Raf-1 is involved in the regulation of the interaction between guanine nucleotide exchange factor and Ha-Ras - Evidences for a function of Raf-1 and phosphatidylinositol 3-kinase upstream to Ras

The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the has effector c-Raf-l on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-l (Raf (1-149) and Raf (51-131)) were used. Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras Raf complex, showed that the binding of Raf and GEF to Ha-Ras GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras GTP by Raf. Consistent with our results, the OFF-stimulated exchange of Ha-Ras GTP was also inhibited by another effector of Ras, the RED (amino acid residues 133-314) of phosphatidylinositol S-kinase p110 alpha. Our data show that Raf-l and phosphatidylinositol I-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effecters and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell.