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Molecular and Cellular Biology, January 2007, p. 31-43, Vol. 27, No. 1
0270-7306/07/$08.00+0     doi:10.1128/MCB.01265-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Differential Regulation of B-Raf Isoforms by Phosphorylation and Autoinhibitory Mechanisms{triangledown}

Isabelle Hmitou,1,2,{ddagger} Sabine Druillennec,1,2,{ddagger} Agathe Valluet,1,2 Carole Peyssonnaux,1,2,{dagger} and Alain Eychène1,2*

Institut Curie, Centre de Recherche, Orsay F-91405, France,1 CNRS, UMR 146, Orsay F-91405, France2

Received 12 July 2006/ Returned for modification 15 August 2006/ Accepted 16 October 2006


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The B-Raf proto-oncogene encodes several isoforms resulting from alternative splicing in the hinge region upstream of the kinase domain. The presence of exon 8b in the B2-Raf8b isoform and exon 9b in the B3-Raf9b isoform 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 show that exons 8b and 9b interfere with the ability of the B-Raf N-terminal region to interact with and inhibit the C-terminal kinase domain, thus modulating the autoinhibition mechanism in an opposite manner. Exons 8b and 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. The S365A mutation increased the activity of all B-Raf isoforms, but the effect on B2-Raf8b was more pronounced. This was correlated to the high level of S365 phosphorylation in this isoform, whereas the B3-Raf9b isoform was poorly phosphorylated on this residue. In contrast, S429 was equally phosphorylated in all B-Raf isoforms, but the S429A mutation activated B2-Raf8b, whereas it inhibited B3-Raf9b. These results indicate that phosphorylation on both S365 and S429 participate in the differential regulation of B-Raf isoforms through distinct mechanisms. Finally, we show that autoinhibition and phosphorylation represent independent but convergent mechanisms accounting for B-Raf regulation by alternative splicing.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The BRAF oncogene encodes a MEK1/2 kinase that was initially identified due to its transduction into the genome of IC10, an acute mitogenic retrovirus able to transform primary cultures of chicken embryonic neuroretina cells (35). Its human ortholog was simultaneously identified in NIH 3T3 cells transfected with Ewing sarcoma DNA (25). In both cases, the B-Raf protein was truncated in its N terminus and the kinase domain was fused to foreign sequences, leading to its constitutive activation. Such a mechanism for B-Raf oncogenic activation was recently reported for a human thyroid papillary carcinoma (7). However, the most widely encountered mode of B-Raf activation in human cancers results from point mutations in the highly conserved glycine-rich loop and activation segment of the kinase domain (10). The V600E substitution, representing the most prevalent mutation, is detected in about 40 to 50% of melanoma and thyroid papillary carcinoma and at lower rates in other human tumors (8, 10, 29, 56). This mutation markedly increases B-Raf basal kinase activity (4, 54), and short interfering RNA-mediated B-Raf depletion in melanoma cells harboring this mutation results in a reversion of the transformed phenotype (4, 22, 28). The role of this mutation in tumor initiation was further confirmed by studies using animal models (30, 37, 42). Although the role of B-Raf protein in oncogenic processes is becoming evident, the regulation of its activity, especially through phosphorylation, is not fully understood. B-Raf displays a higher kinase activity toward its substrate 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-Raf is activated upon binding to GTPases of the Ras family and subsequent phosphorylation of Thr 599 and Ser 602 residues in the activation segment of the kinase domain (36, 44, 54, 58). In resting cells, B-Raf is maintained in an inactive conformation through an autoinhibitory mechanism involving an intramolecular interaction between the kinase domain and the N-terminal regulatory region, which is released upon binding of this domain to GTP-bound Ras (51). This regulatory mechanism was initially described for the related Raf-1 protein (6, 9, 50). However, with the exception of Thr 599 and Ser 602, B-Raf differs from Raf-1 in that it does not require the phosphorylation of additional residues to become activated. In addition, B-Raf activity has been reported to be down-regulated upon phosphorylation on two residues, Ser 365 and Ser 429 (Fig. 1). Serine 365 is located in the CR2 domain and is the equivalent of Ser 259 in Raf-1 and Ser 388 in Drosophila melanogaster Raf (Fig. 1D).Phosphorylation of this residue creates a docking site for 14-3-3 proteins and prevents Raf-1 and D-Raf activation (12, 15, 47). Dephosphorylation of this residue by PP2A is a prerequisite for 14-3-3 displacement and Raf-1 activation by GTP-bound Ras (1, 27, 32, 39). Consequently, several studies demonstrated that mutation of Raf-1 S259 results in an increased kinase activity (13, 14). Similarly, mutation of serine 365 on B-Raf increases its kinase activity (21). This residue is conserved 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 PKA in the phosphorylation of both Raf-1 Ser 259 and B-Raf Ser 365 (12, 15, 31), the relevance of Akt-mediated phosphorylation of these residues remains a matter of debate. In contrast to that by PKA, phosphorylation of B-Raf Ser 365 by Akt has not been demonstrated in vivo, and it is worth noting that sequences surrounding this residue in Drosophila Raf do not match the Akt consensus site (Fig. 1). Likewise, B-Raf Ser 429 phosphorylation by Akt was shown only in vitro and by indirect evidence (21), whereas phosphorylation of this residue by PKA was shown both in vitro and in vivo (31). Interestingly, a residue equivalent to B-Raf Ser 429 is conserved in both Caenorhabditis elegans and Drosophila Raf proteins (Ser 454 and Ser 444, respectively) but is not present in the other vertebrate Raf proteins, Raf-1 and A-Raf, which arose from subsequent gene duplications (Fig. 1).


