Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.
Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser43 and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.
Suhasini
M
M
Department of Medicine, University of California, San Diego, La Jolla, California 92093-0652, USA.
Li
H
H
Lohmann
S M
SM
Boss
G R
GR
Pilz
R B
RB
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D054638:Dual Specificity Phosphatase 1; D004789:Enzyme Activation / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D011905:Genes, ras / Q000235:genetics; D017874:Immediate-Early Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection / Q000235:genetics
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D013873:Thionucleotides; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D004815:Epidermal Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D017869:Cyclic GMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases; D006152:Cyclic GMP