Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras.
Cyclic AMP (cAMP) blocks Raf-1 activation by stimulating its phosphorylation on serine 43 (Ser43), serine 233 (Ser233), and serine 259 (Ser259). We show here that phosphorylation of all three sites blocks Raf-1 binding to Ras.GTP in vivo and that cAMP stimulates binding of 14-3-3 proteins to Ser233 and Ser259. We also show that Raf-1 and protein kinase A (PKA) form a complex in vivo that is disrupted by cAMP and that ablation of PKA by use of small interfering RNA blocks phosphorylation by cAMP. The ability of PKA to block Raf-1 activation is ablated by the PKA inhibitor H89. These studies suggest that Raf-1 and cAMP form a signaling complex in cells. Upon activation of PKA, Raf-1 is phosphorylated and 14-3-3 binds, blocking Raf-1 recruitment to the plasma membrane and preventing its activation.
Dumaz
Nicolas
N
Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom.
Marais
Richard
R
D048948:14-3-3 Proteins; D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors*; D034622:RNA Interference; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013449:Sulfonamides; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D010455:Peptides; D013449:Sulfonamides; D012694:Serine; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide