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.

项目摘要细胞磷酸化水平的扰动与多种疾病状态高度相关。蛋白激酶是负责蛋白质磷酸化的酶，它们在维持蛋白质磷酸化方面发挥着核心作用稳态磷酸化水平，因此已成为有吸引力的药物测试目标。控制蛋白激酶活性的调节机制已经被研究了几十年。激酶除了其催化激酶结构域外还至少有一个蛋白结构域4，并且在许多情况下这些通过与催化域表面进行物理接触，域充当“调节域”，破坏催化必需残基的排列，而分子内调节机制。许多激酶已经被描述，但有许多层的调控缺乏定义。 Maly 和 Fowler 实验室之间的合作揭示了催化上新的假定调控表面这一提议的一个中心假设是，存在相似但不同的 Src 激酶结构域。 Src 激酶家族 (SFK) 其他成员的调控表面会导致激酶差异鉴于 SFK Lck 的参与，底物特异性、定位和总体调节机制。和 Fyn 在 T 细胞发育和成熟胸腺细胞信号传导中的作用，我们希望更好地了解这些如何调节表面有助于生产性 T 细胞受体 (TCR) 信号传导，但尚未系统地研究因此，目标 1 中的实验将确定假定的分子间和分子内调节表面。在 Lck 上（TCR 信号传导中最核心的 SFK）以及 Lck 和 TCR 的两个成员之间使用酵母中的一系列饱和诱变深度突变扫描 (DMS) 复合物（CD45 和 Csk）。目标 2 中的实验将利用目标 1 中获得的 DMS 数据集作为实现最近发表的并行化学选择性分析方法25用于表征动态蛋白质特征 Lck 溶液也将有助于假定的调节表面的功能表征。在目标 1 中发现。最后，目标 3 中的实验将探索磷酸转移酶依赖性和使用新的化学蛋白质组学研究 T 细胞激活中 Lck 和 Fyn 的独立功能技术3 除了揭示有关 Lck 和 Fyn 在调节健康中的作用的基本信息之外。 TCR 信号转导，本文描述的方法是通用的，并且可以应用于研究任何感兴趣的蛋白质。