The amino-terminal B-Raf-specific region mediates calcium-dependent homo- and hetero-dimerization of Raf.
B-Raf is a key regulatory molecule of the mitogen-activated protein kinase kinase (MEK). B-Raf differs from the other Raf isoforms in that it has a long amino-terminal region. By the use of probes based on the principle of fluorescence resonance energy transfer, we found that this amino-terminal B-Raf-specific region is essential for homo-dimerization of B-Raf and hetero-dimerization of B-Raf and c-Raf at the plasma membrane, followed by phosphorylation of Thr118 in the amino-terminal B-Raf-specific region. HeLa cells expressing B-Raf, but not c-Raf, or a B-Raf mutant lacking the B-Raf-specific region, showed enhanced MEK phosphorylation upon stimulation with a calcium agonist. Furthermore, increases in the intracellular calcium concentration were found to be necessary for dimerization and sufficient for the plasma membrane translocation of B-Raf. Notably, in calcium ionophore-stimulated HeLa cells, B-Raf could propagate signals to MEK under the basal level of GTP-Ras. Thus, we propose that the hitherto unidentified function of the B-Raf amino-terminal region is to mediate calcium-dependent activation of B-Raf and the following MEK activation, which may occur in the absence of Ras activation.
Terai
Kenta
K
Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Matsuda
Michiyuki
M
D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
D049452:Green Fluorescent Proteins; D013912:Threonine; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; D002118:Calcium