Growth factor-induced signalling leads to activation of members of the Ras family and subsequent stimulation of different Raf isoforms. Within the mechanism of Raf activation, two isoforms of Raf, cRaf and BRaf, may cooperate. We investigated the relationship between cRaf and BRaf and found that active Ras induced heterodimerization of cRaf and BRaf, an effect that was dependent on the serine residue at position 621 of cRaf. Moreover, we also found that cRaf COOH-terminus constitutively associated with BRaf, whereas the NH2 terminus did not, even in the presence of active Ras. These data suggest that Ras induces the cRaf-BRaf complex formation through the exposure of 14-3-3 binding sites in the COOH-terminus of cRaf. Thus, Ras-induced cRaf-Braf heterodimerization may explain the observed cooperativity of cRaf and BRaf in cells responding to growth factor signals.

Signalling through the MAPK5 cascade is transduced by GTP loading of Ras leading to the activation of Raf kinase. In mammalian cells, there are three isoforms of Raf: Araf, BRaf, and cRaf, all with different tissue distributions (reviewed in Refs. 1 and 2). Although all three of the Raf isoforms share a common function with respect to MEK phosphorylation, studies have shown that these proteins might be differentially activated by oncogenic Ras(3), or have different roles during embryogenesis (4, 5, 6). Recent evidence has indicated that Raf isoforms cooperate to achieve full activation of ERK in response to epidermal growth factor (7). In the mechanism of Ras-induced cRaf activation, it is known that cRaf is translocated to the plasma membrane in which tyrosine phosphorylation (8, 9) and also perhaps homodimerization (10, 11) take place to achieve full catalytic activity of the kinase. Moreover, several studies indicate that Raf activation involves conformational changes, governed by the binding of 14-3-3 proteins to phosphorylated serines in cRaf (12, 13, 14, 15), in a process by which the NH2-terminal regulatory domain unfolds to liberate the catalytic (1, 16). In the present study, we show that active Ras can also induce cRaf heterodimerization with BRaf. We found that the binding site for 14-3-3 proteins at serine 621 in the COOH-terminal half of cRaf is important for heterodimerization, whereas the serine at position 259 in the NH2-terminal half is not, even in the presence of active Ras. These data suggest that Ras induces cRaf-BRaf complex formation through the exposure of COOH-terminal binding sites.

Materials.

All hemagglutinin (HA)-tagged c-Raf1 proteins, including wild type, S259A, S621A, cRafΔ31–303 (cRaf-BXB), cRaf1–330 (cRaf-C4), and Ha-Ras(G12V) were expressed from pcDNA3 (Invitrogen, Germany) vector constructs. Anti-HA monoclonal antibody was purified from hybridoma (12CA5) cell lines (American Type Culture Collection). Anti-c-Raf rabbit polyclonal antibodies were generated against a COOH-terminal peptide of the c-Raf kinase as described previously (17). Protein A agarose was from Roche Biomedical (Germany). Enhanced Chemoluminescence (ECL) reagents, film, and [γ-32P]ATP were purchased from Amersham/Pharmacia (Germany).

Cell Transfections.

Human embryonic kidney, HEK293 cells (American Type Culture Collection) were transfected by Ca2+ phosphate precipitation (3). Equal amounts of plasmids coding for the different cRaf constructs (4 μg DNA/106 cells), BRaf (0.5 μg DNA/106 cells), and Ha-Ras (G12V) (0.05 μg DNA/106 cells) were used in cotransfection experiments, and the total amount of DNA was held constant with plasmid pcDNA3 (Invitrogen). Cells were serum-starved 24 h prior to the experiments.

Cell Lysis and Immunoprecipitation.

Transfected cell pellets were lysed and immunoprecipitated as described previously (3). The immunoprecipitated material was subjected to Raf kinase assays, or the proteins within the immune complexes were separated by SDS-PAGE and analyzed by Western blot for coimmunoprecipitating proteins.

Kinase Assays.

Kinase assays on immunoprecipitated material were performed as has been described using kinase-dead MEK (K97M) as a substrate (3). The extent of MEK phosphorylation was observed from proteins blotted onto nitrocellulose membranes using a phosphorimager (Fuji) or autoradiography.

cRaf and BRaf Heterodimerize in Vivo.

