Date of Award

2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Vacratsis, Panayiotis O.

Keywords

Pure sciences, Phosphatases, Myotubularin related-protein, Dephosphorylation, Proteintyrosine phosphatases

Rights

CC BY-NC-ND 4.0

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 thioloxidation, 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)Pbinding proteins, and increased activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). We have further identified ERK1/2 as kinases capable of phosphorylating MTMR2 Ser58. This resultant ERK1/2 activation may then lead to phosphorylation of MTMR2, destabilizing endosomal interactions through competitive shielding of key residues within the lipid-binding domain required for association, and thus completing a functional negative feedback mechanism to remove MTMR2 from PI(3)P-rich early endosomes. This may, in part, be critical for achieving proper homeostatic endosomal signaling and maturation rates, acting to halt or slow maturation events to allow for increased receptor-signaling responses from these PI(3)P-depleted endosomes until an appropriate threshold is met, triggering ERK1/2-mediated attenuation of the stimulus through MTMR2 phosphorylation

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