Figure 1
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  		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 acid sequence alignment of the region encompassing exons 8, 8b, 9, 9b, and 10 between B-Raf sequences from rat (GenBank accession no. XP_231692), 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 Akt phosphorylation sites are indicated by gray boxes. RBD, Ras binding domain.

 
We previously reported that B-Raf undergoes another level of regulation, through complex alternative splicing (2, 17, 18, 41). Thus, the BRAF gene encodes at least 10 distinct protein isoforms displaying tissue-specific expression in adult mouse (2, 18). These isoforms arise in part from alternative splicing of two exons (8b and 9b) located between the CR2 and CR3 domains (Fig. 1). Exon 9b was initially named exon 10, with respect to the first complete BRAF genomic organization reported in chicken (5), but to avoid confusion, we propose the 9b nomenclature according to the numbering of human BRAF exons. Exon 9b sequences are conserved in all vertebrates since they are present not only in mammalian and avian species but also in mRNAs encoded by BRAF genes from amphibian and fish species (Fig. 1). Exon 8b and 9b sequences are specific for BRAF since they are not conserved in the other vertebrate raf genes (those encoding A-Raf and Raf-1) or in the unique raf ancestor gene in C. elegans and Drosophila. In agreement with an acquired characteristic specific for BRAF following raf gene duplication during evolution, BRAF sequences corresponding to human exons 3 to 10 are clustered within a single exon (exon 2) in Drosophila Raf. We have shown that the presence of these alternative sequences modulates B-Raf biochemical and oncogenic properties (41). Exon 9b increases both the MEK kinase activity and transforming activity of B-Raf, whereas exon 8b has an opposite effect. However, the mechanism of this regulation remained heretofore unknown. In the present study, we show that the presence of exons 8b and 9b modulates the ability of the B-Raf N-terminal region to interact with and inhibit the activity of the C-terminal kinase domain in an opposite manner. Interestingly, Ser 365 and Ser 429 flank these sequences located in the hinge region of B-Raf. By using phospho-specific antibodies and by generating S365A and S429A phosphorylation mutants of the different isoforms, we investigated the role of phosphorylations in the differential regulation of B-Raf isoforms. We found that the presence of exon 8b favors S365 phosphorylation and 14-3-3 binding, whereas the B3-Raf9b isoform containing exon 9b is less efficiently phosphorylated on this residue, in agreement with its elevated activity. To a lesser extent, we observed that S429 phosphorylation differentially regulates the activity of B-Raf, resulting in the activation and inhibition of 8b- and 9b-containing isoforms, respectively. Therefore, both phosphorylation on S365 and S429 residues and autoinhibitory mechanisms are responsible for the differential regulation of B-Raf isoforms.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid constructions. To generate Myc-tagged full-length B-Raf isoforms containing either the S365A or the S429A mutation, a Bsu36I/SphI cassette was mutated by PCR and cloned into pBKS-derived constructs containing B1-Raf, B2-Raf8b, and B3-Raf9b isoform cDNAs (41). The EcoRI fragments containing full-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. The pRcRSV-derived constructs were obtained by subcloning the HindIII fragment from 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). It was generated by amplification of the B-Raf catalytic domain using the following 5' and 3' primers containing HindIII and XhoI sites, respectively: 5'-TTAAGCTTAGCCACCATGAAAACCCTTGGTCGA-3'and 5'-TGCTCGAGCTACTTATCGTCGTCATCCTTGTAATCCTTGAACGCTGCAAATTC-3'. The amplification product was cloned into the HindIII/XhoI sites of pcDNA3 (Invitrogen). The HindIII/XbaI fragment from the resulting pcDNA3/Flag-Cter construct was then subcloned into the pRcRSV and Cla12 vectors (23). pEF/Flag-Cter was obtained by subcloning the ClaI fragment of Cla12/Flag-Cter into the pEF-BOS-CX vector (kindly provided by Jacques Ghysdael). pcDNA3/myc-Nter constructs containing the Myc-tagged N-terminal regulatory domain of B-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-Nter plasmids was then subcloned into the pRcRSV vector to generate pRcRSV/myc-Nter constructs. All of the PCR and cloning procedures were verified by sequencing.

Transfection, Western blotting, and coimmunoprecipitation analysis of HEK293 cells. Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS), 100 mg/ml streptomycin, 100 U/ml penicillin, and 1 mg/ml amphotericin B (Fungizone). Cells were transfected with 500 ng of pcDNA3/myc-B-Raf in 6-mm dishes using Effectene reagent (QIAGEN) according to the manufacturer'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-Cter construct or pRcRSV empty vector. Twenty-four hours after transfection, cells were 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 fluoride, 1 mM sodium orthovanadate, 50 mM NaF, 25 mM ß-glycerophosphate]. Insoluble materials were pelleted by centrifugation at 15,000 x g for 25 min at 4°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 supplemented with 20% FCS, and then lysed as described above. For immunoprecipitation experiments, 100 µl of cell lysate was precipitated with 0.5 µg of mouse anti-Myc (9E10; Santa Cruz Biotechnology) or anti-Flag (M2; Sigma) monoclonal antibody and 40 µl of a 50% slurry of protein A-Sepharose (GE Healthcare). Immunoprecipitates were washed twice with 1 ml of lysis buffer and once with 1 ml 20 mM Tris, pH 8.0, and boiled in Laemmli's sample buffer. They were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane (Millipore), and probed with anti-Flag, anti-Myc, or rabbit polyclonal anti-14-3-3 (K-19; Santa Cruz Biotechnology). Activated forms of MEK or extracellular signal-regulated kinase (ERK) were detected in whole-cell extracts (WCE) by Western blotting using rabbit anti-phospho-MEK1/2 (Ser217/221) (Cell Signaling) or mouse anti-phospho-p42/p44 mitogen-activated protein kinase (Sigma) antibody, respectively. Normalization of cell lysate amounts was achieved by monitoring total ERK expression using rabbit anti-ERK (C-16; Santa Cruz Biotechnology). B-Raf S365 phosphorylation was detected on whole-cell extracts by Western blotting using rabbit polyclonal anti-phospho-S259 Raf-1 (Cell Signaling). B-Raf S429 phosphorylation 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 consensus sequence (Cell Signaling). To further assess the specificity of these antibodies toward phosphorylated S365 and S429, wild-type full-length B-Raf proteins immunoprecipitated with anti-Myc antibody were treated or not treated with 100 U {lambda}-protein phosphatase (Cell Signaling) for 1.5 h at 30°C and then probed with the phospho-specific antibodies. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were used as secondary antibodies, and proteins were visualized 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 Gene Tools software (Syngene).

NR cell proliferation assay. Neuroretina (NR) cell cultures were prepared from 8-day-old Brown Leghorn chicken embryos as previously described (43) and seeded in 100-mm dishes. Cultures were maintained in basal medium Eagle supplemented with 5% FCS, 100 mg/ml streptomycin, 100 U/ml penicillin, 1 mg/ml amphotericin B, and 2 mM glutamine. The mitogenic activity of full-length B-Raf isoforms was assessed by transfecting 20 µg of pRcRSV-derived constructs. The inhibitory effects of the N terminus of B-Raf isoforms on the mitogenic activity of the B-Raf C terminus were assayed by cotransfecting 2 µg of pEF/Flag-Cter and 18 µg of pRcRSV/myc-Nter constructs. Cells were transfected by the calcium phosphate method as previously described, and G418 selection (600 µg/ml) was applied 3 days later for 15 days (43). The cultures were then rinsed with phosphate-buffered saline, and the foci of proliferating cells were stained with 1.0% crystal violet (in 20% ethanol). Quantification of the number and size of the foci was performed using VisionExplorer VA software (Graftec).

PC12 cell differentiation assay. PC12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 6% FCS and 6% horse serum on rat tail collagen-coated dishes. Cells were cotransfected as previously described (13) by using Lipofectamine 2000 reagent, as recommended by the manufacturer (Invitrogen), with 3.0 µg of pcDNA3-derived constructs and 0.2 µg of pEGFP-C3 reporter plasmid encoding enhanced green fluorescent protein (EGFP; BD Biosciences Clontech) to visualize transfected cells. Green fluorescent protein (GFP)-positive cells with one or more growth cone-tipped neurites of >2 cell bodies in length were counted under a fluorescence microscope. Cell differentiation was estimated by the percentage of differentiated cells in total GFP-positive cells.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
B-Raf isoforms are differentially regulated by intramolecular autoinhibition. In order to investigate the role of intramolecular interactions in the regulation of B-Raf isoforms, we generated different constructs as depicted in Fig. 2A. On the one hand, the C-terminal kinase domain, which is common to all B-Raf isoforms, was fused to the Flag tag sequence (Flag-Cter). On the other hand, the N-terminal regulatory region of three distinct B-Raf isoforms, with or without the sequences encoded by alternatively spliced exons, was tagged with the Myc epitope. The B2-Raf8b isoform contains the 12 amino acids encoded by exon 8b, the B3-Raf9b isoform contains the 40 amino acids encoded by exon 9b, and the B1-Raf isoform does not contain any alternatively spliced sequences (41). HEK293 cells were cotransfected with the Flag-Cter construct and each of the myc-Nter constructs. The N-terminal regions of B-Raf isoforms were immunoprecipitated with the Myc antibody, and the immune complexes were then probed for the presence of the C-terminal B-Raf kinase domain by using the Flag antibody. The results presented in Fig. 2B showed that the isolated N terminus and C terminus of B-Raf physically interact, confirming the previous study of Tran et al. (51). However, we reproducibly observed that the N terminus of B2-Raf8b binds more strongly to the C-terminal kinase domain than the N terminus of B1-Raf. In contrast, the interaction with the N terminus of B3-Raf9b appeared to be the weakest. The reciprocal coimmunoprecipitation experiment using the Flag antibody, followed by Western blotting with the Myc antibody, gave rise to similar results and confirmed these differences in the affinities of the N termini of three B-Raf isoforms for the kinase domain (Fig. 2B). To evaluate the consequences of these differences on the ability of the N terminus to repress the activity of the isolated kinase domain, we measured the level of activation of the MEK/ERK pathway induced by the Flag-Cter construct in the presence of the different myc-Nter constructs. As shown in Fig. 2C, activation of both MEK and ERK was more efficiently inhibited by the N terminus of B2-Raf8b, compared to that of B1-Raf, and conversely less inhibited by the N terminus of B3-Raf9b. Therefore, there is a good correlation between the strength of interaction and the ability of the N terminus to inhibit C-terminal activity.


