This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. T cell activation is initiated by a biochemical signal generated upon specific antigen/MHC recognition by the T cell receptor (TCR). The earliest and an obligatory step in T cell activation is the tyrosine phosphorylation of cellular proteins and TCR components. The Src family tyrosine kinases p59fyn (Fyn) and p56lck (Lck) physically interact with the CD3/zeta and CD4/8 molecules, respectively, and genetic experiments have demonstrated their critical roles in early TCR signalling. The Src homology domains (SH3 and SH2) of Fyn and Lck mediate interactions with components of the signalling machinery, and are essential for repression of kinase activity. We have demonstrated a novel physical interaction between SH3 and SH2 domains of Fyn and Lck, which leads to a testable hypothesis that repression of Src-family kinases is mediated by dimerization. We have also demonstrated a ligand-sensitive communication between adjacent Fyn SH3 and SH2 domains, and we hypothesize this to provide a mechanism for regulating the activation-dependent assembly of signalling complexes. Here, we propose approaches to examine these hypotheses in vivo. We will use nondenaturing gel electrophoresis, density gradient centrifugation, chemical cross-linking, two-epitope tagging and yeast two-hybrid interaction systems to assess dimerization of Fyn and Lck in vivo, and test if dimers are modulated by T cell activation. Furthermore, we will use deletional and mutational analyses to demonstrate that dimerization is mediated by SH3-SH2 interaction. We will incorporate the mutations that influence physical and functional SH3-SH2 interactions into full-length Fyn and Lck cDNAs, transfect them into cells and assess the effect of mutations on in vivo biological functions. 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 (a murine hybridoma and a gamma/delta TCR-reconstituted Jurkat), association of mutant proteins with components of signal transduction machinery, their subcellular localization and the status of the regulatory interaction of SH2 with the carboxy 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 better understanding of their oncogenic activation.
