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 tightly regulated by multiple phosphorylation events. Phosphorylation of either tyrosine 340 or 341 in the catalytic domain of Raf-1 has been previously shown to induce the ability of the protein kinase to phosphorylate MEK. By using a combination of mitogenic and enzymatic assays, we found that phosphorylation of the adjacent residue, serine 338, and, to a lesser extent, serine 339 is essential for the biological and enzymatic activities of Raf-1. Replacement of S338 with alanine blocked the ability of prenylated Raf-CX to transform Rat-1 fibroblasts. Similarly, the loss of S338-S339 in Raf-1 prevented protein kinase activation in COS-7 cells by either oncogenic Ras[V12] or v-Src. Consistent with phosphorylation of S338-S339, acidic amino acid substitutions of these residues partially restored transforming activity to Raf-CX, as well as kinase activation of Raf-1 by Ras[V12] or v-Src. Two-dimensional phosphopeptide mapping of wild-type Raf-CX and Raf-CX[A338A339] confirmed the presence of a phosphoserine-containing peptide with the predicted mobility in the wild-type protein which was absent from the mutant. This peptide could be quantitatively precipitated by an antipeptide antibody specific for the 18-residue tryptic peptide containing S338-S339 and was demonstrated to contain only phosphoserine. Phosphorylation of this peptide in Raf-1 was significantly increased by coexpression with Ras[V12]. These data demonstrate that Raf-1 residues 338 to 341 constitute a unique phosphoregulatory site in which the phosphorylation of serine and tyrosine residues contributes to the regulation of Raf by Ras, Src, and Ras-independent membrane localization.
Diaz
B
B
Department of Medicine, Walther Oncology Center, Indiana University School of Medicine, Indianapolis 46202, USA.
Barnard
D
D
Filson
A
A
MacDonald
S
S
King
A
A
Marshall
M
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004789:Enzyme Activation; D005347:Fibroblasts; D011905:Genes, ras / Q000502:physiology*; D016391:Genes, src / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments; D010449:Peptide Mapping; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D011993:Recombinant Fusion Proteins; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf