Date of Award
7-4-2023
Publication Type
Thesis
Degree Name
M.Sc.
Department
Chemistry and Biochemistry
Keywords
DUSP12;Hsp70;hYVH1;Mass Spectrometry;Proteomics;Src
Supervisor
Panayiotis Vacratsis
Rights
info:eu-repo/semantics/openAccess
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Abstract
The protein tyrosine phosphatase hYVH1 (also known as DUSP12) is an atypical member of the DUSP subfamily of PTPs that possesses a unique Cterminal zinc-binding domain and demonstrates functions in cell survival, cell cycle regulation, stress granule disassembly, and ribosome biogenesis. hYVH1 has been shown to associate with the ATPase domain of Hsp70 to amplify its cell survival capability, and recent studies have suggested that this complex can be dissociated by Src kinase phosphorylation. This study demonstrates the relevancy of Src-mediated hYVH1 phosphorylation at the endogenous level, supporting the conclusion that hYVH1 is a novel Src substrate. Label-free quantitative mass spectrometry confirmed a 29% decrease in Hsp70 co-immunoprecipitation in response to Src expression. Orthogonal structural mass spectrometry approaches were used to propose a binding interface on hYVH1 for the interaction with Hsp70, with results from limited proteolysis and differential biotin labelling indicating a peptide region between residues 50-77 on the extreme portion of the N-terminal catalytic domain. Phosphosite mapping of hYVH1 was undertaken to elucidate a mechanism by which Src may abrogate the complex, with targeted mass spectrometry methods helping to identify Tyr35 on hYVH1 as a novel Src phosphorylation site. Upon mutation of Tyr35 to a Glu residue, the hYVH1-Hsp70 complex displayed increased association, suggesting that Src phosphorylation of Tyr35 is not the regulatory mechanism for hYVH1-Hsp70 complex attenuation. These results imply that phosphorylation of Hsp70, not hYVH1, may drive dissociation, although hYVH1 may be required to recruit Src to the complex.
Recommended Citation
Luiso, Adrian A., "Investigating Structural Determinants of the hYVH1-Hsp70 Interaction Using High Resolution Mass Spectrometry" (2023). Electronic Theses and Dissertations. 9339.
https://scholar.uwindsor.ca/etd/9339