Recent evidence demonstrate that cRaf and BRaf cooperate in cells responding to epidermal growth factor (7). We investigated whether cRaf and BRaf are capable of forming heterodimers as a functional basis for the observed cooperativity. Fig. 1 shows, in transient transfection assays using HEK293 cells, that BRaf coimmunoprecipitates with cRaf, and vice versa. The formation of the Raf heterodimer is dependent on the presence of active Ras or serum. Immunoprecipitation of overexpressed cRaf failed to show complexes with endogenous BRaf, even in the presence of oncogenic Ras (Fig. 1,D); however, immunoprecipitation of endogenous BRaf could coimmunoprecipitate transfected cRaf (Fig. 1,C). Compared with expression of endogenous cRaf, the levels of BRaf in HEK293 cells is very low (data not shown), and this might account for the ineffective coimmunoprecipitation of endogenous BRaf with overexpressed cRaf. In kinase assays using recombinant MEK as a substrate, an increased activity was associated with cRaf-BRaf heterodimers (Fig. 1, B and D), this is most likely attributable to the contribution of the active BRaf within the cRaf immunoprecipitate.

GTP-loaded Ras binds Raf at the plasma membrane, in which the kinase becomes further activated by processes that include tyrosine phosphorylation (9). To further investigate the role of Ras in cRaf-BRaf complex formation, we used an oncogenic Ras mutant lacking the COOH-terminal serine residues crucial for the posttranslational modifications necessary to associate it with the plasma membrane [Ha-Ras (G12V, 3S; Ref. 18)]. Cotransfection of this cytosolic mutant of Ras induces cRaf-BRaf heterodimerization in a fashion similar to that observed with Ha-Ras (G12V), but fails to induce Raf kinase activity (compare Fig. 2, Lanes 1–3). Both of the Ras mutants were expressed at the same levels (data not shown). Additionally, plasma membrane-targeted cRaf (cRaf-CAAX; Ref. 9) only associates with BRaf in the presence of Ha-Ras (G12V), or cytosolic Ras (G12V, 3S) (data not shown), which indicates that dimerization can occur independently of activation. The Ras-related G protein Rap1, which presumably activates BRaf but not cRaf (19), induced neither heterodimerization nor Raf kinase activity (Fig. 2, Lane 4). Qiu et al.(20) have recently reported that the expression levels of 14-3-3 proteins are important for Rap1-mediated activation of BRaf. Because we were not able to induce BRaf activation with Rap1 (G12V) in our HEK293 cell system, we assume that there are insufficient 14-3-3 levels for Rap1-induced BRaf activity. Nevertheless, our data indicate that there is sufficient expression of 14-3-3 proteins to promote Ras-induced BRaf-activity and BRaf-cRaf heterodimerization. Taken together, these data suggest that Ras binding, and not Ras-mediated membrane translocation, is the crucial step involved in cRaf-BRaf heterodimerization. Moreover, these data also support the observations of Luo et al.(11) and indicate that dimerization is not sufficient to activate Raf kinase.

Structural Requirements of cRaf/BRaf Heterodimerization.

14-3-3 proteins bind to all of the Raf isoenzymes (12, 13, 21), and are likely to be responsible for maintaining enzyme structure, promoting homo-oligomerization, and facilitating Ras-dependent Raf activation (14, 15). The principle 14-3-3 binding sites on cRaf are the serine residues at positions 259 and 621 (22, 23). In agreement, we found, in yeast two-hybrid assays, that mutation at these sites either reduces or completely eliminates 14-3-3 binding (data not shown). Fig. 3 shows that the Ras-induced coassociation of cRaf (S259A) with BRaf was essentially the same as that observed using wild-type cRaf. In contrast, coassociation of cRaf (S621A) and cRaf (S259A, S621A) with BRaf was significantly reduced. Similar results were observed in experiments in which BRaf was immunoprecipitated: cRaf (S259A) coimmunoprecipitated with BRaf in the presence of active Ras, whereas the coimmunoprecipitation of cRaf (S621A) was inhibited (data not shown). Taken together, these results indicate that the 621 position of cRaf is important for formation of the heterodimers and suggest that 14-3-3 binding to this site is essential for complex formation.

cRafΔ30–303 Constitutively Associates with BRaf.

