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）：响应于许多不同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 (PIP2) 水解为肌醇 1,4,5-三磷酸 (IP3) 和二酰基甘油 (DAG)。反过来，IP3 和 DAG 控制钙通量、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很大差距。此修订后的应用程序的两个目标集中于在 T 细胞受体下游信号传导的背景下调节磷脂酶的单一异构体 PLC?1。 PLC?1 同工型包含许多与 PLC?1 活性调节有关的 Src 同源结构域。确切的调节机制尚不清楚。在我们的初步数据中，我们提出了许多新发现，这些发现为了解 PLC?1 的 Src 同源 2 (SH2) 结构域如何发挥控制磷脂酶活性的作用提供了令人兴奋的见解。 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，该机制涉及 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 磷酸化 PLC?1 中 Y783 所必需的。非常有趣的是，这种相互作用以不依赖于磷酸酪氨酸的方式发生。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 PLC?1 中的另一种非规范 SH2 相互作用，它抑制了与 Itk 的底物对接相互作用。这些调节复合物的结构细节将在拟议的工作过程中得到阐明，并将结果转化为功能测定，以发展对 PLC?1 功能的分子水平理解。鉴于所观察到的 SH2 介导的相互作用的非规范性质，我们预计完成所提出的目标对于正确剖析 PLC?1 在 T 细胞信号传导中的作用将特别有用。迄今为止，对 SH2 结构域在控制 PLC?1 介导的信号传导中的作用的生化和细胞生物学探针仅限于传统的“功能丧失”突变，这些突变只会破坏典型的磷酸酪氨酸识别。公共卫生相关性 该提案旨在了解导致免疫反应激活的特定分子事件。将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方法来限制或增强面对自身免疫、免疫抑制或免疫疾病时的免疫反应。