Regulation of T Cell Signaling: Structural Studies of PLCgamma1

T 细胞信号传导的调节：PLCgamma1 的结构研究

• 批准号: 8260866
• 负责人: AMY H ANDREOTTI
• 金额: $ 31.86万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8260866
• 关键词: 1,2-diacylglycerol; Active Sites; Adopted; Area; Attenuated; Autoimmunity; Binding; Biochemical; Biological; Biological Assay; Biological Models; Catalytic Domain; Cell Surface Receptors; Cells; Chemistry; Collaborations; Complex; Cytoskeleton; Data; Diglycerides; Disease; Docking; Enzymes; Event; Family; Goals; Health; Hydrolysis; Immune System Diseases; Immune response; Immunosuppression; Inositol; Knowledge; Laboratories; Lead; Length; Ligands; Literature; MEKs; Mature T-Lymphocyte; Mediating; Molecular; Molecular Conformation; Mutation; NMR Spectroscopy; Nature; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase; Phospholipase C; Phosphorylation; Phosphotransferases; Phosphotyrosine; Protein Isoforms; Protein Tyrosine Kinase; Ras/Raf; Regulation; Reporting; Research; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Structure; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic Intervention; Tyrosine Phosphorylation; Work; base; design; enzyme substrate complex; extracellular; feeding; immune function; inhibitor/antagonist; innovation; insight; interest; loss of function mutation; mouse model; novel; prevent; professor; programs; protein protein interaction; public health relevance; receptor; release of sequestered calcium ion into cytoplasm; response; second messenger; src Homology Domains; structural biology; therapeutic target; three dimensional structure; thymocyte

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.

描述（由申请人提供）：响应许多不同细胞外受体的信号起始，磷脂酶C（PLC）水解水解磷脂酰肌醇（4,5）双磷酸盐（PIP2）至inositol 1,4,4,5-三磷酸（IP3）（IP3）和二酰甘油（Diamycylglycerol（Dag））。反过来，IP3和DAG控制钙通量，RAS-RAF-MEK-ERK途径的PKC激活和激活。尽管有PLC酶的重要性，但我们的知识上仍然存在有关控制PLC功能的分子决定因素的显着差距。在此修订后，这两个目标的重点是在T细胞受体下游信号传导的背景下，调节磷脂酶Plc？1的单个同工型。 PLC？1同工型包含许多与PLC？1活性相关的SRC同型域。调节的确切机制尚不清楚。在我们的初步数据中，我们提出了许多新发现，这些发现提供了令人兴奋的见解，即PLC？1功能如何控制磷脂酶活性的SRC同型2（SH2）域。在T细胞受体刺激之后，PLC？1是由TEC家族激酶ITK磷酸化的。我们描述了一种前所未有的底物对接机制，该机制涉及ITK的激酶结构域与PLC的羧基末端SH2域之间的直接相互作用？1。这种蛋白质 - 蛋白质相互作用是由ITK在Plc？1中Y783磷酸化所必需的。非常有趣的是，这种相互作用是以磷酸酪氨酸独立的方式出现的。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，另一种非规范的SH2相互作用是plc？1抑制与ITK的底物对接相互作用。这些调节络合物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测定中，以发展对PLC的分子水平理解？1功能。鉴于观察到的SH2介导的相互作用的非规范性质，我们期望提出的目标的完成将在正确剖析plc？1在T细胞信号传导中的作用时特别有用。迄今为止，生化和细胞生物学探针中SH2结构域在控制PLC的作用中的作用仅限于传统的“ Foss of Fosition”突变，仅破坏了规范的磷酸酪氨酸识别。公共卫生相关性该提案旨在了解特定的分子事件，从而激活免疫反应。将研究的分子是调节疾病中免疫反应的主要治疗靶标。因此，该项目的公共卫生相关性与开发新方法有关，以限制或增强面对自身免疫性，免疫抑制或免疫疾病的免疫反应。