Phosphorylation of serine 43 is not required for inhibition of c-Raf kinase by the cAMP-dependent protein kinase

The activity of the serine/threonine kinase c-Raf (Raf) is inhibited by inc reased intracellular cAMP. This is believed to require phosphorylation with the cAMP-dependent protein kinase (PKA), although the mechanism by which P RA inhibits Raf is controversial. We investigated the requirement for PKA p hosphorylation using Raf mutants expressed in HEK293 or NIH 3T3 cells. Phos phopeptide mapping of P-32-labeled Raf (WT) or a mutant lacking a putative PKA phosphorylation site (serine to alanine, S43A) confirmed that serine 43 (Ser(43)) was the major cAMP (forskolin)-stimulated phosphorylation site i n vivo. Interestingly, the EGF-stimulated Raf kinase activity of the S43A m utant was inhibited by forskolin equivalently to that of the WT Raf. Forsko lin also inhibited the activation of an N-terminal deletion mutant Delta 5- 50 Raf completely lacking this phosphorylation site. Although WT Raf was ph osphorylated by PKA, phosphorylation did not inhibit Raf catalytic activity in vitro, nor did forskolin treatment inhibit the activity of an N-termina lly truncated Raf protein (Raf 22W) or a full-length Raf protein (Raf-CAAX) expressed in NIH 3T3 cells. In contrast, forskolin inhibited the EGF-depen dent activation of a Raf isoform (B-Raf), lacking an analogous phosphorylat ion site to Ser43. Thus, these results demonstrate that PKA exerts its inhi bitory effects independently of direct Raf phosphorylation and suggests ins tead that PKA prevents an event required for the EGF-dependent activation o f Raf.