REGULATION OF THE RAS TRANSFORMATION PATHWAY

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The Ras oncoproteins play a critical role in cell growth and differentiation, transducing signals from upstream protein tyrosine kinases (PTKs) to the nuclear transcriptional machinery. In recent years, considerable progress has been made in identifying additional proteins that relay growth stimulatory signals down the "ras pathway". However, interactions between existing proteins are only poorly understood, and it is clear that further regulatory molecules remain to be identified. The goal of this proposal is to characterize the mechanism(s) by which three critical members of the Ras pathway, GAP (GTPase-activating protein), GDS (guanine nucleotide dissociation stimulator), and GRB2 regulate Ras activity and to identify novel molecular associations required for their function. While pl20 GAP is clearly a negative regulator of ras function, its putative role as a downstream target and effector of ras signaling remains controversial. We have recently obtained evidence for such a role and have narrowed this property down to a region that includes the SH3 domain. We will further characterize the role of this region by isolation, deletion and mutational analysis, and identify putative downstream effector(s) of the Ras transformation pathway that interact with GAP-SH3. Recent biochemical and genetic evidence has implicated CDC25 homologs (mCDC25, mSOS1 and 2) as activators of ras function. However, beyond their function as stimulators of the GTP/GDP cycle, little is known about the biological consequences of their interaction with ras proteins. Therefore, we will establish the role of these ras GDSs in mediating normal and oncogenic ras functions, and determine whether deregulated GDS activity may cause transformation in the absence of ras mutations. Finally, recent studies have implicated GRB-2 as the critical link that transmits the mitogenic signal from activated receptor PTKs to ras, and acts to stimulate the SOS exchange factor. However, we have obtained preliminary evidence that the role of GRB2 in intracellular signaling may be more complex, and may involve interactions with proteins other than receptor-PTKs. We have proposed studies to clarify these additional functions of GRB2. Taken together, these studies will provide fundamental information on the mechanisms of Ras activation and may identify additional components involved in regulating the ras pathway.