA reductase, dihydrofolate reductase, phosphodiesterase and HSP90. Searches of OMIM identifies many purine utilizing enzymes that are associated with inborn errors in metabolism. Inhibition of any one of which by a drug could lead to an undesirable side effect. The purinome is therefore somewhat of a drug discovery mixed blessing. It is a rich source of therapeutic targets, but also contains a large collection of diverse proteins whose inhibition could result in an adverse outcome. Drug discovery within the purinome should therefore encompass strategies that enable broad assessment of selectivity across the entire purinome at the earliest stages of the discovery process. In this article we review the purinome within the context of drug discovery and discuss approaches for avoiding off target binding during the discovery/lead optimization process with particular emphasis on use of proteome mining technology.

Authors
Haystead, TAJ
MLA Citation
Haystead, TAJ. "The purinome, a complex mix of drug and toxicity targets." Curr Top Med Chem 6.11 (2006): 1117-1127. (Review)
PMID
16842150
Source
pubmed
Published In
Current Topics in Medicinal Chemistry
Volume
6
Issue
11
Publish Date
2006
Start Page
1117
End Page
1127

Affinity Purification with Natural Immobilized Ligands

This chapter discusses affinity chromatography-based techniques to examine protein interaction between other proteins or small ligands. The first procedure involves the use of toxin microcystin LR (MC-LR) conjugated to a biotin or Sepharose matrix to biochemically examine phosphatase-protein interactions. MC linked to biotin has the advantage of using mild conditions to elute bound proteins and also the holoenzyme components remain intact. Once the ATP-binding proteome is captured, various drugs are applied to the column. If the drug can compete with the natural ligand, the protein is eluted. Proteins are resolved by 1DE and identified by mass spectrometry. Proteome mining involves homogenizing 10 g of tissue in 40 ml of lyses buffer until mixture turns frothy. One needs to combine the supernatant with the ATP-Sepharose and rotate for 30 min at room temperature. MC-biotin has to be prepared fresh and is not reusable as it is lost during elution. It is important to know solubility conditions of the drugs used to elute proteins off the resin. © 2006 Copyright © 2006 Elsevier Inc. All rights reserved.

Authors
Philip, N; Haystead, TA
MLA Citation
Philip, N, and Haystead, TA. "Affinity Purification with Natural Immobilized Ligands." Cell Biology, Four-Volume Set 4 (2006): 265-267.
Source
scival
Published In
Cell Biology, Four-Volume Set
Volume
4
Publish Date
2006
Start Page
265
End Page
267
DOI
10.1016/B978-012164730-8/50218-5

ZIP kinase, a key regulator of myosin protein phosphatase 1.

Two major physiological roles have been defined for zipper interacting protein kinase (ZIPK), regulation of apoptosis in non-muscle cells and regulation of Ca(2+) sensitization in smooth muscle. Although much attention has focused on the role of ZIPK in the regulation of apoptotic events, its roles in smooth muscle are likely to have equal if not greater physiological relevance. We first identified ZIPK as a major protein kinase controlling the phosphorylation of myosin phosphatase (SMPP-1M) and the inhibitor protein CPI17 in smooth muscle. Phosphorylation of SMPP-1M and CPI17 by ZIPK inhibits phosphatase activity towards myosin and causes profound Ca(2+) sensitization and contraction in smooth muscle. ZIPK will also directly phosphorylate both muscle and non-muscle myosin. The highly selective actions of ZIPK in the control of myosin phosphorylation potentially make the enzyme an ideal candidate for the development of novel therapeutics to treat smooth muscle related disorders such as hypertension or asthma.

Authors
Haystead, TAJ
MLA Citation
Haystead, TAJ. "ZIP kinase, a key regulator of myosin protein phosphatase 1." Cell Signal 17.11 (November 2005): 1313-1322. (Review)
PMID
16005610
Source
pubmed
Published In
Cellular Signalling
Volume
17
Issue
11
Publish Date
2005
Start Page
1313
End Page
1322
DOI
10.1016/j.cellsig.2005.05.008

Caspase-3 dependent cleavage and activation of skeletal muscle phosphorylase b kinase.

Phosphorylase b kinase (PhK) is a key enzyme involved in the conversion of glycogen to glucose in skeletal muscle and ultimately an increase in intracellular ATP. Since apoptosis is an ATP-dependent event, we investigated the regulation of skeletal muscle PhK during apoptosis. Incubation of PhK with purified caspase-3 in vitro resulted in the highly selective cleavage of the regulatory alpha subunit and resulted in a 2-fold increase in PhK activity. Edman protein sequencing of a stable 72 kD amino-terminal fragment and a 66 kD carboxy-terminal fragment revealed a specific caspase-3 cleavage site within the alpha subunit at residue 646 (DWMD G). Treatment of differentiated C2C12 mouse muscle myoblasts with the inducers of apoptosis staurosporine, TPEN, doxorubicin, or UV irradiation resulted in the disappearance of the alpha subunit of PhK as determined by immunoblotting, as well as a concurrent increase in caspase-3 activity. Moreover, induction of apoptosis by TPEN resulted in increased phosphorylase activity and sustained ATP levels throughout a 7 h time course. However, induction of apoptosis with staurosporine, also a potent PhK inhibitor, led to a rapid loss in phosphorylase activity and intracellular ATP, suggesting that PhK inhibition by staurosporine impairs the ability of apoptotic muscle cells to generate ATP. Thus, these studies indicate that PhK may be a substrate for caspase regulation during apoptosis and suggest that activation of this enzyme may be important for the generation of ATP during programmed cell death.

Authors
Hilder, TL; Carlson, GM; Haystead, TAJ; Krebs, EG; Graves, LM
MLA Citation
Hilder, TL, Carlson, GM, Haystead, TAJ, Krebs, EG, and Graves, LM. "Caspase-3 dependent cleavage and activation of skeletal muscle phosphorylase b kinase." Mol Cell Biochem 275.1-2 (July 2005): 233-242.
PMID
16335803
Source
pubmed
Published In
Molecular and Cellular Biochemistry
Volume
275
Issue
1-2
Publish Date
2005
Start Page
233
End Page
242

Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation (vol 280, pg 9363, 2005)

Authors
Graves, PR; Winkfield, KM; Haystead, TAJ
MLA Citation
Graves, PR, Winkfield, KM, and Haystead, TAJ. "Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation (vol 280, pg 9363, 2005)." JOURNAL OF BIOLOGICAL CHEMISTRY 280.24 (June 17, 2005): 23424-23424.
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
280
Issue
24
Publish Date
2005
Start Page
23424
End Page
23424

Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation.

Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase implicated in cell death and smooth muscle contractility, but its mechanism of regulation is unknown. We have identified six phosphorylation sites in ZIPK that regulate both its enzyme activity and localization, including Thr180, Thr225, Thr265, Thr299, Thr306, and Ser311. Mutational analysis showed that phosphorylation of Thr180 in the kinase activation T-loop, Thr225 in the substrate-binding groove, and Thr265 in kinase subdomain X is essential for full ZIPK autophosphorylation and activity toward exogenous substrates. Abrogation of phosphorylation of Thr299, Thr306, and Ser311 had little effect on enzyme activity, but mutation of Thr299 and Thr300 to alanine resulted in redistribution of ZIPK from the cytosol to the nucleus. Mutation of Thr299 alone to alanine caused ZIPK to assume a diffuse cellular localization, whereas T299D redistributed the enzyme to the cytoplasm. C-terminal truncations of ZIPK at amino acid 273 or 342 or mutation of the leucine zipper motif increased ZIPK activity toward exogenous substrates by severalfold, suggesting a phosphorylation-independent autoinhibitory role for the C-terminal domain. Additionally, mutation of the leucine zipper reduced the ability of ZIPK to oligomerize and also caused ZIPK to relocalize from the cytoplasm to the nucleus in vivo. Together, our findings show that ZIPK is positively regulated by phosphorylation within its kinase domain and that it contains an inhibitory C-terminal domain that controls enzyme activity, localization, and oligomerization.

Authors
Graves, PR; Winkfield, KM; Haystead, TAJ
MLA Citation
Graves, PR, Winkfield, KM, and Haystead, TAJ. "Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation." J Biol Chem 280.10 (March 11, 2005): 9363-9374.
PMID
15611134
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
10
Publish Date
2005
Start Page
9363
End Page
9374
DOI
10.1074/jbc.M412538200

Mammalian septins regulate microtubule stability through interaction with the microtubule-binding protein MAP4

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4. © 2005 by The American Society for Cell Biology.

Authors
Kremer, BE; Haystead, T; Macara, IG
MLA Citation
Kremer, BE, Haystead, T, and Macara, IG. "Mammalian septins regulate microtubule stability through interaction with the microtubule-binding protein MAP4." Molecular Biology of the Cell 16.10 (2005): 4648-4659.
PMID
16093351
Source
scival
Published In
Molecular Biology of the Cell
Volume
16
Issue
10
Publish Date
2005
Start Page
4648
End Page
4659
DOI
10.1091/mbc.E05-03-0267

Erratum: Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation (Journal of Biological Chemistry (2005) 280 (9363-9374))

Authors
Graves, PR; Winkfield, KM; Haystead, TAJ
MLA Citation
Graves, PR, Winkfield, KM, and Haystead, TAJ. "Erratum: Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation (Journal of Biological Chemistry (2005) 280 (9363-9374))." Journal of Biological Chemistry 280.24 (2005): 23424--.
Source
scival
Published In
Journal of Biological Chemistry
Volume
280
Issue
24
Publish Date
2005
Start Page
23424-

The proteome

Authors
Datto, MB; Haystead, TAJ
MLA Citation
Datto, MB, and Haystead, TAJ. "The proteome." (December 20, 2004): 153-178. (Chapter)
Source
scopus
Publish Date
2004
Start Page
153
End Page
178

Changes in magnetization transfer MRI correlate with spreading depression-induced astroglial reactivity and increased protein expression in mice.

OBJECTIVE: Gliosis refers to a range of glial cell transformations that vary according to specific brain pathologic states. Disease, however, is not a prerequisite for gliosis because glial reactivity may also be seen in regions of increased physiologic activity. Our study tests the hypothesis that high-field-strength magnetization transfer MRI is a sensitive technique for detecting transient glial reactivity after experimental spreading depression, a relatively benign perturbation unaccompanied by cell injury. MATERIALS AND METHODS: Unilateral neocortical spreading depression was elicited in mouse cerebral hemispheres and confirmed by transcranial blood flow and extracellular potential measurements. After 3 days, mice were imaged at 4 T using magnetization transfer techniques. Astroglial reactivity was determined immunohistochemically, and protein expression in control and experimental hemispheres was compared using proteomic techniques. RESULTS: Sixteen ([mean +/- SD] +/- 3) spreading depressions (n = 10) were recorded in experimental hemispheres. Spreading depression was never observed in control hemispheres. At 3 days, an 8% decrease (p < 0.05, n = 4) in magnetization transfer signal intensity was measured in experimental hemispheres, which was associated with a 37% increase (p < 0.001, n = 4) in the intensity of glial fibrillary acidic protein staining. Proteomic analysis performed 3 days after the induction of spreading depression showed upregulation of at least 56 proteins, including extracellular and intracellular elements. CONCLUSION: Magnetization transfer at 4.0-T MRI is a sensitive method for detecting glial reactivity and changes in protein expression not associated with cell injury. These results suggest magnetization transfer MRI techniques may have potential for detecting glial reactivity in physiologic processes such as learning and in early disease states.

Authors
Lascola, CD; Song, AW; Haystead, TA; Warner, DS; Verleysen, K; Freed, TA; Provenzale, JM
MLA Citation
Lascola, CD, Song, AW, Haystead, TA, Warner, DS, Verleysen, K, Freed, TA, and Provenzale, JM. "Changes in magnetization transfer MRI correlate with spreading depression-induced astroglial reactivity and increased protein expression in mice." AJR Am J Roentgenol 183.6 (December 2004): 1791-1797.
PMID
15547231
Source
pubmed
Published In
AJR. American journal of roentgenology
Volume
183
Issue
6
Publish Date
2004
Start Page
1791
End Page
1797
DOI
10.2214/ajr.183.6.01831791

Identification of phosphorylation sites in human tristetraprolin that affect its electrophoretic mobility

Authors
Cao, H; Lai, WS; Deterding, LJ; Venable, J; Kennington, EA; Haystead, TAJ; III, YJR; Tomer, KB; Blackshear, PJ
MLA Citation
Cao, H, Lai, WS, Deterding, LJ, Venable, J, Kennington, EA, Haystead, TAJ, III, YJR, Tomer, KB, and Blackshear, PJ. "Identification of phosphorylation sites in human tristetraprolin that affect its electrophoretic mobility." MOLECULAR & CELLULAR PROTEOMICS 3.10 (October 2004): S66-S66.
Source
wos-lite
Published In
Molecular & cellular proteomics : MCP
Volume
3
Issue
10
Publish Date
2004
Start Page
S66
End Page
S66

Modulation of smooth muscle contractility by CHASM, a novel member of the smoothelin family of proteins.

Cyclic nucleotides acting through their associated protein kinases, the cGMP- and cAMP-dependent protein kinases, can relax smooth muscles without a change in free intracellular calcium concentration ([Ca2+]i), a phenomenon referred to as Ca2+ desensitization. The molecular mechanisms by which these kinases bring about Ca2+ desensitization are unknown and an understanding of this phenomenon may lead to better therapies for treating diseases involving defects in the contractile response of smooth muscles such as hypertension, bronchospasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Utilizing a combination of real-time proteomics and smooth muscle physiology, we characterized a distinct subset of protein targets for cGMP-dependent protein kinase in smooth muscle. Among those phosphoproteins identified was calponin homology-associated smooth muscle (CHASM), a novel protein that contains a calponin homology domain and shares sequence similarity with the smoothelin family of smooth muscle specific proteins. Recombinant CHASM was found to evoke relaxation in a concentration dependent manner when added to permeabilized smooth muscle. A co-sedimentation assay with actin demonstrated that CHASM does not possess actin binding activity. Our findings indicate that CHASM is a novel member of the smoothelin protein family that elicits Ca2+ desensitization in smooth muscle.

Authors
Borman, MA; MacDonald, JA; Haystead, TAJ
MLA Citation
Borman, MA, MacDonald, JA, and Haystead, TAJ. "Modulation of smooth muscle contractility by CHASM, a novel member of the smoothelin family of proteins." FEBS Lett 573.1-3 (August 27, 2004): 207-213.
PMID
15327999
Source
pubmed
Published In
FEBS Letters
Volume
573
Issue
1-3
Publish Date
2004
Start Page
207
End Page
213
DOI
10.1016/j.febslet.2004.08.002

Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides.

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.

Authors
Wooldridge, AA; MacDonald, JA; Erdodi, F; Ma, C; Borman, MA; Hartshorne, DJ; Haystead, TAJ
MLA Citation
Wooldridge, AA, MacDonald, JA, Erdodi, F, Ma, C, Borman, MA, Hartshorne, DJ, and Haystead, TAJ. "Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides." J Biol Chem 279.33 (August 13, 2004): 34496-34504.
PMID
15194681
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
33
Publish Date
2004
Start Page
34496
End Page
34504
DOI
10.1074/jbc.M405957200

A feedback loop in the polo-like kinase activation pathway.

The Xenopus polo-like kinase Plx1 plays important roles during entry into and exit from mitosis (M phase). Previous studies revealed that Plx1 is activated by phosphorylation on serine and threonine residues, and purification of an activating enzyme from mitotic Xenopus egg extracts led to cloning and characterization of Xenopus polo-like kinase kinase (xPlkk1), which can phosphorylate and activate Plx1 in vitro. In the present study, a positive feedback loop between Plx1 and xPlkk1 was shown to result in each kinase phosphorylating and activating the other. Sequencing of radiolabeled xPlkk1 after phosphorylation by Plx1 in vitro identified three phosphorylation sites each spaced three amino acids apart, two of which have the consensus acidic-X-pSer-hydrophobic described for other polo-like kinase substrates. In addition, endogenous xPlkk1 in oocytes was phosphorylated on these sites in M phase but not in interphase. A mutant xPlkk1 in which these three amino acids were changed to alanine (xPlkk1(SA3)) was unable to be phosphorylated or activated in vitro by Plxl. Depletion of Plx1 from oocyte extracts prior to stimulation of the G(2)/M transition blocked the activation of xPlkk1, but depletion of xPlkk1 before stimulation did not block Plx1 activation. These results indicate that xPlkk1 may function downstream as a target of Plx1 rather than as an upstream activating kinase during the G(2)/M transition.

Authors
Erikson, E; Haystead, TAJ; Qian, Y-W; Maller, JL
MLA Citation
Erikson, E, Haystead, TAJ, Qian, Y-W, and Maller, JL. "A feedback loop in the polo-like kinase activation pathway." J Biol Chem 279.31 (July 30, 2004): 32219-32224.
PMID
15166215
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
31
Publish Date
2004
Start Page
32219
End Page
32224
DOI
10.1074/jbc.M403840200

C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats.

The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem heptad repeats with consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. In yeast, this heptad sequence is repeated about 26 times, and it becomes hyperphosphorylated during transcription predominantly at serines 2 and 5. A network of kinases and phosphatases combine to determine the CTD phosphorylation pattern. We sought to determine the positional specificity of phosphorylation by yeast CTD kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA 3'-end formation. Toward this end, we characterized monoclonal antibodies commonly employed to study CTD phosphorylation patterns and found that the H5 monoclonal antibody reacts with CTD species phosphorylated at Ser2 and/or Ser5. We therefore used antibody-independent methods to study CTDK-I, and we found that CTDK-I phosphorylates Ser5 of the CTD if the CTD substrate is either unphosphorylated or prephosphorylated at Ser2. When Ser5 is already phosphorylated, CTDK-I phosphorylates Ser2 of the CTD. We also observed that CTDK-I efficiently generates doubly phosphorylated CTD repeats; CTD substrates that already contain Ser2-PO(4) or Ser5-PO(4) are more readily phosphorylated CTDK-I than unphosphorylby ated CTD substrates.

Authors
Jones, JC; Phatnani, HP; Haystead, TA; MacDonald, JA; Alam, SM; Greenleaf, AL
MLA Citation
Jones, JC, Phatnani, HP, Haystead, TA, MacDonald, JA, Alam, SM, and Greenleaf, AL. "C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats." J Biol Chem 279.24 (June 11, 2004): 24957-24964.
PMID
15047695
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
24
Publish Date
2004
Start Page
24957
End Page
24964
DOI
10.1074/jbc.M402218200

Kinetic mechanism of quinone oxidoreductase 2 and its inhibition by the antimalarial quinolines.

