# Global Methods for Characterizing and Discovering New Protein Kinase Regulatory Mechanisms

> **NIH NIH F31** · UNIVERSITY OF WASHINGTON · 2022 · $33,596

## Abstract

Project Abstract
Perturbations to cellular phosphorylation levels are highly correlated with a variety of disease states. Because
protein kinases are the enzymes responsible for protein phosphorylation, they play a central role in maintaining
homeostatic phosphorylation levels, and as such have become attractive drug targets. Consequently, the
regulatory mechanisms that govern protein kinase activity have been studied for decades. Roughly half of protein
kinases have at least one protein domain in addition to their catalytic kinase domain4 and in many cases these
domains serve as “regulatory domains” by making physical contacts with surfaces on the catalytic domain,
disrupting the alignment of catalytically necessary residues. While the intramolecular regulatory mechanisms of
many kinases have been delineated, there are many layers of regulation that lack definition. Specifically, a
collaborative effort between the Maly and Fowler labs revealed new putative regulatory surfaces on the catalytic
domain of the long-studied Src kinase. One central hypothesis of this proposal is that there are similar but distinct
regulatory surfaces on other members of Src Family of Kinases (SFKs) which give rise to differences in kinase
substrate specificity, localization, and overall mechanisms of regulation. Given the involvement of the SFKs Lck
and Fyn in T-cell development and mature thymocyte signaling, we would like to better understand how these
regulatory surfaces contribute to productive T cell receptor (TCR) signaling, which has yet to be systematically
explored. Therefore, the experiments in Aim 1 will identify putative inter- and intramolecular regulatory surfaces
on Lck—the most centrally involved SFK in TCR signaling—and between Lck and two members of the TCR
complex (CD45 and Csk) using a series of saturation mutagenesis Deep Mutational Scans (DMS) in yeast.
Experiments in Aim 2 will leverage the DMS dataset obtained in Aim 1 as the foundation for implementing the
recently published Parallel Chemoselective Profiling method25 for characterizing the dynamic protein features of
Lck in solution. This method will also facilitate the functional characterization of the putative regulatory surfaces
discovered in Aim 1. Finally, experiments in Aim 3 will explore the phosphotransferase dependent and
independent functions of both Lck and Fyn in the context of T cell activation using a new chemoproteomic
technology3. In addition to revealing fundamental information about the roles of Lck and Fyn in mediating healthy
TCR signaling, the methods described herein are general, and can be applied to study any protein of interest.

## Key facts

- **NIH application ID:** 10399440
- **Project number:** 5F31GM142170-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Zachary Eugene Potter
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $33,596
- **Award type:** 5
- **Project period:** 2021-07-01 → 2023-03-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10399440

## Citation

> US National Institutes of Health, RePORTER application 10399440, Global Methods for Characterizing and Discovering New Protein Kinase Regulatory Mechanisms (5F31GM142170-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10399440. Licensed CC0.

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