Regulation and role of Raf-1/B-Raf heterodimerization

Abstract

The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.

FIG. 1.

B-Raf and Raf-1 form stimulation-inducible heterodimers. (A) Coimmunoprecipitation of overexpressed Raf proteins. COS-1 cells were transiently transfected with Flag-tagged Raf-1 and HA-tagged B-Raf. Cells were starved overnight and stimulated with TPA for 10 min. Lysates were immunoprecipitated with the Flag antibody and probed for associated B-Raf or immunoprecipitated with the HA antibody and probed for associated Raf-1. (B) Endogenous heterodimers are formed in COS-1 cells. Starved COS-1 cells were stimulated with 100-ng/ml TPA for 10 min or EGF for 5 min. B-Raf/Raf-1 heterodimers were detected by immunoprecipitation with a B-Raf antibody, followed by immunoblotting for Raf-1 and vice versa.

FIG. 2.

Raf-1/B-Raf heterodimers possess increased kinase activity. (A) Experimental scheme for the isolation of Raf-1/B-Raf heterodimers and the corresponding homodimers or monomers. (B) Comparison of the specific in vitro kinase activity of Raf-1/B-Raf heterodimers and the corresponding monomers-homodimers. COS-1 cells were transfected with the indicated expression plasmids and serum starved overnight before treatment with 100-ng/ml TPA for 30 min as indicated. Raf protein complexes were isolated as described in panel A and Materials and Methods. Their kinase activity was determined by in vitro kinase assays with recombinant kinase-negative GST-MEK as substrate. MEK phosphorylation was detected with a phosphospecific MEK antibody. The levels of Raf-1 and B-Raf proteins in the immunoprecipitates were visualized by Western blotting with HA or Flag antibodies. (Bottom) The concentrations of the protein complexes were adjusted to similar B-Raf levels. (C) COS-1 cells were transiently cotransfected with Flag-Raf-1 or Flag-Raf-1 K375M (kinase negative) and the HA B-Raf wild-type, G465E, or K482M (kinase-negative) mutants. Heterodimers and B-Raf monomers-homodimers were isolated; after B-Raf levels were balanced, their kinase activity was determined in vitro. MEK phosphorylation was detected with a phosphospecific MEK antibody.

FIG. 3.

Binding stoichiometries and mapping of interaction domains. (A) Raf proteins were produced by coupled IVT. As indicated, Flag-Raf-1 or HA-B-Raf proteins were immunoprecipitated and analyzed by Western blotting alongside the IVT reactions (input) with Flag-specific (Raf-1) and HA-specific (B-Raf) antibodies. The blots were quantified by laser densitometry, showing that approximately 8% of Raf-1 and 7% of B-Raf produced were recovered in heterodimers. (B) Heterodimers were isolated from TPA (100 ng/ml; 10 min)-stimulated COS cells transfected with Flag-Raf-1 and HA-B-Raf as described in Fig. 2A and analyzed by Western blotting alongside a dilution series of cell lysates. Quantification by laser densitometry showed that Raf-1/B-Raf heterodimers contained, on average, 0.1% of the total cellular B-Raf and 0.3% of the total Raf-1 pool. Shown are representatives of three experiments. (C) B-Raf/Raf-1 interaction requires the Raf-1 kinase domain and is modulated by mutation of K375M or S621. COS-1 cells were cotransfected with B-Raf and the indicated Flag-tagged Raf-1 constructs. FL, full-length; C-term, C-terminal kinase domain. B-Raf immunoprecipitates were stained with anti-Flag antibody to detect bound Raf-1 proteins, followed by the B-Raf H145 antibody, to assure similar loading of B-Raf proteins. To control for equal expression of Flag-Raf-1 proteins, cell lysates were immunoblotted with Flag antibody. (D) Peptide arrays that represent the entire Raf-1 and B-Raf coding sequences as 23mer peptides offset by four amino acids were probed with Raf-1 and B-Raf proteins produced in Sf9 insect cells. Raf-1 and B-Raf proteins are schematically shown with the following motifs indicated: CR1 to CR3, conserved regions; ATP, ATP binding loop; activation loop, delineated by DFG and APE amino acid sequences; RBD, Ras binding domain; CBD, cysteine-rich domain; RKIP, minimal RKIP binding domain. Phosphorylation sites are shown in blue. Proteins used to probe the arrays are boxed, and the mapped sites of sites of interaction are indicated by red (high-affinity) and green (medium-affinity) squares with amino acid numbers.