Quinone oxidoreductase 2 (QR2) purified from human red blood cells was recently shown to be a potential target of the quinoline antimalarial compounds [Graves et al., (2002) Mol. Pharmacol. 62, 1364]. QR2 catalyzes the two-electron reduction of menadione via the oxidation of N-alkylated or N-ribosylated nicotinamides. To investigate the mechanism and consequences of inhibition of QR2 by the quinolines further, we have used steady-state and transient-state kinetics to define the mechanism of QR2. Importantly, we have shown that QR2 when isolated from an overproducing strain of E. coli is kinetically equivalent to the enzyme from the native human red blood cell source. We observe ping-pong kinetics consistent with one substrate/inhibitor binding site that shows selectivity for the oxidation state of the FAD cofactor, suggesting that selective inhibition of the liver versus red blood cell forms of malaria may be possible. The reductant N-methyldihydronicotinamide and the inhibitor primaquine bind exclusively to the oxidized enzyme. In contrast, the inhibitors quinacrine and chloroquine bind exclusively to the reduced enzyme. The quinone substrate menadione, on the other hand, binds nonspecifically to both forms of the enzyme. Single-turnover kinetics of the reductive half-reaction are chemically and kinetically competent and confirm the inhibitor selectivity seen in the steady-state experiments. Our studies shed light on the possible in vivo potency of the quinolines and provide a foundation for future studies aimed at creating more potent QR2 inhibitors and at understanding the physiological significance of QR2.

Authors
Kwiek, JJ; Haystead, TAJ; Rudolph, J
MLA Citation
Kwiek, JJ, Haystead, TAJ, and Rudolph, J. "Kinetic mechanism of quinone oxidoreductase 2 and its inhibition by the antimalarial quinolines." Biochemistry 43.15 (April 20, 2004): 4538-4547.
PMID
15078100
Source
pubmed
Published In
Biochemistry
Volume
43
Issue
15
Publish Date
2004
Start Page
4538
End Page
4547
DOI
10.1021/bi035923w

SLLP2, a novel non-bacteriolytic lysozyme-like protein of human spermatozoa linked to the X chromosome

Authors
Mandal, A; Klotz, KL; Shetty, J; Vemuganti, SA; Coppola, MA; Haystead, TAJ; Flickinger, CJ; Herr, JC
MLA Citation
Mandal, A, Klotz, KL, Shetty, J, Vemuganti, SA, Coppola, MA, Haystead, TAJ, Flickinger, CJ, and Herr, JC. "SLLP2, a novel non-bacteriolytic lysozyme-like protein of human spermatozoa linked to the X chromosome." 2004.
Source
wos-lite
Published In
Biology of Reproduction
Publish Date
2004
Start Page
125
End Page
125

Proteomics, Fluorescence, and Binding Affinity

Authors
Graves, PR; Haystead, TAJ
MLA Citation
Graves, PR, and Haystead, TAJ. "Proteomics, Fluorescence, and Binding Affinity." 2-3 (November 7, 2003): 301-303. (Chapter)
Source
scopus
Volume
2-3
Publish Date
2003
Start Page
301
End Page
303
DOI
10.1016/B978-012124546-7/50534-9

CRP: Cleavage of Radiolabeled Phosphoproteins.

The CRP (Cleavage of Radiolabeled Phosphoproteins) program guides the design and interpretation of experiments to identify protein phosphorylation sites by Edman sequencing of unseparated peptides. Traditionally, phosphorylation sites are determined by cleaving the phosphoprotein and separating the peptides for Edman 32P-phosphate release sequencing. CRP analysis of a phosphoprotein's sequence accelerates this process by omitting the separation step: given a protein sequence of interest, the CRP program performs an in silico proteolytic cleavage of the sequence and reports the predicted Edman cycles in which radioactivity would be observed if a given serine, threonine or tyrosine were phosphorylated. Experimentally observed cycles containing 32P can be compared with CRP predictions to confirm candidate sites and/or explore the ability of additional cleavage experiments to resolve remaining ambiguities. To reduce ambiguity, the phosphorylated residue (P-Tyr, P-Ser or P-Thr) can be determined experimentally, and CRP will ignore sites with alternative residues. CRP also provides simple predictions of likely phosphorylation sites using known kinase recognition motifs. The CRP interface is available at http://fasta.bioch.virginia.edu/crp.

Authors
Mackey, AJ; Haystead, TAJ; Pearson, WR
MLA Citation
Mackey, AJ, Haystead, TAJ, and Pearson, WR. "CRP: Cleavage of Radiolabeled Phosphoproteins." Nucleic Acids Res 31.13 (July 1, 2003): 3859-3861.
PMID
12824437
Source
pubmed
Published In
Nucleic Acids Research
Volume
31
Issue
13
Publish Date
2003
Start Page
3859
End Page
3861

Comparative analysis of the ATP-binding sites of Hsp90 by nucleotide affinity cleavage: a distinct nucleotide specificity of the C-terminal ATP-binding site.

The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that assists both in ATP-independent sequestration of damaged proteins, and in ATP-dependent folding of numerous targets, such as nuclear hormone receptors and protein kinases. Recent work from our lab and others has established the existence of a second, C-terminal nucleotide binding site besides the well characterized N-terminal, geldanamycin-sensitive ATP-binding site. The cryptic C-terminal site becomes open only after the occupancy of the N-terminal site. Our present work demonstrates the applicability of the oxidative nucleotide affinity cleavage in the site-specific characterization of nucleotide binding proteins. We performed a systematic analysis of the nucleotide binding specificity of the Hsp90 nucleotide binding sites. N-terminal binding is specific to adenosine nucleotides with an intact adenine ring. Nicotinamide adenine dinucleotides and diadenosine polyphosphate alarmones are specific N-terminal nucleotides. The C-terminal binding site is much more unspecific-it interacts with both purine and pirimidine nucleotides. Efficient binding to the C-terminal site requires both charged residues and a larger hydrophobic moiety. GTP and UTP are specific C-terminal nucleotides. 2',3'-O-(2,4,6-trinitrophenyl)-nucleotides (TNP-ATP, TNP-GTP) and pyrophosphate access the C-terminal binding site without the need for an occupied N-terminal site. Our data provide additional evidence for the dynamic domain-domain interactions of Hsp90, give hints for the design of novel types of specific Hsp90 inhibitors, and raise the possibility that besides ATP, other small molecules might also interact with the C-terminal nucleotide binding site in vivo.

Authors
Soti, C; Vermes, A; Haystead, TAJ; Csermely, P
MLA Citation
Soti, C, Vermes, A, Haystead, TAJ, and Csermely, P. "Comparative analysis of the ATP-binding sites of Hsp90 by nucleotide affinity cleavage: a distinct nucleotide specificity of the C-terminal ATP-binding site." Eur J Biochem 270.11 (June 2003): 2421-2428.
PMID
12755697
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
270
Issue
11
Publish Date
2003
Start Page
2421
End Page
2428

SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c lysozyme-like protein of human spermatozoa.

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.

Authors
Mandal, A; Klotz, KL; Shetty, J; Jayes, FL; Wolkowicz, MJ; Bolling, LC; Coonrod, SA; Black, MB; Diekman, AB; Haystead, TAJ; Flickinger, CJ; Herr, JC
MLA Citation
Mandal, A, Klotz, KL, Shetty, J, Jayes, FL, Wolkowicz, MJ, Bolling, LC, Coonrod, SA, Black, MB, Diekman, AB, Haystead, TAJ, Flickinger, CJ, and Herr, JC. "SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c lysozyme-like protein of human spermatozoa." Biol Reprod 68.5 (May 2003): 1525-1537.
PMID
12606493
Source
pubmed
Published In
Biology of Reproduction
Volume
68
Issue
5
Publish Date
2003
Start Page
1525
End Page
1537
DOI
10.1095/biolreprod.102.010108

Response to Petri: Can a proteornics strategy be used to identify the anti-malarial activity of chloroquine?

Authors
Haystead, TAJ
MLA Citation
Haystead, TAJ. "Response to Petri: Can a proteornics strategy be used to identify the anti-malarial activity of chloroquine?." TRENDS IN PHARMACOLOGICAL SCIENCES 24.5 (May 2003): 212-213.
Source
wos-lite
Published In
Trends in Pharmacological Sciences
Volume
24
Issue
5
Publish Date
2003
Start Page
212
End Page
213
DOI
10.1016/S0168-6147(03)00072-5

Proteomic analysis of calcium/calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic preferences.

The multifunctional calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV) are closely related by primary sequence and predicted to have similar substrate specificities based on peptide studies. We identified a fragment of p300-(1-117) that is a substrate of both kinases, and through both mutagenesis and Edman phosphate ((32)P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser(89) ((84)LLRSGSSPNL(93)), fits the expected consensus whereas the CaMKIV site, Ser(24) ((19)SSPALSASAS(28)), is novel. To compare kinase substrate preferences more generally, we employed a proteomic display technique that allowed comparison of complex cell extracts phosphorylated by each kinase in a rapid in vitro assay, thereby demonstrating substrate preferences that overlapped but were clearly distinct. To validate this approach, one of the proteins labeled in this assay was identified by microsequencing as HSP25, purified as a recombinant protein, and examined as a substrate for both CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in kinetics and stoichiometry of the phosphorylation sites, with CaMKI preferring Ser(15) ((10)LLRTPSWGPF(19)) to Ser(85) ((80)LNRQLSSGVS(89)) 3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.

Authors
Corcoran, EE; Joseph, JD; MacDonald, JA; Kane, CD; Haystead, TAJ; Means, AR
MLA Citation
Corcoran, EE, Joseph, JD, MacDonald, JA, Kane, CD, Haystead, TAJ, and Means, AR. "Proteomic analysis of calcium/calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic preferences." J Biol Chem 278.12 (March 21, 2003): 10516-10522.
PMID
12538590
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
12
Publish Date
2003
Start Page
10516
End Page
10522
DOI
10.1074/jbc.M210642200

Phosphoproteome analysis in yeast.

Authors
Ray, R; Haystead, TA
MLA Citation
Ray, R, and Haystead, TA. "Phosphoproteome analysis in yeast." Methods in enzymology 366 (January 2003): 95-103.
PMID
14674242
Source
epmc
Published In
Methods in Enzymology
Volume
366
Publish Date
2003
Start Page
95
End Page
103
DOI
10.1016/s0076-6879(03)66008-8

Mixed peptide sequencing and the FASTF/FASTS algorithms.

Authors
Ray, R; Haystead, TA
MLA Citation
Ray, R, and Haystead, TA. "Mixed peptide sequencing and the FASTF/FASTS algorithms." Methods in enzymology 366 (January 2003): 84-95.
PMID
14674241
Source
epmc
Published In
Methods in Enzymology
Volume
366
Publish Date
2003
Start Page
84
End Page
95
DOI
10.1016/s0076-6879(03)66007-6

A functional proteomics approach to signal transduction.

The purpose of this review is to highlight how proteomics techniques can be used to answer specific questions related to signal transduction in a wide variety of systems. In our laboratory, we utilize proteomic technologies to elucidate signal transduction pathways involved in smooth muscle contraction and relaxation, cell growth and tumorigenesis, and the pathogenesis of malaria. We see the real application of this technology as a tool to enhance the power of existing approaches such as classical yeast and mouse genetics, tissue culture, protein expression systems, and site-directed mutagenesis. Our basic approach is to examine only those proteins that differ by some variable from the control sample. In this way, the number of proteins to be processed by electrophoresis, Edman degradation, or mass spectrometry is greatly reduced. In addition, since only those proteins that change in response to a given biological treatment are analyzed, the experimental outcome provides information about specific signaling pathways. Examples of typical experiments in our laboratory are measurement of changes in protein phosphorylation in response to treatment of cells with growth factors or specific drugs, characterization of proteins associated with a bait protein in a "pull-down" experiment, or measurement of changes in protein expression. Frequently, in these experiments, it is necessary to define complex protein mixtures. To achieve this goal, we utilize a variety of techniques to isolate specific types of proteins or "subproteomes" for further analysis. In this review, we discuss strategies used in our laboratory for studying signaling pathways, including subproteome isolation, proteome mining, and analysis of the phosphoproteome.

Authors
Graves, PR; Haystead, TAJ
MLA Citation
Graves, PR, and Haystead, TAJ. "A functional proteomics approach to signal transduction." Recent Prog Horm Res 58 (2003): 1-24. (Review)
PMID
12795412
Source
pubmed
Published In
Recent progress in hormone research
Volume
58
Publish Date
2003
Start Page
1
End Page
24

Response to Petri: Can a proteomics strategy be used to identify the anti-malarial activity of chloroquine?

Authors
Haystead, TAJ
MLA Citation
Haystead, TAJ. "Response to Petri: Can a proteomics strategy be used to identify the anti-malarial activity of chloroquine?." Trends in Pharmacological Sciences 24.5 (2003): 212-213.
Source
scival
Published In
Trends in Pharmacological Sciences
Volume
24
Issue
5
Publish Date
2003
Start Page
212
End Page
213
DOI
10.1016/S0165-6147(03)00072-5

[7] Mixed Peptide Sequencing and the FASTF/FASTS Algorithms

Authors
Ray, R; Haystead, TAJ
MLA Citation
Ray, R, and Haystead, TAJ. "[7] Mixed Peptide Sequencing and the FASTF/FASTS Algorithms." Methods in Enzymology 366 (2003): 84-95.
Source
scival
Published In
Methods in Enzymology
Volume
366
Publish Date
2003
Start Page
84
End Page
95
DOI
10.1016/S0076-6879(03)66007-6

[8] Phosphoproteome Analysis in Yeast

Authors
Ray, R; Haystead, TAJ
MLA Citation
Ray, R, and Haystead, TAJ. "[8] Phosphoproteome Analysis in Yeast." Methods in Enzymology 366 (2003): 95-103.
Source
scival
Published In
Methods in Enzymology
Volume
366
Publish Date
2003
Start Page
95
End Page
103
DOI
10.1016/S0076-6879(03)66008-8

Discovery of novel targets of quinoline drugs in the human purine binding proteome.

The quinolines have been used in the treatment of malaria, arthritis, and lupus for many years, yet the precise mechanism of their action remains unclear. In this study, we used a functional proteomics approach that exploited the structural similarities between the quinoline compounds and the purine ring of ATP to identify quinoline-binding proteins. Several quinoline drugs were screened by displacement affinity chromatography against the purine binding proteome captured with gamma-phosphate-linked ATP-Sepharose. Screening of the human red blood cell purine binding proteome identified two human proteins, aldehyde dehydrogenase 1 (ALDH1) and quinone reductase 2 (QR2). In contrast, no proteins were detected upon screening of the Plasmodium falciparum purine binding proteome with the quinolines. In a complementary approach, we passed cell lysates from mice, red blood cells, or P. falciparum over hydroxychloroquine- or primaquine-Sepharose. Consistent with the displacement affinity chromatography screen, ALDH and QR2 were the only proteins recovered from mice and human red blood cell lysate and no proteins were recovered from P. falciparum. Furthermore, the activity of QR2 was potently inhibited by several of the quinolines in vitro. Our results show that ALDH1 and QR2 are selective targets of the quinolines and may provide new insights into the mechanism of action of these drugs.

Authors
Graves, PR; Kwiek, JJ; Fadden, P; Ray, R; Hardeman, K; Coley, AM; Foley, M; Haystead, TAJ
MLA Citation
Graves, PR, Kwiek, JJ, Fadden, P, Ray, R, Hardeman, K, Coley, AM, Foley, M, and Haystead, TAJ. "Discovery of novel targets of quinoline drugs in the human purine binding proteome." Molecular pharmacology 62.6 (December 2002): 1364-1372.
PMID
12435804
Source
epmc
Published In
Molecular pharmacology
Volume
62
Issue
6
Publish Date
2002
Start Page
1364
End Page
1372
DOI
10.1124/mol.62.6.1364

Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase.

A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr(709), and two other sites at Thr(695) and Thr(495). One of the sites for cAMP-dependent protein kinase (PKA) was Ser(694). Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr(695)-->Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr(695) was the major inhibitory site, Thr(709) had only a slight inhibitory effect and Ser(694) had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.

Authors
Murányi, A; MacDonald, JA; Deng, JT; Wilson, DP; Haystead, TAJ; Walsh, MP; Erdodi, F; Kiss, E; Wu, Y; Hartshorne, DJ
MLA Citation
Murányi, A, MacDonald, JA, Deng, JT, Wilson, DP, Haystead, TAJ, Walsh, MP, Erdodi, F, Kiss, E, Wu, Y, and Hartshorne, DJ. "Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase." Biochem J 366.Pt 1 (August 15, 2002): 211-216.
PMID
12030846
Source
pubmed
Published In
The Biochemical journal
Volume
366
Issue
Pt 1
Publish Date
2002
Start Page
211
End Page
216
DOI
10.1042/BJ20020401

Smooth muscle myosin phosphatase-associated kinase induces Ca2+ sensitization via myosin phosphatase inhibition.

Smooth muscle calcium sensitization reflects an inhibition of myosin light chain phosphatase (SMPP-1m) activity; however, the underlying mechanisms are not well understood. SMPP-1m activity can be modulated through phosphorylation of the myosin targeting subunit (MYPT1) by the endogenous myosin phosphatase-associated kinase, MYPT1 kinase (MacDonald, J. A., Borman, M. A., Muranyi, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2419-2424). Recombinant chicken gizzard MYPT1 (M130) was phosphorylated in vitro by a recombinant MYPT1 kinase, and the sites of phosphorylation were identified as Thr(654), Ser(808), and Thr(675). Introduction of recombinant MYPT1 kinase elicited a calcium-independent contraction in beta-escin-permeabilized rabbit ileal smooth muscle. Using an antibody that specifically recognizes MYPT1 phosphorylated at Thr(654) (M130 numbering), we determined that this calcium-independent contraction was correlated with an increase in MYPT1 phosphorylation. These results indicate that SMPP-1m phosphorylation by MYPT1 kinase is a mechanism of smooth muscle calcium sensitization.

Authors
Borman, MA; MacDonald, JA; Murányi, A; Hartshorne, DJ; Haystead, TAJ
MLA Citation
Borman, MA, MacDonald, JA, Murányi, A, Hartshorne, DJ, and Haystead, TAJ. "Smooth muscle myosin phosphatase-associated kinase induces Ca2+ sensitization via myosin phosphatase inhibition." J Biol Chem 277.26 (June 28, 2002): 23441-23446.
PMID
11976330
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
26
Publish Date
2002
Start Page
23441
End Page
23446
DOI
10.1074/jbc.M201597200

A strategy for the rapid identification of phosphorylation sites in the phosphoproteome.

