TCR SIGNALLING AND FYN SH3/SH2 INTERACTION

酪氨酸磷酸化是T细胞中最早也是必不可少的步骤 激活。Src家族酪氨酸激酶p59tyn(Fyn)和p56lck(Lck) 是启动T细胞受体酪氨酸磷酸化的关键 (TCR)触发。基础抑制和依赖激活的相互作用 与信号蛋白的同源(SH3和SH2)结构域 芬恩和洛克。我们已经演示了SH3之间的一种新的物理相互作用 以及Fyn和Lck的SH2结构域，这导致了这样的假设 对Fyn和Lck的抑制是通过二聚化介导的。我们还有 演示了相邻的Fyn SH3和Fyn SH3之间的配体敏感通信 Sh2结构域，我们假设它提供了一种机制来调节 依赖于激活的信号复合体的组装。在这里，我们将使用 非变性凝胶电泳，密度梯度离心法， 化学交联、双表位标记和酵母双杂交 相互作用以评估Fyn和Lck在体内的二聚化，测试IF二聚体 受T细胞激活的调节，并使用缺失和突变 分析评估二聚化是由SH3-SH2相互作用介导的。 全长Fyn和Lck cDNA，携带影响 物理和功能SH3-SH2的相互作用，将被转化为 评估SH3-SH2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在 成纤维细胞，Jurkat人类T细胞TCR信号的增强， 两个抗原特异性T细胞的抗原反应性增加， 突变蛋白与信号转导成分的关系 机械，它们的亚细胞定位和调节的地位 SH2与C端磷酸酪氨酸的相互作用。加在一起，这些 分析应该有助于确定新的SH3-SH2的生物学作用 TCR信令中的相互作用。如果SH3-SH2相互作用依赖 二聚化可以证明，它将提供一个新的范式 了解激活Src家族激酶的受体所传递的信号。 阐明TCR信号转导机制应有助于分析 艾滋病患者T细胞免疫功能缺陷与T细胞激活不当的关系 自身免疫力。对Src家族酪氨酸激酶调控的洞察可能 也提供了对它们致癌活性的更好的理解。

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.