EFFECTOR DOMAIN MUTATIONS DISSOCIATE P21(RAS) EFFECTOR FUNCTION AND GTPASE-ACTIVATING PROTEIN-INTERACTION

The GTPase activity of p21ras is stimulated by GTPase-activating prote ins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also propos ed effectors of ras. We have sought activating substitutions in c-H-ra s in the region encoding the effector domain, on the rationale that su ch mutations would dissociate effector function from negative regulati on by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaire d. The results support the idea that the p21 structural requirements f or effector function and GAP interaction are quite different and sugge st that some molecule(s) other than p120GAP serves as the ras effector . In contrast to the results obtained with p120GAP, the Pro-34-->Arg p 21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.