Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601.
The Raf kinase family serves as a central intermediate to relay signals from Ras to ERK. The precise molecular mechanism for Raf activation is still not fully understood. Here we report that phosphorylation of Thr598 and Ser601, which lie between kinase subdomains VII and VIII, is essential for B-Raf activation by Ras. Substitution of these residues by alanine (B-RafAA) abolished Ras-induced B-Raf activation without altering the association of B-Raf with other signaling proteins. Phosphopeptide mapping and immunoblotting with phospho-specific antibodies confirmed that Thr598 and Ser601 are in vivo phosphorylation sites induced by Ras. Furthermore, replacement of these two sites by acidic residues (B-RafED) renders B-Raf constitutively active. Con sistent with these data, B-RafAA and B-RafED exhibited diminished and enhanced ability, respectively, to stimulate ERK activation and Elk-dependent transcription. Moreover, functional studies revealed that B-RafED was able to promote NIH 3T3 cell transformation and PC12 cell differentiation. Since Thr598 and Ser601 are conserved in all Raf family members from Caenorhabditis elegans to mammals, we propose that phosphorylation of these two residues may be a general mechanism for Raf activation.
Zhang
B H
BH
Department of Biological Chemistry and Institute of Gerontology, University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA.
Guan
K L
KL
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002454:Cell Differentiation; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D017124:Conserved Sequence / Q000235:genetics; D004789:Enzyme Activation; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D048369:MAP Kinase Kinase 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D036224:Receptor, EphB4; D036081:Receptors, Eph Family; D014158:Transcription, Genetic; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D048948:14-3-3 Proteins; D029742:Caenorhabditis elegans Proteins; D018841:HSP90 Heat-Shock Proteins; C106207:par-5 protein, C elegans; D010769:Phosphothreonine; D010768:Phosphoserine; D014446:Tyrosine 3-Monooxygenase; D020794:Receptor Protein-Tyrosine Kinases; D036224:Receptor, EphB4; D036081:Receptors, Eph Family; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D015689:Oncogene Protein p21(ras)