Figure 2
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  		FIG. 2. The N terminus of B-Raf isoforms binds differentially to the C-terminal kinase domain. (A) Schematic representation of the Flag-Cter and myc-Nter B-Raf constructs. (B) Results of coimmunoprecipitations of the B-Raf N- and C-terminal domains in HEK293 cells. Cells were cotransfected with the Flag-Cter construct and each of the three B-Raf myc-Nter constructs depicted in panel A (B1, B28b, or B39b). Cell extracts were immunoprecipitated (IP) with either anti-myc or anti-Flag antibody, and immune complexes were then immunoblotted (WB) with both antibodies. Transfection efficiency was monitored by direct Western blotting of WCE. Quantification of three independent experiments is shown. The percentages were calculated using the highest value as 100% (B28b). (C) Differential inhibitory effect of the N termini of isoforms on MEK/ERK activation induced by the N-terminal kinase domain. The phosphorylation/activation of both MEK1/2 and ERK1/2 by the Flag-Cter construct was assayed in the absence or presence of myc-Nter constructs, by Western blotting of cell extracts from cotransfected HEK293 cells, using phospho-specific antibodies (P-MEK and P-ERK) as indicated. Transfection efficiency was monitored by direct Western blotting with anti-Myc and anti-Flag antibodies. The loading control was performed using an anti-ERK1/2 antibody (lower panel). Quantification of three independent experiments is shown. The percentages were calculated using the highest value as 100% (control Cter-B-Raf alone). RBD, Ras binding domain. C-ter, C terminus; N-ter, N terminus; IgG, immunoglobulin G.

 
We next investigated whether these differences in intramolecular interactions could modulate B-Raf biological activity. B-Raf was initially identified thanks to the ability of its isolated kinase domain to transform primary cultures of chicken embryonic NR cells upon retroviral transduction (35). Indeed, the NR cell system represents a sensitive indicator for the detection of mitogenic properties, even in the absence of gross morphological alterations (13, 41, 43). These primary cultures can be maintained in a nondividing state for several weeks in the presence of serum growth factors. Constitutive expression of activated oncogenes, such as Ras and Raf, promotes sustained NR cell division that results in the formation of foci of dividing cells (11, 35, 43). Therefore, we used this system to compare the abilities of the different myc-Nter constructs to inhibit the transforming potential of Flag-Cter B-Raf. NR cells dissected from 8-day-old chicken embryos were cotransfected with Flag-Cter and each of the myc-Nter constructs, and cultures were then examined for the presence of foci of proliferating cells 2 weeks after G418 selection. As shown in Fig. 3, the Flag-Cter protein induced the formation of numerous and large foci of dividing cells. In contrast, a strong inhibition of cell proliferation was observed in the three cultures coexpressing the N terminus of B-Raf isoforms, demonstrating the ability of this domain to repress the biological activity of the kinase domain. However, the highest level of inhibition was observed with the N terminus of B2-Raf8b, whereas that of B3-Raf9b was less efficient. In conclusion, the presence of exon 8b sequences increases the binding of the B-Raf N terminus to the kinase domain, thereby inhibiting the ability of the latter to induce MEK/ERK activation and NR cell transformation. In contrast, exon 9b sequences exert an opposite effect, resulting in a lower level of inhibition.


Figure 3
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  		FIG. 3. Differential inhibition of proliferating activity of the B-Raf kinase domain by the N termini of isoforms in NR cells. Primary cultures of NR cells were cotransfected with pEF/Flag-Cter and pRcRSV/myc-Nter constructs or empty pRcRSV as indicated. After selection for G418-resistant cells, the foci of proliferating NR cells were stained with crystal violet. The area of the plates covered in cells is indicated in cm2 below each plate. The percentages were calculated using the highest value as 100% (control Cter-B-Raf alone). The data presented are representative of three independent experiments.

 
B-Raf isoforms are differentially regulated by phosphorylation. Owing to the presence of two regulatory phosphorylation sites (S365 and S429) in the vicinity of exons 8b and 9b (Fig. 1), we wanted to investigate whether alternative splicing could interfere with B-Raf regulation through the phosphorylation of these residues. We first characterized phospho-specific antibodies able to detect phosphorylation on either S365 or S429. To this aim, we tested a panel of commercially available antibodies and identified two which specifically recognized phosphorylated S365 or S429 (Fig. 4A). The phospho-S365 antibody was initially described by the manufacturer to be specific for Raf-1 serine 259 phosphorylation but, in our hands, also detected B-Raf serine 365 phosphorylation. With respect to S429, we took advantage of the fact that the sequence surrounding this residue perfectly matched the consensus R/K-R/K-X-S/T for PKA phosphorylation and selected an antibody that was specifically raised against this consensus. Since this antibody was prone to recognizing a large number of PKA substrates in total cell extracts, we first immunoprecipitated B-Raf before Western blotting. As shown in Fig. 4, both antibodies were highly specific for the phosphorylated forms of either S365 or S429 since they failed to detect B-Raf after phosphatase treatment (Fig. 4B) or mutation of the corresponding residue to alanine (Fig. 4A). These phospho-specific antibodies were further used to analyze the basal level of B-Raf isoform phosphorylation of both residues in HEK293 cells. Interestingly, the results presented in Fig. 4C showed that S365 phosphorylation was increased in B2-Raf8b whereas it was strongly decreased in B3-Raf9b, compared to that in B1-Raf. In contrast, the level of S429 phosphorylation remained unchanged in the presence of alternatively spliced sequences.


Figure 4
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  		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, as indicated. To detect phosphorylation on S365, whole-cell extracts were immunoblotted (WB) with an antibody raised against phosphorylated S259 of Raf-1. To detect phosphorylation on S429, protein extracts were immunoprecipitated (IP) with the anti-Myc antibody and the immune complexes were analyzed by Western blotting using an anti-phospho-PKA substrate antibody raised against the R/K-R/K-X-S/T consensus sequence. (B) Protein extracts from HEK293 cells transfected with Myc-tagged B1-Raf were immunoprecipitated with anti-Myc antibody. The immune complexes were then treated or not treated with {lambda}-protein phosphatase ({lambda}-PPase) and analyzed by Western blotting using phospho-S365 (P-365) and phospho-S429 (P-429) antibodies. (C) S365 and S429 phosphorylation of full-length (left panel) or N-terminal (N-ter) (right panel) B-Raf isoforms (B1, B2, and B3) was analyzed as for panel A. Quantification of three independent experiments is shown. (D) Differential interaction of B-Raf isoforms with endogenous 14-3-3 proteins. HEK293 cells were transfected with full-length Myc-tagged B-Raf isoforms, and protein extracts were immunoprecipitated with anti-Myc antibody (IP myc). The immune complexes were analyzed by Western blotting using an anti-14-3-3 antibody. The amount of immunoprecipitated B-Raf proteins was verified using the anti-Myc antibody. Transfection efficiency and loading were monitored using anti-Myc and anti-14-3-3 antibodies, respectively, on WCE. Quantification of three independent experiments is shown.

