REQUIREMENT FOR RAS IN RAF ACTIVATION IS OVERCOME BY TARGETING RAF TOTHE PLASMA-MEMBRANE

A CONSERVED tyrosine kinase-activated signal transduction pathway has recently been identified that comprises the plasma membrane-bound smal l guanine-nucleotide-binding protein Ras and the protein kinases Raf, MAP-kinase kinase and MAP kinase(1,2). GTP-bound Ras interacts directl y with the amino-terminal regulatory domain of Raf(3-8), but although Ras and Raf can be coimmunoprecipitated from ligand-stimulated cells(9 ,10), Ras-GTP does not stimulate the kinase activity of Raf in vitro(6 ). Furthermore, we have failed to detect Ras in preparations of active detergent-solubilized Raf, demonstrating that once it is activated, R af does not require Ras. Whereas Raf is normally cytosolic, in cells e xpressing active Ras, Raf is associated with the plasma membrane. This led us to investigate whether Ras is required to localize Raf to the plasma membrane in order for Raf to become activated. We fused the mem brane localization signal of K-Ras(4B) to the carboxy terminus of Raf. This protein is constitutively active and can be further activated by epidermal growth factor, independently of Ras. Our results indicate t hat Ras functions as a regulated, membrane-bound anchor for Raf, and t hat other signal(s) also contribute to Raf activation.