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.
