Author ORCID Identifier

https://orcid.org/0000-0003-2153-9200

Date of Award

Spring 5-24-2024

Document Type

Thesis (Ph.D.)

Department or Program

Biochemistry and Cell Biology

First Advisor

Arminja Kettenbach

Second Advisor

Charles Barlowe

Third Advisor

James Moseley

Abstract

Reversible phosphorylation is a crucial regulatory mechanism of cellular signaling pathways. Being the most prevalent post-translational modification (PTM) in the cells, with over 75% of all proteins detected to be phosphorylated, phosphorylation regulates a significant number of important cellular processes that have implications in various diseases. Phosphorylation is carried out by protein kinases, which have been extensively studied. However, the opposite reaction, carried out by protein phosphatases, has lagged significantly, exposing a gap of knowledge that is required to be investigated to delineate the kinase-substrate-phosphatase relationship. Phosphoprotein phosphatase family (PPPs), containing seven members of phospho-Serine (pS) and phospho-Threonine (pT) phosphatases, is of particular interest due to its involvement in over 90% of all pS/pT dephosphorylation events, and is the focus of this thesis. To address this gap of knowledge, we leveraged PPPs mechanism of substrate recruitment through the recognition of short linear motifs (SLiMs) and developed a PP1-specific targeting peptide (PhosTAP) that binds specifically to PP1 to compete with endogenous regulatory subunits, inducing hyperphosphorylation of SLiMs-dependent substrates that can be detected and analyzed by quantitative mass spectrometry-based proteomics. This study further confirmed and delineated phosphosite-directed motif preference of PP1 and identified Haspin, an important mitotic kinase, as a novel direct substrate of PP1. To further define the phosphorylation motif signatures of PPPs, we developed a human-derived phosphopeptide library that allows for the investigation of phosphosite-directed motif preference of major phosphoprotein phosphatases: PP2A-B55, PP2A-B56, PP1, and CN. Through a time-series in vitro phosphatase assay and selective chemical inhibition coupled with mass spectrometry-based proteomics, we determined distinctive motif preferences for each phosphatase and established the role of regulatory subunits in influencing substrate specificities.

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