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Modifications, Methods for Analysis, and Mass Spectrometry University of Windsor Scholarship at UWindsor Electronic Theses and Dissertations Theses, Dissertations, and Major Papers 2013 Functional Regulation of Dual Specificity Phosphatases: Modifications, Methods for Analysis, and Mass Spectrometry Christopher Bonham University of Windsor Follow this and additional works at: https://scholar.uwindsor.ca/etd Recommended Citation Bonham, Christopher, "Functional Regulation of Dual Specificity Phosphatases: Modifications, Methods for Analysis, and Mass Spectrometry" (2013). Electronic Theses and Dissertations. 4942. https://scholar.uwindsor.ca/etd/4942 This online database contains the full-text of PhD dissertations and Masters’ theses of University of Windsor students from 1954 forward. These documents are made available for personal study and research purposes only, in accordance with the Canadian Copyright Act and the Creative Commons license—CC BY-NC-ND (Attribution, Non-Commercial, No Derivative Works). Under this license, works must always be attributed to the copyright holder (original author), cannot be used for any commercial purposes, and may not be altered. Any other use would require the permission of the copyright holder. Students may inquire about withdrawing their dissertation and/or thesis from this database. For additional inquiries, please contact the repository administrator via email ([email protected]) or by telephone at 519-253-3000ext. 3208. Functional Regulation of Dual Specificity Phosphatases: Modifications, Methods for Analysis, and Mass Spectrometry Christopher A. Bonham A Dissertation submitted to the Faculty of Graduate Studies through the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Windsor Windsor, Ontario, Canada. 2013 © 2013 Christopher A. Bonham Functional Regulation of Dual Specificity Phosphatases: Modifications, Methods for Analysis, and Mass Spectrometry Christopher A. Bonham APPROVED BY: ______________________________________________ N.K. Tonks, External Program Examiner Cold Spring Harbor Laboratory ______________________________________________ L.A. Porter Department of Biological Sciences ______________________________________________ S. Pandey Department of Chemistry and Biochemistry ______________________________________________ B. Mutus Department of Chemistry and Biochemistry ______________________________________________ P.O. Vacratsis, Advisor Department of Chemistry and Biochemistry September 13, 2013 DECLARATION OF CO-AUTHORSHIP / PREVIOUS PUBLICATION I. Co-Authorship Declaration I hereby declare that this thesis incorporates material that is result of joint research, as follows: Chapter 3 - Collaboration of Christopher A. Bonham with Adam Faccenda as co-first authors, and Dr. Xueji Zhang, under the supervision of Dr. Panayiotis O. Vacratsis and Dr. Bulent Mutus from the University of Windsor. A.F. synthesized the gold nanoparticles, performed nitric oxide analysis, SDS-PAGE and sample preparation. C.A.B. performed all MS experiments including sample preparation for mass spectrometry (MS) and differential labeling [Scheme 2] sample preparation and analysis. C.A.B., A.F., and B.M. wrote and prepared the manuscript. C.A.B. and A.F. performed revisions and edits. P.O.V. and X.Z. provided experimental insights. Chapter 4 - Collaboration of Christopher A. Bonham with Dr. Aaron J. Steevensz as co- first authors, and Quidi Geng, under the supervision of Dr. Panayiotis O. Vacratsis from the University of Windsor. C.A.B. and P.O.V. were principal investigators of the research and co-corresponding authors. C.A.B. designed all experimental hypotheses. C.A.B. and A.J.S. performed all experiments, A.J.S. prepared mercury-immobilized affinity chromatography resin. C.A.B., A.J.S. and Q.G. performed mass spectrometry analysis. C.A.B. wrote and prepared the manuscript, A.J.S. and Q.G. prepared the figures. P.O.V. provided experimental insights and edited the manuscript. Chapter 6 - Collaboration of Christopher A. Bonham with Norah E. Franklin as co-first authors, and Besa Xhabija, under the supervision of Dr. Panayiotis O. Vacratsis from the University of Windsor. C.A.B. designed, isolated, purified and characterized α-pSer58 MTMR2 antibody. C.A.B. and N.E.F. optimized and performed in vitro kinase assays respectively. C.A.B. optimized siRNA of ERK1/2 conditions, performed knock-down immunoblot (IB) experiments and endogenous serum starve immunoprecipitation (IP) and IBs. B.X. performed siRNA and serum starve immunfluorescence (IFA). N.E.F. iii performed MAPK inhibition IPs, IBs and IFAs. C.A.B. performed hydrogen-deuterium exchange mass spectrometry experiments, epitope availability IPs and IBs, and PH- GRAM domain deletion IFAs. P.O.V., C.A.B. and N.E.F. wrote