Edman phosphate ((32)P) release sequencing provides a high sensitivity means of identifying phosphorylation sites in proteins that complements mass spectrometry techniques. We have developed a bioinformatic assessment tool, the cleavage of radiolabeled protein (CRP) program, which enables experimental identification of phosphorylation sites via (32)P labeling and Edman degradation of cleaved proteins obtained at femtomole levels. By observing the Edman cycle(s) in which radioactivity is found, candidate phosphorylation sites are identified by determining which residues occur at the observed number of cycles downstream from a peptide cleavage site. In cases where more than one residue could be responsible for the observed radioactivity, additional experiments with cleavage reagents having alternative specificities may resolve the ambiguity. Given a protein sequence and a cleavage site, CRP performs these experiments in silico, identifying resolved sites based on user-supplied experimental data, as well as suggesting combinations of reagents for additional analyses. Analysis of the PhosphoBase protein sequence database suggests that CRP data from two cleavage experiments can be used to identify unambiguously 60% of known phosphorylation sites. Data from additional cleavage experiments may increase the overall coverage to 70% of known sites. By comparing theoretical data obtained from the CRP program with (32)P release data obtained from an Edman sequencer, a known phosphorylation site was identified unambiguously and correctly. In addition, our results show that in vivo phosphorylation sites can be determined routinely by differential proteolysis analysis and Edman cycling with less than 1 fmol of protein and 1000 cpm.

Authors
MacDonald, JA; Mackey, AJ; Pearson, WR; Haystead, TAJ
MLA Citation
MacDonald, JA, Mackey, AJ, Pearson, WR, and Haystead, TAJ. "A strategy for the rapid identification of phosphorylation sites in the phosphoproteome." Mol Cell Proteomics 1.4 (April 2002): 314-322.
PMID
12096113
Source
pubmed
Published In
Molecular & cellular proteomics : MCP
Volume
1
Issue
4
Publish Date
2002
Start Page
314
End Page
322

Molecular biologist's guide to proteomics.

The emergence of proteomics, the large-scale analysis of proteins, has been inspired by the realization that the final product of a gene is inherently more complex and closer to function than the gene itself. Shortfalls in the ability of bioinformatics to predict both the existence and function of genes have also illustrated the need for protein analysis. Moreover, only through the study of proteins can posttranslational modifications be determined, which can profoundly affect protein function. Proteomics has been enabled by the accumulation of both DNA and protein sequence databases, improvements in mass spectrometry, and the development of computer algorithms for database searching. In this review, we describe why proteomics is important, how it is conducted, and how it can be applied to complement other existing technologies. We conclude that currently, the most practical application of proteomics is the analysis of target proteins as opposed to entire proteomes. This type of proteomics, referred to as functional proteomics, is always driven by a specific biological question. In this way, protein identification and characterization has a meaningful outcome. We discuss some of the advantages of a functional proteomics approach and provide examples of how different methodologies can be utilized to address a wide variety of biological problems.

Authors
Graves, PR; Haystead, TAJ
MLA Citation
Graves, PR, and Haystead, TAJ. "Molecular biologist's guide to proteomics." Microbiol Mol Biol Rev 66.1 (March 2002): 39-63. (Review)
PMID
11875127
Source
pubmed
Published In
Microbiology and molecular biology reviews : MMBR
Volume
66
Issue
1
Publish Date
2002
Start Page
39
End Page
63

Caspase-dependent cleavage of carbamoyl phosphate synthetase II during apoptosis.

Carbamoyl phosphate synthetase II (CPSII) is part of carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Herein, we identify CAD as a substrate for caspase-3 degradation in both in vitro and in vivo models of apoptosis. Withdrawal of interleukin-3 or incubation with staurosporine (STS) or doxorubicin (Dox) resulted in proteolytic cleavage of CAD in a myeloid precursor cell line (32D) or in a cell line over-expressing CAD. The rapid decline in the CPSII activity paralleled the degradation of CAD and preceded the appearance of Annexin-V-stained apoptotic cells and DNA fragmentation. These events correlated closely with the activation of caspase-3 in these cells and were prevented by the cell-permeable caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone. Moreover, the incubation of purified CAD with recombinant caspase-3 in vitro generated CAD fragments that were similar to those obtained in vivo. Edman sequencing revealed that two of the major caspase-3 cleavage sites occurred at the sequences EAVD/G and VACD/G within the catalytic (B2) and allosteric (B3) domains of CAD, thus providing a potential mechanism for the rapid inactivation of CPSII during apoptosis. Consistent with this, an enhanced loss of the intracellular pyrimidines (UTP and CTP) was observed in response to STS or DOX-induced apoptosis. Therefore, these studies show that CAD is a novel target for caspase-dependent regulation during apoptosis and suggest that the selective inactivation of pyrimidine nucleotide synthesis accompanies the process of apoptosis.

Authors
Huang, M; Kozlowski, P; Collins, M; Wang, Y; Haystead, TA; Graves, LM
MLA Citation
Huang, M, Kozlowski, P, Collins, M, Wang, Y, Haystead, TA, and Graves, LM. "Caspase-dependent cleavage of carbamoyl phosphate synthetase II during apoptosis." Mol Pharmacol 61.3 (March 2002): 569-577.
PMID
11854437
Source
pubmed
Published In
Molecular pharmacology
Volume
61
Issue
3
Publish Date
2002
Start Page
569
End Page
577

Getting more from less: algorithms for rapid protein identification with multiple short peptide sequences.

We describe two novel sequence similarity search algorithms, FASTS and FASTF, that use multiple short peptide sequences to identify homologous sequences in protein or DNA databases. FASTS searches with peptide sequences of unknown order, as obtained by mass spectrometry-based sequencing, evaluating all possible arrangements of the peptides. FASTF searches with mixed peptide sequences, as generated by Edman sequencing of unseparated mixtures of peptides. FASTF deconvolutes the mixture, using a greedy heuristic that allows rapid identification of high scoring alignments while reducing the total number of explored alternatives. Both algorithms use the heuristic FASTA comparison strategy to accelerate the search but use alignment probability, rather than similarity score, as the criterion for alignment optimality. Statistical estimates are calculated using an empirical correction to a theoretical probability. These calculated estimates were accurate within a factor of 10 for FASTS and 1000 for FASTF on our test dataset. FASTS requires only 15-20 total residues in three or four peptides to robustly identify homologues sharing 50% or greater protein sequence identity. FASTF requires about 25% more sequence data than FASTS for equivalent sensitivity, but additional sequence data are usually available from mixed Edman experiments. Thus, both algorithms can identify homologues that diverged 100 to 500 million years ago, allowing proteomic identification from organisms whose genomes have not been sequenced.

Authors
Mackey, AJ; Haystead, TAJ; Pearson, WR
MLA Citation
Mackey, AJ, Haystead, TAJ, and Pearson, WR. "Getting more from less: algorithms for rapid protein identification with multiple short peptide sequences." Mol Cell Proteomics 1.2 (February 2002): 139-147.
PMID
12096132
Source
pubmed
Published In
Molecular & cellular proteomics : MCP
Volume
1
Issue
2
Publish Date
2002
Start Page
139
End Page
147

SLLP1, a novel non-bacteriolytic c lysozyme-like protein in the acrosome of human spermatozoa.

Authors
Mandal, A; Klotz, KL; Shetty, J; Jayes, FL; Wolkowicz, MJ; Bolling, LC; Coonrod, SA; Black, MB; Diekman, AB; Haystead, TAJ; Flickinger, CJ; Herr, JC
MLA Citation
Mandal, A, Klotz, KL, Shetty, J, Jayes, FL, Wolkowicz, MJ, Bolling, LC, Coonrod, SA, Black, MB, Diekman, AB, Haystead, TAJ, Flickinger, CJ, and Herr, JC. "SLLP1, a novel non-bacteriolytic c lysozyme-like protein in the acrosome of human spermatozoa." BIOLOGY OF REPRODUCTION 66 (2002): 113-114.
Source
wos-lite
Published In
Biology of Reproduction
Volume
66
Publish Date
2002
Start Page
113
End Page
114

Site-specific phosphorylation and point mutations of telokin modulate its Ca2+-desensitizing effect in smooth muscle.

Forskolin and 8-bromoguanosine 3'-5'-cyclic monophosphate (8-Br-cGMP) induce phosphorylation of Ser-13 of telokin and relaxation of smooth muscle at constant calcium. Comparison with the effect of wild type with aspartate (D; to mimic phosphorylation) and alanine (A; non-phosphorylatable) mutants of telokin showed that the S13D mutant was more effective than wild type in relaxing smooth muscle at constant calcium. The efficacy of the Ser-13A, S12A, and S12D mutants was not significantly different from that of wild-type telokin. The effect of neither S13D nor Ser-13A was affected by 8-Br-cGMP, whereas the effect of wild type, S12A, and S12D was enhanced by 8-Br-cGMP, indicating the specificity of Ser-13 charge modification. Mutation of Ser-19 (a mitogen-activated protein kinase site) showed the S19A to be more effective than, and S19D to be not different from, wild-type telokin. The effect of both mutants was slightly enhanced by 8-Br-cGMP. A truncated (residues 1-142) form lacking the acidic C terminus had the same relaxant effect as wild-type telokin, whereas the C-terminal peptide (residues 142-155) had no effect. We conclude that site-specific modification of the N terminus modulates the Ca2+ -desensitizing effect of telokin on force.

Authors
Walker, LA; MacDonald, JA; Liu, X; Nakamoto, RK; Haystead, TA; Somlyo, AV; Somlyo, AP
MLA Citation
Walker, LA, MacDonald, JA, Liu, X, Nakamoto, RK, Haystead, TA, Somlyo, AV, and Somlyo, AP. "Site-specific phosphorylation and point mutations of telokin modulate its Ca2+-desensitizing effect in smooth muscle." J Biol Chem 276.27 (July 6, 2001): 24519-24524.
PMID
11346659
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
27
Publish Date
2001
Start Page
24519
End Page
24524
DOI
10.1074/jbc.M103560200

Angiotensin II upregulation of rRNA transcription in vascular smooth muscle cells: Identification of Upstream Binding Factor (UBF) phosphorylation sites

Authors
Lin, CH; Ficarro, S; Haystead, TAJ; Hunt, DF; Owens, GK
MLA Citation
Lin, CH, Ficarro, S, Haystead, TAJ, Hunt, DF, and Owens, GK. "Angiotensin II upregulation of rRNA transcription in vascular smooth muscle cells: Identification of Upstream Binding Factor (UBF) phosphorylation sites." ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 21.4 (April 2001): 684-684.
Source
wos-lite
Published In
Arteriosclerosis, Thrombosis, and Vascular Biology
Volume
21
Issue
4
Publish Date
2001
Start Page
684
End Page
684

Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase.

Phosphorylation of CPI-17 and PHI-1 by the MYPT1-associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST-rN-ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI-17 (but not PHI-1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI-17 were identified as Thr 38 and Ser 12 using Edman sequencing with (32)P release and a point mutant of Thr 38.

Authors
MacDonald, JA; Eto, M; Borman, MA; Brautigan, DL; Haystead, TA
MLA Citation
MacDonald, JA, Eto, M, Borman, MA, Brautigan, DL, and Haystead, TA. "Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase." FEBS Lett 493.2-3 (March 30, 2001): 91-94.
PMID
11287002
Source
pubmed
Published In
FEBS Letters
Volume
493
Issue
2-3
Publish Date
2001
Start Page
91
End Page
94

Caspase-dependent cleavage of the pyrimidine biosynthetic enzyme CAD during apoptosis

Authors
Graves, LM; Huang, M; Kozlowski, P; Collins, M; Haystead, TAJ
MLA Citation
Graves, LM, Huang, M, Kozlowski, P, Collins, M, and Haystead, TAJ. "Caspase-dependent cleavage of the pyrimidine biosynthetic enzyme CAD during apoptosis." FASEB JOURNAL 15.4 (March 7, 2001): A25-A25.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
15
Issue
4
Publish Date
2001
Start Page
A25
End Page
A25

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase.

Ca(2+) sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by "mixed-peptide" Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca(2+) sensitizing kinase cascade.

Authors
MacDonald, JA; Borman, MA; Murányi, A; Somlyo, AV; Hartshorne, DJ; Haystead, TA
MLA Citation
MacDonald, JA, Borman, MA, Murányi, A, Somlyo, AV, Hartshorne, DJ, and Haystead, TA. "Identification of the endogenous smooth muscle myosin phosphatase-associated kinase." Proc Natl Acad Sci U S A 98.5 (February 27, 2001): 2419-2424.
PMID
11226254
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
5
Publish Date
2001
Start Page
2419
End Page
2424
DOI
10.1073/pnas.041331498

A Role for the Ppz Ser/Thr Protein Phosphatases in the Regulation of Translation Elongation Factor 1Bα

In vivo 32P-labeled yeast proteins from wild type and ppz1 ppz2 phosphatase mutants were resolved by bidimensional electrophoresis. A prominent phosphoprotein, which in ppz mutants showed a marked shift to acidic regions, was identified by mixed peptide sequencing as the translation elongation factor 1Bα (formerly eEF1β. An equivalent shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1. Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable residue and showed that its phosphorylation was increased in ppz cells. Pull-down experiments using a glutathione S-transferase (GST)-EF1Bα fusion version allowed to identify Ppz1 as an in vivo interacting protein. Cells lacking Ppz display a higher tolerance to known translation inhibitors, such as hygromycin and paromomycin, and enhanced readthrough at all three nonsense codons, suggesting that translational fidelity might be affected. Overexpression of a GST-EF1Bα fusion counteracted the growth defect associated to high levels of Ppz1 and this effect was essentially lost when the phosphorylatable Ser-86 is replaced by Ala. Therefore, the Ppz phosphatases appear to regulate the phosphorylation state of EF1Bα in yeast, and this may result in modification of the translational accuracy.

Authors
Nadal, ED; Fadden, RP; Ruiz, A; Haystead, T; Ariño, J
MLA Citation
Nadal, ED, Fadden, RP, Ruiz, A, Haystead, T, and Ariño, J. "A Role for the Ppz Ser/Thr Protein Phosphatases in the Regulation of Translation Elongation Factor 1Bα." Journal of Biological Chemistry 276.18 (2001): 14829-14834.
PMID
11278758
Source
scival
Published In
Journal of Biological Chemistry
Volume
276
Issue
18
Publish Date
2001
Start Page
14829
End Page
14834
DOI
10.1074/jbc.M010824200

Borg proteins control septin organization and are negatively regulated by Cdc42

The Cdc42 GTPase binds to numerous effector proteins that control cell polarity, cytoskeletal remodelling and vesicle transport. In many cases the signalling pathways downstream of these effectors are not known. Here we show that the Cdc42 effectors Borg1 to Borg3 bind to septin GTPases. Endogenous septin Cdc10 and Borg3 proteins can be immunoprecipitated together by an anti-Borg3 antibody. The ectopic expression of Borgs disrupts normal septin organization. Cdc42 negatively regulates this effect and inhibits the binding of Borg3 to septins. Borgs are therefore the first known regulators of mammalian septin organization and provide an unexpected link between the septin and Cdc42 GTPases.

Authors
Joberty, G; Perlungher, RR; Sheffield, PJ; Kinoshita, M; Noda, M; Haystead, T; Macara, IG
MLA Citation
Joberty, G, Perlungher, RR, Sheffield, PJ, Kinoshita, M, Noda, M, Haystead, T, and Macara, IG. "Borg proteins control septin organization and are negatively regulated by Cdc42." Nature Cell Biology 3.10 (2001): 861-866.
PMID
11584266
Source
scival
Published In
Nature Cell Biology
Volume
3
Issue
10
Publish Date
2001
Start Page
861
End Page
866
DOI
10.1038/ncb1001-861

Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle.

The Ca(2+)-independent acceleration of dephosphorylation of the regulatory light chain of smooth muscle myosin and relaxation of smooth muscle by telokin are enhanced by cyclic nucleotide-activated protein kinase(s) [Wu et al. (1998) J. Biol. Chem. 273, 11362-113691. The purpose of this study was to determine the in vivo site(s) and in vitro rates of telokin phosphorylation and to evaluate the possible effects of sequential phosphorylation by different kinases. The in vivo site(s) of phosphorylation of telokin were determined in rabbit smooth muscles of longitudinal ileum and portal vein. Following stimulation of ileum with forskolin (20 microM) the serine at position 13 was the only amino acid to exhibit increased phosphorylation. Rabbit portal vein telokin was phosphorylated on both Ser-13 and -19 as a result of forskolin and GTPgammaS stimulation in vivo. Point mutation of Ser-13 (to Ala or Asp) abolished in vitro phosphorylation by cyclic nucleotide-dependent protein kinases.

Authors
MacDonald, JA; Walker, LA; Nakamoto, RK; Gorenne, I; Somlyo, AV; Somlyo, AP; Haystead, TA
MLA Citation
MacDonald, JA, Walker, LA, Nakamoto, RK, Gorenne, I, Somlyo, AV, Somlyo, AP, and Haystead, TA. "Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle." FEBS Lett 479.3 (August 18, 2000): 83-88.
PMID
10981712
Source
pubmed
Published In
FEBS Letters
Volume
479
Issue
3
Publish Date
2000
Start Page
83
End Page
88

Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression.

Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr --> Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

Authors
Mothe-Satney, I; Yang, D; Fadden, P; Haystead, TA; Lawrence, JC
MLA Citation
Mothe-Satney, I, Yang, D, Fadden, P, Haystead, TA, and Lawrence, JC. "Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression." Mol Cell Biol 20.10 (May 2000): 3558-3567.
PMID
10779345
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
10
Publish Date
2000
Start Page
3558
End Page
3567

Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase.

Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression in Saccharomyces cerevisiae. Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose. Here, we explore the functional relationships between Reg1, Glc7, and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1. We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex. Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1. We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex.

Authors
Sanz, P; Alms, GR; Haystead, TA; Carlson, M
MLA Citation
Sanz, P, Alms, GR, Haystead, TA, and Carlson, M. "Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase." Mol Cell Biol 20.4 (February 2000): 1321-1328.
PMID
10648618
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
4
Publish Date
2000
Start Page
1321
End Page
1328

Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density.

Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.

Authors
Terry-Lorenzo, RT; Inoue, M; Connor, JH; Haystead, TA; Armbruster, BN; Gupta, RP; Oliver, CJ; Shenolikar, S
MLA Citation
Terry-Lorenzo, RT, Inoue, M, Connor, JH, Haystead, TA, Armbruster, BN, Gupta, RP, Oliver, CJ, and Shenolikar, S. "Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density." J Biol Chem 275.4 (January 28, 2000): 2439-2446.
PMID
10644697
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
4
Publish Date
2000
Start Page
2439
End Page
2446

Site-specific phosphorylation of telokin modulates its relaxant effect on smooth muscle.

