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J. Biol. Chem., Vol. 280, Issue 17, 17027-17037, April 29, 2005
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Interacts with the Non-phosphorylated AF-1 Domain of Retinoid Receptor 
(RAR) and Represses RAR-mediated Transcription*
From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, Unité Mixte de Recherche 7104, 67404 Illkirch Cedex, France
Received for publication, February 4, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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as a new cofactor that interacts with the N-terminal A/B domain of the RAR
isotype. Vinexin is a multiple SH3 motif-containing protein associated with the cytoskeleton and also present in the nucleus. We demonstrate that vinexin colocalizes with RAR in the nucleus and interacts with the non-phosphorylated form of the AF-1 domain of RAR. We also show that this interaction is prevented upon phosphorylation of the AF-1 domain. Using F9 cells stably overexpressing vinexin or vinexin knockdown by RNA interference, we demonstrate that vinexin is an inhibitor of RAR-mediated transcription. We propose a model in which phosphorylation of the AF-1 domain controls RAR-mediated transcription through triggering the dissociation of vinexin . |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, RAR, and RAR) and the retinoid X receptors (RXR, RXR, and RXR), which function as ligand-dependent heterodimeric RAR/RXR transcription activators (4–6). RARs and RXRs exhibit a conserved modular structure (see Fig. 1A) with a central DNA-binding domain and two activation domains (AF-1 and AF-2) that synergize for the activation of RA target genes.
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1, Ser79 in RAR1, and Ser68 in RAR2) (see Fig. 1A) by the Cdk7 subunit of TFIIH, which has cyclin H-dependent kinase activity. This phosphorylation process, which has been extensively studied especially in the case of RAR (10), plays a critical role in the response to RA.
However, in the particular case of the RAR isotype, phosphorylation by TFIIH, although necessary, is not sufficient. Indeed, to be transcriptionally active, RAR needs to be also phosphorylated at an additional nearby residue (Ser77 in RAR1 and Ser66 in RAR2) (see Fig. 1A) by p38 MAPK subsequent to its activation by RA (11, 12). As phosphorylation by both TFIIH and p38 MAPK is required for the activation of RAR-controlled genes (2), we hypothesized that phosphorylation of the N-terminal AF-1 domain might regulate the dissociation and/or association of proteins involved in blocking or stimulating RAR activity. With this aim, we performed yeast two-hybrid screening experiments to characterize new cofactors interacting with the phosphorylated or non-phosphorylated forms of the AF-1 domain of RAR and therefore regulating RAR activity. By this approach, we isolated vinexin as a partner for the non-phosphorylated AF-1 domain of RAR. Vinexin is a recently identified cytoskeletal protein that exists as two isoforms, vinexin and vinexin (13). Both proteins are devoid of any enzymatic activity, but regulate cell adhesion/cytoskeleton organization as well as signal transduction pathways (13–15). They share a common C-terminal sequence containing three SH3 domains that bind proline-rich sequences (16). Vinexin has an additional N-terminal sequence containing a sorbin homology (SoHo) domain that mediates the translocation of the protein to lipid rafts (17).
In this study, we report that vinexin colocalizes with RAR in the nucleus, interacts with the non-phosphorylated N-terminal AF-1 domain of RAR, and represses RAR-mediated transcription. We also demonstrate that phosphorylation of the AF-1 domain prevents the interaction of vinexin with RAR. Based on these results, we propose that the underlying mechanism for the phosphorylation-dependent transcriptional activity of RAR involves, at least in part, the dissociation of vinexin .
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, hRAR1(S77A/S79A), hRAR
AB, mouse (m) RAR1, mRAR1(S77A), and mRXR1 were described previously (8, 9, 18). The vectors encoding the chimeric protein hRAR1(A/B)-ER(C) without and with RAR Ser77 and Ser79 mutated to alanines were also as described (8, 19). hRAR1(S77E/S79E) in pSG5 was constructed by double PCR amplification to generate an MscI/AvaI fragment containing the appropriate mutations, which was cloned into the same sites of pSG5-RAR1.
The prokaryotic vectors encoding mRAR1 and mRXR1 fused to glutathione S-transferase (GST) in the pGEX-2T plasmid (Amersham Biosciences) were described previously (20). The GST-hRAR1 chimera was constructed by subcloning the BamHI fragment from the corresponding pET3a vector (21) into the same site of pGEX-2T. GST-hRAR1(A/B) was constructed with PCR-amplified A/B fragments that were inserted into BamHI-digested pGEX-2T.
The AF-1 domain of hRAR1 with Ser77 and Ser79 substituted with alanines or glutamic acids was amplified by PCR from the corresponding pSG5 vectors and inserted into the XhoI/BamHI-digested pBTM116mod plasmid, which directs synthesis of LexA-DNA-binding domain fusion proteins in yeast. The AF-1 domain of either wild-type (WT) mRAR1 or mRAR1(S77A) was also inserted into the pBTM116mod plasmid following the same protocol.
