Ras binding opens c-Raf to expose the docking site for mitogen-activated protein kinase kinase

Abstract

A key signalling molecule, c-Raf, is situated downstream from Ras and upstream from the mitogen-activated protein kinase kinase (MEK). We studied the mechanism underlying the signal transduction from Ras to MEK by using probes based on the principle of fluorescence resonance energy transfer. In agreement with previous models, it was found that c-Raf adopted two conformations: open active and closed inactive. Ras binding induced the c-Raf transition from closed to open conformation, which enabled c-Raf to bind to MEK. In the presence of a cytosolic Ras mutant, c-Raf bound to, but failed to phosphorylate, MEK in the cytoplasm. In contrast, the cytosolic Ras mutant significantly enhanced MEK phosphorylation by a membrane-targeted c-Raf. These results demonstrated the essential role of Ras-induced conformational change in MEK activation by c-Raf.

Figure 1

Effects of amino-acid substitutions on the conformation and activity of c-Raf. (A) Schematic representation of Prin–c-Raf. FRET efficiency increases when Prin–c-Raf adopts the closed conformation in the cytosol. Ras binding at the plasma membrane changes the conformation to the open state, a condition under which FRET efficiency is reduced. (B) Schematic representation of amino-acid residues analysed in this study. (C) Conformation index (CFP/YFP) of Cos-1 cells expressing Prin–c-Raf mutants in the presence or absence of H-RasV12 or Akt-active. The bars and lines represent the average and s.d., respectively (n=20). (D) Cos-1 cells expressing Prin–c-Raf mutants in the presence or absence of H-RasV12 or Akt-active were analysed by immunoblotting with anti-c-Raf-phosphoser 259 or anti-c-Raf antibody.

Figure 2

MEK binding to c-Raf in the open conformation. (A) Conformation of Prin–c-Raf in the presence or absence of H-RasV12 or H-RasV12ΔC in Cos-1 cells (n=20). (B) The binding of CFP–c-Raf, CFP–c-RafR89L and c-Raf–CFP to YFP or MEK–YFP was analysed in the presence or absence of H-RasV12ΔC or Rap1V12ΔC in Cos-7 cells by intermolecular FRET. (C) HeLa cells coexpressing H-Ras, CFP–c-Raf and MEK–YFP were stimulated with 25 ng/ml epidermal growth factor and imaged using time-lapse (supplementary video 4 online). Representative images of CFP and corrected FRET (lower panel) are shown. Scale bar, 10 μm. (D) Conformation of membrane-anchored Prin–c-Raf-pm in the presence or absence of H-RasV12 or H-Ras-V12ΔC in Cos-1 cells (n=20). (E) Binding of CFP-pm, CFP–c-Raf-pm, CFP–c-RafR89L-pm, c-Raf–CFP-pm and CFP–c-RafS259A-pm to YFP or MEK–YFP was quantified in the presence or absence of H-RasV12ΔC and Akt-active in HeLa cells by intermolecular FRET. Asterisks indicate the significance of t-test analysis: ^*P<0.01; ^**P<0.001.

Figure 3

MEK activation by c-Raf in the open conformation. (A) In vitro MEK kinase activity of Prin–c-Raf and Prin–c-Raf-pm that were immunoprecipitated from HeLa cells in the presence or absence of RasV12 or H-RasV12ΔC (n=3). (B) HeLa cell lysates were immunoprecipitated for the in vitro MEK kinase assay (IP) or were directly used (TCL) for immunoblotting with anti-GFP (c-Raf), anti-phospho-MEK (pMEK) and anti-Flag antibodies (Ras).

Figure 4

Four states of c-Raf during the signalling process. (A) Semiclosed inactive state: in resting cells, a major fraction of c-Raf adopts semiclosed conformation. (B) Open inactive state: on stimulation, Ras–GTP binds to c-Raf and induces MEK binding to c-Raf. (C) Open active state: modifications at plasma membrane such as phosphorylation and lipid binding allow c-Raf to phosphorylate MEK. (D) Closed inactive state: c-Raf phosphorylated on Ser 259 adopts closed conformation by means of 14-3-3 binding and returns to the cytosol. Filled circles represent phospho-tyrosine, phosphoserine and phospho-threonine residues.