The small guanine nucleotide binding protein Ras participates in a growth promoting signal transduction pathway. The mechanism by which interaction of Ras with the protein kinase Raf leads to activation of Raf was studied. Raf was targeted to the plasma membrane by addition of the COOH-terminal localization signals of K-ras. This modified form of Raf (RafCAAX) was activated to the same extent as Raf coexpressed with oncogenic mutant Ras. Plasma membrane localization rather than farnesylation or the presence of the additional COOH-terminal sequence accounted for the activation of RafCAAX. The activation of RafCAAX was completely independent of Ras; it was neither potentiated by oncogenic mutant Ras nor abrogated by dominant negative Ras. Raf, once recruited to the plasma membrane, was not anchored there by Ras; most activated Raf in cells was associated with plasma membrane cytoskeletal elements, not the lipid bilayer. Thus, Ras functions in the activation of Raf by recruiting Raf to the plasma membrane where a separate, Ras-independent, activation of Raf occurs.

Stimulation ofaortic smooth muscle cells with platelet-derived growth factor BB homodimer (PDGF-BB) leads to the rapid activation ofmitogen-activated protein kinase (MAPK) and MAPK kinase (MAPKK). Compounds that increase cAMP and activate protein kinase A (PKA) prostaglandin E2, isoproterenol, cholera toxin, and forskolinwere found to inhibit the PDGF-BB-induced activation of MAPKK and MAPK. Forskolin, but not the inactive analogue 1,9-dideoxyforskolin, inhibited PDGF-BB-stimulated MAPKK and MAPK activation in a dose-dependent manner. PKA antagonism of MAPK signaling was observed at all doses of PDGF-BB or PDGF-AA. PKA did not inhibit MAPKK and MAPK activity in vitro, and MAPKK and MAPK from extracts of forskolin-treated cells could be activated normally with purified Raf-1 and MAPKK, respectively, suggesting that PKA blocked signaling upstrea of MAPKK. Neither PDGF-BBstimulated tyrosine autophosphorylation of the PDGF receptor (3 subunit nor inositol monophosphate accumulation was affected by increased PKA activity, suggesting that PKA inhibits events downstream of the PDGF receptor. This study provides an example of cross talk between two important signaling systems activated by physiological stimuli in smooth muscle cells-namely, the PKA pathway and the growth factoractivated MAPK cascade.The p44 and p42 mitogen-activated protein kinases (MAPKs) (Erkl and Erk2) are central components of a growth factorstimulated protein kinase cascade found in organisms as diverse as mammals and yeast (reviewed in refs.

To identify proteins that may participate in the activation of the protein kinase Raf, proteins that interact with Raf were selected in a two-hybrid screen. Two members of the 14-3-3 protein family were isolated that interacted with both the amino terminal regulatory regions of Raf and the kinase domain of Raf, but did not compete with the guanine nucleotide-binding protein Ras for binding to Raf. 14-3-3 proteins associated with Raf in mammalian cells and accompanied Raf to the membrane in the presence of activated Ras. In yeast cells expressing Raf and MEK, mammalian 14-3-3 beta or 14-3-3 zeta activated Raf to a similar extent as did expression of Ras. Therefore, 14-3-3 proteins may participate in or be required for the regulation of Raf function. These findings suggest a role for 14-3-3 proteins in Raf-mediated signal transduction.

. 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.

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p2l' can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitopetagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-l purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.The serine/threonine kinase Raf-1 is activated by many growth factors, including epidermal growth factor, plateletderived growth factor, erythropoietin, and insulin (1,3,4,6,10,12,13,16,22,23) and is believed to play a central role in cell growth. Several studies have shown that Raf-1 functions downstream of and is required for signalling by receptor and nonreceptor tyrosine kinases and Ras in mammalian cells, in Drosophila melanogaster, and in Caenorhabditis elegans. That Raf-1 acts downstream of Ras is indicated by experiments in which raf-1 antisense RNA and kinase-defective Raf-1 mutants were able to block cell proliferation and transformation by activated Ras (15). The same study also used the expression of raf-1 antisense RNA and kinasedefective Raf-1 mutants to show that Raf-1 is required for the normal growth of the cells. Similarly, the involvement of Ras in the mitogen-activated protein (MAP) kinase pathway is well established (20,24,26,27,31). However, the mechanism by which Src and Ras activate Raf-1 is not known. It has been suggested that the activation of Raf-1 is mediated by yet another kinase whose activity is stimulated by Ras and Src. The hypothesis that Raf-1 is positively regulated by phosphorylation of serine and/or threonine residues is supported by observations that stimulation of growth factor receptors and expression of membrane-bound oncogene products result in the hyperphosphorylation of Raf-1 and an increase in its kinase activity (3,12,13,15,16,22,23). A more recent study showed protein kinase Cot (PKC)-induced phosphorylation of Raf-1 led to increased autokinase a...