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 动态平衡的磷酸化水平，因此已成为有吸引力的药物靶点。因此， 控制蛋白激酶活性的调控机制已经被研究了几十年。大约一半的蛋白质 除了它们的催化激酶域之外，激酶还有至少一个蛋白结构域，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起到调节结构域的作用， 打乱催化必需残基的排列。而分子内调控机制 许多激酶已经被描绘出来，有许多层的调控缺乏定义。具体地说，一个 Maly和Fowler实验室之间的合作揭示了催化剂上新的假定调控表面 研究已久的Src激酶的结构域。这一提议的一个中心假设是，有相似但不同的 引起激酶差异的Src家族(SFK)其他成员的调节表面 底物的专一性、局部性和整体调控机制。鉴于SFK LCK的参与 而Fyn在T细胞发育和成熟的胸腺细胞信号传递方面，我们想更好地了解这些 调节表面有助于产生T细胞受体(TCR)信号，这一信号尚未系统化 探索过了。因此，目标1中的实验将识别假定的分子间和分子内调控表面。 在LCK上-TCR信令中最核心的SFK-以及LCK和TCR的两个成员之间 在酵母中利用一系列饱和诱变的深层突变扫描(DMS)获得了CD45和CSK复合体。 目标2中的实验将利用在目标1中获得的DMS数据集作为实现 最近发表的平行化学选择图谱方法25用于表征动态蛋白质特征 LCK在溶液中。该方法还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的实验将探索依赖于磷酸转移酶和 利用一种新的化学蛋白质组研究Lck和Fyn在T细胞活化中的独立功能 技术3.除了揭示关于Lck和Fyn在调节健康中的作用的基本信息之外 TCR信号，这里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。

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.