title: Cyclic AMP-dependent kinase regulates Raf-1 kinase mainly by phosphorylation of serine 259.
abstract: The Raf-1 kinase activates the ERK (extracellular-signal-regulated kinase) pathway. The cyclic AMP (cAMP)-dependent protein kinase (PKA) can inhibit Raf-1 by direct phosphorylation. We have mapped all cAMP-induced phosphorylation sites in Raf-1, showing that serines 43, 259, and 621 are phosphorylated by PKA in vitro and induced by cAMP in vivo. Serine 43 phosphorylation decreased the binding to Ras in serum-starved but not in mitogen-stimulated cells. However, the kinase activity of a RafS43A mutant was fully inhibited by PKA. Mutation of serine 259 increased the basal Raf-1 activity and rendered it largely resistant to inhibition by PKA. cAMP increased Raf-1 serine 259 phosphorylation in a PKA-dependent manner with kinetics that correlated with ERK deactivation. PKA also decreased Raf-1 serine 338 phosphorylation of Raf-1, previously shown to be required for Raf-1 activation. Serine 338 phosphorylation of a RafS259A mutant was unaffected by PKA. Using RafS259 mutants we also demonstrate that Raf-1 is the sole target for PKA inhibition of ERK and ERK-induced gene expression, and that Raf-1 inhibition is mediated mainly through serine 259 phosphorylation.
authors.affiliation: The Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, United Kingdom.
authors.forename: Amardeep S
authors.initials: AS
authors.lastname: Dhillon
authors.forename: Claire
authors.initials: C
authors.lastname: Pollock
authors.forename: Helge
authors.initials: H
authors.lastname: Steen
authors.forename: Peter E
authors.initials: PE
authors.lastname: Shaw
authors.forename: Harald
authors.initials: H
authors.lastname: Mischak
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D017930:Genes, Reporter; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010449:Peptide Mapping; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D010726:Phosphodiesterase Inhibitors; D005576:Colforsin; D012694:Serine; D000242:Cyclic AMP; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine