CYCLIC-GMP-DEPENDENT PROTEIN-KINASE INHIBITS THE RAS MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY/

Agents which increase the intracellular cyclic GMP (cGMP) concentratio n and cGMP analogs inhibit cell growth in several different cell types , but it is not known which of the intracellular target proteins of cG MP is tare) responsible for the growth-suppressive effects of cGMP. Us ing baby hamster kidney (BHK) cells, which are deficient in cGMP-depen dent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)gua nosine-3',5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic mo nophosphate inhibit cell growth in cells stably transfected with a G-k inase 1 beta expression vector but not in untransfected cells or in ce lls transfected with a catalytically inactive G-kinase, We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activ ation of mitogen-activated protein ((MAP) kinase and nuclear transloca tion of MAP kinase in G-kinase-expressing cells but not in G-kinase-de ficient cells. Ras activation by EGF was not impaired in G-kinase-expr essing cells treated with cGMP analogs. We show that activation of G-k inase inhibited c-Raf kinase activation and that G-kinase phosphorylat ed c-Raf kinase on Ser(43), both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser(43) uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser(43) was insensi tive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase acti vity by cGMP and G-kinase, suggesting that Ser(43) in c-Raf is the maj or target for regulation by G-kinase, Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser(43) phosphorylation site of c-Raf is no t conserved in B-Raf. Activation of G-kinase induced MAP kinase phosph atase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by c GMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinas e on Ser(43) and thereby inhibiting its activation and (ii) by inducin g MAP kinase phosphatase 1 expression.