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

Abstract

The activity of the serine/threonine kinase c-Raf(Raf) is inhibited by increased intracellular cAMP. This is believed to require phosphorylation with the cAMP-dependent protein kinase (PKA), although the mechanism by which PKA inhibits Raf is controversial. We investigated the requirement for PKA phosphorylation using Raf mutants expressed in HEK293 or NIH 3T3 cells. Phosphopeptide mapping of 32P-labeled Raf (WT) or a mutant lacking a putative PKA phosphorylation site (serine to alanine, S43A) confirmed that serine 43 (Ser43) was the major cAMP (forskolin)-stimulated phosphorylation site in vivo. Interestingly, the EGF-stimulated Raf kinase activity of the S43A mutant was inhibited by forskolin equivalently to that of the WT Raf. Forskolin also inhibited the activation of an N-terminal deletion mutant Δ5-50 Raf completely lacking this phosphorylation site. Although WT Raf was phosphorylated by PKA, phosphorylation did not inhibit Raf catalytic activity in vitro, nor did forskolin treatment inhibit the activity of an N-terminally truncated Raf protein (Raf 22W) or a full-length Raf protein (Raf-CAAX) expressed in NIH 3T3 cells. In contrast, forskolin inhibited the EGF-dependent activation of a Raf isoform (B-Raf), lacking an analogous phosphorylation site to Ser43. Thus, these results demonstrate that PKA exerts its inhibitory effects independently of direct Raf phosphorylation and suggests instead that PKA prevents an event required for the EGF-dependent activation of Raf.