Fyn and Rap1 in T cell Activation and Immune Response

描述：（由申请人提供）本提案的目标是研究 涉及小GTP结合蛋白Rap 1的信号通路在T 细胞功能对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，第一次显示出这种途径是 在活化的Src激酶下游使用。我们发现了这个途径， 一种激活Src激酶的新方法，一种利用天然配体的方法， 其结合Src-激酶SH 3结构域并激活Src激酶信号传导。 具体来说，我们使用了一种我们分离的蛋白质，Sin，一种Src和Fyn-SH 3结合蛋白， 蛋白表达截短的组成型活性形式Sin的小鼠 （SindeltaC）表现出缺陷性T细胞增殖。我们建议调查 SindeltaC介导的T细胞抑制的机制 激活，并评估SindeltaC转基因小鼠的免疫应答 都暴露了具体来说，我们将研究是否抑制效果的 SindeltaC表达对T细胞功能的影响是由于信号传导的诱导 涉及SindeltaC介导的Fyn激活的级联反应，SindeltaC Rap 1 GTPase的磷酸化和激活，和/或Rap 1介导的 抑制T细胞增殖。为了测试这个模型，我们将执行 以下实验：1）我们将使用Fyn缺失和SindeltaC突变小鼠， 解决Fyn和SindeltaC相互作用是否需要Rap 1激活， 抑制T细胞增殖。2)我们将产生一个积极的 Rap 1突变体直接显示该G蛋白抑制T细胞活化。 3)我们将使用表达SindeltaC的小鼠来确定Rapl激活是否 通过干扰正常Ras信号传导阻断T细胞增殖， IL-2的转录激活。4)我们将检查CD 4+辅助细胞和CD 8 + SindeltaC转基因小鼠的细胞毒性T细胞应答，并测试是否 SindeltaC介导的Fyn和Rapl激活与T细胞无能相关， vivo.

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.