 
Phosphorylation of the residue equivalent to S365 in Raf proteins was shown to create a docking site for 14-3-3 proteins (12, 15, 47). Therefore, we analyzed the ability of B-Raf isoforms to bind 14-3-3. HEK293 cells were transfected with Myc-tagged B1-Raf, B2-Raf8b, or B3-Raf9b, and cell lysates were immunoprecipitated with the anti-Myc antibody. Immunoprecipitates were then blotted with an anti-14-3-3 antibody. As shown in Fig. 4D, a correlation was observed between the level of S365 phosphorylation and the efficacy of B-Raf isoforms to bind endogenous 14-3-3, B2-Raf8b, and B3-Raf9b, being the most and less efficient, respectively.

We next examined the effect of S365A or S429A mutations on the ability of B-Raf isoforms to activate the MEK/ERK pathway. HEK293 cells were transfected with constructs expressing either wild-type (WT) or mutated B-Raf isoforms on these residues, and activation of MEK1/2 and ERK1/2 was analyzed using phospho-specific antibodies. As shown in Fig. 5A, WT B-Raf isoforms differ in their abilities to activate the MEK/ERK pathway, in agreement with our previous studies demonstrating that these isoforms display differential MEK kinase activity (41). Therefore, we used this assay to analyze the effect of S365A and S429A mutations on B-Raf isoforms both in serum-starved cells and in cells stimulated with FCS for 5 min (Fig. 5B). While mutation of S365 into alanine clearly increased the activity of the three isoforms, mutation of S429, however, did not result in significant changes in the activity of the isoforms under either condition. Therefore, S365 phosphorylation negatively regulates B-Raf isoform activity, a mechanism likely involving 14-3-3 binding, as described for other Raf proteins. The effect of S429 phosphorylation, at this step, remained unclear.


Figure 5
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  		FIG. 5. The S365A mutation increases MEK/ERK activation induced by B-Raf isoforms. (A) Comparison of the abilities of full-length B-Raf isoforms 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 was performed using an anti-ERK1/2 antibody (lower panel). Quantification of three independent experiments is shown. (B) Effect of S365A and S429A mutations on ERK activation induced by B-Raf isoforms. HEK293 cells transfected with full-length wild-type B-Raf isoforms (B1, B2, and B3) or their S365A and S429A mutants were serum starved and stimulated or not stimulated with 20% serum (FCS) for 5 min. ERK1/2 activation was analyzed as for panel A. Quantification of three independent experiments is shown.

 
To further analyze the effect of S365A and S429A mutations on the biological activity of B-Raf isoforms, we took advantage of the NR cell system. We previously reported that this system was sensitive enough to detect mitogenic activity of B-Raf in the absence of protein truncation or mutation and to reveal differences in the biological activity of WT B-Raf isoforms (41). In addition, this system proved to be useful for detecting a gain of function induced by mutation of S259 in the related Raf-1 protein (13). In agreement with our previous studies, B2-Raf8b and B3-Raf9b exhibited diminished and enhanced mitogenic activities, respectively, compared to B1-Raf (Fig. 6) (41). Interestingly, the S365A mutation markedly increased the mitogenic activities of all B-Raf isoforms and abolished the differences in their activities (Fig. 6). The difference in mitogenic activity observed between WT and S365A proteins was more pronounced for B2-Raf8b than for B3-Raf9b. Therefore, a correlation exists between the abilities of distinct B-Raf isoforms to become phosphorylated on S365A, to bind 14-3-3 through this residue, and to induce NR cell proliferation.


Figure 6
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  		FIG. 6. The S365A and S429A mutations differentially affect proliferating activities of B-Raf isoforms in NR cells. Primary cultures of NR cells were transfected with pRcRSV/myc-derived constructs encoding full-length B-Raf isoforms (B1, B2, and B3), either WT or mutated on S365 or S429, as indicated. The empty pRcRSV vector was used as a control. After selection for G418-resistant cells, the foci of proliferating NR cells were stained with crystal violet. Quantification was performed as for Fig. 3. The data presented are representative of four independent experiments.

