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: 7615554
• 负责人: AMY H ANDREOTTI
• 金额: $ 32.56万
• 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 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7615554
• 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; Face; Family; Goals; 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; Ras/Raf; Receptor Protein-Tyrosine Kinases; 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）：响应于在许多不同的细胞外受体处的信号起始，磷脂酶C（PLC）将磷脂酰肌醇（4，5）二磷酸（PIP 2）水解为肌醇1，4，5-三磷酸（IP 3）和甘油二酯（DAG）。反过来，IP 3和DAG控制钙流动、PKC活化和Ras-Raf-MEK-ERK途径的活化。尽管PLC酶的重要性，有一个显着的差距，在我们的知识有关的分子决定因素，控制PLC功能。这两个目标，在这个reversed应用程序集中在一个单一的异构体的磷脂酶，PLC？1，在T细胞受体下游信号传导的背景下。PLC？1异构体含有一些Src同源结构域，已牵连在PLC的调节？1活动。确切的调节机制尚不清楚。在我们的初步数据中，我们提出了一些新的发现，提供了令人兴奋的见解如何Src同源2（SH 2）域的PLC？1的功能来控制磷脂酶活性。在T细胞受体刺激后，PLC？1被Tec家族激酶Itk磷酸化。我们描述了一个前所未有的基板对接机制，涉及的Itk的激酶结构域和PLC的羧基末端SH 2结构域之间的直接相互作用？1.这种蛋白质-蛋白质相互作用是PLC中Y 783磷酸化所必需的。1、Itk相当有趣的是，这种相互作用以磷酸酪氨酸独立的方式发生，而不是SH 2介导的结合事件的预期结果。进一步的初步数据显示另一个非典型的SH 2相互作用whithin PLC？1，其抑制与Itk的基底对接相互作用。这些监管复合物的结构细节将阐明拟议的工作过程中，并将结果转移到功能检测开发PLC的分子水平的理解？1功能。鉴于观察到的SH 2介导的相互作用的非典型性质，我们预计，完成拟议的目标将是特别有用的，在适当解剖PLC的作用？1在T细胞信号传导中。迄今为止，生物化学和细胞生物学探讨了SH 2结构域在控制PLC中的作用？1介导的信号传导仅限于传统的“功能丧失”突变，其仅破坏典型的磷酸酪氨酸识别。公共卫生相关性本提案旨在了解导致免疫应答激活的特定分子事件。将要研究的分子是在疾病背景下调节免疫反应的主要治疗靶点。因此，该项目的公共卫生相关性涉及开发新的方法，以限制或增强面对自身免疫，免疫抑制或免疫性疾病的免疫反应。