INTERACTION OF RAS AND RAF IN INTACT MAMMALIAN-CELLS UPON EXTRACELLULAR STIMULATION

It has recently been shown that Ras proteins interact directly with Ra f serine/threonine kinases in vitro and in the yeast two-hybrid system , leading to speculation that Raf proteins function as effecters for R es. Here it is demonstrated that the endogenous Raf-1 protein co-immun oprecipitates with Ras from mammalian cells when the non-neutralizing anti-Ras monoclonal antibody Y13-238 is used. The formation of a Ras-R af complex is absolutely dependent on prior treatment of the cells wit h a stimulus that activates Ras: phorbol ester or anti-T cell receptor antibody in the case of human peripheral blood T lymphoblasts, or epi dermal growth factor in the case of Rat-1 fibroblasts. Up to 3% of cel lular Raf-1 can be found in association with Ras. The association is n ot competed by addition of exogenous GST-Raf to the cell lysates and i s therefore unlikely to be due to Ras-Raf binding after cell lysis. Sp ecific interaction of Ras and Raf therefore occurs in intact mammalian cells in response to stimuli that cause Res to become GTP-bound.