Authors
Walker, LA; MacDonald, JA; Somlyo, AV; Nakamoto, RK; Haystead, TAJ; Somlyo, AP
MLA Citation
Walker, LA, MacDonald, JA, Somlyo, AV, Nakamoto, RK, Haystead, TAJ, and Somlyo, AP. "Site-specific phosphorylation of telokin modulates its relaxant effect on smooth muscle." BIOPHYSICAL JOURNAL 78.1 (January 2000): 23A-23A.
Source
wos-lite
Published In
Biophysical Journal
Volume
78
Issue
1
Publish Date
2000
Start Page
23A
End Page
23A

An inhibitor of p160 Rho-kinase blocks MARCKS phosphorylation in vascular smooth muscle

Authors
Gorenne, I; Haystead, TAJ; Somlyo, AS; Somlyo, AP
MLA Citation
Gorenne, I, Haystead, TAJ, Somlyo, AS, and Somlyo, AP. "An inhibitor of p160 Rho-kinase blocks MARCKS phosphorylation in vascular smooth muscle." MOLECULAR BIOLOGY OF THE CELL 10 (November 1999): 247A-247A.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
10
Publish Date
1999
Start Page
247A
End Page
247A

Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome.

Protein phosphatase 1 (Glc7p) and its binding protein Reg1p are essential for the regulation of glucose repression pathways in Saccharomyces cerevisiae. In order to identify physiological substrates for the Glc7p-Reg1p complex, we examined the effects of deletion of the REG1 gene on the yeast phosphoproteome. Analysis by two-dimensional phosphoprotein mapping identified two distinct proteins that were greatly increased in phosphate content in reg1Delta mutants. Mixed peptide sequencing identified these proteins as hexokinase II (Hxk2p) and the E1alpha subunit of pyruvate dehydrogenase. Consistent with increased phosphorylation of Hxk2p in response to REG1 deletion, fractionation of yeast extracts by anion-exchange chromatography identified Hxk2p phosphatase activity in wild-type strains that was selectively lost in the reg1Delta mutant. The phosphorylation state of Hxk2p and Hxk2p phosphatase activity was restored to wild-type levels in the reg1Delta mutant by expression of a LexA-Reg1p fusion protein. In contrast, expression of LexA-Reg1p containing mutations at phenylalanine in the putative PP-1C-binding site motif (K/R)(X)(I/V)XF was unable to rescue Hxk2p dephosphorylation in intact yeast or restore Hxk2p phosphatase activity. These results demonstrate that Reg1p targets PP-1C to dephosphorylate Hxk2p in vivo and that the motif (K/R)(X) (I/V)XF is necessary for its PP-1 targeting function.

Authors
Alms, GR; Sanz, P; Carlson, M; Haystead, TA
MLA Citation
Alms, GR, Sanz, P, Carlson, M, and Haystead, TA. "Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome." EMBO J 18.15 (August 2, 1999): 4157-4168.
PMID
10428955
Source
pubmed
Published In
EMBO Journal
Volume
18
Issue
15
Publish Date
1999
Start Page
4157
End Page
4168
DOI
10.1093/emboj/18.15.4157

Identification of kinases involved in the growth factor-induced increases in phosphorylation of the rRNA transcription factor Upstream Binding Factor (UBF).

Authors
Kihm, AJ; Haystead, TAJ; Owens, GK
MLA Citation
Kihm, AJ, Haystead, TAJ, and Owens, GK. "Identification of kinases involved in the growth factor-induced increases in phosphorylation of the rRNA transcription factor Upstream Binding Factor (UBF)." FASEB JOURNAL 13.4 (March 12, 1999): A388-A388.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
13
Issue
4
Publish Date
1999
Start Page
A388
End Page
A388

Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein.

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF in vivo in response to stimulation with different growth factors or in vitro with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF-TBP binding depended on the C-terminal 'acidic tail' of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.

Authors
Kihm, AJ; Hershey, JC; Haystead, TA; Madsen, CS; Owens, GK
MLA Citation
Kihm, AJ, Hershey, JC, Haystead, TA, Madsen, CS, and Owens, GK. "Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein." Proc Natl Acad Sci U S A 95.25 (December 8, 1998): 14816-14820.
PMID
9843972
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
95
Issue
25
Publish Date
1998
Start Page
14816
End Page
14820

Rapid identification of protein phosphatase 1-binding proteins by mixed peptide sequencing and data base searching. Characterization of a novel holoenzymic form of protein phosphatase 1.

Microcystin-affinity chromatography was used to purify 15 protein phosphatase 1 (PP1)-binding proteins from the myofibrillar fraction of rabbit skeletal muscle. To reduce the time and amount of material required to identify these proteins, proteome analysis by mixed peptide sequencing was developed. Proteins are resolved by SDS-polyacrylamide gel electrophoresis, electroblotted to polyvinylidene fluoride membrane, and stained. Bands are sliced from the membrane, cleaved briefly with CnBr, and applied without further purification to an automated Edman sequencer. The mixed peptide sequences generated are sorted and matched against the GenBank using two new programs, FASTF and TFASTF. This technology offers a simple alternative to mass spectrometry for the subpicomolar identification of proteins in polyacrylamide gels. Using this technology, all 15 proteins recovered in PP-1C affinity chromatography were sequenced. One of the proteins, PP-1bp55, was homologous to human myosin phosphatase, MYPT2. A second, PP-1bp80, identified in the EST data bases, contained a putative PP-1C binding site and a nucleotide binding motif. Further affinity purification over ATP-Sepharose isolated PP-1bp80 in a quaternary complex with PP-1C and two other proteins, PP-1bp29 and human p20. Recombinant PP-1bp80 also bound PP-1C and suppressed its activity toward a variety of substrates, suggesting that the protein is a novel regulatory subunit of PP-1.

Authors
Damer, CK; Partridge, J; Pearson, WR; Haystead, TA
MLA Citation
Damer, CK, Partridge, J, Pearson, WR, and Haystead, TA. "Rapid identification of protein phosphatase 1-binding proteins by mixed peptide sequencing and data base searching. Characterization of a novel holoenzymic form of protein phosphatase 1." J Biol Chem 273.38 (September 18, 1998): 24396-24405.
PMID
9733729
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
38
Publish Date
1998
Start Page
24396
End Page
24405

Phosphorylation of the translational regulator, PHAS-I, by protein kinase CK2.

The primary site in PHAS-I for phosphorylation by protein kinase CK2 in vitro was identified as Ser111. A relatively small amount of phosphorylation of Ser99 was also detected, and mutating Ser99 to Ala in PHAS-I slightly decreased phosphorylation by CK2 in vitro. In contrast, mutating Ser111 to Ala almost abolished phosphorylation, confirming Ser111 as the preferred site for CK2. Phosphorylation of Ser111 did not decrease binding of PHAS-I to eIF4E, and results of peptide mapping experiments with PHAS-I immunoprecipitated from 32P-labeled adipocytes indicated that Ser111 was not phosphorylated in cells. These results support the conclusion that CK2 is not involved in the control of PHAS-I.

Authors
Fadden, P; Haystead, TA; Lawrence, JC
MLA Citation
Fadden, P, Haystead, TA, and Lawrence, JC. "Phosphorylation of the translational regulator, PHAS-I, by protein kinase CK2." FEBS Lett 435.1 (September 11, 1998): 105-109.
PMID
9755868
Source
pubmed
Published In
FEBS Letters
Volume
435
Issue
1
Publish Date
1998
Start Page
105
End Page
109

Acceleration of myosin light chain dephosphorylation and relaxation of smooth muscle by telokin. Synergism with cyclic nucleotide-activated kinase.

Incorporation of 32P into telokin, a smooth muscle-specific, 17-18-kDa, acidic (pI 4.2-4.4) protein, was increased by forskolin (20 microM) in intact rabbit ileum smooth muscle (ileum) and by 8-bromo-cyclic GMP (100 microM) in alpha-toxin-permeabilized ileum. Native telokin (5-20 microM), purified from turkey gizzard, and recombinant rabbit telokin, expressed in Escherichia coli and purified to >90% purity, induced dose-dependent relaxation, associated with a significant decrease in regulatory myosin light chain phosphorylation, without affecting the rate of thiophosphorylation of regulatory myosin light chain of ileum permeabilized with 0.1% Triton X-100. Endogenous telokin was lost from ileum during prolonged permeabilization (>20 min) with 0.1% Triton X-100, and the time course of loss was correlated with the loss of 8-bromo-cyclic GMP-induced calcium desensitization. Recombinant and native gizzard telokins were phosphorylated, in vitro, by the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and p42/44 mitogen-activated protein kinase; the recombinant protein was also phosphorylated by calmodulin-dependent protein kinase II. Exogenous cGMP-dependent protein kinase (0.5 microM) activated by 8-bromo-cyclic GMP (50 microM) phosphorylated recombinant telokin (10 microM) when added concurrently to ileum depleted of its endogenous telokin, and their relaxant effects were mutually potentiated. Forskolin (20 microM) also increased phosphorylation of telokin in intact ileum. We conclude that telokin induces calcium desensitization in smooth muscle by enhancing myosin light chain phosphatase activity, and cGMP- and/or cAMP-dependent phosphorylation of telokin up-regulates its relaxant effect.

Authors
Wu, X; Haystead, TA; Nakamoto, RK; Somlyo, AV; Somlyo, AP
MLA Citation
Wu, X, Haystead, TA, Nakamoto, RK, Somlyo, AV, and Somlyo, AP. "Acceleration of myosin light chain dephosphorylation and relaxation of smooth muscle by telokin. Synergism with cyclic nucleotide-activated kinase." J Biol Chem 273.18 (May 1, 1998): 11362-11369.
PMID
9556631
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
18
Publish Date
1998
Start Page
11362
End Page
11369

Okadaic acid in New Zealand sponges: Detection by cytotoxicity, protein phosphatase inhibition and immunoassay techniques

Using a combination of P388 cytotoxicity testing, protein phosphatase enzyme inhibition assays and an ELISA immunoassay, okadaic acid, the causative agent of diarrhetic shellfish poisoning (DSP), has been found in Raspalia agminata, and detected in two other species of New Zealand sponges.

Authors
Perry, NB; Ellis, G; Blunt, JW; Haystead, TAJ; Lake, RJ; Munro, MHG
MLA Citation
Perry, NB, Ellis, G, Blunt, JW, Haystead, TAJ, Lake, RJ, and Munro, MHG. "Okadaic acid in New Zealand sponges: Detection by cytotoxicity, protein phosphatase inhibition and immunoassay techniques." Natural Product Letters 11.4 (1998): 305-312.
Source
scival
Published In
Natural Product Letters
Volume
11
Issue
4
Publish Date
1998
Start Page
305
End Page
312

The mammalian target of rapamycin phosphorylates sites having a (Ser/Thr)-Pro motif and is activated by antibodies to a region near its COOH terminus.

The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [gamma-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the phosphorylation of PHAS-I in cells.

Authors
Brunn, GJ; Fadden, P; Haystead, TA; Lawrence, JC
MLA Citation
Brunn, GJ, Fadden, P, Haystead, TA, and Lawrence, JC. "The mammalian target of rapamycin phosphorylates sites having a (Ser/Thr)-Pro motif and is activated by antibodies to a region near its COOH terminus." J Biol Chem 272.51 (December 19, 1997): 32547-32550.
PMID
9405468
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
51
Publish Date
1997
Start Page
32547
End Page
32550

The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.

Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the gamma-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1-221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.

Authors
Grenert, JP; Sullivan, WP; Fadden, P; Haystead, TA; Clark, J; Mimnaugh, E; Krutzsch, H; Ochel, HJ; Schulte, TW; Sausville, E; Neckers, LM; Toft, DO
MLA Citation
Grenert, JP, Sullivan, WP, Fadden, P, Haystead, TA, Clark, J, Mimnaugh, E, Krutzsch, H, Ochel, HJ, Schulte, TW, Sausville, E, Neckers, LM, and Toft, DO. "The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation." J Biol Chem 272.38 (September 19, 1997): 23843-23850.
PMID
9295332
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
38
Publish Date
1997
Start Page
23843
End Page
23850

Activation of MAPK by MEK is enhanced by a thioltransferase

Authors
Scott, A; Errede, B; Haystead, TAJ
MLA Citation
Scott, A, Errede, B, and Haystead, TAJ. "Activation of MAPK by MEK is enhanced by a thioltransferase." FASEB JOURNAL 11.9 (July 31, 1997): A1359-A1359.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1359
End Page
A1359

Fluorescent labeling of phosphoserines in tissue proteins.

Authors
Long, W; Hillier, TA; Haystead, TAJ; Barrett, EJ
MLA Citation
Long, W, Hillier, TA, Haystead, TAJ, and Barrett, EJ. "Fluorescent labeling of phosphoserines in tissue proteins." DIABETES 46 (May 1997): 766-766.
Source
wos-lite
Published In
Diabetes
Volume
46
Publish Date
1997
Start Page
766
End Page
766

Identification of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes.

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser64, the preferred site of MAP kinase. Binding of the Ala64 mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser64 control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [gamma-32P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which 32P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82. In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr36 remained bound to eIF-4E, indicating that phosphorylation of Thr36 is insufficient for dissociation of the PHAS-I.eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.

Authors
Fadden, P; Haystead, TA; Lawrence, JC
MLA Citation
Fadden, P, Haystead, TA, and Lawrence, JC. "Identification of phosphorylation sites in the translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes." J Biol Chem 272.15 (April 11, 1997): 10240-10247.
PMID
9092573
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
15
Publish Date
1997
Start Page
10240
End Page
10247

Telokin relaxes smooth muscle and is phosphorylated, in situ

Authors
Wu, X; Haystead, TAJ; Somlyo, AV; Somlyo, AP
MLA Citation
Wu, X, Haystead, TAJ, Somlyo, AV, and Somlyo, AP. "Telokin relaxes smooth muscle and is phosphorylated, in situ." BIOPHYSICAL JOURNAL 72.2 (February 1997): TU228-TU228.
Source
wos-lite
Published In
Biophysical Journal
Volume
72
Issue
2
Publish Date
1997
Start Page
TU228
End Page
TU228

PHAS proteins as mediators of the actions of insulin, growth factors and cAMP on protein synthesis and cell proliferation.

PHAS-I and PHAS-II are members of a newly discovered family of proteins that regulate translation initiation. PHAS-I is expressed in a wide variety of cell types, but it is highest in adipocytes, where protein synthesis is markedly increased by insulin. PHAS-II is highest in liver and kidney, where very little PHAS-I is found. PHAS proteins bind to eIF-4E, the mRNA cap-binding protein, and inhibit translation of capped mRNA in vitro and in cells. In rat adipocytes PHAS-I is phosphorylated in at least five sites, all of which conform to the consensus, (Ser/Thr)-Pro. Both PHAS proteins are phosphorylated in response to insulin or growth factors, such as EGF, PDGF and IGF-1. Phosphorylation in the appropriate site(s) promotes dissociation of PHAS/eIF-4E complexes. This allows eIF-4E to bind to eIF-4G (p220), thereby increasing the amount of the eIF-4F complex and the rate of translation initiation. Increasing cAMP promotes PHAS-I dephosphorylation and increases binding to eIF-4E. Unlike PHAS-I, PHAS-II is readily phosphorylated by PKA in vitro, suggesting that regulation of the two proteins differs. However, increasing cAMP in cells also promotes dephosphorylation of PHAS-II. Thus, PHAS proteins appear to be key mediators not only of the stimulatory effects of insulin and growth factors on protein synthesis, but also of the inhibitory effects of cAMP. Moreover, by controlling eIF-4E PHAS proteins may be involved in the control of cell proliferation, as increasing eIF-4E is mitogenic and can even cause malignant transformation of cells. MAP kinase readily phosphorylates both PHAS-I and PHAS-II in vitro, but inhibiting activation of MAP kinase does not attenuate the effects of insulin on increasing phosphorylation of the PHAS proteins in adipocytes or skeletal muscle. MAP kinase phosphorylates neither PHAS-I nor PHAS-II at a significant rate when the proteins are bound to eIF-4E. Therefore, the role of MAP kinase in promoting the dissociation of PHAS/eIF-4E complexes is not clear. Of several protein kinases tested, only casein kinase-II phosphorylated PHAS-I when it was bound eIF-4E. Indeed, the bound form of PHAS-I was phosphorylated more rapidly than the free form. However, it is unlikely that casein kinase II regulates either PHAS protein, as the major site (Ser111) in PHAS-I phosphorylated by casein kinase II in vitro is not phosphorylated in adipocytes, and PHAS-II is not a substrate for casein kinase-II. Pharmacological and genetic evidence indicates that the mTOR/p70S6K pathway is involved in the control of PHAS-I and -II. Thus, PHAS proteins may be mediators of the effects of this pathway on protein synthesis and cell proliferation.

Authors
Lawrence, JC; Fadden, P; Haystead, TA; Lin, TA
MLA Citation
Lawrence, JC, Fadden, P, Haystead, TA, and Lin, TA. "PHAS proteins as mediators of the actions of insulin, growth factors and cAMP on protein synthesis and cell proliferation." Adv Enzyme Regul 37 (1997): 239-267. (Review)
PMID
9381973
Source
pubmed
Published In
Advances in Enzyme Regulation
Volume
37
Publish Date
1997
Start Page
239
End Page
267

Endothelial cell-conditioned medium downregulates smooth muscle contractile protein expression

Smooth muscle cells (SMC) within atherosclerotic lesions proliferate and exhibit phenotypic modulation, but the contribution of vascular endothelium to this process is poorly understood. Our aim was to examine the effects of endothelial cell-conditioned medium (ECCM) on vascular SMC growth and differentiation. Rat aortic ECCM stimulated a ninefold increase in [3H]thymidine incorporation and downregulated smooth muscle-specific myosin heavy chain and α-actin synthesis in rat aortic SMC. These effects were not inhibited by antibodies to platelet-derived growth factor (PDGF)-BB or PDGF- AB or with a PDGF β-receptor subunit. Treatment with PDGF-BB (at a concentration found in ECCM), PDGF-AA, basic fibroblast growth factor, endothelin-1, or transforming growth factor-β did not reproduce these effects. The ECCM activities were sensitive to heat and trypsinization, were >30 kDa in molecular mass, and bound weakly to heparin-Sepharose. Our data indicate that cultured endothelial cells produce a factor(s) that downregulates contractile protein expression in SMC, which may contribute to SMC dedifferentiation and proliferation.

