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Global Methods for Characterizing and Discovering New Protein Kinase Regulatory Mechanisms

表征和发现新蛋白激酶调节机制的全局方法

基本信息

批准号：
10230912
负责人：
Zachary Eugene Potter
金额：
$ 4.06万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10230912
关键词：
Active Sites Address Affect Area Binding Biochemical Biological Assay Biophysics Catalytic Domain Cellular biology Collaborations Complex Coupled Data Set Development Disease Distant Drug Targeting Enzymes Foundations Goals Health Human Human Genome Immune system Length Lymphocyte-Specific p56LCK Tyrosine Protein Kinase Maps Mass Spectrum Analysis Mature Thymocyte Mediating Methodology Methods Modeling Molecular Molecular Conformation Mutagenesis Mutation PTPRC gene Phosphorylation Phosphotransferases Play Protein Kinase Proteins Publishing Receptor Activation Receptor Signaling Regulation Role Series Signal Transduction Signal Transduction Pathway Solvents Structure Substrate Specificity Surface T Cell Receptor Signaling Pathway T-Cell Activation T-Cell Development T-Cell Receptor Technology Tertiary Protein Structure Training Yeasts base biophysical properties chemoproteomics experimental study genetic regulatory protein genome sciences inhibitor/antagonist insight interest intermolecular interaction male member mutation screening novel protein protein interaction scaffold src-Family Kinases

项目摘要

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.

项目摘要