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

T细胞受体(TCR)受抗原/MHC或受 抗受体抗体会引发一系列事件，包括 蛋白酪氨酸激酶的活化与磷酸化和活化 磷脂酶CG1(PLCG1)。反过来，PLCG1介导的磷脂酰肌醇 (PI)水解控制钙动员和蛋白激酶C 淋巴因子分泌和细胞的两种必备事件--激活 扩散。PLCG1结构含有两个Src-Homology(SH)2结构域 作为酪氨酸磷酸化蛋白质的对接部位。这个 这些结构域在T细胞PLCG1激活中的作用尚不清楚。通过 作为其他分子的对接地点，它们很可能扮演着 PLCG1与表面受体或其他调节分子偶联的作用 它控制着PLCG1的激活和区划。 该项目的总体目标是建立一个结构-功能 T淋巴细胞中PLCG1活化的关系。 特定的目标是识别与之相互作用的T细胞蛋白 T细胞PLCG1亚区及其在PLCG1激活中的作用 细胞。 该项目的战略包括： 1.表达PLCG1结构域作为融合蛋白进行筛选 候选结合蛋白； 2.T细胞表达的PLCG1表位突变分析 淋巴细胞； 3.PLCG1基因缺陷细胞受体信号的重建。 PLCG1GST-SH2(N)结构域与一个38 kDa的磷酸蛋白结合，而 SH2(C)结构域结合了几个磷酸蛋白。一个SH2(N)缺陷域 在人Jurkat T细胞中表达的PLCG1突变体不与p38结合， 对TCR参与的反应略有磷酸化。基因的突变 Sh2(C)对TCR诱导的PLCG1磷酸化无影响。药理作用 过钒酸盐的治疗绕过了受体并导致PLCG1 磷酸化与SH2结构域突变无关。因此，PLCG1 可以被交替的、SH2(N)结构域不依赖的磷酸化 机制(S)。将野生型(WT)和突变型PLCG1蛋白 转化到DT40来源的PLCG1/2缺陷鸡B细胞系。与一致 在Jurkat细胞中观察到的结果只有WT和SH2(C)结构域 突变体，而不是SH2(N)结构域突变体，酪氨酸被磷酸化 对抗原受体结合的反应。而WT蛋白 重组抗原受体诱导的PI水解，既不是SH2结构域 突变体重组了这一活动。这些数据表明PLCG1 SH2 域服务于离散的功能。需要SH2(N)域，并且 足以使抗原受体诱导的PLCG1酪氨酸磷酸化， 而这两个结构域都是PLCG1被酶激活所必需的 抗原受体。

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.