Authors
Vernon, SM; Campos, MJ; Haystead, T; Thompson, MM; DiCorleto, PE; Owens, GK
MLA Citation
Vernon, SM, Campos, MJ, Haystead, T, Thompson, MM, DiCorleto, PE, and Owens, GK. "Endothelial cell-conditioned medium downregulates smooth muscle contractile protein expression." American Journal of Physiology - Cell Physiology 272.2 41-2 (1997): C582-C591.
PMID
9124302
Source
scival
Published In
American journal of physiology. Cell physiology
Volume
272
Issue
2 41-2
Publish Date
1997
Start Page
C582
End Page
C591

Activation of MAPK by MEK is enhanced by a thioltransferase

Recently we purified to homogeneity a MEK enhancing factor (MEF) that stimulates the rate of MAPK phosphorylation by MEK. In the presence of MKF, molar equivalents of MEK to MAPK were sufficient for MAPK phosphorylation to stoichiometric levels. MEE was identified as thioltransferase. a member of a family of thiol reducing enzymes that catalyze thiol-disullide interchange among proteins and are involved in the regulation of the redox sta tus of glutatbione. Thioitransferases mediate thiol-disulfide excliange via thenactive -site CPYCXX. X being any basic amino acid. STE5 was found to have a similar motif, CFKCKK. in the arnino terminus region that is responsible for EUS3 binding. Because such an active site is unique to thioltransferases. we investigated its role in STE5 function. Using a yeast plasmid containing the entire coding region of STE5. wo mutated the two cysteinos to serines, individually and concurrently. To mimic the yeast thioltransferase and remove the charged lysine between the cysteines, we mutated GTKC to CPYC. We measured ihe ability of the mutants to respond to pheromone in two wavs. Firstly, we expressed the mutagenized STE5 in a STE5 deletion mutant yeast strain and assayed for ability to suppress the mating defect. Secondly, we used a halobioassay to assess pheromone induced Gl arrest and recovery. Since thioltransferase enhances MAPK activation in the mammalian pathway, we assayed for the inability of the mutant STE5 to facilitate FUS3 phosphory lation of STET. The results of these, studies suggest a mechanism for STE5 function in yeast.

Authors
Scott, A; Erredc, B; Haystead, TAJ
MLA Citation
Scott, A, Erredc, B, and Haystead, TAJ. "Activation of MAPK by MEK is enhanced by a thioltransferase." FASEB Journal 11.9 (1997): A1358-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1358

Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography.

Biotinylated microcystin was used to affinity purify over avidin-Sepharose the entire cellular content of active forms of protein phosphatase (PP) 1 and 2A holoenzymes present in three subcellular fractions of skeletal muscle. Biotinylated microcystin displayed IC50 values in the nM range against PP-1C (1.58 +/- 0.6 nM S.E., n = 3), PP-2AC (0.63 +/- 0.2 nM S.E., n = 3) and SMPP-1M (5.9 +/- 1.3 S.E., n = 3). Subsequent anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis of the microcystin-biotin eluates of the three fractions revealed a complex pattern of proteins associated with PP-1C and PP-2AC. Far Western analysis and the rebinding interaction with recombinant PP-1C distinguished proteins in the eluates that bound PP-1C from those that bound PP-2AC. In Far Western analysis, 29 distinct proteins were identified to bind PP-1C. Significantly, these same proteins, plus seven others, were also recovered from the isothiocyanate eluates from microcystin-Sepharose by a rebinding interaction with PP-1C-microcystin-biotin. The number of proteins and range of novel molecular masses (18-125 kDa) identified to interact with PP-1C by these two techniques cannot be accounted for by the previously characterized subunits of PP-1. Our findings further support the concept that PP-1C is regulated in vivo by multiple and distinct substrate-targeting subunits.

Authors
Campos, M; Fadden, P; Alms, G; Qian, Z; Haystead, TA
MLA Citation
Campos, M, Fadden, P, Alms, G, Qian, Z, and Haystead, TA. "Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography." J Biol Chem 271.45 (November 8, 1996): 28478-28484.
PMID
8910475
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
45
Publish Date
1996
Start Page
28478
End Page
28484

Regions of the 110-kDa regulatory subunit M110 required for regulation of myosin-light-chain-phosphatase activity in smooth muscle.

To characterize the in situ interactions between the subunits (regulatory 110 kDa, M110; 21-kDa, M21 and catalytic, 37-kDa, PP1c) of smooth muscle myosin phosphatase (SMPP-1M), we determined, in Triton-X-100-permeabilized rabbit portal vein contracted with microcystin-LR, the ability of the following fragments of M110 to regulate relaxation induced by exogenous PP1c: (a) M110 purified from pig bladder; (b) the 72.5-kDa N-terminal fragment expressed from rat kidney cDNA [glutathione-S-transferase-M110-(11-612)-peptide]; (c) a 58-kDa fragment, the N-terminal degradation product of M110 (M58); (d) two fragments expressed from rat aorta cDNA [M110-(1-309)-peptide and M110-(39-309)-peptide]; a synthetic fragment of M110 [M110-(1-38)-peptide]. The M110/M21 complex accelerated approximately 1.6-fold the rate of dephosphorylation of the myosin P-light chain and also relaxation induced by PP1c. The glutathione-S-transferase-M110-(11-612)-peptide and the M58 fragments, as well as the M110-(1-309)-peptide and, at higher concentration, M110-(1-38)-peptide, had similar effects that did not require the M21 subunit. Arachidonic acid, known to dissociate PP1c from the native holoenzyme and inhibit SMPP-1M activity, inhibited the regulatory action of the M110/M21 complex on PP1c activity and, to a lesser extent that of the glutathione-S-transferase-M110-(11-612)-peptide, but not that of the M58 fragment or of the shorter peptides. We conclude that, consistent with in vitro studies [8], the N-terminal sequence (1-309) of the M110 subunit is also sufficient to enhance the activity of PP1c for myosin in muscle. However, its C-terminal half (downstream from the M58 fragment) is required for inhibition by arachidonic acid. In contrast to the effect of the M110 subunit and its fragments, a peptide, corresponding to part of the PP1c-binding site of the regulatory glycogen-binding subunit from skeletal muscle GM [GM-(63-93)-peptide], specifically slowed the relaxation, induced by flash photolysis of diazo-2, of Triton X-100-permeabilized femoral artery strips, and inhibited the holoenzyme-induced relaxation in the portal vein, suggesting that the GM subunit can compete with the regulatory effect of M110 on PP1c in smooth muscle.

Authors
Gailly, P; Wu, X; Haystead, TA; Somlyo, AP; Cohen, PT; Cohen, P; Somlyo, AV
MLA Citation
Gailly, P, Wu, X, Haystead, TA, Somlyo, AP, Cohen, PT, Cohen, P, and Somlyo, AV. "Regions of the 110-kDa regulatory subunit M110 required for regulation of myosin-light-chain-phosphatase activity in smooth muscle." Eur J Biochem 239.2 (July 15, 1996): 326-332.
PMID
8706736
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
239
Issue
2
Publish Date
1996
Start Page
326
End Page
332

Protein phosphatase 2A is involved in regulation of the secondary sustained phase of MAP kinase activity in response to vascular smooth muscle hypertrophic and mitogenic stimuli.

Authors
Hershey, JC; Madsen, CS; Partridge, JM; Owens, GK; Haystead, TAJ
MLA Citation
Hershey, JC, Madsen, CS, Partridge, JM, Owens, GK, and Haystead, TAJ. "Protein phosphatase 2A is involved in regulation of the secondary sustained phase of MAP kinase activity in response to vascular smooth muscle hypertrophic and mitogenic stimuli." FASEB JOURNAL 10.3 (March 8, 1996): 419-419.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
3
Publish Date
1996
Start Page
419
End Page
419

Effects of insulin and diabetes on the association of eukaryotic initiation factor 4E and the translational regulator, PHAS-I, in rat skeletal muscle.

Authors
Jefferson, LS; Kimball, SR; Fadden, P; Haystead, TAJ; Lawrence, JC
MLA Citation
Jefferson, LS, Kimball, SR, Fadden, P, Haystead, TAJ, and Lawrence, JC. "Effects of insulin and diabetes on the association of eukaryotic initiation factor 4E and the translational regulator, PHAS-I, in rat skeletal muscle." FASEB JOURNAL 10.3 (March 8, 1996): 4271-4271.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
3
Publish Date
1996
Start Page
4271
End Page
4271

Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle.

We have investigated the roles of eukaryotic initiation factor 4E (eIF-4E), the cap-binding protein, and the translational regulator, PHAS-I, in the effects of insulin and alloxan-induced diabetes on protein synthesis in rat skeletal muscle. Diabetes increased the amount of eIF-4E found in the inactive PHAS-I.eIF-4E complex by threefold, explaining in part the inhibitory effect of insulin deficiency on translation initiation. Insulin treatment of diabetic rats caused dissociation of the complex, consistent with the action of the hormone on reversing the inhibitory effect of diabetes on translation initiation. The effects of both insulin and diabetes on PHAS-I binding to eIF-4E appeared to be due to changes in PHAS-I phosphorylation. Neither insulin nor diabetes changed the phosphorylation state of eIF-4E. The results indicate that the effects of both insulin and diabetes on protein synthesis in skeletal muscle involve modulation of the interaction of PHAS-I and eIF-4E.

Authors
Kimball, SR; Jefferson, LS; Fadden, P; Haystead, TA; Lawrence, JC
MLA Citation
Kimball, SR, Jefferson, LS, Fadden, P, Haystead, TA, and Lawrence, JC. "Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle." Am J Physiol 270.2 Pt 1 (February 1996): C705-C709.
PMID
8779938
Source
pubmed
Published In
The American journal of physiology
Volume
270
Issue
2 Pt 1
Publish Date
1996
Start Page
C705
End Page
C709

Regulation of smooth muscle myosin light chain phosphatase (SMPP1M) by its 110 kDa (M110) subunit.

Authors
Gailly, P; Johnson, D; Haystead, CMM; Chen, YH; Wu, X; Haystead, TAJ; Somlyo, AP; Cohen, PTW; Somlyo, AV; Cohen, P
MLA Citation
Gailly, P, Johnson, D, Haystead, CMM, Chen, YH, Wu, X, Haystead, TAJ, Somlyo, AP, Cohen, PTW, Somlyo, AV, and Cohen, P. "Regulation of smooth muscle myosin light chain phosphatase (SMPP1M) by its 110 kDa (M110) subunit." BIOPHYSICAL JOURNAL 70.2 (February 1996): WPO26-WPO26.
Source
wos-lite
Published In
Biophysical Journal
Volume
70
Issue
2
Publish Date
1996
Start Page
WPO26
End Page
WPO26

Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle

Authors
Kimball, SR; Jefferson, LS; Fadden, P; Haystead, TAJ; Lawrence, JC
MLA Citation
Kimball, SR, Jefferson, LS, Fadden, P, Haystead, TAJ, and Lawrence, JC. "Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle." AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 270.2 (February 1996): C705-C709.
Source
wos-lite
Published In
American journal of physiology. Cell physiology
Volume
270
Issue
2
Publish Date
1996
Start Page
C705
End Page
C709

Effects of insulin and diabetes on the association of eukaryotic initiation factor 4e and the translational regulator, phas-i, in rat skeletal muscle

Our previous studies have shown that protein synthesis is inhibited in rat skeletal muscle during diabetes and that the inhibition is rapidly reversed by insulin treatment of diabetic rats. In the present study, we have investigated the roles of the cap-binding protein, eukaryotic initiation factor 4E (eIF-4E), and the translational regulator, PHAS-I. in the effects of insulin and alloxaninduced diabetes on protein synthesis in rat skeletal muscle. We found that diabetes increased the amount of eIF-4E present in the inactive PHAS-I-eIF-4E complex by 3-fold, explaining in part the inhibitory effect of insulin deficiency on translation initiation. Two hours after insulin treatment of diabetic rats, the amount of PHAS-I present in the PHAS-I-eIF-4E complex was reduced to control values, consistent with the action of the hormone on reversing the inhibitory effect of diabetes on translation initiation. The effects of both insulin and diabetes on PHAS-I binding to eIF-4E correlated with changes in PHAS-I phosphorylation. Neither insulin nor diabetes changed the phosphorylation state of eIF-4E. The results indicate that the effects of both insulin and diabetes on protein synthesis in skeletal muscle involve modulation of the interaction of PHAS-I and eIF-4E. Supported by NIH grants DK15658 (LSJl and DK28312 and AR41180 (JCL).

Authors
Jefferson, LS; Kimball, SR; Fadden, P; Haystead, TAJ; Jr, JCL
MLA Citation
Jefferson, LS, Kimball, SR, Fadden, P, Haystead, TAJ, and Jr, JCL. "Effects of insulin and diabetes on the association of eukaryotic initiation factor 4e and the translational regulator, phas-i, in rat skeletal muscle." FASEB Journal 10.3 (1996): A739-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
3
Publish Date
1996
Start Page
A739

Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle

We have investigated the roles of eukaryotic initiation factor 4E (eIF-4E), the cap-binding protein, and the translational regulator, PHAS-I, in the effects of insulin and alloxan-induced diabetes on protein synthesis in rat skeletal muscle. Diabetes increased the amount of eIF-4E found in the inactive PHAS-I·eIF-4E complex by threefold, explaining in part the inhibitory effect of insulin deficiency on translation initiation. Insulin treatment of diabetic rats caused dissociation of the complex, consistent with the action of the hormone on reversing the inhibitory effect of diabetes on translation initiation. The effects of both insulin and diabetes on PHAS-I binding to eIF-4E appeared to be due to changes in PHAS-I phosphorylation. Neither insulin nor diabetes changed the phosphorylation state of eIF-4E. The results indicate that the effects of both insulin and diabetes on protein synthesis in skeletal muscle involve modulation of the interaction of PHAS-I and eIF-4E.

Authors
Kimball, SR; Jefferson, LS; Fadden, P; Haystead, TAJ; Jr, JCL
MLA Citation
Kimball, SR, Jefferson, LS, Fadden, P, Haystead, TAJ, and Jr, JCL. "Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle." American Journal of Physiology - Cell Physiology 270.2 39-2 (1996): C705-C709.
Source
scival
Published In
American Journal of Physiology - Cell Physiology
Volume
270
Issue
2 39-2
Publish Date
1996
Start Page
C705
End Page
C709

Protein phosphatase 2a is involved in regulation of the secondary sustained phase of map kinase activity in response to vascular smooth muscle hypertrophic and mitogenic stimuli

Angiotensin II (A-II) and cc-thrombin are growth factors that have been previously shown in this laboratory to induce hypertrophy and mitogenesis of cultured vascular smooth muscle cells (VSMC), respectively. The intracellular signaling pathways through which these factors regulate growth and proliferation of VSMC are complex, but known to involve mitogen-activated protein kinase (MAPK). Furthermore, it has been shown that a sustained activation of MAPK is required for mitogenic growth in fibroblasts (Kahan et al. JBC 267:13369-13375, 1992). In this study, we tested the hypothesis that protein phosphatase 2A (PP2A), a known MAPK phosphatase, regulates MAPK activity differently in response to VSMC hypertrophie and mitogenic stimuli. MAPK and MAPK kinase (MEK) activities in growth arrested VSMC were markedly increased within 5 min. of stimulation with either A-II or a-thrombin. However, only athrombin stimulated a secondary sustained phase of activity. In vitro phosphatase assays with cytosolic extracts and 32P-MAPK showed that the A-II induced dephosphorylation of MAPK was inhibited by okadaic acid (OA), a potent PP2A inhibitor. It was also demonstrated that after 3 H of a-thrombin treatment, there was a significant decrease in PP2A activity in vivo. The a-thrombin-induced sustained increase in MAPK activity was rapidly abolished by treatment with the thrombm antagonists hirudin, and this effect could be reversed with the addition of OA. These data provide evidence that growth factors can differentially affect the level of PP2A activity and thus influence MAPK activity. This in turn may explain the different growth responses (i.e. hypertrophie or mitogenic) of the VSMC.

Authors
Hershev, JC; Madsen, CS; Partridge, JM; Owens, GK; Haystead, TAJ
MLA Citation
Hershev, JC, Madsen, CS, Partridge, JM, Owens, GK, and Haystead, TAJ. "Protein phosphatase 2a is involved in regulation of the secondary sustained phase of map kinase activity in response to vascular smooth muscle hypertrophic and mitogenic stimuli." FASEB Journal 10.3 (1996): A72-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
10
Issue
3
Publish Date
1996
Start Page
A72

Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M.

We have cloned a partial rat kidney cDNA that encodes a 72.5 kDa N terminal fragment of a third isoform of the M110 subunit of phosphatase 1. This new isoform contains an insert in the 542-597 position not present in the M110 previously cloned (Chen et al. (1994) FEBS Lett. 356, 51-55) from the same species. The encoded cDNA was expressed as a soluble GST-fusion protein in E. coli, and its ability to interact with native PP-1C was measured both in vitro and in permeabilized smooth muscle. In vitro, the fusion protein was capable of selectively binding PP-1C and increasing the substrate specificity of the phosphatase towards myosin 13.2 +/- 3.5-fold (S.E. of the mean, n = 3). In permeabilized smooth muscle pretreated with microcystin, the recombinant protein alone (1.0 microM) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 microM) to relax the muscle. These findings show that the N terminal domain of the M110 subunit is the primary site for both PP-1C and myosin binding, and thereby determines myosin specificity. The presence of isoformic variation within this sequence may permit organ/cell specific regulation of phosphorylation sites.

Authors
Haystead, CM; Gailly, P; Somlyo, AP; Somlyo, AV; Haystead, TA
MLA Citation
Haystead, CM, Gailly, P, Somlyo, AP, Somlyo, AV, and Haystead, TA. "Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M." FEBS Lett 377.2 (December 18, 1995): 123-127.
PMID
8543033
Source
pubmed
Published In
FEBS Letters
Volume
377
Issue
2
Publish Date
1995
Start Page
123
End Page
127

PHOSPHORYLATION OF CALDESMON BY MITOGEN-ACTIVATED PROTEIN-KINASE WITH NO EFFECT ON CALCIUM SENSITIVITY IN SMOOTH-MUSCLE

Authors
NIXON, GF; HAYSTEAD, TAJ; SOMLYO, AP; SOMLYO, AV
MLA Citation
NIXON, GF, HAYSTEAD, TAJ, SOMLYO, AP, and SOMLYO, AV. "PHOSPHORYLATION OF CALDESMON BY MITOGEN-ACTIVATED PROTEIN-KINASE WITH NO EFFECT ON CALCIUM SENSITIVITY IN SMOOTH-MUSCLE." BRITISH JOURNAL OF PHARMACOLOGY 116 (December 1995): P279-P279.
Source
wos-lite
Published In
British Journal of Pharmacology
Volume
116
Publish Date
1995
Start Page
P279
End Page
P279

Angiotensin II-induced hypertrophy of rat vascular smooth muscle is associated with increased 18 S rRNA synthesis and phosphorylation of the rRNA transcription factor, upstream binding factor.

Hypertrophy of vascular smooth muscle cells (VSMC) is an important adaptive response of hypertension. Drug intervention studies have implicated a role for angiotensin II (A-II) in the mediation of VSMC hypertrophy in vivo, and A-II is a potent hypertrophic agent for VSMC in culture. Our laboratory has previously shown that A-II-induced hypertrophy of cultured VSMC is due in part to generalized increases in protein synthesis and increased content of rRNA. The aim of the present study was to determine if A-II stimulates rRNA gene synthesis and whether the rRNA transcription factor, upstream binding factor (UBF), is involved. Nuclear run-on analysis demonstrated that A-II induced a greater than 5-fold increase in rRNA gene synthesis within 6 h of stimulation. A-II also stimulated a rapid increase in UBF phosphorylation as well as nucleolar localization, but no changes in the content of UBF. Phosphoamino acid analysis showed that phosphorylation occurred only on serine residue(s). Results demonstrate that increased transcription of ribosomal DNA contributes to the A-II-induced increase in protein synthesis and VSMC hypertrophy, and suggest that an important regulatory event in this pathway may be the phosphorylation and/or nucleolar localization of UBF.

