MECHANISM OF T LYMPHOCYTE ACTIVATION--REGULATION OF PLC GAMMA1 ACTIVATION

T淋巴细胞激活机制--PLC GAMMA1激活的调控

• 批准号: 6161348
• 负责人: E BONVINI
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6161348
• 关键词: T cell receptor; T lymphocyte; antigen receptors; biological signal transduction; chimeric proteins; clone cells; enzyme activity; enzyme structure; gene expression; intermolecular interaction; leukocyte activation /transformation; major histocompatibility complex; phospholipase C; phosphoproteins; phosphorylation; protein structure function; receptor binding; transfection

Perturbation of the T cell receptor (TCR) by antigen/MHC or by anti-receptor antibodies initiates a cascade of events which includes protein tyrosine kinase activation and the phosphorylation and activation of phospholipase Cg1 (PLCg1). In turn, PLCg1-mediated phosphoinositide (PI) hydrolysis controls calcium mobilization and protein kinase C activation, two obligatory events in lymphokine secretions and cell proliferation. PLCg1 structure includes two src-homology (SH) 2 domains that function as docking sites for tyrosine-phosphorylated proteins. The function of these domains in PLCg1 activation in T cells is unknown. By acting as docking sites for other molecules, they are likely to play a role in coupling PLCg1 to surface receptors or other regulatory molecules that control PLCg1 activation and compartmentalization. The overall goal of this project is to establish a structure-function relationship for PLCg1 activation in T lymphocytes. Specific aims are the identification of T-cell proteins that interact with PLCg1 subdomains and the role of these domains in PLCg1 activation in T cells. The project's strategy includes: 1. The expression of PLCg1 domains as fusion proteins to screen for candidate binding proteins; 2. A mutational analysis of an epitope-tagged PLCg1 expressed in T lymphocytes; 3. The reconstitution of receptor signaling in PLCg1-deficient cells. PLCg1 GST-SH2(N) domain bound exclusively a 38 kDa phosphoprotein, while the SH2(C) domain bound several phosphoproteins. A SH2(N) domain-defective PLCg1 mutant expressed in human Jurkat T cells did not bind p38 and was marginally phosphorylated in response to TCR engagement. Mutations of the SH2(C) had no impact of TCR-induced PLCg1 phosphorylation. Pharmacological treatment with pervanadate bypasses the receptors and resulted in PLCg1 phosphorylation irrespective of the SH2 domain mutations. Therefore, PLCg1 could be phosphorylated by alternate, SH2(N) domain-independent mechanism(s). Wild type (WT) and mutant PLCg1 proteins were transfected into a DT40-derived PLCg1/2-defective chicken B cell line. Consistent with the results observed in Jurkat cells, only the WT and SH2(C) domain mutant, but not the SH2(N) domain mutant, were tyrosine phosphorylated in response to antigen receptor engagement. While the WT protein reconstituted antigen receptor-induced PI hydrolysis, neither SH2 domain mutant reconstituted this activity. These data indicate that PLCg1 SH2 domains serve discrete functions. The SH2(N) domain is required and sufficient for antigen receptor-induced PLCg1 tyrosine phosphorylation, while both domains are required for the enzymatic activation of PLCg1 by the antigen receptors.

抗原/MHC或抗原/MHC引起的T细胞受体（TCR）的扰动 抗受体抗体引发一系列事件，包括 蛋白酪氨酸激酶的激活和磷酸化及激活 磷脂酶Cg 1（PLCg 1）。反过来，PLCg 1介导的磷酸肌醇 (PI)水解控制钙动员和蛋白激酶C 激活，淋巴因子分泌和细胞中的两个强制性事件 增殖PLCg 1结构包括两个src同源（SH）2结构域 作为酪氨酸磷酸化蛋白质的对接点。的 这些结构域在T细胞中PLCg 1活化中的功能尚不清楚。通过 作为其他分子的对接点，它们很可能发挥 在将PLCg 1偶联至表面受体或其他调节分子中的作用 控制PLCg 1的激活和区室化。 本项目的总体目标是建立一个结构-功能 与T淋巴细胞中PLCg 1活化的关系。 具体的目标是鉴定T细胞蛋白质， PLCg 1亚结构域及其在T细胞PLCg 1活化中的作用 细胞 该项目的战略包括： 1. PLCg 1结构域作为融合蛋白的表达以筛选 候选结合蛋白; 2.表位标记的PLCg 1在T细胞中表达的突变分析 淋巴细胞; 3. PLCg 1缺陷细胞中受体信号的重建。 PLCg 1 GST-SH 2（N）结构域仅结合38 kDa磷蛋白，而 SH 2（C）结构域结合几种磷蛋白。SH 2（N）结构域缺陷型 在人Jurkat T细胞中表达的PLCg 1突变体不结合p38， 轻微磷酸化以响应TCR接合。突变 SH 2（C）对TCR诱导的PLCg 1磷酸化没有影响。药理 用过钒酸盐处理绕过受体，导致PLCg 1 磷酸化，而与SH 2结构域突变无关。因此，PLCg 1 可被交替的SH 2（N）结构域非依赖性磷酸化 机制。转染野生型（WT）和突变型PLCg 1蛋白 导入DT 40衍生的PLC g1/2缺陷型鸡B细胞系。符合 在Jurkat细胞中观察到的结果，仅WT和SH 2（C）结构域 突变体，而不是SH 2（N）结构域突变体，在 对抗原受体接合的反应。 而WT蛋白 重组抗原受体诱导的PI水解，SH 2结构域 突变体恢复了这种活性。这些数据表明PLCg 1 SH 2 域服务于离散功能。需要SH 2（N）域， 足以用于抗原受体诱导的PLCg 1酪氨酸磷酸化， 而这两个结构域都是酶促激活PLCg 1所必需的， 抗原受体。