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

• 批准号: 10399440
• 负责人: Zachary Eugene Potter
• 金额: $ 3.36万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10399440
• 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; 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; insight; interest; intermolecular interaction; male; member; mutation screening; novel; protein protein interaction; scaffold; src-Family Kinases

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。因为 蛋白激酶是负责蛋白质磷酸化的酶，它们在维持 稳态磷酸化水平，因此已成为有吸引力的药物靶标。因此， 几十年来，控制蛋白激酶活性的调节机制已研究了数十年。大约一半的蛋白质 激酶除了它们的催化激酶结构域外，至少还有一个蛋白质结构域，在许多情况下， 通过与催化域上的表面进行物理接触，域是“调节域”， 破坏催化必要残留物的对齐。而分子内调节机制 许多激酶已经被描述了，有许多缺乏定义的调节层。具体而言， Maly和Fowler Labs之间的协作努力揭示了催化的新推定调节表面 长期研究的SRC激酶的域。该提议的一个核心假设是，有相似但独特的 SRC家族（SFK）其他成员的调节表面会导致激酶的差异 底物特异性，定位和调节的总体机制。鉴于SFKS LCK的参与 和FYN在T细胞发育和成熟的胸腺细胞信号中，我们想更好地了解这些 调节表面有助于生产性T细胞受体（TCR）信号，尚未系统地 探索。因此，AIM 1中的实验将确定推定的分子间和分子内调节表面 在LCK上（最集中参与TCR信号的SFK）以及LCK和两个TCR成员之间 酵母中使用一系列饱和诱变深突变扫描（DMS）的复合物（CD45和CSK）。 AIM 2中的实验将利用AIM 1中获得的DMS数据集作为实施的基础 最近发表的平行化学选择性分析方法25用于表征 LCK在解决方案中。该方法还将促进推定调节表面的功能表征 在AIM 1中发现。最后，AIM 3中的实验将探索磷酸转移酶依赖酶和 LCK和FYN在T细胞激活的背景下使用新的化学蛋白质组学的独立功能 技术3。除了揭示有关LCK和FYN在介导健康中的作用的基本信息 TCR信号，本文所述的方法是一般的，可以应用于研究任何感兴趣的蛋白质。