FIG. 4.

14-3-3 proteins enhance Raf heterodimerization. (A) COS cells were cotransfected with Flag-Raf-1, HA-B-Raf, and increasing amounts of GST-14-3-3. Lysates from serum-starved cells were immunoprecipitated with Flag or HA antibodies as indicated and examined for the presence of Raf-1, B-Raf, and 14-3-3 by immunoblotting with the indicated antibodies. As a control for protein expression, lysates were immunoblotted as well. (B) Raf proteins were produced by coupled IVT. As indicated, increasing amounts of purified GST-14-3-3 produced in E. coli were added. Flag-Raf-1 or HA-B-Raf proteins were immunoprecipitated and analyzed by Western blotting with Flag- and HA-specific antibodies. DN 14-3-3, dimerization-negative 14-3-3 mutant.

FIG. 5.

Heterodimer persistence is regulated by ERK signaling. (A) Treatment of PC12 with the MEK inhibitor U0126 causes an increase in heterodimer association. PC12 cells were serum starved overnight and then incubated with 10 μM U0126 for 1 h before stimulation with NGF for the times indicated. Endogenous heterodimers were detected by immunoprecipitation with a Raf-1 antibody, followed by blotting for B-Raf. Lysates were immunoblotted with antibodies against phospho-ERK to show the inhibition of ERK phosphorylation by U0126 and with antibodies against ERK, B-Raf, and Raf-1 to assure equal loading. (B) The same experiment as that in panel A is shown, with Jurkat T cells stimulated with 100-ng/ml TPA. (C) Dominant negative MEK blocks ERK activation and promotes Raf-1 heterodimerization with B-Raf. COS-1 cells were transfected with vector or MEKAA, a dominant negative MEK-1 mutant where the activating phosphorylation sites S218/S222 are mutated to alanines. Heterodimers were detected by immunoprecipitation of endogenous B-Raf and immunoblotting for endogenous Raf-1. MEKAA efficiently blocked ERK activation, as shown by blotting lysates for phospho-ERK. Lysates were also immunoblotted for the expression of endogenous total ERK, Raf-1, and B-Raf, as well as transfected MEKAA, as indicated. (D) ERK induces phosphorylation of T753 in B-Raf. COS cells were transfected with HA-B-Raf or HA-B-Raf T753A. Cells were serum starved overnight and then incubated with 10 μM U0126 for 1 h before stimulation with 20-ng/ml EGF for the times indicated. B-Raf proteins were immunoprecipitated with HA antibody. Phosphorylation of T753 was detected by immunoblotting with a phosphothreonine-proline-specific antibody. B-Raf proteins were visualized with HA antibody. (E) Sustained heterodimerization of B-Raf T753A with Raf-1. COS-1 cells were transiently transfected with Flag Raf-1 and either HA-B-Raf or the HA-B-Raf T753A mutant. Cells were starved overnight and stimulated with 20-ng/ml EGF over a time course of 0 to 120 min. Cells were lysed, immunoprecipitated with an HA antibody, and blotted for associated Raf-1.

FIG. 6.

Mutation of B-Raf T753 enhances the differentiation of PC12 cells. (A) PC12 cells were transiently cotransfected with GFP plus either empty vector, B-Raf WT, or B-Raf T753A. Cells were serum starved overnight and stimulated with EGF or NGF for 36 h. Cells were fixed with formaldehyde and photographed under a fluorescent microscope. Six random pictures of each condition were taken, and the percentage of differentiated, GFP-positive cells was calculated. Cells with neurites at least two cell bodies long were considered differentiated. (B) The results of three independent experiments were plotted as a bar graph. Statistical significance was evaluated by Student's paired t test.