Fyn and Rap1 in T cell Activation and Immune Response

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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.