The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras

The serine/threonine kinase Raf-1 is an essential component of the MAPK cas cade. Activation of Raf-1 by extracellular signals is initiated by associat ion with intracellular membranes. Recruitment of Raf-1 to membranes has bee n reported to be mediated by direct association with Res and by the phospho lipase D product phosphatidic acid (PA). Here we report that insulin stimul ation of HIRcB fibroblasts leads to accumulation of Res, Raf-1, phosphoryla ted MEK, phosphorylated MAPK, and PA on endosomal membranes, Mutations that disrupt Raf-PA interactions prevented recruitment of Raf-1 to membranes, w hereas disruption of Ras-Raf interactions did not affect agonist-dependent translocation, Expression of a dominant-negative Pas mutant did not prevent insulin-dependent Raf-1 translocation, but inhibited phosphorylation of MA PK Finally, the PA-binding region of Raf-1 was sufficient to target green f luorescent protein to membranes, and its overexpression blocked recruitment of Raf-1 to membranes and disrupted insulin dependent MAPK phosphorylation , These results indicate that agonist-dependent Raf-1 translocation is prim arily mediated by a direct interaction with PA and is independent of associ ation with Ras.