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

EMBO J. 2006 Aug 9;25(15):3556-64. doi: 10.1038/sj.emboj.7601241. Epub 2006 Jul 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism*
  • Calcium / pharmacology
  • Calcium Signaling* / drug effects
  • Cell Membrane / metabolism
  • Dimerization
  • Fluorescence Resonance Energy Transfer
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Mitogen-Activated Protein Kinase Kinases / metabolism
  • Models, Biological
  • Phosphorylation
  • Proto-Oncogene Proteins B-raf / chemistry*
  • Proto-Oncogene Proteins B-raf / genetics
  • Proto-Oncogene Proteins B-raf / metabolism*
  • Proto-Oncogene Proteins c-raf / metabolism
  • Threonine / metabolism
  • ras Proteins / metabolism

Substances

  • Green Fluorescent Proteins
  • Threonine
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinase Kinases
  • ras Proteins
  • Calcium