Carole Peyssonnaux,1,2† and Alain Eychene1,2*Institut Curie, Centre de Recherche, Orsay F-91405, France,1 and CNRS, UMR 146, Orsay F-91405, France2
Received 12 July 2006/Returned for modification 15 August 2006/Accepted 16 October 2006
The B-Raf proto-oncogene encodes several isoforms resulting from alternative splicing in the hinge regionupstream of the kinase domain. The presence of exon 8b in the B2-Raf8b isoform and exon 9b in the B3-Raf9bisoform differentially regulates B-Raf by decreasing and increasing MEK activating and oncogenic activities,respectively. Using different cell systems, we investigated here the molecular basis of this regulation. We showthat exons 8b and 9b interfere with the ability of the B-Raf N-terminal region to interact with and inhibit theC-terminal kinase domain, thus modulating the autoinhibition mechanism in an opposite manner. Exons 8band 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. The S365Amutation increased the activity of all B-Raf isoforms, but the effect on B2-Raf8b was more pronounced. This wascorrelated to the high level of S365 phosphorylation in this isoform, whereas the B3-Raf9b isoform was poorlyphosphorylated on this residue. In contrast, S429 was equally phosphorylated in all B-Raf isoforms, but theS429A mutation activated B2-Raf8b, whereas it inhibited B3-Raf9b. These results indicate that phosphorylationon both S365 and S429 participate in the differential regulation of B-Raf isoforms through distinct mecha-nisms. Finally, we show that autoinhibition and phosphorylation represent independent but convergent mech-anisms accounting for B-Raf regulation by alternative splicing.
The BRAF oncogene encodes a MEK1/2 kinase that wasinitially identified due to its transduction into the genome ofIC10, an acute mitogenic retrovirus able to transform primarycultures of chicken embryonic neuroretina cells (35). Its hu-man ortholog was simultaneously identified in NIH 3T3 cellstransfected with Ewing sarcoma DNA (25). In both cases, theB-Raf protein was truncated in its N terminus and the kinasedomain was fused to foreign sequences, leading to its consti-tutive activation. Such a mechanism for B-Raf oncogenic acti-vation was recently reported for a human thyroid papillarycarcinoma (7). However, the most widely encountered mode ofB-Raf activation in human cancers results from point muta-tions in the highly conserved glycine-rich loop and activationsegment of the kinase domain (10). The V600E substitution,representing the most prevalent mutation, is detected in about40 to 50% of melanoma and thyroid papillary carcinoma and atlower rates in other human tumors (8, 10, 29, 56). This muta-tion markedly increases B-Raf basal kinase activity (4, 54), andshort interfering RNA-mediated B-Raf depletion in melanomacells harboring this mutation results in a reversion of the trans-formed phenotype (4, 22, 28). The role of this mutation intumor initiation was further confirmed by studies using animalmodels (30, 37, 42). Although the role of B-Raf protein inoncogenic processes is becoming evident, the regulation of its
activity, especially through phosphorylation, is not fully under-stood. B-Raf displays a higher kinase activity toward its sub-strate MEK than the related Raf-1 and A-Raf (34, 40, 41, 45),and its activation requires fewer phosphorylation events (56).Biochemical and structural studies have established that B-Rafis activated upon binding to GTPases of the Ras family andsubsequent phosphorylation of Thr 599 and Ser 602 residues inthe activation segment of the kinase domain (36, 44, 54, 58). Inresting cells, B-Raf is maintained in an inactive conformationthrough an autoinhibitory mechanism involving an intramolec-ular interaction between the kinase domain and the N-terminalregulatory region, which is released upon binding of this do-main to GTP-bound Ras (51). This regulatory mechanism wasinitially described for the related Raf-1 protein (6, 9, 50).However, with the exception of Thr 599 and Ser 602, B-Rafdiffers from Raf-1 in that it does not require the phosphory-lation of additional residues to become activated. In addition,B-Raf activity has been reported to be down-regulated uponphosphorylation on two residues, Ser 365 and Ser 429 (Fig. 1).Serine 365 is located in the CR2 domain and is the equivalentof Ser 259 in Raf-1 and Ser 388 in Drosophila melanogaster Raf(Fig. 1D). Phosphorylation of this residue creates a dockingsite for 14-3-3 proteins and prevents Raf-1 and D-Raf activa-tion (12, 15, 47). Dephosphorylation of this residue by PP2A isa prerequisite for 14-3-3 displacement and Raf-1 activation byGTP-bound Ras (1, 27, 32, 39). Consequently, several studiesdemonstrated that mutation of Raf-1 S259 results in an in-creased kinase activity (13, 14). Similarly, mutation of serine365 on B-Raf increases its kinase activity (21). This residue isconserved in all Raf family proteins identified thus far (Fig. 1)and is phosphorylated by protein kinases of the AGC family,such as protein kinase A (PKA) and Akt (12, 15, 21, 31, 60).While a number of studies strongly support a critical role for
FIG. 1. Alternative splicing and phosphorylation sites of B-Raf. (A) Schematic representation of B1-Raf, B2-Raf8b, and B3-Raf9b isoforms.(B) B-Raf exon structure and conserved regions (CR1, CR2, and CR3). Exon numbering refers to that of the human BRAF gene. (C) Amino acidsequence alignment of the region encompassing exons 8, 8b, 9, 9b, and 10 between B-Raf sequences from rat (GenBank accession no. XP_231692),
PKA in the phosphorylation of both Raf-1 Ser 259 and B-RafSer 365 (12, 15, 31), the relevance of Akt-mediated phosphor-ylation of these residues remains a matter of debate. In con-trast to that by PKA, phosphorylation of B-Raf Ser 365 by Akthas not been demonstrated in vivo, and it is worth noting thatsequences surrounding this residue in Drosophila Raf do notmatch the Akt consensus site (Fig. 1). Likewise, B-Raf Ser 429phosphorylation by Akt was shown only in vitro and by indirectevidence (21), whereas phosphorylation of this residue by PKAwas shown both in vitro and in vivo (31). Interestingly, a resi-due equivalent to B-Raf Ser 429 is conserved in both Caeno-rhabditis elegans and Drosophila Raf proteins (Ser 454 and Ser444, respectively) but is not present in the other vertebrate Rafproteins, Raf-1 and A-Raf, which arose from subsequent geneduplications (Fig. 1).
