PHOSPHORYLATION OF RAF-1 SERINE-338 SERINE-339 IS AN ESSENTIAL REGULATORY EVENT FOR RAS-DEPENDENT ACTIVATION AND BIOLOGICAL SIGNALING

Activation of the Raf serine/threonine protein kinases is lightly regu lated by multiple phosphorylation events. Phosphorylation of either ty rosine 340 or 341 in the catalytic domain of Raf-1 has been previously shown to induce the ability of the protein kinase to phosphorylate ME K. By using a combination of mitogenic and enzymatic assays, we found that phosphorylation of the adjacent residue, serine 338, and, to a le sser extent, serine 339 is essential for the biological and enzymatic activities of Raf-1. Replacement of S338 with alanine blocked the abil ity of prenylated Raf-CX to transform Rat-1 fibroblasts. Similarly, th e loss of S338-S339 in Raf-1 prevented protein kinase activation in CO S-7 cells by either oncogenic Ras[V12] or v-Src. Consistent with phosp horylation of S338-S339, acidic amino acid substitutions of these resi dues partially restored transforming activity to Raf-CX, as well as ki nase activation of Raf-1 by Ras[V12] or v-Src. Two-dimensional phospho peptide mapping of wild-type Raf-CX and Raf-CX[A338A339] confirmed the presence of a phosphoserine-containing peptide with the predicted mob ility in the wild-type protein which was absent from the mutant. This peptide could be quantitatively precipitated by an antipeptide antibod y specific for the 18-residue tryptic peptide containing S338-S339 and was demonstrated to contain only phosphoserine, Phosphorylation of th is peptide in Raf-1 was significantly increased by coexpression with R as[V12]. These data demonstrate that Raf-1 residues 338 to 341 constit ute a unique phosphoregulatory site in which the phosphorylation of se rine and tyrosine residues contributes to the regulation of Raf by Ras , Src, and Ras-independent membrane localization.