The cDNA of vinexin was amplified by PCR and cloned into the pCX vector driven by the cytomegalovirus immediate-early enhancer and already containing the hemagglutinin, FLAG, and yellow fluorescent protein tags (a gift from T. Lerouge). All constructs were generated using standard cloning procedures and were verified by restriction enzyme analysis and automated DNA sequencing.2
The DR5-tk-CAT and DR1-tk-CAT reporter constructs were described previously (18). The plasmids encoding FLAG-tagged vinexins and were provided by Dr. N. Kioka (13). All-trans-RA was from Sigma. The synthetic RAR (BMS961) and pan-RXR (BMS649) agonists were gifts from Bristol-Myers Squibb Co.
Antibodies—Rabbit polyclonal antibodies raised against the F domain of RAR (antibody RP(F)) and mouse monoclonal antibodies raised against the same F domain (mAb4(F)) or the N-terminal A domain (mAb441(A)) were as described (8, 22). Rabbit polyclonal antibodies specific to RAR phosphorylated at Ser77 or Ser79 were described previously (12). Anti-FLAG monoclonal antibody M2 (immobilized or not on agarose) was obtained from Sigma, and goat anti--actin polyclonal antibody C-11 was from Santa Cruz Biotechnology Inc. Rabbit polyclonal antibodies against LexA were as described (23). Mouse monoclonal antibodies against vinexin were generated using a synthetic peptide corresponding to amino acids 211–223 of mouse vinexin according to standard procedures (22). Cy3-conjugated goat anti-mouse antibodies were from Amersham Biosciences, and Alexa Fluor 488-conjugated goat anti-rabbit antibodies from Molecular Probes, Inc.
Yeast Two-hybrid Screening—Yeast two-hybrid screening was performed as described previously (24) using the L40 reporter strain (trp1 leu2 his3 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ) harboring the HIS3 and lacZ reporter genes, both under the control of LexA-binding sites. The mouse embryo (12.5 days postcoitus) cDNA library in the yeast VP16 acidic activation domain fusion vector pASV3 was described previously (20). It was introduced by lithium acetate transformation into the reporter strain expressing the LexA-RAR1(A/B) fusion proteins from the pBTM116mod vector. Approximately 2 x 106 yeast transformants were screened for their ability to grow on medium lacking histidine and containing 3-amino-1,2,4-triazole (7 mM for RAR(A/B)(S77A/S79A) and 35 mM for RAR(A/B)(S77E/S79E); ICN Pharmaceuticals) and to express -galactosidase. After several rounds of replica plating on selective medium, library plasmids were recovered from the positive clones, amplified, subjected to restriction analysis, and sequenced.
Protein-Protein Interactions Using the Yeast Two-hybrid System— The yeast strain L40 was cotransformed with the plasmids encoding the LexA-RAR1(A/B) fusion protein and the VP16 acidic activation domain fused to the isolated interacting protein. The cells were grown overnight in selective liquid medium containing histidine, and a quantitative -galactosidase assay was performed as described (24).
GST Pull-down Assays—The GST, GST-RAR1, GST-RAR1, GST-RXR1, and GST-RAR1(A/B) proteins were produced in Escherichia coli strain NB42 and purified on glutathione-Sepharose 4B beads (Amersham Biosciences) as described (25). Equimolar amounts of the GST fusion proteins bound to the beads were incubated in GST buffer (50 mM Tris-HCl (pH 8), 0.05% Nonidet P-40, 0.3 mM dithiothreitol, 10 mM MgCl2, and 5% glycerol) containing a protease inhibitor mixture and 150–500 mM NaCl with COS-1 cell extracts expressing the FLAG-vinexin protein. After three washes in GST buffer, bound proteins were recovered in SDS loading buffer, subjected to SDS-10% PAGE, and analyzed by immunoblotting.
Cells, Transfections, Immunoprecipitations, and Chloramphenicol Acetyltransferase (CAT) Assays—COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and transiently transfected using the DMRIE-C reagent (Invitrogen) as described (11). After a 20-h incubation with DNA, the cells were washed and maintained for the indicated times in medium with or without ligand. Cells were harvested, and whole cell extracts were prepared in lysis buffer (25 mM Tris-HCl (pH 8), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, and protease inhibitor mixture). Where mentioned, immunoprecipitation was performed by incubation of the extracts with the indicated monoclonal antibodies in 50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl, 10 mM MgCl2, 0.3 mM dithiothreitol, 5% glycerol, 0.05% Nonidet P-40, 0.5 mg/ml bovine serum albumin, and protease inhibitor mixture with protein G-Sepharose beads. Proteins with or without prior immunoprecipitation were resolved by 10% SDS-PAGE, electrotransferred to nitrocellulose membranes, immunoprobed, and detected by chemiluminescence according to the protocol of Amersham Biosciences. CAT assays were performed using the enzyme-linked immunosorbent assay method (Roche Diagnostics). All results were normalized to equal -galactosidase activity and to the activity of each receptor in the absence of vinexin and without ligand.
