In this paper, we demonstrate for the first time an endogenous interaction between B-Raf and Raf-1 to form heterodimers. The basal level of association is enhanced by mitogen stimulation, and we found this in all cell types analyzed, including PC12, COS-1, HCT116, MCF-7, and T cells. The kinetics of heterodimer formation and persistence are variable, however, depending on the cell type and stimulus used. This suggests that the increase in heterodimerization is part of the activation process of Raf proteins.
Based on immunoprecipitation experiments, <1% of cellular Raf-1 and B-Raf is found in heterodimers. These low stoichiometries under the steady-state conditions of immunoprecipitation could be due to low affinity for each other, fast turnover, or sequestration into different complexes and subcellular compartments. Unfortunately, we were not able to perform quantitative measurements of affinities and on-off rates, as we currently cannot purify full-length Raf proteins to the purity and concentration required for such experiments. The expression yields of Raf-1 and B-Raf in commonly used expression systems, including Sf9 insect cells, Pichia pastoris
, and E. coli
, are very low, and the Raf proteins rapidly lose solubility when deprived of their copurifying chaperones HSP90, HSP70, and CDC37. Nevertheless, the heterodimer seems to be physiologically important, as suggested by its very high catalytic activity, its rather complex regulation, and the observation that a B-Raf mutant, B-Raf T753A, which causes accumulation of the heterodimer, enhances the differentiation of PC12 cells. Interestingly, the KSR1 scaffolding protein, which enhances ERK activation by MEK, also engages only a small fraction of MEK and ERK. Reconstitution experiments of KSR1 knockout cells with increasing amounts of transfected KSR1 showed that ERK activation became maximal when KSR1 was overexpressed ~14 fold above endogenous levels. However, even under these optimal conditions <5% of MEK and ERK were bound to KSR1 (30
). This example shows that even small changes in the efficiency of an activation step within the Raf-MEK-ERK kinase cascade can translate into a strong effect on ERK activity.
It has to be noted that in mitogen-stimulated cells, B-Raf becomes efficiently activated as a MEK kinase, independently of its participation in the heterodimer (Fig. ). Only when adjusted to similar B-Raf levels does the much higher catalytic activity of the heterodimer becomes apparent. Thus, B-Raf in cells may contribute significantly to MEK activation independently of Raf-1. This view is supported by the observation that in Raf-1 knockout cells the activation of the MEK-ERK pathway is apparently normal, presumably due to compensation by B-Raf (15
). Thus, it is attractive to speculate that Raf-1/B-Raf heterodimers modulate the signaling strength or fulfill specialized functions, rather than affecting ERK activation globally. This hypothesis is consistent with our observation that extending the lifetime of the heterodimer by transfecting B-Raf T753A selectively supported NGF-driven differentiation in PC12 cells, which relies on the sustained activation of ERK, but did not change the response to EGF, which produces transient ERK activation without differentiation.
It becomes increasingly apparent that the protein components of signaling pathways are organized in multiprotein complexes that can carry out context-dependent cellular functions, determined by their interaction partners and subcellular localization (18
). Both Raf-1 (37
) and B-Raf (Rushworth et al., unpublished) exist in multiprotein complexes, which when analyzed by mass spectrometry contain >40 proteins, including 14-3-3 proteins and the chaperones HSP90, HSP70, and CDC37, which are important to keep Raf proteins folded and soluble (32
). According to sucrose gradient fractionation, Raf complexes are spread out between 70 and >500 kDa. Cotransfection of Raf-1 shifts B-Raf complexes to higher molecular weights, whereas cotransfection of B-Raf shifts Raf-1 complexes to smaller size (data not shown). Thus, presumably due to changes in other association partners, there is no simple relationship between Raf-1/B-Raf heterodimerization and the size of the respective protein complexes. This raises the question whether the association between Raf-1 and B-Raf is direct or mediated through other proteins. 14-3-3 proteins can increase heterodimerization, but there is also a constitutive or basal level of heterodimerization. Although the impediments to purify full-length Raf proteins to homogeneity prevented association studies with highly purified proteins, several lines of evidence indicate that heterodimerization involves direct interactions between Raf-1 and B-Raf. First, partially pure Raf-1 and B-Raf proteins produced by in vitro transcription-translation (Fig. ) or in Sf9 insect cells (data not shown) readily form heterodimers. Second, partially pure Raf proteins produced in Sf9 insect cells bind to peptide arrays representing the Raf-1 and B-Raf amino acid sequences (Fig. ). Third, the enhancement of heterodimerization conferred by 14-3-3 proteins indicates direct interactions between Raf-1 and B-Raf (see below).
