Our studies have involved ras encoded proteins, which have been analyzed largely by examining proteins that influence the activity of Ras protein. The work emphasizes the complex mechanisms that regulate Ras activity, with the regulators of Ras activity being themselves subject to multiple types of regulation. Recent studies have been concerned with Ras-specific guanine nucleotide exchange factors, which are upstream activators of Ras. These include GRF, which is expressed primarily in brain, and Sos1 and Sos2, which are ubiquitously expressed. When expressed in mouse NIH 3T3 cells, full-length GRF, or fragments that include its catalytic domain, induce Ras-dependent focal transformation. By contrast, neither sos gene is transforming. However, addition of a membrane targeting signal to the N-terminus of Sos1 renders the protein transforming. Mutational analysis of the membrane targeted Sos1 has shown that a pleckstrin homology region in the N-terminus of Sos1 is required for transformation, while the C-terminus of Sos1, which binds the adapter protein Grb-2, is dispensable. Unlike Sos1, addition of the membrane targeting signal to Sos2 does not lead to transformation. Analysis of Sos1 and Sos2 proteins in cells indicates that while Sos1 has a long half-life (greater than 18 hours), the half-life of Sos2 is much shorter (approximately 3 hours). In vitro analysis with rabbit reticulocyte extracts indicates that Sos2 is subject to ubiquitin mediated degradation, while Sos1 is resistant under the same conditions. Thus, the various Ras exchange factors are subject to distinct forms of regulation, including restricted versus ubiquitous expression, differential stability of their encoded proteins, and diverse mechanisms of activation.
