Ras oncogenes are frequently associated with human cancer and activated forms of ras are powerfully transforming in experimental systems. Ras proteins control multiple signaling pathways via multiple effectors. The mechanisms by which those effectors are activated by Ras remain unclear. We have investigated the interaction between Ras and its effector/regulator p120 GAP. We have found that Ras serves to modulate an interamolecular interaction between the catalytic and the regulatory regions of p120 GAP. We are also investigating the molecular mechanisms by which the Ras proteins activate the Raf kinase and Nore1 class of effectors. The ultimate aim of these studies is to allow the design of highly specific small molecule inhibitors of Ras-mediated transformation.We have found that Ras activates Raf by binding to two distinct sites on Raf. Both binding interactions are crtitical in generating a fully activated Raf molecule. We have identified specific residues within the second Ras binding domain that are essential for Ras interaction. Moreover, we have characterized the interplay of 14-3-3 and phosphatidylserine as co-factors in the Ras-mediated activation of Raf. We now have evidence that, as with p120 GAP, Ras serves to release the C-terminal kinase domain of Raf from inhibitory, intramolecular binding contacts in the N-terminal, regulatory domain of Raf. This interaction appears to mediated by 14-3-3 and requires the binding of a lipid co-factor (PS) for full manifestation. Moreover, it appears that the release of the inhibitory intramolecular interaction allows the same binding sites to modulate a kinase domain dimerization event essential for activity.We are currently studying the novel Ras effector Nore1 to determine if Ras also acts to destabilize intramolecular binding events or to influence dimerization.