 
To a lower extent than S365A mutation, S429A mutation also appeared to modestly affect the mitogenic activity of B-Raf isoforms in NR cells. A moderate increase and decrease could be observed for the B2-Raf8b and B3-Raf9b isoforms, respectively (Fig. 6). The observation that the activity of a B-Raf isoform, namely, B3-Raf9b, was inhibited upon S429A mutation was somewhat surprising since a previous study suggested that phosphorylation of B1-Raf on S429 slightly inhibited its activity (21). It should be noted that in these previous studies, the effect of the S429A mutation on B-Raf biological activity was never assessed solely but only in combination with other mutations (21). Therefore, we wanted to confirm these opposite effects of the S429A mutation on B2-Raf8b and B3-Raf9b isoforms in another cell type. Rat pheochromocytoma PC12 cells are well known for undergoing neuronal differentiation upon nerve growth factor treatment through the recruitment of a TrkA/B-Raf/MEK/ERK signaling cascade (26, 44, 49, 52). Accordingly, constitutive activation of a component of this cascade is sufficient to induce neurite outgrowth. As shown in Fig. 7A, overexpression of the wild-type B3-Raf9b isoform was unable to induce PC12 cell differentiation, despite the presence of exon 9b. Similar results were obtained with B1 and B2 isoforms (data not shown). Therefore, we looked at the effect of a gain-of-function mutation on the differentiating activity of B-Raf isoforms. To this aim, T599 and S602 phosphorylation residues in the activation loop of B-Raf (Fig. 1) were mutated into glutamic acid residues. We chose this double modification instead of the constitutively activating V600E mutation or fusion with Ras-CAAX sequence because it has been reported that a B-Raf protein carrying a double-acidic substitution on T599 and S602 residues can be further stimulated by activated Ras or by additional mutations on S365 and S429 (58). The three B-Raf isoforms mutated on both residues induced neurite outgrowth (Fig. 7A). Importantly, the double T599E/S602E gain-of-function mutation preserved the differential activities of B-Raf isoforms. B3-Raf9bEE displayed the highest differentiating activity, whereas B2-Raf8bEE was the least efficient, as shown both by the percentage of differentiated cells (Fig. 7B) and by the complexity of the neuritic network (Fig. 7A). We next examined the effect of an additional S429A mutation on B-RafEE isoform activity. As observed in NR cells with otherwise wild-type proteins (Fig. 6), the S429A mutation reproducibly increased B2-Raf8bEE activity and decreased that of B3-Raf9bEE (Fig. 7B). Finally, we also tested the effect of the S429A mutation in combination with the T599E/S602E gain-of-function mutation in HEK293 cells. As shown in Fig. 7C, the S429A mutation had opposite effects on the abilities of B2-Raf8bEE and B3-Raf9bEE to activate the MEK/ERK pathway.


Figure 7
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  		FIG. 7. Opposing effects of the S429A mutation on PC12 cell differentiation or ERK activation induced by B-Raf isoforms. (A) PC12 cell differentiation induced by B-Raf isoforms carrying the phosphomimetic T599E/S602E double substitution. PC12 cells were cotransfected with a pEGFP reporter plasmid and pcDNA3-derived constructs encoding B-Raf isoforms, either WT or carrying the T599E/S602E double mutation (EE). A representative field for each condition was photographed under an inverted fluorescence microscope. Note that overexpression of WT B3-Raf9b does not induce neurite outgrowth. Similar results were obtained with B1-Raf and B2-Raf8b WT isoforms (data not shown). (B) Effect of the S429A mutation on PC12 cell differentiation induced by B-RafEE isoforms. PC12 cells were transfected as for panel A, and the total number of GFP-positive cells was counted. The indicated percentages were calculated from three independent experiments and represent the ratios between the number of GFP-positive cells undergoing neurite outgrowth and the total number of GFP-positive transfected cells. Statistical significance was evaluated by a Student paired t test (*, P < 0.01). (C) pRcRSV-derived constructs containing the same mutants as in panel B were used to transfect HEK293 cells. ERK1/2 activation was analyzed by Western blotting using anti-phospho-ERK (P-ERK) antibody. Transfection efficiency 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.

 
Taken together, these results show that phosphorylation on both S365 and S429 participates in the differential regulation of B-Raf isoforms through distinct mechanisms. The basal levels of S429 phosphorylation are equivalent in the three isoforms, but the effects of this phosphorylation on B-Raf activity differ: it inhibits B2-Raf8b, whereas it appears to activate B3-Raf9b. S365 phosphorylation inhibits the activities of the three isoforms, but the basal level of phosphorylation of this residue differs for each isoform. Interestingly, the isolated N terminus of B-Raf isoforms was differentially phosphorylated at S365, as in the full-length proteins (Fig. 4C). Therefore, we wondered whether phosphorylation on S365 and S429 could be involved in the differences observed between B-Raf isoforms in the ability of their N termini to inhibit the activity of the kinase domain. To test this hypothesis, we examined the effect of the S365A and S429A mutations on the inhibitory effect of the N terminus of B-Raf isoforms in NR cells. As shown in Fig. 8, mutation of either residue was unable to decrease the ability of the N terminus of B-Raf isoforms to inhibit the mitogenic effect of the isolated kinase domain. In agreement with this, the S365A or S429A mutation did not alter the ability of the N terminus of B-Raf isoforms to coprecipitate with the C-terminal domain in HEK293 cells (Fig. 9). These results suggest that differential B-Raf isoform regulation through phosphorylations and intramolecular interactions proceeds from independent mechanisms.


Figure 8
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  		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 kinase domain in NR cells. Primary cultures of NR cells were cotransfected with pEF/Flag-Cter and pRcRSV/myc-Nter constructs mutated or not mutated on S365 and S429 as indicated. Empty pRcRSV was used as a control. After selection for G418-resistant cells, the foci of proliferating NR cells were stained with crystal violet. Quantification was performed as for Fig. 3. The data presented are representative of three independent experiments. N-ter, N terminus.

