MECHANISM OF T LYMPHOCYTE ACTIVATION--REGULATION OF PLCR1 ACTIVATION

T淋巴细胞激活机制--PLCR1激活的调控

• 批准号: 2569028
• 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/2569028
• 关键词: CD3 molecule; T cell receptor; T lymphocyte; antireceptor antibody; binding proteins; chimeric proteins; clone cells; enzyme activity; enzyme structure; gene expression; gene mutation; intermolecular interaction; leukocyte activation /transformation; major histocompatibility complex; phospholipase C; phosphoproteins; phosphorylation; precipitation; protein structure function; protein tyrosine kinase; receptor binding

Perturbation of the T cell receptor (TCR) by antigen/MHC or by anti-receptor antibodies (Ab) initiates a cascade of events which include protein tyrosine kinase activation and the phosphorylation and activation of phospholipase Cg-1 (PLCg1). PLCg-1 contains a number of src-homology (SH) domains. These domains function as protein docking sites and are likely to play a role in coupling PLCg-1 to the TCR/CD3 complex and in regulating its activity. Our goal is to establish a structure-function relationship for PLCg1 activation in T lymphocytes. Specific aims of this project are: 1). The identification of T-cell proteins that interact with PLCg1 SH domains. 2). The identification of the functional role of PLCg1 SH domains in T-lymphocyte activation. The project's strategy is as follows: 1). Expression of PLCg1 domains as GST- fusion proteins, as a screening tool for the identification of candidate binding proteins. 2). Demonstration of the interaction between native proteins. 3). Characterization of the function of individual domains by mutational anlaysis of an epitope-tagged PLCg1. PLCg1 GST-SH2(N) domain bound a phosphoprotein of about 38 kDa, while the SH2(C) domain bound phosphoproteins of about 38, 75, and 120 kDa and, to a lesser extent, 60, 85 and 100 kDa phosphoproteins. The GST-SH3 domain bound a prominent tyrosine phosphorylated protein of 120 kDa. Phospho- proteins of about 38 and about 120 kDa were coprecipitated with native PLCg1 from CD3-activated cells and a phosphoprotein of about 75 kDa was observed in cells treated with pervanadate, a strong inducer of protein tyrosine phosphorylation used for mimicry of T-cell activation. The 120 kDa SH3 domain-binding phospho- protein was identified as cbl, a protein of unknown function, but rapidly phosphorylated in response to mitogens or Ab-mediated ligation of the receptor. A constitutive interaction between native PLCg1 and cbl was shown by co-precipitation in Jurkat cells and displacement by a peptide encoding an SH3-binding motif of cbl. The function of cbl in T cell activation is addressed as a separate project (Z01 BO 04002-02 LIB). Single amino acid mutations of PLCg1 SH domains were performed on the basis of loss-of-function mutations of conserved amino acids in the SH domains of Grb2. A prominent defect in CD3-induced PLCg1 tyrosine phosphorylation was associated with the mutation of the SH2(N), but not the SH2(C) or SH3 domains. The SH2(N) domain binds exclusively to a about 38 kDa phospho- protein, suggesting that this protein, likely to be the recently cloned protein, Lnk, may be critical for phosphorylation of PLCg1 in response to CD3 ligation. Mutation of PLCg1 SH2(N) domain did not affect tyrosine phosphorylation in response to treatment with pervanadate, suggesting that the mutated PLCg1 could be phosphorylated by alternate mechanism(s).

抗原/MHC或抗原/MHC引起的T细胞受体（TCR）的扰动 抗受体抗体（Ab）启动一系列事件，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）。PLCg-1含有许多src同源性， (SH)域.这些结构域作为蛋白质对接位点发挥功能， 可能在将PLCg-1偶联至TCR/CD 3复合物以及在 规范其活动。我们的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。 具体目标 该项目是：1）。T细胞相互作用蛋白的鉴定 PLCg 1 SH结构域。2）。确定联合国的职能作用 PLC g1 SH结构域在T淋巴细胞活化中的作用 该项目的战略是 具体如下：1）.作为GST-融合蛋白的PLCg 1结构域的表达， 用于鉴定候选结合蛋白的筛选工具。 2）。天然蛋白质之间相互作用的证明。 3）。 通过突变来表征单个结构域的功能 表位标记的PLCg 1的分析。 PLCg 1 GST-SH 2（N）结构域结合a 约38 kDa的磷蛋白，而SH 2（C）结构域结合 约38、75和120 kDa的磷蛋白，以及在较小程度上，60， 85和100 kDa磷蛋白。 GST-SH 3结构域结合一个突出的 酪氨酸磷酸化蛋白120 kDa。磷蛋白约 38和约120 kDa与来自 CD 3-活化的细胞和约75 kDa的磷蛋白，观察到在 用过钒酸盐（一种强蛋白酪氨酸诱导剂）处理的细胞 用于模拟T细胞活化的磷酸化。 120 kDa SH3 结构域结合磷酸化蛋白被鉴定为CBL， 功能未知，但对有丝分裂原或 Ab介导的受体连接。本构相互作用， 通过Jurkat细胞中的共沉淀显示天然PLCg 1和cbl， 在一些实施方案中，cbl的SH 3结合基序被编码cbl的SH 3结合基序的肽置换。 的 cbl在T细胞活化中的功能是作为一个单独的项目来解决的 (Z01 BO 04002-02 LIB）。 PLCg 1 SH结构域的单个氨基酸突变 是基于保守基因的功能缺失突变进行的。 Grb 2的SH结构域中的氨基酸。 CD 3诱导的免疫缺陷 PLCg 1酪氨酸磷酸化与PLCg 1基因的突变有关。 SH 2（N），但不是SH 2（C）或SH 3结构域。SH 2（N）结构域结合 仅限于约38 kDa的磷酸化蛋白，这表明， 一种蛋白质，可能是最近克隆的蛋白质，Lnk，可能是关键的 用于响应于CD 3连接的PLCg 1的磷酸化。突变 PLCg 1 SH 2（N）结构域不影响酪氨酸磷酸化反应 过钒酸盐治疗，这表明突变的PLCg 1可以 通过替代机制被磷酸化。