COLOCALIZATION OF RAS AND RAL ON THE MEMBRANE IS REQUIRED FOR RAS-DEPENDENT RAL ACTIVATION THROUGH RAL GDP DISSOCIATION STIMULATOR

Ral GDP dissociation stimulator (RalGDS), a putative effector protein of Ras, stimulated the GDP/GTP exchange reaction of the post-tanslatio nally lipid-modified but not the unmodified form of Ral in response to epidermal growth factor in COS cells, The RalGDS action on Ral was en hanced by an active form of Res but not a Ras mutant which was not pos t-translationally modified in the cells, The RalGDS activity was inhib ited by acidic membrane phospholipids such as phosphatidylinositol and phosphatidylserine but not by phosphatidylcholine or phosphatidyletha nolamines in vitro. The posttranslationally modified form but not unmo dified form of Ras, Ral, and Rap were incorporated in liposomes consis ting of these phospholipids. When Ral was incorporated alone in the li posomes, RalGDS did not stimulate the dissociation of GDP from Ral, Wh en Rat was incorporated with the GTP-bound form of Ras in the liposome s, RalGDS stimulated the dissociation of GDP from Ral, while the GDP-b ound form of Ras did not affect the RalGDS action, The Ras-dependent R al activation through RalGDS required the Ras-binding domain of RalGDS , Rap, which shared the same effector loop as Ras, also stimulated the dissociation of GDP from Ral through RalGDS in the liposomes, althoug h Rap did not enhance the RalGDS action in COS cells, Taken together w ith our previous observations that Ras recruits RalGDS to the membrane , these results indicate that the post-translational modifications of Ras and Ral are important for Ras-dependent Ral activation through Ral GDS and that colocalization of Ras and Ral on the membrane is necessar y for Ral activation in intact cells.