 

Figure 9
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  		FIG. 9. The S365A and S429A mutations do not affect the N-terminal/C-terminal interaction of B-Raf isoforms. Coimmunoprecipitations of the B-Raf N- and C-terminal domains in HEK293 cells were performed as described for Fig. 2. Cells were cotransfected with the Flag-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, and immune complexes were then immunoblotted (WB) with either anti-Myc or anti-Flag antibody as indicated. N-ter, N terminus; C-ter, C terminus.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
B-Raf is involved in many physiological and pathological processes (19, 37, 44, 56). Like those of other Raf proteins, B-Raf activity is regulated through complex mechanisms, including inhibitory and activating phosphorylations (56, 58). However, it has been shown that B-Raf requires fewer phosphorylation events than A-Raf and Raf-1 for maximal activation, thereby explaining its higher basal kinase activity (36). We previously reported that B-Raf also differs from the other Raf proteins in vertebrates by another level of regulation involving alternative splicing (41). Thus, exon 9b present in the B3-Raf9b isoform increases both the MEK activity and transforming activity of B-Raf, whereas exon 8b has an opposite effect in B2-Raf8b isoforms. In the present study, we have investigated the molecular basis accounting for these differences. Using different cell systems, we found that both phosphorylation of two serine residues and intramolecular interactions participate in this regulation through distinct mechanisms.

In agreement with a recent report, we showed that the B-Raf N-terminal regulatory region inhibits the activity of the kinase domain (51). Our results indicated that the N terminus of B2-Raf8b has a higher affinity for the C terminus than that of B1-Raf, whereas the N terminus of B3-Raf9b has a lower affinity. Accordingly, the N terminus of B2-Raf8b was more efficient than that of B3-Raf9b at inhibiting MEK/ERK activation and NR cell proliferation induced by the isolated C terminus. We also showed that this ability of the N terminus of B-Raf to bind to and inhibit the kinase domain does not depend on the phosphorylation of S365 or S429. Similar observations were reported for S259, the residue equivalent to S365 in Raf-1 (6). The exact mechanisms by which intramolecular interactions between both domains regulate Raf protein activity currently remain unknown. Several recent studies demonstrated that Raf activation occurs through the formation of multiprotein complexes involving homo- or hetero-oligomerization between Raf family members (20, 38, 48, 55). Mapping of the molecular determinants implicated in oligomerization indicates that some of them are clustered in B-Raf regions that could also be engaged in intramolecular interactions (48). Therefore, the role of N-terminal/C-terminal intramolecular interaction could be to lock B-Raf in a closed conformation that prevents oligomerization and subsequent phosphorylation on the activation loop. This autoinhibition is released upon binding of the B-Raf N-terminal domain to GTP-bound Ras (51). This mechanism was initially proposed for the regulation of Raf-1 activity (6, 9). Using the fluorescence resonance energy transfer technique, Terai and Matsuda recently showed that membrane recruitment of Raf-1 through Ras was required to achieve this conformational change and to activate Raf-1 kinase activity (50). In agreement with this model, the isolated C-terminal kinase domain of B-Raf proteins carrying either a T599E/S602D double substitution or a V600E oncogenic mutation is no longer inhibited by the N terminus, although both domains could still bind together (51). Whether phosphorylation of T599 and S602 results from trans phosphorylation within the oligomers or from phosphorylation by a yet unknown protein kinase at the plasma membrane remains to be determined. In light of these observations, we propose that the presence of exon 9b in B3-Raf9b favors the open active conformation by reducing the autoinhibitory mechanism, thereby explaining the higher MEK activity of this isoform. In contrast, exon 8b, by increasing N-terminal affinity for the kinase domain, would have an opposite effect on downstream MEK activation.

As described above, a key step in Raf activation is the conformational 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 dephosphorylation of a residue equivalent to B-Raf S365 by PP2A, resulting in 14-3-3 displacement (1, 27, 32, 39). Consequently, mutation of Raf-1 S259 results in increased kinase activity (13, 14). We showed here that mutation of S365 into alanine potentiates B-Raf-mediated ERK activation in HEK293 cells, in agreement with a previous report (21). We further demonstrate that it strongly increases B-Raf mitogenic activity in NR cells, an effect similar to what we previously observed for Raf-1 (13). However, the difference in mitogenic activity observed between WT and S365A proteins was more pronounced for B2-Raf8b than for B3-Raf9b. Using a phospho-specific antibody, we found that phosphorylation of S365 was increased in B2-Raf8b and decreased in B3-Raf9b, compared to that in B1-Raf. This was correlated with a larger amount of 14-3-3 proteins associated with B2-Raf8b, compared to that associated with B3-Raf9b. These differences in S365 phosphorylation are independent of the N-terminal/C-terminal intramolecular interaction since they were also observed on the isolated N terminus of B-Raf isoforms. These results suggest that exon 9b-containing isoforms are less dependent on PP2A-mediated dephosphorylation for their membrane recruitment than exon 8b-containing isoforms. However, alternative, but not mutually exclusive, hypotheses exist concerning the role of the S259 residue in Raf-1 activity (13) and one cannot exclude that they could be applied to B-Raf as well. Nevertheless, phosphorylation of S365 appears to be a major determinant for the differential regulation of B-Raf isoforms.

In the course of this study, we identified S429 as a second phosphorylation site differentially regulating B-Raf isoform activity. The role of this phosphorylation has been poorly documented so far, mainly because the effect of the S429A mutation was never assessed solely but only in combination with mutations on S365 and T440 (21). Using a phospho-specific antibody, we demonstrate for the first time that this residue is indeed phosphorylated in cells. In contrast with S365, S429 was found equally phosphorylated in the three B-Raf isoforms tested. However, mutation of this residue into alanine indicated that it could differentially regulate B-Raf isoforms. The observed 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 in HEK293 cells, it increased and decreased mitogenic activities of B2-Raf8b and B3-Raf9b, respectively, in the NR cell system. A similar differential effect was also observed in the PC12 cell differentiation assay, where the mutation could be tested only in combination with the double phosphomimetic T599E/S602E substitution. As mentioned above, this double mutation renders B-Raf insensitive to the autoinhibition mechanism. Therefore, it is conceivable that the contribution of S429 phosphorylation is minor compared to that of autoinhibition and becomes easier to detect in the absence of the latter. Accordingly, an effect of the S429A mutation could be also detected in HEK293 cells in the context of the T599E/S602E substitution. The mechanism by which S429 phosphorylation differentially regulates B-Raf isoform activity is currently unknown. Given that this residue was reported to be phosphorylated by Akt, at least in vitro, it could be proposed to serve as a docking site for 14-3-3 when phosphorylated. A cryptic binding site was recently described for Raf-1, upon S233 phosphorylation (15). However, B-Raf S429 is not located in the same region as Raf-1 S233 and does not match to either strong or weak 14-3-3 binding consensus sequences (57). Our data also indicate that the S429A mutation does not impinge on the N-terminal/C-terminal interaction. Interestingly, however, serine 429 is closed to two key residues of the B-Raf kinase domain: S446, which is constitutively phosphorylated, and D448 (Fig. 1). The negative charges provided by these residues appear to be important for maintaining the kinase domain in an active conformation. For example, D448 forms an electrostatic interaction with R506 of the {alpha}C-helix, thereby stabilizing the small lobe of the kinase domain (54). The presence of either exon 8b or exon 9b could differentially modify the local conformation and relationship between phosphorylated S429 and this region of the small lobe, resulting in opposite effects. Finally, we do not exclude that S429 phosphorylation regulates a yet unknown B-Raf function that would be uncoupled from its MEK kinase activity.

Whatever the mechanisms by which phosphorylation of S365 and S429 differentially regulates B-Raf isoforms, it is noteworthy that both residues can be targeted by the same protein kinase. Thus, different members of the AGC kinase family have been proposed to phosphorylate these residues, including PKA, Akt, and SGK (21, 31, 59). While the ability of Akt and SGK to phosphorylate B-Raf has not been firmly demonstrated in vivo, their implication in Raf-1 regulation is still a matter of debate. More convincing are the data suggesting that PKA can directly phosphorylate both Raf proteins in cells (12, 15, 31, 33). B-Raf phosphorylation by PKA in vitro clearly inhibits its activity (33). Paradoxically, in cell types of neuroectodermic origin (neuronal and neural crest-derived cells), elevation of intracellular cyclic AMP (cAMP), which normally activates PKA, results in B-Raf and ERK activation (3, 16, 33, 46, 53). The mechanisms by which cAMP activates B-Raf remain controversial and might be cell type dependent since both Rap1-dependent and -independent pathways have been reported (16, 44). These observations have led to a model in which B-Raf is resistant to PKA-mediated inhibition upon cAMP increase in cells (16, 33). With respect to this, it is interesting to underline that B3-Raf9b should be the most resistant isoform to PKA-mediated inhibition since, on the one hand, it is less phosphorylated on S365 than the other isoforms and, on the other hand, its activity is increased upon S429 phosphorylation. In contrast, B2-Raf8b should be less resistant because exon 8b favors S365 phosphorylation, while S429 phosphorylation decreases its activity. Finally, we cannot exclude that kinases other than Akt and PKA could also phosphorylate these residues, raising the possibility of presently unknown physiological regulations of B-Raf isoforms.

Taken together, the results presented in this study show that alternative splicing modulates B-Raf activity through distinct but convergent mechanisms. This suggests that exons 8b and 9b impose structural constraints in the hinge region of the kinase, resulting in opposite effects on the intramolecular autoinhibition, the sensitivity to S365 inhibiting phosphorylation, and the consequence of S429 phosphorylation. Structural studies are required to support this model, but our attempts to purify full-length B-Raf isoforms suitable for X-ray analysis using bacterial or baculovirus systems were unsuccessful, as also observed by other groups (48, 54). The only multidomain kinases for which a three-dimensional structure of the full-length protein is available are members of the Src family. Interestingly, these kinases are also autoinhibited by their N termini. It has been demonstrated that the linker region immediately upstream of the kinase domain, which is in close contact with both the SH3 domain and the small lobe of the kinase, plays a key role during the transition between inactive and active conformations of Src (24). Given the structural similarities between B-Raf and Src family kinase domains (36, 54), it is tempting to speculate that the presence of alternatively spliced exons in the B-Raf hinge region impinges on a similar mode of conformational regulation.

Owing to this complex mode of regulation and to the conservation of B-Raf alternatively spliced exons through evolution, future directions will focus on the specific role of B-Raf isoforms during development and oncogenesis.


   ACKNOWLEDGMENTS
 
We thank Brian Rudkin for helpful advice on PC12 cells. We also thank Carole Burns, Andrew Doedens, Celio Pouponnot, and Nathalie Rocques for comments on the manuscript.

This work was funded by the Centre National de la Recherche Scientifique, by the Institut Curie, and by grants from the Ligue Nationale Contre le Cancer (Comité de l'Essonne) and INCA (melanoma network). I.H. was supported by fellowships from the Ligue Nationale Contre le Cancer and the Association pour la Recherche sur le Cancer. A.E. and S.D. are INSERM investigators.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institut Curie-Recherche, Laboratoire 110, Centre Universitaire, 91405 Orsay Cédex, France. Phone: 33-1 69 86 30 74. Fax: 33-1 69 07 45 25. E-mail: Alain.Eychene{at}curie.u-psud.fr. Back

{triangledown} Published ahead of print on 30 October 2006. Back

{ddagger} These authors contributed equally to this work. Back

{dagger} Present address: Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, MC-0377, La Jolla, CA 92093-0377. Back


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 DISCUSSION
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Molecular and Cellular Biology, January 2007, p. 31-43, Vol. 27, No. 1
0270-7306/07/$08.00+0     doi:10.1128/MCB.01265-06
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