PHYSICAL ASSOCIATION WITH RAS ENHANCES ACTIVATION OF MEMBRANE-BOUND RAF (RAFCAAX)

The transforming activity of artificially membrane-targeted Raf1 sugge sts that Pas-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation, Cellular has is concentrated in th e caveolae, a microdomain of the plasma membrane that is highly enrich ed in caveolin, glycosylphosphatidylinositol-anchored proteins, and si gnal transduction molecules, Growth factor stimulation recruits Raf1 t o this membrane domain, Whether Pas simply promotes Raf1 association w ith caveolae membranes or also modulates subsequent activation events is presently unclear, We have identified a ras variant, ras12V,37G, th at does not interact with Raf1 but does interact with a mutant raf1, r af1(257L), To examine the role of Pas in the activation of membrane-bo und Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V,37G, Cell fractionation localized bo th raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of ra s12V,37G expression; however, coexpression of ras12V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by inducti on of cellular transformation, increased Raf kinase activity, and indu ction of activated MAP kinase. These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from mem brane recruitment.