Global Methods for Characterizing and Discovering New Protein Kinase Regulatory Mechanisms

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，它们在维持蛋白质磷酸化中起核心作用。 稳态磷酸化水平，因此已经成为有吸引力的药物靶标。因此 控制蛋白激酶活性的调节机制已经研究了几十年。大约一半的蛋白质 激酶除了它们的催化激酶结构域4之外，还具有至少一个蛋白质结构域，并且在许多情况下，这些 结构域通过与催化结构域上的表面进行物理接触而充当“调节结构域”， 破坏催化必需残基的排列。虽然分子内的调节机制， 虽然已经描述了许多激酶，但仍有许多缺乏定义的调节层。特别是 Maly和Fowler实验室之间的合作努力揭示了催化剂上新的假定调节表面， 研究已久的Src激酶。这一提议的一个核心假设是， Src激酶家族（SFK）其他成员的调节表面，其引起激酶 底物特异性、定位和整体调控机制。考虑到SFK Lck的参与 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 调节表面有助于生产性T细胞受体（TCR）信号传导，其尚未被系统地研究。 探讨了因此，目标1中的实验将鉴定推定的分子间和分子内调节表面 在Lck上-TCR信号传导中最中心参与的SFK-以及Lck和TCR的两个成员之间 在酵母中使用一系列饱和诱变深度突变扫描（DMS）对复合物（CD 45和Csk）进行突变。 目标2中的实验将利用目标1中获得的DMS数据集作为实施 最近发表的平行化学选择性分析方法25用于表征 溶液中的Lck。这种方法也将促进推定的调节表面的功能表征 发现于Aim 1。最后，目标3中的实验将探索磷酸转移酶依赖性和 Lck和Fyn在T细胞活化背景下的独立功能，使用新的化学蛋白质组学 技术3.除了揭示关于Lck和Fyn在介导健康中的作用的基本信息之外， 为了研究TCR信号传导，本文所述的方法是通用的，并且可以应用于研究任何感兴趣的蛋白质。

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.