The long-term objective of this research project is to elucidate the function of mammalian ras proteins. The ras gene family has been implicated in the control of normal cell proliferation, and mutated forms of these genes are the most frequently identified oncogenes in an array of human cancers. The goal of the proposed studies is to define the role of ras proteins in signal transduction using T cell activation as a model system. We will first develop functional assays for the involvement of ras proteins in receptor mediated T lymphocyte activation focusing on activating signals that are delivered via the T cell antigen receptor (TCR). Two functional assays win be established: (1) ligand-induced increase in the rate of GTP/GDP exchange on ras proteins and (2) induction of immunologic functions (interleukin-2 and interleukin 2 receptor expression) by activated ras proteins. Using these assays we will then investigate the functional position of ras proteins in relation to specific components of the TCR-mediated signalling pathway. The general approach will involve interfering with the signalling pathway at selected points to allow the identification of upstream regulators and downstream effects of ras proteins. These functional analyses will be complemented by biochemical studies aimed at the isolation and characterization of cellular proteins that interact with ras proteins in T lymphocytes. Using chemical crosslinking and coimmunoprecipitation as methods to probe protein-protein interaction, we will identify proteins that form physical complexes with P21ras. Development of antibodies against these proteins and isolation of DNA clones encoding these proteins will allow the characterization of their physical and biological properties. The functional characterization of ras proteins in T lymphocytes will have broad implications for the understanding of their mode of action as signal transducing proteins in normal and transformed cells.
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