We previously reported that B-Raf undergoes another levelof regulation, through complex alternative splicing (2, 17, 18,41). Thus, the BRAF gene encodes at least 10 distinct proteinisoforms displaying tissue-specific expression in adult mouse(2, 18). These isoforms arise in part from alternative splicing oftwo exons (8b and 9b) located between the CR2 and CR3domains (Fig. 1). Exon 9b was initially named exon 10, withrespect to the first complete BRAF genomic organization re-ported in chicken (5), but to avoid confusion, we propose the9b nomenclature according to the numbering of human BRAFexons. Exon 9b sequences are conserved in all vertebrates sincethey are present not only in mammalian and avian species butalso in mRNAs encoded by BRAF genes from amphibian andfish species (Fig. 1). Exon 8b and 9b sequences are specific forBRAF since they are not conserved in the other vertebrate rafgenes (those encoding A-Raf and Raf-1) or in the unique rafancestor gene in C. elegans and Drosophila. In agreement withan acquired characteristic specific for BRAF following raf geneduplication during evolution, BRAF sequences correspondingto human exons 3 to 10 are clustered within a single exon (exon2) in Drosophila Raf. We have shown that the presence of thesealternative sequences modulates B-Raf biochemical and onco-genic properties (41). Exon 9b increases both the MEK kinaseactivity and transforming activity of B-Raf, whereas exon 8bhas an opposite effect. However, the mechanism of this regu-lation remained heretofore unknown. In the present study, weshow that the presence of exons 8b and 9b modulates theability of the B-Raf N-terminal region to interact with andinhibit the activity of the C-terminal kinase domain in an op-posite manner. Interestingly, Ser 365 and Ser 429 flank thesesequences located in the hinge region of B-Raf. By using phos-pho-specific antibodies and by generating S365A and S429Aphosphorylation mutants of the different isoforms, we investi-gated the role of phosphorylations in the differential regulationof B-Raf isoforms. We found that the presence of exon 8bfavors S365 phosphorylation and 14-3-3 binding, whereas theB3-Raf9b isoform containing exon 9b is less efficiently phos-phorylated on this residue, in agreement with its elevated ac-
tivity. To a lesser extent, we observed that S429 phosphoryla-tion differentially regulates the activity of B-Raf, resulting inthe activation and inhibition of 8b- and 9b-containing isoforms,respectively. Therefore, both phosphorylation on S365 andS429 residues and autoinhibitory mechanisms are responsiblefor the differential regulation of B-Raf isoforms.
MATERIALS AND METHODS
Plasmid constructions. To generate Myc-tagged full-length B-Raf isoformscontaining either the S365A or the S429A mutation, a Bsu36I/SphI cassette wasmutated by PCR and cloned into pBKS-derived constructs containing B1-Raf,B2-Raf8b, and B3-Raf9b isoform cDNAs (41). The EcoRI fragments containingfull-length B-Raf cDNAs were then subcloned into the pcDNA3-myc vector(Invitrogen). The Myc-tagged B-Raf isoforms mutated on the activation loop(T599E/S602E) were generated similarly, using a SphI/NsiI cassette. ThepRcRSV-derived constructs were obtained by subcloning the HindIII fragmentfrom pcDNA3/myc-B-Raf constructs described above into the pRcRSV vector(Invitrogen). The Flag-Cter construct encodes the last 330 amino acids of B-Raf(from Met 438) fused to the Flag epitope sequence at its C terminus (Fig. 1). Itwas generated by amplification of the B-Raf catalytic domain using the following5� and 3� primers containing HindIII and XhoI sites, respectively: 5�-TTAAGCTTAGCCACCATGAAAACCCTTGGTCGA-3� and 5�-TGCTCGAGCTACTTATCGTCGTCATCCTTGTAATCCTTGAACGCTGCAAATTC-3�. The am-plification product was cloned into the HindIII/XhoI sites of pcDNA3(Invitrogen). The HindIII/XbaI fragment from the resulting pcDNA3/Flag-Cterconstruct was then subcloned into the pRcRSV and Cla12 vectors (23). pEF/Flag-Cter was obtained by subcloning the ClaI fragment of Cla12/Flag-Cter intothe pEF-BOS-CX vector (kindly provided by Jacques Ghysdael). pcDNA3/myc-Nter constructs containing the Myc-tagged N-terminal regulatory domain ofB-Raf isoforms mutated or not mutated on S365 and S429 (amino acids 1 to 443)(Fig. 1) were generated by subcloning the EcoRI/AccI fragment from pcDNA3/myc-B-Raf plasmids into pcDNA3. The XbaI fragment of pcDNA3/myc-Nterplasmids was then subcloned into the pRcRSV vector to generate pRcRSV/myc-Nter constructs. All of the PCR and cloning procedures were verified by se-quencing.
Transfection, Western blotting, and coimmunoprecipitation analysis ofHEK293 cells. Human embryonic kidney 293 (HEK293) cells were maintained inDulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum(FCS), 100 mg/ml streptomycin, 100 U/ml penicillin, and 1 mg/ml amphotericinB (Fungizone). Cells were transfected with 500 ng of pcDNA3/myc-B-Raf in6-mm dishes using Effectene reagent (QIAGEN) according to the manufactur-er’s instructions. In cotransfection experiments, either 100 ng of pcDNA3/Flag-Cter or 10 ng of pEF/Flag-Cter was transfected with 500 ng of pRcRSV/myc-Cterconstruct or pRcRSV empty vector. Twenty-four hours after transfection, cellswere lysed in 0.3 ml of Triton lysis buffer [20 mM Tris, pH 8, 100 mM NaCl, 0.5%Triton X-100, 2 mg/ml aprotinin, 1 mM 4-(2-aminoethyl)-benzene-sulfonyl fluo-ride, 1 mM sodium orthovanadate, 50 mM NaF, 25 mM �-glycerophosphate].Insoluble materials were pelleted by centrifugation at 15,000 � g for 25 min at4°C. When indicated, cells were serum starved 24 h after transfection for 8 h,stimulated for 5 min with Dulbecco’s modified Eagle’s medium supplementedwith 20% FCS, and then lysed as described above. For immunoprecipitationexperiments, 100 �l of cell lysate was precipitated with 0.5 �g of mouse anti-Myc(9E10; Santa Cruz Biotechnology) or anti-Flag (M2; Sigma) monoclonal anti-body and 40 �l of a 50% slurry of protein A-Sepharose (GE Healthcare).Immunoprecipitates were washed twice with 1 ml of lysis buffer and once with 1ml 20 mM Tris, pH 8.0, and boiled in Laemmli’s sample buffer. They were thenresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, trans-ferred onto a polyvinylidene difluoride membrane (Millipore), and probed withanti-Flag, anti-Myc, or rabbit polyclonal anti-14-3-3 (K-19; Santa Cruz Biotech-nology). Activated forms of MEK or extracellular signal-regulated kinase (ERK)were detected in whole-cell extracts (WCE) by Western blotting using rabbitanti-phospho-MEK1/2 (Ser217/221) (Cell Signaling) or mouse anti-phospho-
mouse (2), human (2), quail (17), Xenopus laevis (accession no. BAD01470), Tetraodon nigroviridis (accession no. CAF96750), and zebra fish(accession no. BAD16728). (D) Amino acid sequence alignment between vertebrate Raf (Raf-1, A-Raf, and B-Raf), Drosophila (D-Raf), and C.elegans (Ce-Raf) proteins in the regions surrounding B-Raf S365 and S429 phosphorylation sites. Consensus sequences for PKA and Aktphosphorylation sites are indicated by gray boxes. RBD, Ras binding domain.
p42/p44 mitogen-activated protein kinase (Sigma) antibody, respectively. Nor-malization of cell lysate amounts was achieved by monitoring total ERK expres-sion using rabbit anti-ERK (C-16; Santa Cruz Biotechnology). B-Raf S365phosphorylation was detected on whole-cell extracts by Western blotting usingrabbit polyclonal anti-phospho-S259 Raf-1 (Cell Signaling). B-Raf S429 phos-phorylation was detected by immunoprecipitation of cell lysates with the anti-Myc antibody, followed by Western blotting using a rabbit monoclonal anti-phospho-PKA substrate antibody raised against the R/K-R/K-X-S/T consensussequence (Cell Signaling). To further assess the specificity of these antibodiestoward phosphorylated S365 and S429, wild-type full-length B-Raf proteins im-munoprecipitated with anti-Myc antibody were treated or not treated with 100 U�-protein phosphatase (Cell Signaling) for 1.5 h at 30°C and then probed with thephospho-specific antibodies. Horseradish peroxidase-conjugated anti-rabbit oranti-mouse antibodies were used as secondary antibodies, and proteins werevisualized by enhanced chemiluminescence (SuperSignal West Dura reagent;Pierce) using either autoradiography or a charge-coupled-device camera(GeneGnome bioimaging system; Syngene). Signals were quantified using GeneTools software (Syngene).
NR cell proliferation assay. Neuroretina (NR) cell cultures were preparedfrom 8-day-old Brown Leghorn chicken embryos as previously described (43) andseeded in 100-mm dishes. Cultures were maintained in basal medium Eaglesupplemented with 5% FCS, 100 mg/ml streptomycin, 100 U/ml penicillin, 1mg/ml amphotericin B, and 2 mM glutamine. The mitogenic activity of full-length B-Raf isoforms was assessed by transfecting 20 �g of pRcRSV-derivedconstructs. The inhibitory effects of the N terminus of B-Raf isoforms on themitogenic activity of the B-Raf C terminus were assayed by cotransfecting 2 �gof pEF/Flag-Cter and 18 �g of pRcRSV/myc-Nter constructs. Cells were trans-fected by the calcium phosphate method as previously described, and G418selection (600 �g/ml) was applied 3 days later for 15 days (43). The cultures werethen rinsed with phosphate-buffered saline, and the foci of proliferating cellswere stained with 1.0% crystal violet (in 20% ethanol). Quantification of thenumber and size of the foci was performed using VisionExplorer VA software(Graftec).
PC12 cell differentiation assay. PC12 cells were cultured in Dulbecco’s mod-ified Eagle’s medium supplemented with 6% FCS and 6% horse serum on rat tailcollagen-coated dishes. Cells were cotransfected as previously described (13) byusing Lipofectamine 2000 reagent, as recommended by the manufacturer (In-vitrogen), with 3.0 �g of pcDNA3-derived constructs and 0.2 �g of pEGFP-C3reporter plasmid encoding enhanced green fluorescent protein (EGFP; BD Bio-sciences Clontech) to visualize transfected cells. Green fluorescent protein(GFP)-positive cells with one or more growth cone-tipped neurites of �2 cellbodies in length were counted under a fluorescence microscope. Cell differ-entiation was estimated by the percentage of differentiated cells in totalGFP-positive cells.
RESULTS
B-Raf isoforms are differentially regulated by intramolecu-lar autoinhibition. In order to investigate the role of intramo-lecular interactions in the regulation of B-Raf isoforms, wegenerated different constructs as depicted in Fig. 2A. On theone hand, the C-terminal kinase domain, which is common toall B-Raf isoforms, was fused to the Flag tag sequence (Flag-Cter). On the other hand, the N-terminal regulatory region ofthree distinct B-Raf isoforms, with or without the sequencesencoded by alternatively spliced exons, was tagged with theMyc epitope. The B2-Raf8b isoform contains the 12 aminoFIG. 2. The N terminus of B-Raf isoforms binds differentially to
the C-terminal kinase domain. (A) Schematic representation of theFlag-Cter and myc-Nter B-Raf constructs. (B) Results of coimmuno-precipitations of the B-Raf N- and C-terminal domains in HEK293cells. Cells were cotransfected with the Flag-Cter construct and each ofthe three B-Raf myc-Nter constructs depicted in panel A (B1, B28b, orB39b). Cell extracts were immunoprecipitated (IP) with either anti-mycor anti-Flag antibody, and immune complexes were then immuno-blotted (WB) with both antibodies. Transfection efficiency was moni-tored by direct Western blotting of WCE. Quantification of threeindependent experiments is shown. The percentages were calculatedusing the highest value as 100% (B28b). (C) Differential inhibitoryeffect of the N termini of isoforms on MEK/ERK activation induced bythe N-terminal kinase domain. The phosphorylation/activation of bothMEK1/2 and ERK1/2 by the Flag-Cter construct was assayed in the
absence or presence of myc-Nter constructs, by Western blotting of cellextracts from cotransfected HEK293 cells, using phospho-specific an-tibodies (P-MEK and P-ERK) as indicated. Transfection efficiency wasmonitored by direct Western blotting with anti-Myc and anti-Flagantibodies. The loading control was performed using an anti-ERK1/2antibody (lower panel). Quantification of three independent experi-ments is shown. The percentages were calculated using the highestvalue as 100% (control Cter-B-Raf alone). RBD, Ras binding domain.C-ter, C terminus; N-ter, N terminus; IgG, immunoglobulin G.
acids encoded by exon 8b, the B3-Raf9b isoform contains the40 amino acids encoded by exon 9b, and the B1-Raf isoformdoes not contain any alternatively spliced sequences (41).HEK293 cells were cotransfected with the Flag-Cter constructand each of the myc-Nter constructs. The N-terminal regionsof B-Raf isoforms were immunoprecipitated with the Myc an-tibody, and the immune complexes were then probed for thepresence of the C-terminal B-Raf kinase domain by using theFlag antibody. The results presented in Fig. 2B showed thatthe isolated N terminus and C terminus of B-Raf physicallyinteract, confirming the previous study of Tran et al. (51).However, we reproducibly observed that the N terminus ofB2-Raf8b binds more strongly to the C-terminal kinase domainthan the N terminus of B1-Raf. In contrast, the interactionwith the N terminus of B3-Raf9b appeared to be the weakest.The reciprocal coimmunoprecipitation experiment using theFlag antibody, followed by Western blotting with the Mycantibody, gave rise to similar results and confirmed these dif-ferences in the affinities of the N termini of three B-Raf iso-forms for the kinase domain (Fig. 2B). To evaluate the conse-quences of these differences on the ability of the N terminus torepress the activity of the isolated kinase domain, we measuredthe level of activation of the MEK/ERK pathway induced bythe Flag-Cter construct in the presence of the different myc-Nter constructs. As shown in Fig. 2C, activation of both MEKand ERK was more efficiently inhibited by the N terminus ofB2-Raf8b, compared to that of B1-Raf, and conversely lessinhibited by the N terminus of B3-Raf9b. Therefore, there is agood correlation between the strength of interaction and theability of the N terminus to inhibit C-terminal activity.
We next investigated whether these differences in intramo-lecular interactions could modulate B-Raf biological activity.B-Raf was initially identified thanks to the ability of its isolatedkinase domain to transform primary cultures of chicken em-bryonic NR cells upon retroviral transduction (35). Indeed, theNR cell system represents a sensitive indicator for the detec-tion of mitogenic properties, even in the absence of grossmorphological alterations (13, 41, 43). These primary culturescan be maintained in a nondividing state for several weeks inthe presence of serum growth factors. Constitutive expressionof activated oncogenes, such as Ras and Raf, promotes sus-tained NR cell division that results in the formation of foci ofdividing cells (11, 35, 43). Therefore, we used this system tocompare the abilities of the different myc-Nter constructs toinhibit the transforming potential of Flag-Cter B-Raf. NR cellsdissected from 8-day-old chicken embryos were cotransfectedwith Flag-Cter and each of the myc-Nter constructs, and cul-tures were then examined for the presence of foci of prolifer-ating cells 2 weeks after G418 selection. As shown in Fig. 3, theFlag-Cter protein induced the formation of numerous andlarge foci of dividing cells. In contrast, a strong inhibition ofcell proliferation was observed in the three cultures coexpress-ing the N terminus of B-Raf isoforms, demonstrating the abil-ity of this domain to repress the biological activity of the kinasedomain. However, the highest level of inhibition was observedwith the N terminus of B2-Raf8b, whereas that of B3-Raf9b wasless efficient. In conclusion, the presence of exon 8b sequencesincreases the binding of the B-Raf N terminus to the kinasedomain, thereby inhibiting the ability of the latter to induceMEK/ERK activation and NR cell transformation. In contrast,
exon 9b sequences exert an opposite effect, resulting in a lowerlevel of inhibition.
B-Raf isoforms are differentially regulated by phosphoryla-tion. Owing to the presence of two regulatory phosphorylationsites (S365 and S429) in the vicinity of exons 8b and 9b (Fig. 1),we wanted to investigate whether alternative splicing couldinterfere with B-Raf regulation through the phosphorylation ofthese residues. We first characterized phospho-specific anti-bodies able to detect phosphorylation on either S365 or S429.To this aim, we tested a panel of commercially available anti-bodies and identified two which specifically recognized phos-phorylated S365 or S429 (Fig. 4A). The phospho-S365 anti-
FIG. 3. Differential inhibition of proliferating activity of the B-Rafkinase domain by the N termini of isoforms in NR cells. Primarycultures of NR cells were cotransfected with pEF/Flag-Cter andpRcRSV/myc-Nter constructs or empty pRcRSV as indicated. Afterselection for G418-resistant cells, the foci of proliferating NR cellswere stained with crystal violet. The area of the plates covered in cellsis indicated in cm2 below each plate. The percentages were calculatedusing the highest value as 100% (control Cter-B-Raf alone). The datapresented are representative of three independent experiments.
body was initially described by the manufacturer to be specificfor Raf-1 serine 259 phosphorylation but, in our hands, alsodetected B-Raf serine 365 phosphorylation. With respect toS429, we took advantage of the fact that the sequence sur-rounding this residue perfectly matched the consensus R/K-R/K-X-S/T for PKA phosphorylation and selected an antibodythat was specifically raised against this consensus. Since thisantibody was prone to recognizing a large number of PKAsubstrates in total cell extracts, we first immunoprecipitatedB-Raf before Western blotting. As shown in Fig. 4, both anti-bodies were highly specific for the phosphorylated forms ofeither S365 or S429 since they failed to detect B-Raf afterphosphatase treatment (Fig. 4B) or mutation of the corre-sponding residue to alanine (Fig. 4A). These phospho-specificantibodies were further used to analyze the basal level ofB-Raf isoform phosphorylation of both residues in HEK293cells. Interestingly, the results presented in Fig. 4C showed thatS365 phosphorylation was increased in B2-Raf8b whereas itwas strongly decreased in B3-Raf9b, compared to that in B1-Raf. In contrast, the level of S429 phosphorylation remainedunchanged in the presence of alternatively spliced sequences.
