TCR SIGNALLING AND FYN SH3/SH2 INTERACTION

TCR 信号传导和 FYN SH3/SH2 相互作用

• 批准号: 2748770
• 负责人: Hamid Band
• 金额: $ 30.17万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-02 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2748770
• 关键词: T cell receptor; T lymphocyte; biological signal transduction; laboratory mouse; leukocyte activation /transformation; molecular cloning; phosphorylation; protein tyrosine kinase; tissue /cell culture; transfection

Tyrosine phosphorylation is the earliest and an obligatory step in T cell activation. The Src family tyrosine kinases p59tyn (Fyn) and p56lck (Lck) are critical for initiating tyrosine phosphorylation upon T cell receptor (TCR) triggering. The basal repression and activation-dependent interaction with signalling proteins require the Src homology (SH3 and SH2) domains of Fyn and Lck. We have demonstrated a novel physical interaction between SH3 and SH2 domains of Fyn and Lck, which leads to the hypothesis that repression of Fyn and Lck is mediated by dimerization. We have also demonstrated a ligand-sensitive communication between adjacent Fyn SH3 and SH2 domains, which we hypothesize to provide a mechanism to regulate activation-dependent assembly of signalling complexes. Here, we will use nondenaturing gel electrophoresis, density gradient centrifugation, chemical cross-linking, two-epitope tagging and yeast two-hybrid interaction to assess dimerization of Fyn and Lck in vivo, test if dimers are modulated by T cell activation, and use deletional and mutational analyses to assess that dimerization is mediated by SH3-SH2 interaction. Full-length Fyn and Lck cDNAs, carrying mutations that influence the physical and functional SH3-SH2 interactions, will be transfected into cells to assess the in vivo biological functions of SH3-SH2 interactions. Thus, we will assess kinase activity and transforming potential in fibroblasts, enhancement of TCR signalling in Jurkat human T cells, increase in antigen-responsiveness of two antigen-specific T cells, association of mutant proteins with components of signal transduction machinery, their subcellular localization and the status of the regulatory interaction of SH2 with the C-terminal phosphotyrosine. Together, these analyses should help establish the biological roles of the novel SH3-SH2 interactions in TCR signalling. If SH3-SH2 interaction-dependent dimerization can be demonstrated, it will provide a new paradigm to understand signalling through receptors that activate Src-family kinases. Elucidation of the mechanisms of TCR signalling should facilitate analyses of defective T cell immunity in AIDS and inappropriate T cell activation in autoimmunity. Insights into regulation of Src-family tyrosine kinases may also provide a better understanding of their oncogenic activation.

酪氨酸磷酸化是T细胞活化的最早和必不可少的步骤， activation. Src家族酪氨酸激酶p59 tyn（Fyn）和p56 lck（Lck） 是启动T细胞受体酪氨酸磷酸化的关键 (TCR)触发。基础阻遏和激活依赖的相互作用 与信号蛋白的同源性需要Src同源性（SH 3和SH 2）结构域， Fyn和Lck。我们已经证明了一种新的物理相互作用SH 3 和Fyn和Lck的SH 2结构域，这导致了以下假设， Fyn和Lck的阻遏由二聚化介导。 我们还 证明了相邻的Fyn SH 3之间的配体敏感性通信， SH 2结构域，我们假设它提供了一种调节 信号复合物的活化依赖性组装。在这里，我们将使用 非变性凝胶电泳，密度梯度离心， 化学交联、双表位标签和酵母双杂交 相互作用以评估体内Fyn和Lck的二聚化，测试是否为二聚体 受T细胞活化的调节，并使用缺失和突变 分析以评估二聚化由SH 3-SH 2相互作用介导。 全长Fyn和Lck cDNA，携带影响细胞增殖的突变， 物理和功能性SH 3-SH 2相互作用，将被转染到 细胞来评估SH 3-SH 2相互作用的体内生物学功能。 因此，我们将评估激酶活性和转化潜力， 成纤维细胞，增强Jurkat人T细胞中的TCR信号传导， 两种抗原特异性T细胞的抗原应答性增加， 突变蛋白与信号转导组分的关联 机制，它们的亚细胞定位和调节的状态， SH 2与C-末端磷酸酪氨酸的相互作用。所有这些 分析应该有助于建立新的SH 3-SH 2的生物学作用， TCR信号的相互作用。 如果SH 3-SH 2相互作用依赖 二聚化可以证明，它将提供一个新的范例， 理解通过激活Src家族激酶的受体的信号传导。 TCR信号传导机制的阐明将有助于分析 艾滋病中T细胞免疫缺陷和 自身免疫对Src家族酪氨酸激酶调节的认识可能 也提供了更好的了解他们的致癌激活。