MOLECULAR MECHANISM OF T LYMPHOCYTE ACTIVATION

T淋巴细胞激活的分子机制

• 批准号: 3748256
• 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/3748256
• 关键词: CD3 molecule; T cell receptor; T lymphocyte; chimeric proteins; enzyme activity; glutathione transferase; isozymes; leukocyte activation /transformation; phospholipase C; precipitation; protein purification

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 includes protein tyrosine kinase activation, tyr osine phosphorylation of an inositol phospholipid (InsPL)-specific phospholipase C (PLC) isozyme, PLC gamma-1 , activation of InsPL hydrolysis, and the generation of second messengers that control protein kinase C activation and calcium mobilization. The objective of this study is to elucidate the mechanism coupling PLC gamma-1 to the TCR/CD3 complex and regulating its activity and effector function. A number of functional subdomains constitutes PLC gamma-1. Some of these domains are homologous to amino acid modules initially defined in the src gene product. These src-homology domains (SH) function as docking sites in protein/protein interactions: SH2 recognizes tyrosine phosphorylated peptides, while SH3 interacts with proline-rich sequences of certain proteins. PLC gamma-1 contains two SH2 and one SH3, whose function is currently unknown. We have expressed both SH2 and the SH3 of PLC gamma-1 (individually or in combination) as fusion proteins (FP) with the glutathione S-transferase (GST) from S. iaponicum. By screening with an anti-phosphotyrosine (PY) Ab, at least two phosphoproteins were detected by coprecipitation with the carboxy-terminal SH2 from lysates of activated human Jurkat T leukemia cells or a mouse T lymphocyte cell line. One distinct phosphoprotein was recognized by the GST-SH3. No detectable phosphoprotein was precipitated with the NH2-terminal SH2 or from non-activated cells. Additive but not synergistic effects on phosphoprotein recruitment was observed with the GST-FP encompassing multiple SH domains. The phosphoprotein recognized by the GST-SH3 was identified by immunoreactivity as c-cbl, a transcription factor. Although cbl phosphorylation was activation-dependent, its interaction with GST- SH3 was not. An additional non-phosphorylated protein coprecipitated with GST-SH3 and competed with cbl for SH3 binding. Identification of this protein and the GST-SH2-precipitated proteins is in progress. Additional PLC gamma-1 subdomains are also tested for their role in protein recruitment and PLC regulation.

抗原对T细胞受体（TCR）/CD 3复合物的扰动（在免疫组化中） 适当的MHC的背景）或抗受体抗体（Ab） 启动包括蛋白酪氨酸激酶在内的级联反应 肌醇磷脂的酪氨酸磷酸化活化 （InsPL）特异性磷脂酶C（PLC）同工酶，PLC γ-1，活化 的InsPL水解，并产生第二信使，控制 蛋白激酶C激活和钙动员。的目标 本研究旨在阐明PLC γ-1与 TCR/CD 3复合物及其活性和效应功能的调节。一 许多功能亚结构域构成PLC γ-1。其中一些 结构域与src中最初定义的氨基酸模块同源 基因产物这些src同源结构域（SH）作为对接位点发挥功能 在蛋白质/蛋白质相互作用中：SH 2识别酪氨酸磷酸化 肽，而SH 3与某些富含脯氨酸的序列相互作用， proteins. PLC gamma-1包含两个SH 2和一个SH 3，其功能是 目前未知。我们表达了PLC γ-1的SH 2和SH 3， （单独或组合）作为与 谷胱甘肽S-转移酶（GST）。Iaponicum。通过筛选， 抗磷酸酪氨酸（PY）抗体，至少检测到两种磷蛋白 通过与来自以下的裂解物的羧基末端SH 2共沉淀， 活化的人Jurkat T白血病细胞或小鼠T淋巴细胞 线一个独特的磷蛋白被GST-SH 3识别。没有 可检测的磷蛋白与NH 2-末端SH 2或 非活化细胞。 相加而非协同效应 在GST-FP中观察到磷蛋白募集， 多个SH结构域。GST-SH 3识别的磷蛋白是 通过免疫反应性鉴定为c-cbl，一种转录因子。虽然 cbl磷酸化是激活依赖性的，它与GST- SH 3没有。一种额外的非磷酸化蛋白质与 GST-SH 3，并与cbl竞争SH 3结合。鉴定此 蛋白质和GST-SH 2沉淀蛋白质正在进行中。额外 还测试了PLC γ-1亚结构域在蛋白质中的作用。 招聘和PLC监管。