the manuscript, C.A.B. and B.X. performed revision experiments, writing and editing. I am aware of the University of Windsor Senate Policy on Authorship and I certify that I have properly acknowledged the contribution of other researchers to my thesis, and have obtained written permission from each of the co-author(s) to include the above material(s) in my thesis. I certify that, with the above qualification, this thesis, and the research to which it refers, is the product of my own work. II. Declaration of Previous Publication This thesis includes four original papers that have been previously published/submitted for publication in peer-reviewed journals, as follows: Thesis Chapter Publication title/full citation Publication status Chapter 2 Bonham, C.A. & Vacratsis, P.O., Redox Published regulation of the dual specificity phosphatase human YVH1 through disulfide bond formation, J. Biol. Chem. 284 (2009) 22853-22864. Chapter 3 ‡Faccenda, A., ‡Bonham, C.A., Vacratsis, P.O., Published Zhang, X., Mutus, B., Gold nanoparticle enrichment method for identifying S-nitrosylation and S-glutathionylation sites in proteins, J. Am. Chem. Soc. 132 (2010) 11392-11394. ‡Co-authorship iv Chapter 4 ‡†Bonham, C.A., ‡Steevensz, A.J., Geng, Q., Published †Vacratsis, P.O., Investigating Redox Regulation of Protein Tyrosine Phosphatases using Low pH Thiol Labeling and Enrichment Strategies Coupled to MALDI-TOF Mass Spectrometry, Methods (2013) August 23, [epub ahead of print] http://dx.doi.org/10.1016/j.ymeth.2013.08.014. ‡Co-authorship, †Co-corresponding authorship Chapter 6 ‡Franklin, N.E., ‡Bonham, C.A., Xhabija, B., Published Vacratsis, P.O., Differential phosphorylation of the phosphoinositide-3-phosphate MTMR2 regulates its association with early endosomal subtypes, J. Cell Sci. 126 (2013) 1333-1344. ‡Co-authorship I certify that I have obtained a written permission from the copyright owner(s) to include the above published material(s) in my thesis. I certify that the above material describes work completed during my registration as graduate student at the University of Windsor. I declare that, to the best of my knowledge, my thesis does not infringe upon anyone’s copyright nor violate any proprietary rights and that any ideas, techniques, quotations, or any other material from the work of other people included in my thesis, published or otherwise, are fully acknowledged in accordance with the standard referencing practices. Furthermore, to the extent that I have included copyrighted material that surpasses the bounds of fair dealing within the meaning of the Canada Copyright Act, I certify that I have obtained a written permission from the copyright owner(s) to include such material(s) in my thesis. I declare that this is a true copy of my thesis, including any final revisions, as approved by my thesis committee and the Graduate Studies office, and that this thesis has not been submitted for a higher degree to any other University or Institution. v ABSTRACT Protein tyrosine phosphatases (PTPs) are a diverse family of signaling molecules capable of dynamic modes of post-translational regulation. Using a combination of chemical labeling and enrichment, targeted structural mass spectrometry (MS), and immunochemistry, we have discovered unique regulatory mechanisms among two members of the Class I PTP sub-group, VH1-like dual specificity phosphatases (DUSPs). Human YVH1 (hYVH1 or DUSP12) was found to be reversibly regulated by a variety of cellular oxidants, resulting in concomitant enzymatic inactivation and zinc ejection through the formation of disulfide bonds. This was one of the first accounts of PTP oxidation outside of the active site cleft, and of disulfide exchange reactions among PTPs to prevent irreversible oxidation of the active site. Furthermore, using gold nanoparticles and mercury-immobilized metal affinity chromatography, we developed novel methods for studying PTP thiol-oxidation, which can be readily applied to alternative redox- regulated biomolecules and systems biology applications. Similarly, we have optimized the development of a library of multifaceted, low pH thiol-labeling and enrichment reagents for application in quantitative analysis of biological thiols by MS. Additionally, we have uncovered a mechanism which may participate in regulating sub-cellular localization of the lipid phosphatase myotubularin related-protein 2 (MTMR2). Specifically, we have evidence that dephosphorylation of MTMR2 Ser58 increases accessibility of key residues within a putative lipid-binding domain, which may lead to its stabilized localization to substrate-rich endosomes. Here, MTMR2 depletes phosphatidylinositol 3-phosphate (PI(3)P) substrate molecules, causing mis-localization of resident PI(3)P-binding
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