The amino-terminal B-Raf-specific region mediates calcium-dependent homo- and hetero-dimerization of Raf

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Abstract
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.