The pathway involving the signalling protein p21Ras propagates a range of extracellular signals from receptors on the cell membrane to the cytoplasm and nucleus. The Ras proteins regulate many effectors, including members of the Raf family of protein kinases. Ras-dependent activation of Raf-1 at the plasma membrane involves phosphorylation events, protein-protein interactions and structural changes. Phosphorylation of serine residues 338 or 339 in the catalytic domain of Raf-1 regulates its activation in response to Ras, Src and epidermal growth factor. Here we show that the p21-activated protein kinase Pak3 phosphorylates Raf-1 on serine 338 in vitro and in vivo. The p21-activated protein kinases are regulated by the Rho-family GTPases Rac and Cdc42. Our results indicate that signal transduction through Raf-1 depends on both Ras and the activation of the Pak pathway. As guanine-nucleotide-exchange activity on Rac can be stimulated by a Ras-dependent phosphatidylinositol-3-OH kinase, a mechanism could exist through which one Ras effector pathway can be influenced by another.

Protein kinase C (PKC) regulates activation of the Raf-1 signaling cascade by growth factors, but the mechanism by which this occurs has not been elucidated. Here we report that one mechanism involves dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1. Classic and atypical but not novel PKC isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP Ser-153 phosphorylation by PKC either in vitro or in response to 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor causes release of RKIP from Raf-1, whereas mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC can rescue inhibition of the mitogen-activated protein (MAP) kinase signaling cascade by wild-type but not mutant S153V RKIP. Taken together, these results constitute the first model showing how phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase signaling cascade and may represent a general mechanism for the regulation of MAP kinase pathways.The MAP 1 kinase cascade, an evolutionarily conserved signaling module, stimulates numerous biological processes including growth and differentiation. The known elements of the pathway include a MAP kinase kinase kinase that phosphorylates and activates a MAP kinase kinase, which, in turn, phosphorylates the threonine-X-tyrosine (TXY) activation domain of MAP kinase (reviewed in Ref. 1). The first characterized subfamily of MAP kinases, termed extracellular signal-regulated kinases (ERKs), is activated by growth factors and other stimuli via a cascade involving Ras, Raf-1 kinase, and MEK/ERK kinase (MEK). Activation of MAP kinase is under exquisite regulatory control, particularly at the level of Raf-1 activation. The N-terminal regulatory domain of Raf-1 interacts with Ras leading to dephosphorylation at negative regulatory sites, conformational changes to expose the kinase domain, and subsequent phosphorylation at activating sites such as serine 338 (Ser-338) and tyrosine 341 (Tyr-341) (reviewed in Ref.2). A variety of studies have shown that protein kinase C (PKC) isozymes are also capable of activating Raf-1 (3-5) and/or the downstream MEK (6), but the mechanism has not been elucidated.The PKC family of serine/threonine kinases are key mediators of several physiological processes including growth, death, differentiation, and transformation (reviewed in Ref. 7). There are three major classes of PKCs that are distinguished by their physiological activators. The classical PKCs (␣, ␤I, ␤II, and ␥) require both Ca 2ϩ and diacylglycerol (DAG) for activation whereas the novel PKCs (␦, ⑀, , and ) are Ca 2ϩ -independent but still require DAG. Both of these classes of PKCs are activated by phorbol esters that mimic the DAG stimulus. In contrast, the atypical PKCs, and /, are Ca 2ϩ -, DAG-, and phorbol ester-independent. Not only are PKCs able to activate Raf-1, but in a number of cell systems they are required for the activation of ERKs by growth factors (8 -11).Multiple hypotheses have been proposed to explain how PKCs activate the ERK cascade, including direct phos...

. These data demonstrate that Raf-1 residues 338 to 341 constitute a unique phosphoregulatory site in which the phosphorylation of serine and tyrosine residues contributes to the regulation of Raf by Ras, Src, and Ras-independent membrane localization.

We have previously shown that inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the activation of extracellular signal-regulated kinase (Erk) following engagement of integrin/fibronectin receptors and that Raf is the critical target of PI 3-kinase regulation [1]. To investigate how PI 3-kinase regulates Raf, we examined sites on Raf1 required for regulation by PI 3-kinase and explored the mechanisms involved in this regulation. Serine 338 (Ser338), which was critical for fibronectin stimulation of Raf1, was phosphorylated in a PI 3-kinase-dependent manner following engagement of fibronectin receptors. In addition, fibronectin activation of a Raf1 mutant containing a phospho-mimic mutation (S338D) was independent of PI 3-kinase. Furthermore, integrin-induced activation of the serine/threonine kinase Pak-1, which has been shown to phosphorylate Raf1 Ser338, was also dependent on PI 3-kinase activity and expression of a kinase-inactive Pak-1 mutant blocked phosphorylation of Raf1 Ser338. These results indicate that PI 3-kinase regulates phosphorylation of Raf1 Ser338 through the serine/threonine kinase Pak. Thus, phosphorylation of Raf1 Ser338 through PI 3-kinase and Pak provides a co-stimulatory signal which together with Ras leads to strong activation of Raf1 kinase activity by integrins.