Authors
Hershey, JC; Hautmann, M; Thompson, MM; Rothblum, LI; Haystead, TA; Owens, GK
MLA Citation
Hershey, JC, Hautmann, M, Thompson, MM, Rothblum, LI, Haystead, TA, and Owens, GK. "Angiotensin II-induced hypertrophy of rat vascular smooth muscle is associated with increased 18 S rRNA synthesis and phosphorylation of the rRNA transcription factor, upstream binding factor." J Biol Chem 270.42 (October 20, 1995): 25096-25101.
PMID
7559641
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
42
Publish Date
1995
Start Page
25096
End Page
25101

Purification of a 12,020-dalton protein that enhances the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase.

We have purified 3500-fold from rabbit skeletal muscle a 12,020-Da mitogen-activated protein kinase kinase (MEK)-enhancing factor (MEF) that stimulates both mitogen-activated protein kinase (MAPK) autophosphorylation and the rate (24-fold) at which the enzyme is phosphorylated by MEK in vitro. This was manifest by the finding that in the presence of MEF, molar equivalents of MEK to MAPK were sufficient to produce fully phosphorylated (2.1 +/- 0.4 mol/mol; S.D., n = 3) and activated MAPK. This contrasted with the 40:1 molar excess ratio of MEK to MAPK required to produce fully phosphorylated and activated MAPK in the absence of MEF. Phosphoamino acid analysis revealed that in the presence of MEF, phosphorylation of MAPK by MEK was ordered, with Tyr-185 phosphorylation preceding Thr-183 phosphorylation. However, the rate at which Thr-183 was phosphorylated relative to Tyr-185 was greatly increased. The finding that MEF stimulated MAPK autophosphorylation and increased its ability to be phosphorylated by MEK suggests a mechanism of action in which MEF interacts with MAPK to alter its conformation.

Authors
Scott, A; Haystead, CM; Haystead, TA
MLA Citation
Scott, A, Haystead, CM, and Haystead, TA. "Purification of a 12,020-dalton protein that enhances the activation of mitogen-activated protein (MAP) kinase by MAP kinase kinase." J Biol Chem 270.41 (October 13, 1995): 24540-24547.
PMID
7592672
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
41
Publish Date
1995
Start Page
24540
End Page
24547

Phosphorylation of caldesmon by mitogen-activated protein kinase with no effect on Ca2+ sensitivity in rabbit smooth muscle.

1. Recombinant, activated mitogen-activated protein kinase (3.3 microM; p42mapk) phosphorylated caldesmon in phasic (rabbit portal vein) and tonic (rabbit femoral artery) smooth muscle strips permeabilized with Triton X-100. 2. Phosphorylation of caldesmon by p42mapk neither induced contraction of relaxed smooth muscle nor affected the Ca2+ sensitivity of submaximally contracted permeabilized phasic or tonic smooth muscle.

Authors
Nixon, GF; Iizuka, K; Haystead, CM; Haystead, TA; Somlyo, AP; Somlyo, AV
MLA Citation
Nixon, GF, Iizuka, K, Haystead, CM, Haystead, TA, Somlyo, AP, and Somlyo, AV. "Phosphorylation of caldesmon by mitogen-activated protein kinase with no effect on Ca2+ sensitivity in rabbit smooth muscle." J Physiol 487 ( Pt 2) (September 1, 1995): 283-289.
PMID
8558463
Source
pubmed
Published In
The Journal of Physiology
Volume
487 ( Pt 2)
Publish Date
1995
Start Page
283
End Page
289

Quantitative and selective fluorophore labeling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary electrophoresis and laser-induced fluorescence.

Reaction conditions were defined for the selective quantitative derivatization and fluorophore labeling of phosphoserine residues on peptides and proteins. Phosphoserine was derivatized with 1,2-ethanedithiol using a modification of the reaction conditions defined by R. C. Clark and J. Dijkstra (1967) Int. J. Biochem. 11, 577-585 and H. E. Meyer, E. Hoffman-Posorke, H. Korte, and M. G. Heilmeyer (1986) FEBS Lett. 204, 61-66 for stabilizing the phosphoamino acid during Edman degradation reactions. Following derivatization, the thiol-serine residues were coupled to fluorescence by iodoacetate reaction. Characterization by capillary zone electrophoresis and laser-induced fluorescence allowed quantitation of phosphoserine content of peptides and proteins at < 75 amol. In three separate experiments, the overall reaction efficiency for 1,2-ethanedithiol derivatization of phosphoserine was estimated at 89.27 +/- 2.44% (SDM). Subsequent coupling of the derivatized serine residue with 6-iodoacetamidofluoroscein was estimated at > 98% efficiency. Fluorescent probe tagging of phosphoamino acids on proteins and peptides offers direct quantitative evaluation of cellular phosphorylation states at the attomole level in tissue samples derived from plants, animals, and humans, without the use of radioisotopes, antibodies, or mass spectrometry.

Authors
Fadden, P; Haystead, TA
MLA Citation
Fadden, P, and Haystead, TA. "Quantitative and selective fluorophore labeling of phosphoserine on peptides and proteins: characterization at the attomole level by capillary electrophoresis and laser-induced fluorescence." Anal Biochem 225.1 (February 10, 1995): 81-88.
PMID
7539987
Source
pubmed
Published In
Analytical Biochemistry
Volume
225
Issue
1
Publish Date
1995
Start Page
81
End Page
88
DOI
10.1006/abio.1995.1111

raf-induced kinases may be necessary but are not sufficient for ras-p21-protein induction of oocyte maturation

Authors
Haspel, J; Dent, P; Haystead, T; Brandt-Rauf, PW; Chung, D; Weinstein, IB; Nishimura, S; Yamaizumi, Z; Pincus, MR
MLA Citation
Haspel, J, Dent, P, Haystead, T, Brandt-Rauf, PW, Chung, D, Weinstein, IB, Nishimura, S, Yamaizumi, Z, and Pincus, MR. "raf-induced kinases may be necessary but are not sufficient for ras-p21-protein induction of oocyte maturation." Medical Science Research 23.7 (1995): 455-457.
Source
scival
Published In
Medical Science Research
Volume
23
Issue
7
Publish Date
1995
Start Page
455
End Page
457

PHAS-I: A new player in the regulation of protein synthesis by insulin and growth factors

Authors
Lawrence, JC; Lin, TA; Kong, XM; Haystead, TAJ; Hu, CB
MLA Citation
Lawrence, JC, Lin, TA, Kong, XM, Haystead, TAJ, and Hu, CB. "PHAS-I: A new player in the regulation of protein synthesis by insulin and growth factors." 1995.
Source
wos-lite
Published In
International Congress Series
Volume
1100
Publish Date
1995
Start Page
622
End Page
629

Purification and characterization of the mammalian myosin light chain phosphatase holoenzyme. The differential effects of the holoenzyme and its subunits on smooth muscle.

We have purified to homogeneity from the myofibrillar fraction of pig bladder a mammalian heterotrimeric form of PP-1, SMPP-1M. Purified pig bladder SMPP-1M is similar in composition and substrate specificity to avian gizzard PP-1M reported by Alessi et al. (Alessi, D., Macdougall, L. K., Sola, M. M., Ikebe, M., and Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035) and consists of the catalytic subunit of PP-1 (37 kDa) and two other equimolar subunits of 130 and 20 kDa. The properties of SMPP-1M and the role of its regulatory subunits in the dephosphorylation of myosin and in the initiation of relaxation were characterized both in vitro and in smooth muscle. We show that the relaxant effect of the catalytic subunit in smooth muscle is markedly potentiated by the addition of the regulatory subunits of SMPP-1M. Our findings demonstrate that SMPP-1M is the major phosphatase dephosphorylating myosin in mammalian smooth muscle and that myosin dephosphorylation is regulated in vivo via targeting subunits that specifically alter the substrate specificity of PP-1C toward myosin.

Authors
Shirazi, A; Iizuka, K; Fadden, P; Mosse, C; Somlyo, AP; Somlyo, AV; Haystead, TA
MLA Citation
Shirazi, A, Iizuka, K, Fadden, P, Mosse, C, Somlyo, AP, Somlyo, AV, and Haystead, TA. "Purification and characterization of the mammalian myosin light chain phosphatase holoenzyme. The differential effects of the holoenzyme and its subunits on smooth muscle." J Biol Chem 269.50 (December 16, 1994): 31598-31606.
PMID
7989330
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
50
Publish Date
1994
Start Page
31598
End Page
31606

PHAS-I as a link between mitogen-activated protein kinase and translation initiation.

PHAS-I is a heat-stable protein (relative molecular mass approximately 12,400) found in many tissues. It is rapidly phosphorylated in rat adipocytes incubated with insulin or growth factors. Nonphosphorylated PHAS-I bound to initiation factor 4E (eIF-4E) and inhibited protein synthesis. Serine-64 in PHAS-I was rapidly phosphorylated by mitogen-activated (MAP) kinase, the major insulin-stimulated PHAS-I kinase in adipocyte extracts. Results obtained with antibodies, immobilized PHAS-I, and a messenger RNA cap affinity resin indicated that PHAS-I did not bind eIF-4E when serine-64 was phosphorylated. Thus, PHAS-I may be a key mediator of the stimulation of protein synthesis by the diverse group of agents and stimuli that activate MAP kinase.

Authors
Lin, TA; Kong, X; Haystead, TA; Pause, A; Belsham, G; Sonenberg, N; Lawrence, JC
MLA Citation
Lin, TA, Kong, X, Haystead, TA, Pause, A, Belsham, G, Sonenberg, N, and Lawrence, JC. "PHAS-I as a link between mitogen-activated protein kinase and translation initiation." Science 266.5185 (October 28, 1994): 653-656.
PMID
7939721
Source
pubmed
Published In
Science
Volume
266
Issue
5185
Publish Date
1994
Start Page
653
End Page
656

PHAS-1 AS A LINK BETWEEN MITOGEN-ACTIVATED PROTEIN-KINASE AND TRANSLATION INITIATION

Authors
LIN, TA; KONG, XM; HAYSTEAD, TAJ; PAUSE, A; BELSHAM, G; SONENBERG, N; LAWRENCE, JC
MLA Citation
LIN, TA, KONG, XM, HAYSTEAD, TAJ, PAUSE, A, BELSHAM, G, SONENBERG, N, and LAWRENCE, JC. "PHAS-1 AS A LINK BETWEEN MITOGEN-ACTIVATED PROTEIN-KINASE AND TRANSLATION INITIATION." SCIENCE 266.5185 (October 28, 1994): 653-656.
Source
wos-lite
Published In
Science
Volume
266
Issue
5185
Publish Date
1994
Start Page
653
End Page
656
DOI
10.1126/science.7939721

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes.

PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors. To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein (MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.

Authors
Haystead, TA; Haystead, CM; Hu, C; Lin, TA; Lawrence, JC
MLA Citation
Haystead, TA, Haystead, CM, Hu, C, Lin, TA, and Lawrence, JC. "Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes." J Biol Chem 269.37 (September 16, 1994): 23185-23191.
PMID
8083223
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
37
Publish Date
1994
Start Page
23185
End Page
23191

Purification of the AMP-activated protein kinase on ATP-gamma-sepharose and analysis of its subunit structure.

The AMP-activated protein kinase has been purified by affinity chromatography on ATP-gamma-Sepharose. A proportion of the activity can be eluted using AMP, while the remainder is eluted using ATP. The AMP eluate contains three polypeptides of 63, 38 and 35 kDa (p63, p38 and p35) in a molar ratio (by Coomassie blue binding) close to 1:1:1. p63 was previously identified as the AMP-binding catalytic subunit [Carling, D., Clarke, P. R., Zammit, V. A. & Hardie, D. G. (1989) Eur. J. Biochem. 186, 129-136]. All three polypeptides exactly comigrate both on native gel electrophoresis and on gel filtration, suggesting that p38 and p35 are additional subunits. Estimation of Stokes radius (5.4-5.8 nm) by gel filtration, and sedimentation coefficient (7.9-8.4 S) by glycerol gradient centrifugation, suggest that the kinase has an asymmetric structure with a native molecular mass for the complex of 190 +/- 10 kDa. Thus the native enzyme appears to be a heterotrimer with a p63/p38/p35 (1:1:1) structure. Despite the fact that the ATP eluate has a higher specific activity than the AMP eluate (3.5 +/- 0.2 vs 2.3 +/- 0.2 mumol.min-1.mg-1), it appears to be less pure, containing p63, p38 and p35 plus other polypeptides. Experiments examining the effects of protein phosphatase-2A and kinase kinase, and analysis by Western blotting with anti-p63 antibody, suggests that the AMP eluate is entirely in the low-activity dephosphorylated form, while the ATP eluate is a mixture of that form and the high-activity phosphorylated form. As well as establishing the subunit structure of the AMP-activated protein kinase, these results suggest that the kinase can bind to ATP-gamma-Sepharose through either the allosteric (AMP/ATP) site or the catalytic (ATP) site, and that phosphorylation by the kinase kinase increases the affinity for the latter site.

Authors
Davies, SP; Hawley, SA; Woods, A; Carling, D; Haystead, TA; Hardie, DG
MLA Citation
Davies, SP, Hawley, SA, Woods, A, Carling, D, Haystead, TA, and Hardie, DG. "Purification of the AMP-activated protein kinase on ATP-gamma-sepharose and analysis of its subunit structure." Eur J Biochem 223.2 (July 15, 1994): 351-357.
PMID
8055903
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
223
Issue
2
Publish Date
1994
Start Page
351
End Page
357

Insulin activates a novel adipocyte mitogen-activated protein kinase kinase kinase that shows rapid phasic kinetics and is distinct from c-Raf.

Treatment of adipocytes with insulin or phorbol 12-myristate 13-acetate (PMA) results in transient activation of mitogen-activated protein kinase kinase (MEK) (Tmax = 90 s) and mitogen-activated protein kinase (MAPK) (Tmax = 300 s). We have identified a novel insulin-stimulated MEK kinase (I-MEKK) in the 100,000 x g infranatant that shows rapid phasic kinetics that temporally precede that of MEK. Maximal activation of I-MEKK occurs within 20 +/- 5 s (S.D., n = 3) followed by complete inactivation by 30 +/- 10 s (S.D., n = 3). I-MEKK was characterized by anion-exchange and gel filtration chromatography and separated into two distinct activities of approximately 56 kDa that phosphorylated and activated MEK. I-MEKKs did not co-elute on anion exchange with c-Raf or 73-kDa MEK kinase (MEKK), suggesting they are distinct enzymes. Protein phosphatase 2A inactivated both I-MEKKs in vitro and in the intact cell okadaic acid blocked inactivation in the presence of insulin. These results suggest activation of I-MEKK involves phosphorylation on serine or threonine residues. I-MEKK was not activated by PMA, suggesting that in adipocytes the enzyme represents a divergence point between signal transduction pathways mediated by insulin and those activating protein kinase C.

Authors
Haystead, CM; Gregory, P; Shirazi, A; Fadden, P; Mosse, C; Dent, P; Haystead, TA
MLA Citation
Haystead, CM, Gregory, P, Shirazi, A, Fadden, P, Mosse, C, Dent, P, and Haystead, TA. "Insulin activates a novel adipocyte mitogen-activated protein kinase kinase kinase that shows rapid phasic kinetics and is distinct from c-Raf." J Biol Chem 269.17 (April 29, 1994): 12804-12808.
PMID
8175693
Source
pubmed
Published In
The Journal of biological chemistry
Volume
269
Issue
17
Publish Date
1994
Start Page
12804
End Page
12808

Gamma-phosphate-linked ATP-sepharose for the affinity purification of protein kinases. Rapid purification to homogeneity of skeletal muscle mitogen-activated protein kinase kinase.

Recently, Sowadski and colleagues [Knighton, D.R., Zheng, J., Eyck, L.F.T., Ashford, V.A., Xuong, N., Taylor, S.S. & Sowadski, J.M. (1991) Science 407, 407-420] reported the structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase (cyclic A kinase), MgATP and a 20-residue inhibitor peptide, at a resolution of 0.27 nm. This structure has since been refined to 0.2-nm resolution and the orientation of the nucleotide and interactions of MgATP with numerous conserved residues at the active site defined [Zheng, J., Knighton, D.R., Eyck, L.F.T., Karlsson, R., Xuong, N., Taylor, S.S. & Sowadski, J.M. (1993) Biochemistry, in the press]. These studies revealed that the adenosine portion of ATP is buried deep within the catalytic cleft, with the alpha, beta and gamma phosphates protruding towards the opening of the cleft. The unique spatial positioning of MgATP within the catalytic cleft of cyclic A kinase and its interactions with conserved amino acids found in all protein kinases, led us to reconsider the use of ATP as an affinity ligand for the purification of these enzymes. In this paper, we describe a straightforward method for the synthesis of [gamma-32P]adenosine-5'-(gamma-4-aminophenyl)triphosphate for the covalent linkage of ATP to Sepharose through its gamma phosphate. In the presence of 20 microM ATP, adenosine-5'-(gamma-4-aminophenyl)triphosphate exhibited apparent Ki values of 103.6, 75.18, 176.28 and 120.00 microM against cyclic A kinase, mitogen-activated protein kinase (p42mapk), mitogen-activated protein kinase kinase and p60c-src, respectively. To illustrate the effectiveness of adenosine-5'-(gamma-4-aminophenyl)triphosphate-Sepharose as an affinity column for protein kinases, we have used the resin to purify rabbit skeletal muscle mitogen-activated protein kinase kinase over 19000-fold to homogeneity.

Authors
Haystead, CM; Gregory, P; Sturgill, TW; Haystead, TA
MLA Citation
Haystead, CM, Gregory, P, Sturgill, TW, and Haystead, TA. "Gamma-phosphate-linked ATP-sepharose for the affinity purification of protein kinases. Rapid purification to homogeneity of skeletal muscle mitogen-activated protein kinase kinase." Eur J Biochem 214.2 (June 1, 1993): 459-467.
PMID
8513796
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
214
Issue
2
Publish Date
1993
Start Page
459
End Page
467

Functional expression of a MAP kinase kinase in COS cells and recognition by an anti-STE7/byr1 antibody.

