MOLECULAR MECHANISM OF T LYMPHOCYTE ACTIVATION

T细胞受体（TCR）/CD 3复合物在免疫细胞中的抗原扰动 适当的MHC背景下或通过抗受体抗体（Ab） 启动包括蛋白酪氨酸激酶在内的级联反应 活化，肌醇磷脂的磷酸化 （InsPL）特异性磷脂酶C（PLC）同工酶，PLC γ-1，激活 的InsPL水解，并产生第二信使，控制 蛋白激酶C激活和Ca 2+动员。PLC γ-1含有 许多独立的亚结构域，包括src-同源性（SH）和 plekstrin-homology（PH）结构域。这些结构域作为对接点发挥作用 在蛋白质/蛋白质相互作用中， 在将PLC γ-1偶联至TCR/CD 3复合物和调节其 活动重组谷胱甘肽S-转移酶融合蛋白 包含单个或多个PLC γ-1亚结构域的化合物用于 筛选结合蛋白。许多磷蛋白相互作用 特异性地与单个SH结构域结合。A磷蛋白类 与SH 3结构域结合的120 kDa的蛋白质被鉴定为 c-cbl原癌基因的产物，一种功能未知的蛋白质，但 参与响应TCR信号传导的早期激活事件， 表明其快速磷酸化的酪氨酸激酶， 有丝分裂原或抗体介导的受体连接。的 两种天然蛋白质之间的相互作用被证明是由 用抗p120 c-cbl的抗血清共沉淀PLC γ-1 并用SH 3结合肽封闭。高水平表达 在瞬时转染的人Jurkat T白血病细胞中， 获得并将用作模型来评估p120 c-cBL功能。在 此外，PLC γ-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结构域，并将用于 结合PLC γ-1 SH突变体的表达来评估其 在体内蛋白质/蛋白质相互作用和T细胞活化中的作用。

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.