Phosphatidylinositol 3-kinase regulates Raf1 through Pak phosphorylation of serine 338

We have previously shown that inhibition of phosphatidylinositol (PI) 3 kin ase severely attenuates the activation of extracellular signal-regulated ki nase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [I], To investigate ho w PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulat ion by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf 1, was phosphorylated in a PI 3-kinase-dependent manner following engagemen t of fibronectin receptors. In addition, fibronectin activation of a Raf1 m utant containing a phospho-mimic mutation (S338D) was independent of PI 3-k inase. Furthermore, integrin-induced activation of the serine/threonine kin ase Pak-l,which has been shown to phosphorylate Raf1 Ser338, was also depen dent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 muta nt blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3 -kinase regulates phosphorylation of Raf1 Ser338 through the serine/threoni ne kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Pas leads to stro ng activation of Raf1 kinase activity by integrins.