Date of Award

Fall 2021

Publication Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

P.O. Vacratsis

Second Advisor

B. Mutus

Third Advisor

S. Ananvoranich

Keywords

Protein tyrosine phosphatase, Cellular stressors, Cellular translational fitness, Mechanism of hYVH1

Rights

info:eu-repo/semantics/openAccess

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

Abstract

The protein tyrosine phosphatase (PTP) superfamily is a major segment of the signal transduction landscape, responsible for regulating the biomolecular phosphorylation status of the cell. Diverse PTP subclasses exist, some of which are understudied and whose cellular functions are not yet fully elucidated. YVH1, an atypical PTP of the dual-specificity phosphatase (DUSP) subclass, is a pleiotropic enzyme with no known substrate. Human YVH1 (hYVH1) protects cells from cellular stressors, including heat shock and oxidative stress, regulates the cell cycle, disassembles stress granules, and acts as 60S ribosome biogenesis factor. Additionally, heat shock protein 70 (Hsp70) has been found to be a binding partner of hYVH1. The functional significance of this interaction includes improving the cell survival phenotype, but further details remain to be resolved, including their temporal and spatial regulation in vivo. In this study, we investigate cellular effects of the recently discovered novel Src-mediated phosphorylation site at tyrosine 179 on hYVH1. First, this phosphorylation event negatively regulates the ability of hYVH1 to perform its stress granule disassembly function. Phosphorylated hYVH1 also displays enhanced shuttling to the nucleus, in contrast to its typical steady-state localization pattern that presents as a more cytoplasmic distribution. Biochemical evidence suggests that this phosphorylation event induces a higher level of cellular translational fitness, due to its increased binding to the 60S ribosome. Quantitative proteomics reveal that upon Src-mediated phosphorylation of hYVH1, formation of ribosomal species that represent stalled intermediates are attenuated through the alteration of associating factors that promote translational repression. Furthermore, the robust interaction of hYVH1 and Hsp70 is disrupted upon Src phosphorylation. Using limited proteolysis, we propose a putative binding interface consisting of residues 68-77 on hYVH1 and 326-361 on Hsp70.

Collectively, we demonstrate the relevance of the Src-mediated phosphorylation event at tyrosine 179 on the subcellular localization of hYVH1, its disassembly function at stress granules, and the interaction between hYVH1 and Hsp70. Most notably, we have identified Src phosphorylation of hYVH1 as increasing the ability of hYVH1 to perform its 60S ribosome biogenesis role, thereby increasing cellular translational fitness and allowing for fine-tuning of protein synthesis. As the ribosome continues to emerge as a major scaffold structure for integrating various inputs regarding cellular homeostasis, insights into the mechanism of hYVH1 are essential.

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