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. Myristoylated Sos2 is non-trans- forming under the same conditions. The lack of transforming activity is correlated with the endogenous mouse Sos2 protein or its myristoylated version having a much shorter half-life than the mouse Sos1 protein. This relative instability correlates with the Sos2 protein being efficiently targeted for ubiquitin-dependent degradation under conditions where Sos1 is not ubiquitinated and stable. Thus, the various Ras exchange factors are subject to distinct forms of regulation, including restricted versus ubiquitous expression, differen- tial stability of their encoded proteins, and diverse mechanisms of activation. We have also found that the N-terminus of Sos1 plays a critical role for the biologic and signaling activity of Sos1. Specifically, the Dbl and pleckstrin homology (PH) domains, located in the N-terminus, each contribute to the activity of membrane localized Sos1. The N-terminus of Sos1 can act as a dominant inhibitory mutant for endogenous Sos1, with most of its inhibitory activity localized to the Dbl and PH domains. Stable association between Sos1 and the EGF receptor, which correlates with endogenous Sos1 activation in respose to EGF, requires the Sos N- terminus, in addition to the Sos C-terminus, which is known to bind to the receptor via adapter molecules.