Phosphorylation of the residue equivalent to S365 in Rafproteins was shown to create a docking site for 14-3-3 proteins(12, 15, 47). Therefore, we analyzed the ability of B-Raf iso-forms to bind 14-3-3. HEK293 cells were transfected with Myc-tagged B1-Raf, B2-Raf8b, or B3-Raf9b, and cell lysates wereimmunoprecipitated with the anti-Myc antibody. Immunopre-cipitates were then blotted with an anti-14-3-3 antibody. Asshown in Fig. 4D, a correlation was observed between the levelof S365 phosphorylation and the efficacy of B-Raf isoforms tobind endogenous 14-3-3, B2-Raf8b, and B3-Raf9b, being themost and less efficient, respectively.
We next examined the effect of S365A or S429A mutationson the ability of B-Raf isoforms to activate the MEK/ERKpathway. HEK293 cells were transfected with constructs ex-pressing either wild-type (WT) or mutated B-Raf isoforms onthese residues, and activation of MEK1/2 and ERK1/2 wasanalyzed using phospho-specific antibodies. As shown in Fig.5A, WT B-Raf isoforms differ in their abilities to activate theMEK/ERK pathway, in agreement with our previous studiesdemonstrating that these isoforms display differential MEKkinase activity (41). Therefore, we used this assay to analyzethe effect of S365A and S429A mutations on B-Raf isoformsboth in serum-starved cells and in cells stimulated with FCS for5 min (Fig. 5B). While mutation of S365 into alanine clearlyincreased the activity of the three isoforms, mutation of S429,however, did not result in significant changes in the activity ofthe isoforms under either condition. Therefore, S365 phosphor-ylation negatively regulates B-Raf isoform activity, a mecha-nism likely involving 14-3-3 binding, as described for other Rafproteins. The effect of S429 phosphorylation, at this step, re-mained unclear.
To further analyze the effect of S365A and S429A mutationson the biological activity of B-Raf isoforms, we took advantageof the NR cell system. We previously reported that this systemwas sensitive enough to detect mitogenic activity of B-Raf inthe absence of protein truncation or mutation and to revealdifferences in the biological activity of WT B-Raf isoforms(41). In addition, this system proved to be useful for detectinga gain of function induced by mutation of S259 in the related
FIG. 4. B-Raf isoforms are differentially phosphorylated on S365.(A) Characterization of phospho-S365 (P-365)- and phospho-S429 (P-429)-specific antibodies. HEK293 cells were transfected with the full-length Myc-tagged B1-Raf isoform or its mutants, S365A and S429A, asindicated. To detect phosphorylation on S365, whole-cell extracts wereimmunoblotted (WB) with an antibody raised against phosphorylatedS259 of Raf-1. To detect phosphorylation on S429, protein extracts wereimmunoprecipitated (IP) with the anti-Myc antibody and the immunecomplexes were analyzed by Western blotting using an anti-phospho-PKAsubstrate antibody raised against the R/K-R/K-X-S/T consensus sequence.(B) Protein extracts from HEK293 cells transfected with Myc-taggedB1-Raf were immunoprecipitated with anti-Myc antibody. The immunecomplexes were then treated or not treated with �-protein phosphatase(�-PPase) and analyzed by Western blotting using phospho-S365 (P-365)and phospho-S429 (P-429) antibodies. (C) S365 and S429 phosphoryla-tion of full-length (left panel) or N-terminal (N-ter) (right panel) B-Rafisoforms (B1, B2, and B3) was analyzed as for panel A. Quantification ofthree independent experiments is shown. (D) Differential interaction ofB-Raf isoforms with endogenous 14-3-3 proteins. HEK293 cells weretransfected with full-length Myc-tagged B-Raf isoforms, and protein ex-tracts were immunoprecipitated with anti-Myc antibody (IP myc). Theimmune complexes were analyzed by Western blotting using an anti-14-3-3 antibody. The amount of immunoprecipitated B-Raf proteins wasverified using the anti-Myc antibody. Transfection efficiency and loadingwere monitored using anti-Myc and anti-14-3-3 antibodies, respectively,on WCE. Quantification of three independent experiments is shown.
Raf-1 protein (13). In agreement with our previous studies,B2-Raf8b and B3-Raf9b exhibited diminished and enhancedmitogenic activities, respectively, compared to B1-Raf (Fig. 6)(41). Interestingly, the S365A mutation markedly increased themitogenic activities of all B-Raf isoforms and abolished thedifferences in their activities (Fig. 6). The difference in mito-genic activity observed between WT and S365A proteins wasmore pronounced for B2-Raf8b than for B3-Raf9b. Therefore,a correlation exists between the abilities of distinct B-Raf iso-forms to become phosphorylated on S365A, to bind 14-3-3through this residue, and to induce NR cell proliferation.
To a lower extent than S365A mutation, S429A mutationalso appeared to modestly affect the mitogenic activity of B-Raf isoforms in NR cells. A moderate increase and decreasecould be observed for the B2-Raf8b and B3-Raf9b isoforms,respectively (Fig. 6). The observation that the activity of aB-Raf isoform, namely, B3-Raf9b, was inhibited upon S429Amutation was somewhat surprising since a previous study sug-gested that phosphorylation of B1-Raf on S429 slightly inhib-ited its activity (21). It should be noted that in these previousstudies, the effect of the S429A mutation on B-Raf biologicalactivity was never assessed solely but only in combination with
FIG. 5. The S365A mutation increases MEK/ERK activation induced by B-Raf isoforms. (A) Comparison of the abilities of full-length B-Rafisoforms to induce MEK/ERK activation in HEK293 cells. Cells were transfected with full-length Myc-tagged B-Raf isoforms (B1, B2, and B3),and the activation of both MEK1/2 and ERK1/2 was analyzed by Western blotting (WB) using anti-phospho-MEK (P-MEK) and anti-phospho-ERK (P-ERK) antibodies, respectively. Transfection efficiency was monitored by Western blotting with anti-Myc antibody. The loading control wasperformed using an anti-ERK1/2 antibody (lower panel). Quantification of three independent experiments is shown. (B) Effect of S365A andS429A mutations on ERK activation induced by B-Raf isoforms. HEK293 cells transfected with full-length wild-type B-Raf isoforms (B1, B2, andB3) or their S365A and S429A mutants were serum starved and stimulated or not stimulated with 20% serum (FCS) for 5 min. ERK1/2 activationwas analyzed as for panel A. Quantification of three independent experiments is shown.
