DISTINCT MECHANISMS MEDIATE THE INITIAL AND SUSTAINED PHASES OF INTEGRIN-MEDIATED ACTIVATION OF THE RAF MEK/MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE/

Integrin-mediated adhesion to the extracellular matrix activates the c anonical mitogen-activated protein kinase cascade, although the exact mechanism is not fully resolved. We show that integrin-mediated activa tion of Raf-l, an upstream regulator of mitogen-activated protein kina se, occurs in two phases. Efficient early activation of Raf required R af-Ras interaction but was not affected by protein kinase C (PKC) inhi bitors, while a lower, sustained level of activity was independent of Raf-Ras interaction but was reduced by PEC inhibitors. The combination of PEC inhibition and lack of Ras binding completely blocked integrin -mediated Raf activity. The activity of a membrane-bound Raf mutant th at is deficient in Ras binding (Raf-R89L-CAAX) was also regulated by a dhesion. Raf-R89L-CAAX activity was low in nonadherent cells, was rapi dly stimulated to wild-type levels by cell. adhesion, and remained at nearly maximal levels longer than wild-type activity. The activation o f wild-type and mutant Raf proteins was ablated by cytochalasin D, dem onstrating that cytoskeletal organization is required for activation o f Raf, even when targeted to the membrane. These data suggest distinct initial and sustained phases of integrin-mediated Raf activation that require Raf membrane localization and possibly PKC activity, respecti vely, and that integrin-mediated adhesion may regulate a cytoskeleton- associated factor(s) responsible for Raf activation.