We further investigated the structural requirements of cRaf-BRaf complex formation by comparing the ability of the regulatory cRaf NH2 terminus (cRaf-C4) with the catalytic cRaf COOH-terminus (cRaf-BXB) to associate with BRaf. Fig. 4,A shows that cRaf-BXB constitutively associates with BRaf, and coexpression of active Ras does not appear to enhance this association. One possible reason for this observed constitutive association is that cRaf-BXB lacks the Ras binding domain, and its activity is not regulated by active Ras. In contrast to cRaf-BXB, the cRaf-C4 region, which contains the Ras-binding domain of cRaf, does not heterodimerize with BRaf, even in the presence of Ras (G12V) (Fig. 4 B). In the presented experiment the expression of cRaf-C4 is hampered in the absence of active Ras, possibly owing to the dominant-negative effect on cell growth by cRaf-C4. Nevertheless, the increased expression of cRaf-C4 in the presence of Ras (G12V) and the lack of heterodimeric complexes with coexpressed BRaf suggest that the NH2 terminus of cRaf is not responsible for the association with BRaf. This is supported by the above data showing that the S621A mutation in cRaf inhibits the formation of the complex.

To our knowledge, this is the first study to show Raf dimerization without the need of artificial dimerization constructs. In previous studies, cRaf chimeras were constructed to include regions that would bind bivalent drugs (10, 11). Both of these studies indicated that Raf dimerization was potentially an important step in the mechanism of Ras-induced Raf activation. The present work supports this view and presents the novel finding that active Ras is the agent that is able to promote Raf dimerization. Our observations were made using a transfected cell system and, although endogenous BRaf coimmunoprecipitated transfected cRaf, we were unable to show endogenous cRaf-BRaf heterodimers, even in the presence of oncogenic Ras or in Ras-transformed cells (data not shown). The reasons for this are not clear and may be attributable to the relative amounts of Raf homo- and heterodimers. The process of cRaf-BRaf complex formation is likely to involve the binding of active Ras and not membrane translocation of the kinases, because membrane-targeted cRaf did not associate with BRaf in the absence of Ras (G12V) and because cytosolic Ras (G12V, 3S) also induced heterodimer formation. Our data indicate that the 14-3-3 binding site at position 621 of cRaf is important for dimerization, an observation supported by experiments showing that cRaf-BXB constitutively associates with BRaf, whereas complex formation between BRaf and cRaf-C4 did not occur under any condition. These results also lend themselves to supporting the role of the NH2 terminus of Raf in the regulation of kinase activity (16). That is, the NH2 terminus of cRaf regulates Raf activity through controlling the exposure of COOH-terminal sites, such as S621, for 14-3-3 protein-mediated dimerization. Interestingly, mutation at this site renders cRaf inactive in response to virtually all stimuli (24, 25, and data not shown), which suggests that dimerization is an important feature of Raf kinase activity.

Recently, it was demonstrated that active Ras dimerizes at the plasma membrane, but that this is not sufficient to induce full Raf activation (26). Other studies have shown dimerization of the downstream targets of Raf kinases, such as ERK (27, 28). Together, these studies add support to our presented data and suggest a model of signal amplification through the induction of a macromolecular complex of associated kinase dimers.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

Supported by Deutsche Forschungsgemeinschaft grants We2023/3-1 (to C. K. W.) and 642/3-2, SFB487 (to U. R. R.).

                        
5

The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase.

Fig. 1.

Active Ras induces heterodimerization of cRaf and BRaf. Immunoblots of lysates from HEK293 cells transfected with the indicated constructs: A and B, with BRaf and variably with HA-cRaf and oncogenic Ras; C and D, with HA-cRaf and variably with oncogenic Ras. A, anti-BRaf immunoprecipitates showing coassociation with transfected cRaf. B, anti-HA immunoprecipitates from the same lysate as in A, showing expression of HA-cRaf, coassociated BRaf, and kinase activity of the immunoprecipitated cRaf. C, anti-BRaf immunoprecipitates showing coassociation of endogenous BRaf with transfected cRaf. D, anti-HA immunoprecipitates from the same lysate as in C, showing expression of transfected HA-cRaf, and kinase activity. Coimmunoprecipitation of endogenous BRaf was not observed in this part of the experiment.

Fig. 1.