other mutations (21). Therefore, we wanted to confirm theseopposite effects of the S429A mutation on B2-Raf8b and B3-Raf9b isoforms in another cell type. Rat pheochromocytomaPC12 cells are well known for undergoing neuronal differen-tiation upon nerve growth factor treatment through the re-cruitment of a TrkA/B-Raf/MEK/ERK signaling cascade (26,44, 49, 52). Accordingly, constitutive activation of a componentof this cascade is sufficient to induce neurite outgrowth. Asshown in Fig. 7A, overexpression of the wild-type B3-Raf9b
isoform was unable to induce PC12 cell differentiation, despitethe presence of exon 9b. Similar results were obtained with B1and B2 isoforms (data not shown). Therefore, we looked at theeffect of a gain-of-function mutation on the differentiatingactivity of B-Raf isoforms. To this aim, T599 and S602 phos-phorylation residues in the activation loop of B-Raf (Fig. 1)were mutated into glutamic acid residues. We chose this dou-ble modification instead of the constitutively activating V600E
mutation or fusion with Ras-CAAX sequence because it hasbeen reported that a B-Raf protein carrying a double-acidicsubstitution on T599 and S602 residues can be further stimu-lated by activated Ras or by additional mutations on S365 andS429 (58). The three B-Raf isoforms mutated on both residuesinduced neurite outgrowth (Fig. 7A). Importantly, the doubleT599E/S602E gain-of-function mutation preserved the differ-ential activities of B-Raf isoforms. B3-Raf9b
EE displayed thehighest differentiating activity, whereas B2-Raf8b
EE was theleast efficient, as shown both by the percentage of differenti-ated cells (Fig. 7B) and by the complexity of the neuriticnetwork (Fig. 7A). We next examined the effect of an addi-tional S429A mutation on B-RafEE isoform activity. As ob-served in NR cells with otherwise wild-type proteins (Fig. 6),the S429A mutation reproducibly increased B2-Raf8b
EE activ-ity and decreased that of B3-Raf9b
EE (Fig. 7B). Finally, we alsotested the effect of the S429A mutation in combination withthe T599E/S602E gain-of-function mutation in HEK293 cells.As shown in Fig. 7C, the S429A mutation had opposite effectson the abilities of B2-Raf8b
EE and B3-Raf9bEE to activate the
MEK/ERK pathway.Taken together, these results show that phosphorylation on
both S365 and S429 participates in the differential regulationof B-Raf isoforms through distinct mechanisms. The basallevels of S429 phosphorylation are equivalent in the threeisoforms, but the effects of this phosphorylation on B-Rafactivity differ: it inhibits B2-Raf8b, whereas it appears to acti-vate B3-Raf9b. S365 phosphorylation inhibits the activities ofthe three isoforms, but the basal level of phosphorylation ofthis residue differs for each isoform. Interestingly, the isolatedN terminus of B-Raf isoforms was differentially phosphory-lated at S365, as in the full-length proteins (Fig. 4C). There-fore, we wondered whether phosphorylation on S365 and S429could be involved in the differences observed between B-Rafisoforms in the ability of their N termini to inhibit the activityof the kinase domain. To test this hypothesis, we examined theeffect of the S365A and S429A mutations on the inhibitoryeffect of the N terminus of B-Raf isoforms in NR cells. Asshown in Fig. 8, mutation of either residue was unable todecrease the ability of the N terminus of B-Raf isoforms toinhibit the mitogenic effect of the isolated kinase domain. Inagreement with this, the S365A or S429A mutation did notalter the ability of the N terminus of B-Raf isoforms to copre-cipitate with the C-terminal domain in HEK293 cells (Fig. 9).These results suggest that differential B-Raf isoform regulationthrough phosphorylations and intramolecular interactions pro-ceeds from independent mechanisms.
DISCUSSION
B-Raf is involved in many physiological and pathologicalprocesses (19, 37, 44, 56). Like those of other Raf proteins,B-Raf activity is regulated through complex mechanisms, in-cluding inhibitory and activating phosphorylations (56, 58).However, it has been shown that B-Raf requires fewer phos-phorylation events than A-Raf and Raf-1 for maximal activa-tion, thereby explaining its higher basal kinase activity (36).We previously reported that B-Raf also differs from the otherRaf proteins in vertebrates by another level of regulation in-volving alternative splicing (41). Thus, exon 9b present in the
FIG. 6. The S365A and S429A mutations differentially affect pro-liferating activities of B-Raf isoforms in NR cells. Primary cultures ofNR cells were transfected with pRcRSV/myc-derived constructs en-coding full-length B-Raf isoforms (B1, B2, and B3), either WT ormutated on S365 or S429, as indicated. The empty pRcRSV vector wasused as a control. After selection for G418-resistant cells, the foci ofproliferating NR cells were stained with crystal violet. Quantificationwas performed as for Fig. 3. The data presented are representative offour independent experiments.
B3-Raf9b isoform increases both the MEK activity and trans-forming activity of B-Raf, whereas exon 8b has an oppositeeffect in B2-Raf8b isoforms. In the present study, we haveinvestigated the molecular basis accounting for these differ-ences. Using different cell systems, we found that both phos-phorylation of two serine residues and intramolecular interac-tions participate in this regulation through distinctmechanisms.
