Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak

Activation of the protein kinase Raf-l is a complex process involving association with the GTP-bound form of Pas (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation (reviewed in [1]), We have reported previously that p21-activated kinase 3 (Pak3) upregulates Raf-l through direct phosphorylation on Ser338 [2]. Here, we investigated the origin of the signal for Pak-mediated Raf-l activation by examining the role of the small GTPases Cdc42, Pac and Pas, and of phosphatidylinositol (PI) 3-kinase, Pak3 acted synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-l, Raf-CX, a membrane-localized Raf-l mutant, and Raf-l mutants defective in Pas binding. Raf-l mutants defective in Pas binding were also readily activated by RasV12. This indirect activation of Raf-l by Pas was blocked by a dominant-negative mutant of Pak, implicating an alternative Pas effector pathway in Pak-mediated Raf-l activation. Subsequently, we show that Pak-mediated Raf-l activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase, In addition, p85 Delta a mutant of the Pt a-kinase regulatory subunit, inhibited the stimulated activity of Raf-l, Pharmacological inhibitors of PI 3-kinase also blocked both activation and Ser338 phosphorylation of Raf-l induced by epidermal growth factor (EGF). Thus, Raf-l activation by Pas is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Pas effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1.