Fyn and Rap1 in T cell Activation and Immune Response

描述：(申请人提供)这项建议的目的是研究 小分子GTP结合蛋白Rap1信号通路在T细胞中的作用 细胞功能。进行这些实验的兴趣是基于我们的研究 在一个细胞培养系统中，首次表明这一途径是 在激活的Src激酶的下游利用。我们用以下方法确定了这条途径 一种新的激活Src-Kinase的方法，一种利用自然配体， 它与Src-kinaseSH3结构域结合并激活Src-kinase1信号通路。 具体地说，我们使用了我们分离的一种蛋白质Sin，它是一种与Src和Fyn-SH3结合的蛋白质 蛋白。表达截短的、构成活性形式的SIN的小鼠 (SindeltaC)，表现为T细胞增殖缺陷。我们建议调查 SindeltaC介导的T细胞抑制机制 并评价SindeltaC转基因小鼠的免疫应答 都受到了威胁。具体地说，我们将研究是否有抑制作用 SindeltaC表达对T细胞功能的影响是由于一种信号的诱导 涉及SindeltaC介导的Fyn、SindeltaC激活的级联 RAP1 GTP酶的磷酸化和激活，和/或RAP1介导的 抑制T细胞增殖。为了测试此模型，我们将执行 以下实验：1)我们将使用Fyn Null和SindeltaC突变小鼠 解决是否需要FYN和SindeltaC交互RAP1激活和 抑制T细胞增殖。2)我们将产生一个结构性活跃的 RAP突变体L直接表明这种G蛋白抑制T细胞的激活。 3)我们将使用SindeltaC表达的小鼠来确定RAPL的激活 通过干扰正常的RAS信号和抑制T细胞的增殖 IL-2的转录激活。4)我们将检查CD4辅助细胞和CD8 SindeltaC转基因小鼠的细胞毒性T细胞反应及检测 SindeltaC介导的Fyn和RAPL激活与T细胞无能相关 活着。

DESCRIPTION:(provided by applicant) The goal of this proposal is to study the role of a signaling pathway involving the small GTP-binding protein Rap 1, in T cell function. Interest in pursuing these experiments is based on our studies in a cell culture system that showed for the first time that this pathway is utilized downstream of activated Src kinases. We identified this pathway using a novel approach to activate Src-kinases, one that utilizes natural ligands, which bind to Src-kinase SH3 domains and activate Src kinase signaling. Specifically, we used a protein we isolated, Sin, a Src- and Fyn-SH3-binding protein. Mice expressing a truncated, constitutively active form of Sin (SindeltaC), exhibit defective T cell proliferation. We propose to investigate the mechanisms responsible for the SindeltaC-mediated inhibition of T cell activation and to evaluate if the immune responses of SindeltaC transgenic mice are compromised. Specifically, we will examine whether the inhibitory effect of SindeltaC expression on T cell function is due to the induction of a signaling cascade that involves SindeltaC-mediated activation of Fyn, SindeltaC phosphorylation and activation of the Rap1 GTPase, and/or Rap1-mediated inhibition of T cell proliferation. To test this model we will perform the following experiments: 1) we will use Fyn null and SindeltaC mutant mice to address whether Fyn and SindeltaC interaction is required Rap1 activation and inhibition of T cell proliferation. 2) We will generate a constitutively active Rap l mutant to directly show that this G-protein inhibits T cell activation. 3) We will use SindeltaC-expressing mice to determine whether Rapl activation blocks T cell proliferation by interfering with normal Ras signaling and transcriptional activation of IL-2. 4) We will examine the CD4+ helper and CD8+ cytotoxic T cell responses in SindeltaC transgenic mice and test whether SindeltaC-mediated activation of Fyn and Rapl correlates with T cell anergy in vivo.