In higher eukaryotes, the Ras and Raf-1 proto-oncoproteins transduce growth and differentiation signals initiated by tyrosine kinases. The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) are shown to interact, directly in vitro and in a yeast expression system. Raf-1 (1-257) binds GTP-Ras in preference to GDP-Ras, and inhibits Ras-GAP activity. Mutations in and around the Ras effector domain impair Ras binding to Raf-1 (1-257) and Ras transforming activity in parallel.

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...

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.

Activation of the Ras–MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane–proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf–MEK1–MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

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The c-Raf-1 proto-oncoprotein is a Ras-GTP-regulated protein kinase that associates in situ with 14-3-3 proteins, which are naturally dimeric. In COS cells, recombinant Raf is found in oligomeric assemblies. To examine whether induced oligomerization can alter Raf kinase activity, sequences encoding the FK506-binding protein FKBP12 were fused to the amino terminus of c-Raf-1, introducing a binding site for FK506. Oligomerization of recombinant FKBP-Raf in situ, induced by the addition of the dimeric FK506 derivative FK1012A, activated Raf kinase activity at least half as well as epidermal growth factor (EGF). As with EGF, activation of FKBP-Raf by FK1012A is entirely Ras-GTP dependent. Thus oligomerization of Raf per se promotes Raf activation through a Ras-dependent mechanism.

Mast cells generated from Rac2-deficient (-/-) mice demonstrated defective actin-based functions, including adhesion, migration, and degranulation. Rac2(-/-) mast cells generated lower numbers and less mast cell colonies in response to growth factors and were deficient in vivo. Rac2(-/-) mast cells demonstrated a significant reduction in growth factor-induced survival, which correlated with the lack of activation of Akt and significant changes in the expression of the Bcl-2 family members BAD and Bcl-XL, in spite of a 3-fold induction of Rac1 protein. These results suggest that Rac2 plays a unique role in multiple cellular functions and describe an essential role for Rac2 in growth factor-dependent survival and expression of BAD/Bcl-XL.