Active Ras induces heterodimerization of cRaf and BRaf. Immunoblots of lysates from HEK293 cells transfected with the indicated constructs: A and B, with BRaf and variably with HA-cRaf and oncogenic Ras; C and D, with HA-cRaf and variably with oncogenic Ras. A, anti-BRaf immunoprecipitates showing coassociation with transfected cRaf. B, anti-HA immunoprecipitates from the same lysate as in A, showing expression of HA-cRaf, coassociated BRaf, and kinase activity of the immunoprecipitated cRaf. C, anti-BRaf immunoprecipitates showing coassociation of endogenous BRaf with transfected cRaf. D, anti-HA immunoprecipitates from the same lysate as in C, showing expression of transfected HA-cRaf, and kinase activity. Coimmunoprecipitation of endogenous BRaf was not observed in this part of the experiment.

Close modal
Fig. 2.

Role of Ras in promoting cRaf-BRaf complex formation. Immunoblots of lysates from HEK293 cells transfected with HA-cRaf, BRaf, and the indicated constructs. Lysates were immunoprecipitated with anti-BRaf antibodies and analyzed for cRaf coimmunoprecipitation, BRaf expression, and catalytic activity using kinase-inactive MEK as a substrate. Upper panel, the coimmunoprecipitation of cRaf in the presence of different Ras constructs; middle panels, expression of transfected BRaf or HA-cRaf in the same sample; bottom panel, the autoradiograph of phosphorylated MEK. The “cytosolic” version of Ras [Ras (G12V, 3S)] fails to activate transfected Raf.

Fig. 2.

Role of Ras in promoting cRaf-BRaf complex formation. Immunoblots of lysates from HEK293 cells transfected with HA-cRaf, BRaf, and the indicated constructs. Lysates were immunoprecipitated with anti-BRaf antibodies and analyzed for cRaf coimmunoprecipitation, BRaf expression, and catalytic activity using kinase-inactive MEK as a substrate. Upper panel, the coimmunoprecipitation of cRaf in the presence of different Ras constructs; middle panels, expression of transfected BRaf or HA-cRaf in the same sample; bottom panel, the autoradiograph of phosphorylated MEK. The “cytosolic” version of Ras [Ras (G12V, 3S)] fails to activate transfected Raf.

Close modal
Fig. 3.

Requirement of serine 621 in cRaf for dimerization with BRaf. Immunoblots of lysates from HEK293 cells transfected with the indicated constructs. Lysates were immunoprecipitated with anti-HA antibodies and were analyzed for BRaf coimmunoprecipitation with HA-cRaf. Upper band in middle panel, a nonspecific reaction of the anti-HA antibody used for the analysis of transfected HA-cRaf expression. Bottom panel, expression of transfected BRaf in the same sample.

Fig. 3.

Requirement of serine 621 in cRaf for dimerization with BRaf. Immunoblots of lysates from HEK293 cells transfected with the indicated constructs. Lysates were immunoprecipitated with anti-HA antibodies and were analyzed for BRaf coimmunoprecipitation with HA-cRaf. Upper band in middle panel, a nonspecific reaction of the anti-HA antibody used for the analysis of transfected HA-cRaf expression. Bottom panel, expression of transfected BRaf in the same sample.

Close modal
Fig. 4.

Role of the NH2 and COOH termini of cRaf for complex formation with BRaf. Immunoblots of lysates from HEK293 cells transfected with the indicated constructs. A, Anti-HA immunoprecipitates (IP) showing expression of transfected cRaf-BXB and coassociation of BRaf. B, Anti-HA immunoprecipitates showing coimmunoprecipitation of BRaf with full-length cRaf but not with cRaf-C4. Bottom panels (A and B), BRaf expression in the same samples. kD, Mr in thousands.

Fig. 4.

Role of the NH2 and COOH termini of cRaf for complex formation with BRaf. Immunoblots of lysates from HEK293 cells transfected with the indicated constructs. A, Anti-HA immunoprecipitates (IP) showing expression of transfected cRaf-BXB and coassociation of BRaf. B, Anti-HA immunoprecipitates showing coimmunoprecipitation of BRaf with full-length cRaf but not with cRaf-C4. Bottom panels (A and B), BRaf expression in the same samples. kD, Mr in thousands.

Close modal

We thank Dr C Block (MPI Dortmund, Germany) for providing Ha-Ras (G12V) and Ha-Ras (G12V, 3S) in pcDNA3. We also thank Barbara Bauer, Renate Metz, and Doreen Stötzer for excellent technical assistance.

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