Regulation of T Cell Signaling: Structural Studies of PLCgamma1

描述(申请人提供)：磷脂酶C(PLC)响应于一些不同细胞外受体的信号启动，将磷脂酰肌醇(4，5)二磷酸(PIP2)水解成肌醇1，4，5-三磷酸(IP3)和二酰甘油(DAG)。反过来，IP3和DAG控制钙离子流量、PKC激活和Ras-Raf-MEK-ERK通路的激活。尽管PLC酶很重要，但我们对控制PLC功能的分子决定因素的了解还存在着很大的差距。在这一改进的应用中，两个目的集中在调节磷脂酶的单一异构体，PLC？1，在T细胞受体下游信号传递的背景下。PLC？1亚型包含许多与PLC？1活性调控有关的Src同源结构域。监管的确切机制尚不清楚。在我们的初步数据中，我们提出了一些新的发现，这些发现为PLC？1的Src同源2(SH2)结构域如何控制磷脂酶活性提供了令人兴奋的见解。在T细胞受体刺激后，PLC？1被Tec家族激酶ITK磷酸化。我们描述了一种前所未有的底物对接机制，它涉及到ITK的激酶结构域和PLC？1的羧基末端SH2结构域之间的直接相互作用。这种蛋白质-蛋白质相互作用是ITK对PLC？1中的Y783进行磷酸化所必需的。非常有趣的是，相互作用是以磷酸酪氨酸不依赖的方式发生的；而不是SH2介导的结合事件的预期结果。进一步的初步数据显示了另一个非典型的SH2相互作用，而PLC？1抑制了底物与ITK的对接相互作用。这些调控复合体的结构细节将在拟议的工作过程中阐明，并将结果转移到功能分析中，以发展对PLC？1功能的分子水平的理解。鉴于所观察到的SH2介导的相互作用的非典型性，我们预计完成所提出的目标将对正确剖析PLC？1在T细胞信号中的作用特别有用。到目前为止，关于SH2结构域在控制PLC？1介导的信号转导中的作用的生化和细胞生物学探测仅限于传统的“功能缺失”突变，这些突变只会扰乱典型的磷酸酪氨酸识别。公共卫生相关性这项建议旨在了解导致免疫反应激活的特定分子事件。将被研究的分子是在疾病背景下调节免疫反应的主要治疗目标。因此，该项目的公共卫生相关性涉及开发新的方法，以限制或加强面对自身免疫、免疫抑制或免疫性疾病的免疫反应。

DESCRIPTION (provided by applicant): In response to signal initiation at a number of different extracellular receptors, phospholipase C (PLC) hydrolyzes phosphatidyl inositol (4,5)bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). In turn, IP3 and DAG control calcium flux, PKC activation and activation of the Ras-Raf-MEK-ERK pathway. In spite of the importance of the PLC enzymes, there is a significant gap in our knowledge regarding the molecular determinants that control PLC function. The two aims in this revsed application focus on the regulation of a single isoform of the phospholipases, PLC?1, in the context of signaling downstream of the T cell receptor. The PLC?1 isoform contains a number of Src homolgy domains that have been implicated in regulation of PLC?1 activity. The precise mechanism of regulation is not known. In our preliminary data we present a number of new findings that provide exciting insights into how the Src homolgy 2 (SH2) domains of PLC?1 function to control phospholipase activity. Following T cell receptor stimulation, PLC?1 is phosphorylated by the Tec family kinase Itk. We describe an unprecedented substrate docking mechanism that involves a direct interaction between the kinase domain of Itk and the carboxy-terminal SH2 domain of PLC?1. This protein-protein interaction is required for phosphorylation of Y783 in PLC?1 by Itk. Quite interesting is the fact that the interaction occurs in a phosphotyrosine-independent fashion; not the expected result for an SH2 mediated binding event. Further preliminary data show another noncanonical SH2 interaction whithin PLC?1 that inhibits the substrate docking interaction with Itk. The structural details of these regulatory complexes will be elucidated during the course of the proposed work and the results transferred into functional assays to develop a molecular level understanding of PLC?1 function. Given the non-canonical nature of the observed SH2 mediated interactions we expect that completion of the proposed aims will be particularly useful in properly dissecting the role of PLC?1 in T cell signaling. To date, biochemical and cell biological probes into the role of the SH2 domains in controlling PLC?1 mediated signaling have been limited to traditional 'loss-of-funtion' mutations that only disrupt canonical phosphotyrosine recognition. PUBLIC HEALTH RELEVANCE This proposal aims to understand specific molecular events leading to activation of the immune response. The molecules that will be studied are prime therapeutic targets for modulating an immune response in the context of disease. Thus, the public health relevance of the project relates to developing new ways to either limit or enhance the immune response in the face of autoimmunity, immunosuppression or immunological diseases.