Mitogen-activated protein (MAP) kinases p42mapk and p44mapk are activated by dual tyrosine and threonine phosphorylation in vivo. Both MAPKs are phosphorylated and activated in vitro by an activator recently identified as a protein-tyrosine/threonine kinase. We have isolated a putative cDNA for a MAP kinase kinase (MAPKK) and determined its structure [Proc. Natl. Acad. Sci. USA, in press]. The protein encoded by this cDNA shares sequence homology with two yeast protein kinases byr1 and STE7. We now report that stimulation with serum of COS cells expressing this shares sequence homology with two yeast protein kinases byr1 and STE7. We now report that stimulation with serum of COS cells expressing this protein amplifies MAPK activator activity markedly. The increased activity co-migrates during chromatography with the expressed 45 kDa protein, recognized by an anti-STE7/byr1 antibody, and is abrogated by treatment with phosphatase 2A. Thus, this cDNA encodes a functional MAPKK. The anti-STE7/byr1 antibody also recognized a 46 kDa COS cell protein that was resolved from the expressed MAPKK by anion-exchange chromatography. This immunoreactive protein co-eluted with endogenous MAPKK activity, suggesting identification of the immunoreactive band as monkey MAPKK.

Authors
Haystead, CM; Wu, J; Gregory, P; Sturgill, TW; Haystead, TA
MLA Citation
Haystead, CM, Wu, J, Gregory, P, Sturgill, TW, and Haystead, TA. "Functional expression of a MAP kinase kinase in COS cells and recognition by an anti-STE7/byr1 antibody." FEBS Lett 317.1-2 (February 8, 1993): 12-16.
PMID
8428620
Source
pubmed
Published In
FEBS Letters
Volume
317
Issue
1-2
Publish Date
1993
Start Page
12
End Page
16

THE ROLE OF PROTEIN PHOSPHATASES IN THE SUPPRESSION OF THE MAP KINASE SIGNAL TRANSDUCTION PATHWAY

Authors
HAYSTEAD, TAJ
MLA Citation
HAYSTEAD, TAJ. "THE ROLE OF PROTEIN PHOSPHATASES IN THE SUPPRESSION OF THE MAP KINASE SIGNAL TRANSDUCTION PATHWAY." JOURNAL OF CELLULAR BIOCHEMISTRY (January 9, 1993): 310-310.
Source
wos-lite
Published In
Journal of Cellular Biochemistry
Publish Date
1993
Start Page
310
End Page
310

Molecular structure of a protein-tyrosine/threonine kinase activating p42 mitogen-activated protein (MAP) kinase: MAP kinase kinase.

MAP kinases p42mapk and p44mapk participate in a protein kinase cascade(s) important for signaling in many cell types and contexts. Both MAP kinases are activated in vitro by MAP kinase kinase, a protein-tyrosine and threonine kinase. A MAP kinase kinase cDNA was isolated from a rat kidney library by using peptide sequence data we obtained from MAP kinase kinase isolated from rabbit skeletal muscle. The deduced sequence, containing 393 amino acids (predicted mass, 43.5 kDa), is most similar to byr1 (Bypass of ras1), a yeast protein kinase functioning in the mating pathway induced by pheromones in Schizosaccharomyces pombe. An unusually large insert is present in MAP kinase kinase between domains IX and X and may contribute to protein-protein interactions with MAP kinase. Major (2.7 kilobases) and minor (1.7 kilobases) transcripts are widely expressed in rat tissues and appear to be derived from a single gene.

Authors
Wu, J; Harrison, JK; Vincent, LA; Haystead, C; Haystead, TA; Michel, H; Hunt, DF; Lynch, KR; Sturgill, TW
MLA Citation
Wu, J, Harrison, JK, Vincent, LA, Haystead, C, Haystead, TA, Michel, H, Hunt, DF, Lynch, KR, and Sturgill, TW. "Molecular structure of a protein-tyrosine/threonine kinase activating p42 mitogen-activated protein (MAP) kinase: MAP kinase kinase." Proc Natl Acad Sci U S A 90.1 (January 1, 1993): 173-177.
PMID
8380494
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
90
Issue
1
Publish Date
1993
Start Page
173
End Page
177

γ-Phosphate-linked ATP-Sepharose for the affinity purification of protein kinases. Rapid purification to homogeneity of skeletal muscle mitogen-activated protein kinase kinase

Recently, Sowadski and colleagues [Knighton, D.R., Zheng, J., Eyck, L.F.T., Ashford, V.A., Xuong, N., Taylor, S.S. and Sowadski, J.M. (1991) Science 407, 407-420] reported the structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase (cyclic A kinase), MgATP and a 20-residue inhibitor peptide, at a resolution of 0.27 nm. This structure has since been refined to 0.2-nm resolution and the orientation of the nucleotide and interactions of MgATP with numerous conserved residues at the active site defined [Zheng, J., Knighton, D.R., Eyck, L.F.T., Karlsson, R., Xuong, N., Taylor, S.S. and Sowadski, J.M. (1993) Biochemistry, in the press]. These studies revealed that the adenosine portion of ATP is buried deep within the catalytic cleft, with the α, β and γ phosphates protruding towards the opening of the cleft. The unique spatial positioning of MgATP within the catalytic cleft of cyclic A kinase and its interactions with conserved amino acids found in all protein kinases, led us to reconsider the use of ATP as an affinity ligand for the purification of these enzymes. In this paper, we describe a straightforward method for the synthesis of [γ-32P]adenosine-5'-(γ-4-aminophenyl)triphosphate for the covalent linkage of ATP to Sepharose through its γ phosphate. In the presence of 20 μM ATP, adenosine-5'-(γ-4-aminophenyl)triphosphate exhibited apparent K(i) values of 103.6, 75.18, 176.28 and 120.00 μM against cyclic A kinase, mitogen-activated protein kinase (p42(mapk)), mitogen-activated protein kinase kinase and p60(c-src), respectively. To illustrate the effectiveness of adenosine-5'-(γ-4-aminophenyl)triphosphate-Sepharose as an affinity column for protein kinases, we have used the resin to purify rabbit skeletal muscle mitogen-activated protein kinase kinase over 19000-fold to homogeneity.

Authors
Haystead, CMM; Gregory, P; Sturgill, TW; Haystead, TAJ
MLA Citation
Haystead, CMM, Gregory, P, Sturgill, TW, and Haystead, TAJ. "γ-Phosphate-linked ATP-Sepharose for the affinity purification of protein kinases. Rapid purification to homogeneity of skeletal muscle mitogen-activated protein kinase kinase." European Journal of Biochemistry 214.2 (1993): 459-467.
Source
scival
Published In
European Journal of Biochemistry
Volume
214
Issue
2
Publish Date
1993
Start Page
459
End Page
467

Activation of MAP kinase by a dual specificity Tyr/Thr kinase.

Authors
Wu, J; Michel, H; Dent, P; Haystead, T; Hunt, DF; Sturgill, TW
MLA Citation
Wu, J, Michel, H, Dent, P, Haystead, T, Hunt, DF, and Sturgill, TW. "Activation of MAP kinase by a dual specificity Tyr/Thr kinase." Advances in second messenger and phosphoprotein research 28 (1993): 219-225.
PMID
8398407
Source
scival
Published In
Advances in second messenger and phosphoprotein research
Volume
28
Publish Date
1993
Start Page
219
End Page
225

Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro.

Mitogen-activated protein (MAP) kinases are 42- and 44-kD serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation in cells stimulated with mitogens and growth factors. MAP kinase and the protein kinase that activates it (MAP kinase kinase) were constitutively activated in NIH 3T3 cells infected with viruses containing either of two oncogenic forms (p35EC12, p3722W) of the c-Raf-1 protein kinase. The v-Raf proteins purified from cells infected with EC12 or 22W viruses activated MAP kinase kinase from skeletal muscle in vitro. Furthermore, a bacterially expressed v-Raf fusion protein (glutathione S-transferase-p3722W) also activated MAP kinase kinase in vitro. These findings suggest that one function of c-Raf-1 in mitogenic signaling is to phosphorylate and activate MAP kinase kinase.

Authors
Dent, P; Haser, W; Haystead, TA; Vincent, LA; Roberts, TM; Sturgill, TW
MLA Citation
Dent, P, Haser, W, Haystead, TA, Vincent, LA, Roberts, TM, and Sturgill, TW. "Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro." Science 257.5075 (September 4, 1992): 1404-1407.
PMID
1326789
Source
pubmed
Published In
Science
Volume
257
Issue
5075
Publish Date
1992
Start Page
1404
End Page
1407

Ordered phosphorylation of p42mapk by MAP kinase kinase.

Preparation of milligram amounts of [32P]p42mapk, phosphorylated at Tyr185 or diphosphorylated at Tyr185/Thr183, for use as specific protein phosphatase substrates is described. Tyr- but not Thr-phosphorylated p42mapk, accumulates when ATP is limiting. Furthermore, Tyr185-phosphorylated p42mapk exhibits an apparent 10-fold decrease in apparent Km (46.6 +/- 6.6 nM) for MAP kinase kinase compared to that for the dephospho form (approximately 476 nM). We conclude that Tyr185 precedes Thr183 phosphorylation, and that this is prerequisite, dramatically increasing the affinity of p42mapk for MAP kinase kinase.

Authors
Haystead, TA; Dent, P; Wu, J; Haystead, CM; Sturgill, TW
MLA Citation
Haystead, TA, Dent, P, Wu, J, Haystead, CM, and Sturgill, TW. "Ordered phosphorylation of p42mapk by MAP kinase kinase." FEBS Lett 306.1 (July 13, 1992): 17-22.
PMID
1628739
Source
pubmed
Published In
FEBS Letters
Volume
306
Issue
1
Publish Date
1992
Start Page
17
End Page
22

Renaturation and partial peptide sequencing of mitogen-activated protein kinase (MAP kinase) activator from rabbit skeletal muscle

Mitogen-activated protein kinase (MAP kinase) activator was purified 2000-fold from skeletal muscle, and proteins which co-purified with the activator were analysed after SDS/PAGE by renaturation and partial sequencing. Activity tor tyrosine and threonine phosphorylation of MAP kinase was present in two bands of approx. 48 and 46 kDa, which have sequence similarity to small GTP-binding protein p25 GDP dissociation inhibitor and protein kinases (PBS2, SPK1+, STE7, BYR1) respectively.

Authors
Wu, J; Michel, H; Rossomando, A; Haystead, T; Shabanwitz, J; Hunt, DF; Sturgill, TW
MLA Citation
Wu, J, Michel, H, Rossomando, A, Haystead, T, Shabanwitz, J, Hunt, DF, and Sturgill, TW. "Renaturation and partial peptide sequencing of mitogen-activated protein kinase (MAP kinase) activator from rabbit skeletal muscle." Biochemical Journal 285.3 (1992): 701-705.
PMID
1379797
Source
scival
Published In
Biochemical Journal
Volume
285
Issue
3
Publish Date
1992
Start Page
701
End Page
705

Activation of messenger-independent protein kinases in wild-type and phorbol ester-resistant EL4 thymoma cells.

Phorbol esters, acting via activation of the protein kinase C family of protein serine/threonine kinases, are able to exert profound effects on various cellular functions. In this study, we used the EL4 thymoma cell line to study the potential role of "downstream" protein serine/threonine kinases in cellular responses to phorbol esters. In wild-type EL4 cells, addition of phorbol ester caused a rapid activation of kinase activity toward RRLSSLRA (S6P). This increased activity was maintained for at least 15 min but diminished to control levels by 60 min. Activation of a myelin basic protein (MBP) kinase was also seen in response to phorbol ester. In a variant EL4 cell line in which phorbol ester does not induce interleukin 2 transcription, phorbol ester failed to activate either the S6P kinase or MBP kinase. Partial purification of the activated S6P and MBP kinases from wild-type cells showed that they represent separate enzymes that are distinct from protein kinase C. Although the variant cells had reduced levels of protein kinase C as compared with the wild-type cells, the amount of membrane-bound enzyme increased in response to phorbol 12-myristate 13-acetate in both wild-type and variant cells. Treatment of intact cells with phorbol ester resulted in phosphorylation of some of the same protein substrates in both cell lines. Okadaic acid, a phosphatase inhibitor, increased S6P and MBP kinase activities in both wild-type and variant cells. Thus, phorbol ester failed to activate the S6P and MBP kinases in the variant cells even though these cells express activatable protein kinase C, S6P kinase, and MBP kinase. Two protein kinase inhibitors, staurosporine and H-7, inhibited the activity of all three kinases in vitro, while a peptide inhibitor (PKC 19-31) showed specificity for protein kinase C. In summary, these results suggest that activation of messenger-independent protein kinases may be critical for certain protein kinase C-dependent responses.

Authors
Meier, KE; Licciardi, KA; Haystead, TA; Krebs, EG
MLA Citation
Meier, KE, Licciardi, KA, Haystead, TA, and Krebs, EG. "Activation of messenger-independent protein kinases in wild-type and phorbol ester-resistant EL4 thymoma cells." J Biol Chem 266.3 (January 25, 1991): 1914-1920.
PMID
1988454
Source
pubmed
Published In
The Journal of biological chemistry
Volume
266
Issue
3
Publish Date
1991
Start Page
1914
End Page
1920

Use of okadaic acid to inhibit protein phosphatases in intact cells.

Authors
Hardie, DG; Haystead, TA; Sim, AT
MLA Citation
Hardie, DG, Haystead, TA, and Sim, AT. "Use of okadaic acid to inhibit protein phosphatases in intact cells." Methods Enzymol 201 (1991): 469-476.
PMID
1658560
Source
pubmed
Published In
Methods in Enzymology
Volume
201
Publish Date
1991
Start Page
469
End Page
476

Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal.

Treatment of adipocytes with okadaic acid (a specific inhibitor of type 1 and 2a protein phosphatases) resulted in a rapid 8-10-fold stimulation of cell extract myelin basic protein (MBP) kinase activity (t1/2 = 10 min) and kinase activity toward a synthetic peptide RRLSSLRA (S6 peptide) (t1/2 = 5 min). Insulin brought about a smaller stimulation of these two activities (t1/2 = 2.5 min). MBP kinase activity from cells treated with okadaic acid or insulin was resolved by anion exchange chromatography into two well defined peaks; S6 peptide kinase activity was less well resolved. The two partially purified MBP kinases were inactivated by the protein tyrosine phosphatase CD45 or by protein phosphatase 2a (PP-2a). In contrast, partially purified S6 peptide kinase activity was inactivated only by PP-2a or protein phosphatase 1 (PP-1). Furthermore, a 38-kDa protein which co-eluted with one peak of MBP kinase and a 42-kDa protein which co-eluted with the other peak of MBP kinase were phosphorylated on tyrosine after treatment with okadaic acid. These findings illustrate several important points concerning regulation of MBP and S6 peptide kinases. First, these protein kinases are regulated by phosphorylation, and, second, in the absence of hormonal stimuli their activities are strongly suppressed by protein phosphatases. Lastly, the increased tyrosine phosphorylation accompanying the activation of MBP kinases following okadaic acid treatment suggests a role for PP-2a in events that are mediated by tyrosine phosphorylation.

Authors
Haystead, TA; Weiel, JE; Litchfield, DW; Tsukitani, Y; Fischer, EH; Krebs, EG
MLA Citation
Haystead, TA, Weiel, JE, Litchfield, DW, Tsukitani, Y, Fischer, EH, and Krebs, EG. "Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes. The role of protein phosphatase 2a in attenuation of the signal." J Biol Chem 265.27 (September 25, 1990): 16571-16580.
PMID
2168895
Source
pubmed
Published In
The Journal of biological chemistry
Volume
265
Issue
27
Publish Date
1990
Start Page
16571
End Page
16580

Roles of the AMP-activated and cyclic-AMP-dependent protein kinases in the adrenaline-induced inactivation of acetyl-CoA carboxylase in rat adipocytes.

1. In isolated rat adipocytes, acetyl-CoA carboxylase is inactivated by treatment of the cells with adrenaline or the beta-agonist isoproterenol, but not by the alpha-agonist phenylephrine. The inactivation is stable during purification in the presence of protein phosphatase inhibitors, and is associated with a 30-40% increase in the labelling of enzyme isolated from 32P-labelled cells. 2. Increased phosphorylation occurs within peptide T1, which was identified by sequencing to be the peptide Ser-Ser77-Met-Ser79-Gly-Leu-His-Leu-Val-Lys, containing Ser-77 (phosphorylated by cyclic-AMP-dependent protein kinase) and Ser-79 (phosphorylated by the AMP-activated protein kinase). Analysis of the release of radioactivity as free phosphate during Edman degradation of peptide T1 revealed that all of the phosphate was in Ser-79 in both basal and hormone- or agonist-stimulated cells. Treatment of adipocytes with various agents which activate cyclic-AMP-dependent protein kinase by receptor-independent mechanisms (forskolin, cyclic AMP analogues, isobutylmethylxanthine) also produced inactivation of acetyl-CoA carboxylase and increased phosphorylation at Ser-79. 3. The (Rp)-[thio]phosphate analogue of cyclic AMP, which is an antagonist of binding of cyclic AMP to the regulatory subunit of cyclic-AMP-dependent protein kinase, opposes the effect of adrenaline on phosphorylation and inactivation of acetyl-CoA carboxylase. Together with the effects of isobutylmethylxanthine and the stimulatory cyclic AMP analogues, this strongly indicates that cyclic-AMP-dependent protein kinase is an essential component of the signal transduction pathway, although clearly it does not directly phosphorylate acetyl-CoA carboxylase. 4. As shown by okadaic acid inhibition, greater than 95% of the acetyl-CoA carboxylase phosphatase activity in extracts of rat adipocytes or liver is accounted for by protein phosphatase-2A, with less than 5% attributable to protein phosphatase-1. Inhibition of protein phosphatase-1 via phosphorylation of inhibitor-1 is therefore unlikely to be the mechanism by which cyclic-AMP-dependent protein kinase indirectly increases phosphorylation of acetyl-CoA carboxylase. Various other potential mechanisms are discussed.

Authors
Haystead, TA; Moore, F; Cohen, P; Hardie, DG
MLA Citation
Haystead, TA, Moore, F, Cohen, P, and Hardie, DG. "Roles of the AMP-activated and cyclic-AMP-dependent protein kinases in the adrenaline-induced inactivation of acetyl-CoA carboxylase in rat adipocytes." Eur J Biochem 187.1 (January 12, 1990): 199-205.
PMID
1688796
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
187
Issue
1
Publish Date
1990
Start Page
199
End Page
205

Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism.

Okadaic acid is a polyether derivative of 38-carbon fatty acid, and is implicated as the causative agent of diarrhetic shellfish poisoning. It is a potent tumour promoter that is not an activator of protein kinase C, but is a powerful inhibitor of protein phosphatases-1 and -2A (PP1 and PP2A) in vitro. We report here that okadaic acid rapidly stimulates protein phosphorylation in intact cells, and behaves like a specific protein phosphatase inhibitor in a variety of metabolic processes. Our results indicate that PP1 and PP2A are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo. We also find that okadaic acid mimics the effect of insulin on glucose transport in adipocytes, which suggests that this process is stimulated by a serine/threonine phosphorylation event.

