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 ( Ser(43\)), serine 233 (Ser(233)), and serine 259 (Ser(259)). 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 Ser(233) and Ser(259). 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.