In agreement with a recent report, we showed that the B-RafN-terminal regulatory region inhibits the activity of the kinasedomain (51). Our results indicated that the N terminus ofB2-Raf8b has a higher affinity for the C terminus than that ofB1-Raf, whereas the N terminus of B3-Raf9b has a lower af-finity. Accordingly, the N terminus of B2-Raf8b was more ef-ficient than that of B3-Raf9b at inhibiting MEK/ERK activa-tion and NR cell proliferation induced by the isolated Cterminus. We also showed that this ability of the N terminus ofB-Raf to bind to and inhibit the kinase domain does not de-pend on the phosphorylation of S365 or S429. Similar obser-vations were reported for S259, the residue equivalent to S365in Raf-1 (6). The exact mechanisms by which intramolecularinteractions between both domains regulate Raf protein activ-ity currently remain unknown. Several recent studies demon-strated that Raf activation occurs through the formation ofmultiprotein complexes involving homo- or hetero-oligomer-ization between Raf family members (20, 38, 48, 55). Mappingof the molecular determinants implicated in oligomerizationindicates that some of them are clustered in B-Raf regions thatcould also be engaged in intramolecular interactions (48).Therefore, the role of N-terminal/C-terminal intramolecularinteraction could be to lock B-Raf in a closed conformationthat prevents oligomerization and subsequent phosphorylationon the activation loop. This autoinhibition is released uponbinding of the B-Raf N-terminal domain to GTP-bound Ras(51). This mechanism was initially proposed for the regulationof Raf-1 activity (6, 9). Using the fluorescence resonance en-ergy transfer technique, Terai and Matsuda recently showed
FIG. 7. Opposing effects of the S429A mutation on PC12 cell dif-ferentiation or ERK activation induced by B-Raf isoforms. (A) PC12cell differentiation induced by B-Raf isoforms carrying the phospho-mimetic T599E/S602E double substitution. PC12 cells were cotrans-fected with a pEGFP reporter plasmid and pcDNA3-derived con-structs encoding B-Raf isoforms, either WT or carrying the T599E/S602E double mutation (EE). A representative field for each conditionwas photographed under an inverted fluorescence microscope. Notethat overexpression of WT B3-Raf9b does not induce neurite out-growth. Similar results were obtained with B1-Raf and B2-Raf8b WTisoforms (data not shown). (B) Effect of the S429A mutation on PC12cell differentiation induced by B-RafEE isoforms. PC12 cells weretransfected as for panel A, and the total number of GFP-positive cellswas counted. The indicated percentages were calculated from threeindependent experiments and represent the ratios between the numberof GFP-positive cells undergoing neurite outgrowth and the total num-ber of GFP-positive transfected cells. Statistical significance was eval-uated by a Student paired t test (*, P � 0.01). (C) pRcRSV-derivedconstructs containing the same mutants as in panel B were used totransfect HEK293 cells. ERK1/2 activation was analyzed by Westernblotting using anti-phospho-ERK (P-ERK) antibody. Transfection ef-ficiency was monitored by Western blotting with anti-Myc antibody.The loading control was performed using an anti-ERK1/2 antibody.Quantification of two independent experiments is shown below.
that membrane recruitment of Raf-1 through Ras was requiredto achieve this conformational change and to activate Raf-1kinase activity (50). In agreement with this model, the isolatedC-terminal kinase domain of B-Raf proteins carrying either aT599E/S602D double substitution or a V600E oncogenic mu-tation is no longer inhibited by the N terminus, although bothdomains could still bind together (51). Whether phosphoryla-tion of T599 and S602 results from trans phosphorylationwithin the oligomers or from phosphorylation by a yet un-known protein kinase at the plasma membrane remains to bedetermined. In light of these observations, we propose that thepresence of exon 9b in B3-Raf9b favors the open active con-formation by reducing the autoinhibitory mechanism, therebyexplaining the higher MEK activity of this isoform. In contrast,exon 8b, by increasing N-terminal affinity for the kinase do-
main, would have an opposite effect on downstream MEKactivation.
As described above, a key step in Raf activation is the con-formational change that relieves autoinhibition through Ras-mediated recruitment of the kinase at the plasma membrane.Such a recruitment of Raf-1 and D-Raf requires dephosphor-ylation of a residue equivalent to B-Raf S365 by PP2A, result-ing in 14-3-3 displacement (1, 27, 32, 39). Consequently, mu-tation of Raf-1 S259 results in increased kinase activity (13,14). We showed here that mutation of S365 into alanine po-tentiates B-Raf-mediated ERK activation in HEK293 cells, inagreement with a previous report (21). We further demon-strate that it strongly increases B-Raf mitogenic activity in NRcells, an effect similar to what we previously observed for Raf-1(13). However, the difference in mitogenic activity observed
FIG. 8. The S365A and S429A mutations do not affect the inhibitory effect of the B-Raf N terminus on the proliferating activity of the kinasedomain in NR cells. Primary cultures of NR cells were cotransfected with pEF/Flag-Cter and pRcRSV/myc-Nter constructs mutated or notmutated on S365 and S429 as indicated. Empty pRcRSV was used as a control. After selection for G418-resistant cells, the foci of proliferatingNR cells were stained with crystal violet. Quantification was performed as for Fig. 3. The data presented are representative of three independentexperiments. N-ter, N terminus.
between WT and S365A proteins was more pronounced forB2-Raf8b than for B3-Raf9b. Using a phospho-specific anti-body, we found that phosphorylation of S365 was increased inB2-Raf8b and decreased in B3-Raf9b, compared to that in B1-Raf. This was correlated with a larger amount of 14-3-3 pro-teins associated with B2-Raf8b, compared to that associatedwith B3-Raf9b. These differences in S365 phosphorylation areindependent of the N-terminal/C-terminal intramolecular in-teraction since they were also observed on the isolated Nterminus of B-Raf isoforms. These results suggest that exon9b-containing isoforms are less dependent on PP2A-mediateddephosphorylation for their membrane recruitment than exon8b-containing isoforms. However, alternative, but not mutuallyexclusive, hypotheses exist concerning the role of the S259residue in Raf-1 activity (13) and one cannot exclude that theycould be applied to B-Raf as well. Nevertheless, phosphoryla-tion of S365 appears to be a major determinant for the differ-ential regulation of B-Raf isoforms.
