MOLECULAR MECHANISM OF T LYMPHOCYTE ACTIVATION

T细胞受体(TCR)/CD3复合体受抗原干扰的研究 适当的MHC的背景或通过抗受体抗体(Ab) 启动一系列事件，包括蛋白酪氨酸激酶 肌醇磷脂的活化、磷酸化 (InsPL)特异性磷脂酶C(PLC)同工酶，PLC，Gamma-1，激活 InsPL水解和控制第二信使的产生 蛋白激酶C的激活与钙离子动员。PLC Gamma-1含有 一些独立的子域，包括src同源(SH)和 Plekstrin同源(PH)结构域。这些域起到停靠站点的作用 在蛋白质/蛋白质的相互作用中，很可能发挥关键作用 在将PLCGamma-1偶联到TCR/CD3复合体并调节其 活动。重组谷胱甘肽S-转移酶融合蛋白 包括单个或多个PLCGamma-1亚域用于 筛选结合蛋白。多个相互作用的磷酸蛋白 特异性地检测到单个SH结构域。A磷蛋白 与SH3结构域结合的120 kDa为该蛋白 原癌基因c-cbl的产物，一种功能未知的蛋白质，但 参与响应TCR信号的早期激活事件，AS 通过酪氨酸激酶的快速磷酸化反应来指示 到有丝分裂原或抗体介导的受体连接。这个 这两种天然蛋白质之间的相互作用通过 P120c-CBL抗血清与PLCGamma-1共沉淀 并用SH3结合的多肽进行阻断。高水平的表达 C-CBL在瞬时转染人Jurkat T白血病细胞中的表达 获得并将用于评估p120c-CBL功能的AS模型。在……里面 另外，PLC-1 SH在Jurkat细胞中的瞬时表达 已经获得了作为独立蛋白质的亚域，并将用于 结合PLCGamma-1 SH突变体的表达 在体内蛋白质/蛋白质相互作用和T细胞激活中的作用。

Perturbation of the T cell receptor (TCR)/CD3 complex by antigen in the context of the appropriate MHC or by anti-receptor antibodies (Ab) initiates a cascade of events which include protein tyrosine kinase activation, the phosphorylation of an inositol phospholipid (InsPL)-specific phospholipase C (PLC) isozyme, PLCgamma-1, activation of InsPL hydrolysis, and the generation of second messengers that control protein kinase C activation and Ca2+ mobilization. PLCgamma-1 contains a number of independent sub-domains, including src-homology (SH) and plekstrin-homology (PH) domains. These domains function as docking sites in protein/protein interactions and are likely to play a critical role in coupling PLCgamma-1 to the TCR/CD3 complex and in regulating its activity. Recombinant glutathione S-transferase fusion proteins encompassing individual or multiple PLCgamma-1 sub-domains were used to screen for binding proteins. A number of phosphoproteins interacting specifically with individual SH domains were detected. A phosphoproteins of 120 kDa binding to the SH3 domain was identified as the protein product of the c-cbl proto-oncogene, a protein of unknown function, but involved in early activation events in response to TCR signaling, as indicated by its rapid phosphorylation by tyrosine kinases in response to mitogens or antibody-mediated ligation of the receptor. The interaction between the two native proteins was demonstrated by co-precipitating PLCgamma-1 with an anti-serum directed against p120c-cbl and blocking with an SH3-binding peptide. High level of expression of c-cbl in transiently transfected human Jurkat T leukemia cells have been obtained and will be used a as model to assess p120c-cbl function. In addition, transient expression in Jurkat cells of PLCgamma-1 SH subdomains as independent proteins has been obtained and will be used in conjunction with the expression of PLCgamma-1 SH mutants to asses their role in protein/protein interactions in vivo and T cell activation.