Authors
Haystead, TA; Sim, AT; Carling, D; Honnor, RC; Tsukitani, Y; Cohen, P; Hardie, DG
MLA Citation
Haystead, TA, Sim, AT, Carling, D, Honnor, RC, Tsukitani, Y, Cohen, P, and Hardie, DG. "Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism." Nature 337.6202 (January 5, 1989): 78-81.
PMID
2562908
Source
pubmed
Published In
Nature
Volume
337
Issue
6202
Publish Date
1989
Start Page
78
End Page
81
DOI
10.1038/337078a0

Epidermal growth factor alters metabolism of inositol lipids and activity of protein kinase C in mouse embryo palate mesenchyme cells.

Epidermal growth factor (EGF) stimulated mouse embryo palate mesenchyme (MEPM) cells (1) to incorporate [32P]O4(3-) into phosphatidylinositol (PI), phosphatidylcholine, and phosphatidic acid over a period of 60 min; 2) to incorporate [32P]O4(3-) into polyphosphoinositides as a function of time; and 3) to incorporate [32P]O4(-3) into PI, only, as a function of concentration when the period of stimulation was kept short. EGF stimulated the release of radiolabeled inositol phosphates from MEPM cells that had been radiolabeled with [3H]myoinositol. The release of inositol 1-phosphate was sustained over a period of at least 60 min, whereas the release of inositol 1,4-bisphosphate and inositol trisphosphate peaked during the first 10 min of stimulation. EGF also stimulated phosphorylation of an Mr 80,000 protein whose pI, phosphopeptide map, and phosphoamino acid pattern were identical to those of an Mr 80,000 protein phosphorylated in response to phorbol 12-myristate 13-acetate. Mobilization or metabolism of arachidonic acid was not stimulated under the same conditions that permitted EGF to alter inositol lipid metabolism. We interpret these data to mean that 1) in contrast to the findings with some cell lines, alterations in inositol lipid metabolism may be part of the signalling mechanism for EGF in embryonic cells; 2) EGF is capable of activating inositol-dependent signalling pathways leading to activation of protein kinase C in MEPM cells; and 3) mobilization and metabolism of arachidonic acid are not an inherent part of this signalling mechanism.

Authors
Chepenik, KP; Haystead, TA
MLA Citation
Chepenik, KP, and Haystead, TA. "Epidermal growth factor alters metabolism of inositol lipids and activity of protein kinase C in mouse embryo palate mesenchyme cells." J Craniofac Genet Dev Biol 9.3 (1989): 285-301.
PMID
2613862
Source
pubmed
Published In
Journal of craniofacial genetics and developmental biology
Volume
9
Issue
3
Publish Date
1989
Start Page
285
End Page
301

Analysis of sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Comparison with sites phosphorylated by casein kinase-2 and the calmodulin-dependent multiprotein kinase.

We have examined the sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P-labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the peptide containing the sites phosphorylated in vitro by casein kinase-2. The other may correspond to the 'I' site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme-bound phosphate (equivalent to less than 0.1 mol phosphate/mol 240-kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic-AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase respectively. We have sequenced peptides T4a and T4b from acetyl-CoA carboxylase derived from control and insulin-treated adipocytes, and also after phosphorylation in vitro with casein kinase-2 and the calmodulin-dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe-Ile-Ile-Gly-Ser4-Val-Ser5-Gln-Asp-Asn-Ser6-Glu-Asp -Glu-Ile-Ser-Asn-Leu-. Site 5 was phosphorylated by the calmodulin-dependent protein kinase and site 6 by casein kinase-2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P-labelling of T4a. We were unable to isolate sufficient quantities of the other insulin-sensitive peptide to determine its sequence. Our results are consistent with the idea that insulin activates either casein kinase-2, or a protein kinase which has the same specificity as casein kinase-2. The function of this modification is not clear, since phosphorylation by casein kinase-2 has no direct effect on acetyl-CoA carboxylase activity.

Authors
Haystead, TA; Campbell, DG; Hardie, DG
MLA Citation
Haystead, TA, Campbell, DG, and Hardie, DG. "Analysis of sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Comparison with sites phosphorylated by casein kinase-2 and the calmodulin-dependent multiprotein kinase." Eur J Biochem 175.2 (August 1, 1988): 347-354.
PMID
2900140
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
175
Issue
2
Publish Date
1988
Start Page
347
End Page
354

Insulin and phorbol ester stimulate phosphorylation of acetyl-CoA carboxylase at similar sites in isolated adipocytes. Lack of correspondence with sites phosphorylated on the purified enzyme by protein kinase C.

1. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) stimulates fatty acid synthesis from glucose in isolated adipocytes with a half-maximal effect at 0.72 microM. In seven batches of cells, the maximal effects of TPA and insulin were 8.5 +/- 1.1-fold and 27.1 +/- 2.1-fold respectively. Insulin also stimulated fatty acid synthesis from acetate 8.9 +/- 0.5-fold (three experiments), but TPA did not significantly increase fatty acid synthesis from this precursor. 2. In contrast to insulin, TPA treatment of isolated adipocytes did not produce an activation of acetyl-CoA carboxylase which was detectable in crude cell extracts. 3. The total phosphate content of acetyl-CoA carboxylase, isolated from adipocytes in the presence of protein phosphatase inhibitors, was estimated by 32P-labelling experiments to be 2.6 +/- 0.1 (5), 3.4 +/- 0.2 (5), and 3.8 +/- 0.2 (3) mol/mol subunit for enzyme from control, insulin- and TPA-treated cells respectively. Insulin and TPA stimulated phosphorylation within the same two tryptic peptides. 4. Purified acetyl-CoA carboxylase is phosphorylated in vitro by protein kinase C at serine residues which are recovered in three tryptic peptides, i.e. peptide T1, which appears to be identical with the peptide Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys phosphorylated by cyclic-AMP-dependent protein kinase, and peptides Ta and Tb, which have the sequences Ile-Asp-Ser(P)-Gln-Arg and Lys-Ile-Asp-Ser(P)-Gln-Arg respectively, and which appear to be derived from a single site by alternative cleavages. None of these correspond to the peptides whose 32P-labelling increase in response to insulin or TPA. Peptides Ta/Tb are not significantly phosphorylated in isolated adipocytes, even after insulin or TPA treatment. Peptide T1 is phosphorylated in isolated adipocytes, but this phosphorylation is not altered by insulin or TPA. 5. These results show that TPA mimics the effect of insulin on phosphorylation, but not activation, of acetyl-CoA carboxylase, i.e. that these two events can be dissociated. In addition, phorbol ester stimulates phosphorylation of acetyl-CoA carboxylase in isolated adipocytes, but this is not catalyzed directly by protein kinase C, and acetyl-CoA carboxylase does not appear to be a physiological substrate for this kinase.

Authors
Haystead, TA; Hardie, DG
MLA Citation
Haystead, TA, and Hardie, DG. "Insulin and phorbol ester stimulate phosphorylation of acetyl-CoA carboxylase at similar sites in isolated adipocytes. Lack of correspondence with sites phosphorylated on the purified enzyme by protein kinase C." Eur J Biochem 175.2 (August 1, 1988): 339-345.
PMID
2900139
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
175
Issue
2
Publish Date
1988
Start Page
339
End Page
345

REGULATION OF ACETYL-COA CARBOXYLASE AND HMG-COA REDUCTASE BY AMP-ACTIVATED PROTEIN-KINASE

Authors
HARDIE, DG; CARLING, D; HAYSTEAD, TAJ; MUNDAY, MR; CAMPBELL, DG
MLA Citation
HARDIE, DG, CARLING, D, HAYSTEAD, TAJ, MUNDAY, MR, and CAMPBELL, DG. "REGULATION OF ACETYL-COA CARBOXYLASE AND HMG-COA REDUCTASE BY AMP-ACTIVATED PROTEIN-KINASE." FASEB JOURNAL 2.6 (March 25, 1988): A1855-A1855.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
2
Issue
6
Publish Date
1988
Start Page
A1855
End Page
A1855

EVIDENCE FOR CASEIN KINASE-2 MEDIATED PHOSPHORYLATION OF ACETYL-COA CARBOXYLASE IN INSULIN-TREATED ADIPOCYTES

Authors
HAYSTEAD, TAJ; HARDIE, DG
MLA Citation
HAYSTEAD, TAJ, and HARDIE, DG. "EVIDENCE FOR CASEIN KINASE-2 MEDIATED PHOSPHORYLATION OF ACETYL-COA CARBOXYLASE IN INSULIN-TREATED ADIPOCYTES." FASEB JOURNAL 2.5 (March 20, 1988): A993-A993.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
2
Issue
5
Publish Date
1988
Start Page
A993
End Page
A993

ACTIVATION OF ACETYL-COA CARBOXYLASE BY INSULIN-ALLOSTERIC EFFECT OR PROTEIN-PHOSPHORYLATION

Authors
HAYSTEAD, TAJ; HARDIE, DG
MLA Citation
HAYSTEAD, TAJ, and HARDIE, DG. "ACTIVATION OF ACETYL-COA CARBOXYLASE BY INSULIN-ALLOSTERIC EFFECT OR PROTEIN-PHOSPHORYLATION." ARCHIVES INTERNATIONALES DE PHYSIOLOGIE DE BIOCHIMIE ET DE BIOPHYSIQUE 95.5 (December 1987): B209-B209.
Source
wos-lite
Published In
Archives Internationales de Physiologie et de Biochimie
Volume
95
Issue
5
Publish Date
1987
Start Page
B209
End Page
B209

Evidence that activation of acetyl-CoA carboxylase by insulin in adipocytes is mediated by a low-Mr effector and not by increased phosphorylation.

The activation of acetyl-CoA carboxylase (measured in a crude supernatant fraction) caused by insulin treatment of adipocytes was completely unaffected by the addition of a large amount of highly purified protein phosphatase to the supernatant fraction. Under the same conditions the inhibition of acetyl-CoA carboxylase by adrenaline was totally reversed. Experiments with 32P-labelled adipocytes showed that insulin increased the total phosphorylation of acetyl-CoA carboxylase from 2.7 to 3.5 molecules of phosphate/240 kDa subunit, and confirmed that this increase was partially accounted for by phosphorylation within a specific peptide (the 'I-site' peptide). Protein phosphatase treatment of the crude supernatant fractions removed over 80% of the 32P radioactivity from the enzyme and removed all detectable radioactivity from the I-site peptide. The effect of insulin on acetyl-CoA carboxylase activity, but not the effect on phosphorylation, was lost on purification of the enzyme on avidin-Sepharose. The effect on enzyme activity was also lost if crude supernatant fractions were subjected to rapid gel filtration after treatment under conditions of high ionic strength, similar to those used in the avidin-Sepharose procedure. These results show that, although insulin does increase the phosphorylation of acetyl-CoA carboxylase at a specific site, this does not cause enzyme activation. They suggest instead that activation of the enzyme by insulin is mediated by a tightly bound low-Mr effector which dissociates from the enzyme at high ionic strength.

Authors
Haystead, TA; Hardie, DG
MLA Citation
Haystead, TA, and Hardie, DG. "Evidence that activation of acetyl-CoA carboxylase by insulin in adipocytes is mediated by a low-Mr effector and not by increased phosphorylation." Biochem J 240.1 (November 15, 1986): 99-106.
PMID
2881538
Source
pubmed
Published In
The Biochemical journal
Volume
240
Issue
1
Publish Date
1986
Start Page
99
End Page
106

The role of phosphorylation/dephosphorylation of acetyl-CoA carboxylase in the regulation of mammalian fatty acid biosynthesis.

Authors
Munday, MR; Haystead, TA; Holland, R; Carling, DA; Hardie, DG
MLA Citation
Munday, MR, Haystead, TA, Holland, R, Carling, DA, and Hardie, DG. "The role of phosphorylation/dephosphorylation of acetyl-CoA carboxylase in the regulation of mammalian fatty acid biosynthesis." Biochem Soc Trans 14.3 (June 1986): 559-562.
PMID
2874088
Source
pubmed
Published In
Biochemical Society transactions
Volume
14
Issue
3
Publish Date
1986
Start Page
559
End Page
562

Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay.

Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect on acetyl-CoA carboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.

Authors
Haystead, TA; Hardie, DG
MLA Citation
Haystead, TA, and Hardie, DG. "Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay." Biochem J 234.2 (March 1, 1986): 279-284.
PMID
2872882
Source
pubmed
Published In
The Biochemical journal
Volume
234
Issue
2
Publish Date
1986
Start Page
279
End Page
284

Further consideration of the learning impairment after aceperone in the marmoset: effects of the drug on shape and colour discrimination and on an alternation task.

Ten marmosets (Callithrix jacchus) learned to discriminate between pairs of small grey objects differing only in shape or small plain plaques differing only in colour, in a Wisconsin General Test Apparatus. Each day, each animal was presented with three consecutive visual discrimination problems in the order shape-colour-shape or colour-shape-colour. After aceperone, an alpha-noradrenergic antagonist, animals were impaired at learning the first but not the subsequent tasks of each trio. These results suggest that the previously observed impairment [10] on the first of a pair of object discrimination tasks after aceperone is a consequence of disruption of a mechanism common to both shape and colour discrimination learning. The fact that there is no impairment on task 2 in a dimension differing from task 1 suggests that the deficit is not one of attending to, or switching attention to, the appropriate visual dimension. Three further marmosets were trained to perform an alternation task and tested under aceperone. No impairment in performance was seen, suggesting that a variety of cognitive skills other than stimulus-reward association were intact. We conclude that the impairment following aceperone is a dysfunction of processes involved in association formation, but that it is one which is manifest only when the animal is faced with a type of task which has not recently been performed and that it can be overcome with persistence even the animal encounters novel stimuli.

Authors
Baker, HF; Ridley, RM; Haystead, TA; Crow, TJ
MLA Citation
Baker, HF, Ridley, RM, Haystead, TA, and Crow, TJ. "Further consideration of the learning impairment after aceperone in the marmoset: effects of the drug on shape and colour discrimination and on an alternation task." Pharmacol Biochem Behav 18.5 (May 1983): 701-704.
PMID
6222386
Source
pubmed
Published In
Pharmacology Biochemistry and Behavior
Volume
18
Issue
5
Publish Date
1983
Start Page
701
End Page
704

A new approach to the role of noradrenaline in learning: problem-solving in the marmoset after alpha-noradrenergic receptor blockade.

Nine marmosets (Callithrix jacchus) were tested on a variety of visual discrimination learning tasks in a Wisconsin General Test Apparatus with or without alpha-noradrenergic receptor blockade achieved by the administration of aceperone. After aceperone, animals were found to be severely and consistently impaired at learning the first task of each test session and to be impaired on new and repeated reversal learning. They were, however, unimpaired on learning another similar task in each test session and on performance of a well-learnt task. Results were interpreted as evidence for defective association formation which can be compensated for by suitable priming or practice.

Authors
Ridley, RM; Haystead, TA; Baker, HF; Crow, TJ
MLA Citation
Ridley, RM, Haystead, TA, Baker, HF, and Crow, TJ. "A new approach to the role of noradrenaline in learning: problem-solving in the marmoset after alpha-noradrenergic receptor blockade." Pharmacol Biochem Behav 14.6 (June 1981): 849-855.
PMID
6114497
Source
pubmed
Published In
Pharmacology Biochemistry and Behavior
Volume
14
Issue
6
Publish Date
1981
Start Page
849
End Page
855

An analysis of visual object reversal learning in the marmoset after amphetamine and haloperidol.

The effect of amphetamine and haloperidol pretreatment on visual object reversal learning was assessed in the marmoset. Amphetamine induced perseverative responding demonstrated by high reversal learning scores and worse than chance performance in the early stages of reversal. This perseverative responding was prevented by pretreatment with haloperidol. Haloperidol, either alone or in conjunction with amphetamine caused a mild, non-perseverative impairment on reversal learning only.

Authors
Ridley, RM; Haystead, TA; Baker, HF
MLA Citation
Ridley, RM, Haystead, TA, and Baker, HF. "An analysis of visual object reversal learning in the marmoset after amphetamine and haloperidol." Pharmacol Biochem Behav 14.3 (March 1981): 345-351.
PMID
6785766
Source
pubmed
Published In
Pharmacology Biochemistry and Behavior
Volume
14
Issue
3
Publish Date
1981
Start Page
345
End Page
351

An involvement of dopamine in higher order choice mechanisms in the monkey.

Low doses of amphetamine induce choice perseveration in an object discrimination task under conditions where such perseveration either increases or decreases the number of rewards obtained as compared to chance performance. Neither stereotyped motor actions nor repetitive choice of position contributed to this effect which could be blocked by pre-treatment with the neuroleptic haloperidol. These results demonstrate that higher order choice mechanisms may involve dopamine systems in the primate.

Authors
Ridley, RM; Haystead, TA; Baker, HF
MLA Citation
Ridley, RM, Haystead, TA, and Baker, HF. "An involvement of dopamine in higher order choice mechanisms in the monkey." Psychopharmacology (Berl) 72.2 (1981): 173-177.
PMID
6782607
Source
pubmed
Published In
Psychopharmacology
Volume
72
Issue
2
Publish Date
1981
Start Page
173
End Page
177

Perseverative behaviour after amphetamine; dissociation of response tendency from reward association.

Low doses of amphetamine were found to alter the ability of marmosets to take account of changes in reward values of object stimuli in a visual discrimination task. Under amphetamine, animals changed their motor responses and stimulus choice in order to preserve the acquired reward value or meaning of certain stimuli. These results suggest that the perseverative effect of amphetamine on behaviour is due to impaired cognitive flexibility rather than to an enhancement of motor habit.

Authors
Ridley, RM; Baker, HF; Haystead, TA
MLA Citation
Ridley, RM, Baker, HF, and Haystead, TA. "Perseverative behaviour after amphetamine; dissociation of response tendency from reward association." Psychopharmacology (Berl) 75.3 (1981): 283-286.
PMID
6798619
Source
pubmed
Published In
Psychopharmacology
Volume
75
Issue
3
Publish Date
1981
Start Page
283
End Page
286

"Go here-to there" performance after amphetamine: the importance of the response requirement in successive discrimination.

Marmosets were trained on a task involving simultaneous and successive visual discrimination performance where responses were required on all trials. Performance of this task was not affected by low doses of amphetamine. From this it is concluded that amphetamine does not cause a narrowing of attention and that the disruptive effect of amphetamine on the "go-no go" successive discrimination task already reported is due to a loss of response inhibition rather than to difficulties in the recognition of stimuli presented without a comparison stimulus.

Authors
Ridley, RM; Weight, ML; Haystead, TA; Baker, HF
MLA Citation
Ridley, RM, Weight, ML, Haystead, TA, and Baker, HF. ""Go here-to there" performance after amphetamine: the importance of the response requirement in successive discrimination." Psychopharmacology (Berl) 69.3 (1980): 271-273.
PMID
6774366
Source
pubmed
Published In
Psychopharmacology
Volume
69
Issue
3
Publish Date
1980
Start Page
271
End Page
273
Show More