In the course of this study, we identified S429 as a secondphosphorylation site differentially regulating B-Raf isoform ac-tivity. The role of this phosphorylation has been poorly docu-mented so far, mainly because the effect of the S429A mutationwas never assessed solely but only in combination with muta-tions on S365 and T440 (21). Using a phospho-specific anti-body, we demonstrate for the first time that this residue isindeed phosphorylated in cells. In contrast with S365, S429 wasfound equally phosphorylated in the three B-Raf isoformstested. However, mutation of this residue into alanine indi-cated that it could differentially regulate B-Raf isoforms. Theobserved effects of the S429A mutation in otherwise WT B-Raf
proteins remained moderate and dependent on the cell system.While it had barely detectable effects on ERK activation inHEK293 cells, it increased and decreased mitogenic activitiesof B2-Raf8b and B3-Raf9b, respectively, in the NR cell system.A similar differential effect was also observed in the PC12 celldifferentiation assay, where the mutation could be tested onlyin combination with the double phosphomimetic T599E/S602Esubstitution. As mentioned above, this double mutation ren-ders B-Raf insensitive to the autoinhibition mechanism. There-fore, it is conceivable that the contribution of S429 phosphor-ylation is minor compared to that of autoinhibition andbecomes easier to detect in the absence of the latter. Accord-ingly, an effect of the S429A mutation could be also detected inHEK293 cells in the context of the T599E/S602E substitution.The mechanism by which S429 phosphorylation differentiallyregulates B-Raf isoform activity is currently unknown. Giventhat this residue was reported to be phosphorylated by Akt, atleast in vitro, it could be proposed to serve as a docking site for14-3-3 when phosphorylated. A cryptic binding site was re-cently described for Raf-1, upon S233 phosphorylation (15).However, B-Raf S429 is not located in the same region asRaf-1 S233 and does not match to either strong or weak 14-3-3binding consensus sequences (57). Our data also indicate thatthe S429A mutation does not impinge on the N-terminal/C-terminal interaction. Interestingly, however, serine 429 isclosed to two key residues of the B-Raf kinase domain: S446,which is constitutively phosphorylated, and D448 (Fig. 1). Thenegative charges provided by these residues appear to be im-portant for maintaining the kinase domain in an active confor-mation. For example, D448 forms an electrostatic interactionwith R506 of the C-helix, thereby stabilizing the small lobe ofthe kinase domain (54). The presence of either exon 8b or exon9b could differentially modify the local conformation and re-lationship between phosphorylated S429 and this region of thesmall lobe, resulting in opposite effects. Finally, we do notexclude that S429 phosphorylation regulates a yet unknownB-Raf function that would be uncoupled from its MEK kinaseactivity.
Whatever the mechanisms by which phosphorylation of S365and S429 differentially regulates B-Raf isoforms, it is notewor-thy that both residues can be targeted by the same proteinkinase. Thus, different members of the AGC kinase familyhave been proposed to phosphorylate these residues, includingPKA, Akt, and SGK (21, 31, 59). While the ability of Akt andSGK to phosphorylate B-Raf has not been firmly demon-strated in vivo, their implication in Raf-1 regulation is still amatter of debate. More convincing are the data suggesting thatPKA can directly phosphorylate both Raf proteins in cells (12,15, 31, 33). B-Raf phosphorylation by PKA in vitro clearlyinhibits its activity (33). Paradoxically, in cell types of neuro-ectodermic origin (neuronal and neural crest-derived cells),elevation of intracellular cyclic AMP (cAMP), which normallyactivates PKA, results in B-Raf and ERK activation (3, 16, 33,46, 53). The mechanisms by which cAMP activates B-Raf re-main controversial and might be cell type dependent since bothRap1-dependent and -independent pathways have been re-ported (16, 44). These observations have led to a model inwhich B-Raf is resistant to PKA-mediated inhibition uponcAMP increase in cells (16, 33). With respect to this, it isinteresting to underline that B3-Raf9b should be the most
FIG. 9. The S365A and S429A mutations do not affect the N-terminal/C-terminal interaction of B-Raf isoforms. Coimmunoprecipi-tations of the B-Raf N- and C-terminal domains in HEK293 cells wereperformed as described for Fig. 2. Cells were cotransfected with theFlag-Cter construct and each of the three B-Raf myc-Nter constructs(B1, B28b, or B39b) mutated or not mutated on either S365 or S429.Cell extracts were immunoprecipitated with anti-Flag antibody, andimmune complexes were then immunoblotted (WB) with either anti-Myc or anti-Flag antibody as indicated. N-ter, N terminus; C-ter, Cterminus.
resistant isoform to PKA-mediated inhibition since, on the onehand, it is less phosphorylated on S365 than the other isoformsand, on the other hand, its activity is increased upon S429phosphorylation. In contrast, B2-Raf8b should be less resistantbecause exon 8b favors S365 phosphorylation, while S429phosphorylation decreases its activity. Finally, we cannot ex-clude that kinases other than Akt and PKA could also phos-phorylate these residues, raising the possibility of presentlyunknown physiological regulations of B-Raf isoforms.
Taken together, the results presented in this study show thatalternative splicing modulates B-Raf activity through distinctbut convergent mechanisms. This suggests that exons 8b and 9bimpose structural constraints in the hinge region of the kinase,resulting in opposite effects on the intramolecular autoinhibi-tion, the sensitivity to S365 inhibiting phosphorylation, and theconsequence of S429 phosphorylation. Structural studies arerequired to support this model, but our attempts to purifyfull-length B-Raf isoforms suitable for X-ray analysis usingbacterial or baculovirus systems were unsuccessful, as also ob-served by other groups (48, 54). The only multidomain kinasesfor which a three-dimensional structure of the full-length pro-tein is available are members of the Src family. Interestingly,these kinases are also autoinhibited by their N termini. It hasbeen demonstrated that the linker region immediately up-stream of the kinase domain, which is in close contact withboth the SH3 domain and the small lobe of the kinase, plays akey role during the transition between inactive and active con-formations of Src (24). Given the structural similarities be-tween B-Raf and Src family kinase domains (36, 54), it istempting to speculate that the presence of alternatively splicedexons in the B-Raf hinge region impinges on a similar mode ofconformational regulation.
Owing to this complex mode of regulation and to the con-servation of B-Raf alternatively spliced exons through evolu-tion, future directions will focus on the specific role of B-Rafisoforms during development and oncogenesis.
ACKNOWLEDGMENTS
We thank Brian Rudkin for helpful advice on PC12 cells. We alsothank Carole Burns, Andrew Doedens, Celio Pouponnot, and NathalieRocques for comments on the manuscript.
This work was funded by the Centre National de la RechercheScientifique, by the Institut Curie, and by grants from the Ligue Na-tionale Contre le Cancer (Comite de l’Essonne) and INCA (melanomanetwork). I.H. was supported by fellowships from the Ligue NationaleContre le Cancer and the Association pour la Recherche sur le Cancer.A.E. and S.D. are INSERM investigators.
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