Interestingly, Raf-1/B-Raf heterodimerization is regulated. We describe here two mechanisms, 14-3-3 and ERK feedback phosphorylation. 14-3-3 proteins are ubiquitously expressed adaptor proteins that typically dock to phosphoserine or phosphothreonine residues, although phosphorylation-independent modes of binding have been previously described (40
). Despite a wealth of literature describing the interaction of 14-3-3 proteins with Raf-1 and B-Raf, their role in Raf protein regulation is still unclear. A simplified consensus view suggests that 14-3-3 proteins can stabilize both inactive and activated conformations of Raf proteins (33
). Our results show that 14-3-3 can enhance Raf-1/B-Raf heterodimerization. This is dependent upon the intactness of the dimerization site within 14-3-3 proteins, suggesting that in this scenario 14-3-3 acts as a true bridging molecule that cross-links Raf-1 to B-Raf. Consistent with this view is the fact that the mutation of S621, a 14-3-3 binding site in Raf-1, reduced heterodimerization. However, this reduction also could be related to an inability to bind ATP, as the kinase-negative Raf-1 K375M mutant (which is defective in ATP binding) also exhibited a similar reduction of association. S621 is also a Raf-1 autophosphorylation site (24
), and its mutation renders Raf-1 inactive. Thus, the classical mutational approach breaks down; without structural information, it will be difficult if not impossible to distinguish between these possibilities.
Further, when studying what regulated the Raf-1/B-Raf interaction, we noticed that the MEK inhibitor U0126 and two different Raf inhibitors stabilized the concentration of Raf-1/B-Raf heterodimers in all cell lines examined, albeit with different kinetics and efficiencies (Fig. and data not shown). Our findings suggest a dual role for mitogens in Raf-1/B-Raf heterodimerization. First, mitogens induce heterodimer formation independent of ERK, but once they are established they destabilize them via an ERK-mediated pathway. The destabilization of Raf-1/B-Raf heterodimers seems to be mediated at least in part by ERK phosphorylating B-Raf on T753, thus constituting a direct negative feedback. T753 is located within a main interaction site where Raf-1 binds to B-Raf (Fig. ); it is conceivable that phosphorylation at this site disrupts the interaction. The possibility that the phosphorylation of T753 and the adjacent S750 are part of a negative ERK feedback loop has been raised by Brummer et al. (3
). Here, we show that a target of this feedback is the destabilization of the Raf-1/B-Raf heterodimer. This coupling of stimulation with a negative feedback could serve as a timing device that ensures that heterodimers are dissociated after an appropriate response time. It should be noted that ERK also phosphorylates Raf-1 as part of a negative feedback loop (11
). Whether this may influence heterodimer formation has not been investigated here.
The fact that the formation of the Raf-1/B-Raf heterodimer is regulated on several levels suggests that it fulfils a physiological function that is either qualitatively or quantitatively different from that of the corresponding homodimers or monomers. One such function was inferred from results that B-Raf mutants with low levels of kinase activity could form heterodimers with Raf-1 and efficiently stimulate the ERK pathway if Raf-1 was present in the cell (36
). As low-kinase-activity B-Raf mutations are found in human tumors, this observation indicates a potentially important role for Raf-1 in transformation by B-Raf mutants. In the study by Wan et al. (36
), the kinase activity of the Raf-1/B-Raf heterodimers was not determined directly. Thus, it remained open whether this enhancement was due to heterodimerization or whether B-Raf stimulated Raf-1 activity through another mechanism. Our finding that the Raf-1/B-Raf heterodimer has enhanced kinase activity supports the former interpretation. Importantly, B-Raf mutants with low intrinsic kinase activity could stimulate the kinase activity of the heterodimer to an extent similar to that of wild-type B-Raf. The in vitro heterodimer kinase assays show that the biochemical effect of forming heterodimers is to vastly elevate the catalytic activity of the heterodimer and that Raf-1 makes a major contribution to this. Recent work has suggested that in many cells the major MEK phosphorylating protein is B-Raf (15
) and that the comparably low kinase activity of Raf-1 lends itself to a scaffolding role (28
) or a role in signaling to other, as-yet-unidentified substrates (14
). Our results suggest that Raf-1 can play an important role in signaling to MEK by a process that requires B-Raf, but in a purely interactive way rather than as a kinase. The detailed mechanism is not clear at present. According to our results, B-Raf activity seems not to be required for the superactivation of Raf-1 in the heterodimer, suggesting that B-Raf confers a conformational change on Raf-1 that allows its stimulation, or it brings a protein into the complex which can activate Raf-1. Interestingly, the same seems to hold true in the reverse, as the kinase-negative Raf-1 K375M mutant could efficiently induce MEK phosphorylation via B-Raf (Fig. ). Thus, either kinase-competent Raf isoform is sufficient to confer high catalytic activity on the Raf-1/B-Raf heterodimer. In Raf-1−/−
fibroblasts, ERK activation is normal in response to all stimuli tested, which has been attributed to compensation by B-Raf (15
). This finding suggests that wild-type B-Raf does not rely on Raf-1 or that it can use an alternative mechanism to enhance ERK activation. Interestingly, our recent proteomics experiments have shown that A-Raf can also be coimmunoprecipitated with both B-Raf and Raf-1 (unpublished data). Thus, other combinations of Raf heterodimers or indeed heterotrimers may form, and A-Raf may be able to substitute for Raf-1 as a heterodimerization partner for B-Raf. A-Raf and Raf-1 have recently been shown to cooperate in the transient activation of ERK, although sustained activation was unaffected (21
). Interestingly, the Raf-1 and A-Raf double knockout slowed down proliferation, suggesting that Raf-1 or A-Raf are redundant for this function, possibly because either one can cooperate with B-Raf.
Thus, Raf isozyme heterodimerization begins to emerge as an important regulatory motif and opens a new avenue of research toward understanding the regulation of Raf signaling.