Our broad objective is to trace the pathways utilized by receptor tyrosine kinases to regulate cell proliferation and to understand how these pathways are altered in malignancy. Previous studies, from this and other laboratories, have shown that specific cell proteins bind to phosphorylated tyrosine residues in activated receptor tyrosine kinases. The bound proteins regulate different signaling pathways. One pathway leads to the activation of the membrane-associated GTPase, Ras, by promoting the release of GDP from Ras and its replacement by GTP. In its GTP state, Ras binds protein kinases of the Raf family. The membrane-associated Ras-Raf complex is a substrate for one or more other regulatory inputs that together stimulate Raf kinase activity. One known substrate of Raf is responsible for MAP kinase activation, but Raf may have other substrates. We have identified three other proteins capable of specific interactions with RasGTP. These proteins are: a Ral guanine nucleotide dissociation stimulator (GDS), a protein related to the RalGDS, and a protein related to the catalytic (P110) subunits of phosphoinositide 3-kinases. We propose to confirm that these proteins interact with Ras GTP in the cell, determine the biochemical functions of these proteins, and evaluate their roles in oncogenic transformation by activated mutants of Ras. Another goal of this proposal is to identify residues on Ras and Raf involved in binding and generate tools for selective activation of the Raf pathway and other Ras-dependent pathways.
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