F9 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum as described (26). F9 cells ablated for RAR (RAR–/–) and re-expressing either WT RAR or RAR mutated at its phosphorylation sites were described previously (26–28). To establish stable lines overexpressing vinexin , F9 cells were electroporated with the pCX construct along with a plasmid conferring neomycin resistance. After 24–36 h, the cells were selected with neomycin for 10 days as described (26, 27) and analyzed for the expression of the transgene by quantitative reverse transcription (RT)-PCR and immunoblotting. Several clones were isolated and one, V(5), was selected and used in these experiments. Cytosolic and nuclear extracts were prepared as described (12).
Immunofluorescence—COS-1 cells cotransfected with the FLAG-vinexin ( or ) and RAR expression vectors were seeded onto glass coverslips coated with 0.1% gelatin. After 16 h, the cells were fixed with 2% paraformaldehyde in phosphate-buffered saline, permeabilized with 0.1% Triton X-100, and saturated with 5% bovine serum albumin in phosphate-buffered saline. The cells were then incubated with rabbit polyclonal antibodies against RAR (antibody RP(F)) and mouse monoclonal anti-FLAG antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit and/or Cy3-conjugated goat anti-mouse secondary antibodies. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (Sigma), and the coverslips were mounted on glass slides. The cells were analyzed by fluorescence microscopy using an epifluorescence microscope or a confocal laser scanning microscope.
RNA Isolation and Real-time RT-PCR—Total RNAs were isolated using the guanidinium thiocyanate method, and aliquots (50 ng) were subjected to quantitative real-time RT-PCR using the Light-Cycler (Roche) and the qRT-PCR&GO one-step kit (Qbiogene, Inc.). Transcript levels were normalized according to 36B4 transcripts, which are unresponsive to RA. The oligonucleotide sequences were as follows: 36B4, 5'-GAGGTCACTGTGCCAGCTCA-3' and 5'-GAAGGTGTACTCAGTCTCCA-3'; CYP26, 5'-TAAGGAGACCCTGCGATTGA-3' and 5'-TGAGGCACTATAAAGCGGTCG-3'; RAR2, 5'-TGGTGTTCTAGCACCCAGTT-3' and 5'-AAACGATTCCATGCAGTCGT-3'; Hoxa-1, 5'-AACCCAAAGGTATTCATTCTTTCA-3' and 5'-ATGTTAAGACCCGTAAACTCTGCT-3'; Stra4, 5'-TGTGCTGGTTCATGACAACTC-3' and 5'-TGGAGCTGATTCGAGACTGTT-3'; cellular RA-binding protein II (CRABP-II), 5'-AACCTCCACCACTGTGCGAA-3' and 5'-AGGCAGTTCTTGGACCCGTA-3'; Hoxb-1, 5'-TGACCAGTTCTCTCGAAGAC-3' and 5'-CTCTCTAAGCTCAAAGGCAC-3'; hepatocyte nuclear factor (HNF) 3, 5'-TGGCGTAGGACATGTTGAAG-3' and 5'-GCATGAGAGCAACGACTGGA-3'; HNF1, 5'-CCTGTACACT TGGTACGTCA-3' and 5'-GAACCAGTTGTAGACACGGA-3'; and vinexin, 5'-AGCCACTAGCCGTCCCATAA-3' and 5'-CGTCACATTGCTGCATGACA-3'.
Small Interfering RNA (siRNA)—The 19-nucleotide RNA oligonucleotides corresponding to vinexin with a 3'-dTdT overhang (UGAGGACGAGCUGGAACUUdTdT and dTdTACUCCUGCUCGACCUUGAA) were designed, synthesized, and annealed (to generate duplex siRNA) by Eurogentec S. A. The siRNA corresponding to vinexin was transfected into F9 cells at a final concentration of 50 nM using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 48-h post-transfection (with an intermediate re-transfection at 24 h), the cells were treated with vehicle or RA. At the indicated times, the cells were harvested and subjected to RNA and protein analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in Yeast Two-hybrid Screening—The yeast two-hybrid system was used to isolate cDNAs encoding mouse proteins interacting with the N-terminal domain of RAR. The N-terminal A/B domain of hRAR1in which the two phosphorylatable serine residues had been substituted with alanines, RAR(A/B)(S77A/S79A) (Fig. 1B), was used as bait in a screening of a mouse embryo cDNA library. We isolated a clone growing on selective medium and expressing -galactosidase (Fig. 1C) that contained a 1.5-kb cDNA insert homologous to mouse vinexin (GenBankTM/EBI accession number AF064806
[GenBank]
) (13). This cDNA fragment (designated RAR(A/B)-BP) contained the 3'-terminal half of the open reading frame encoding mouse vinexin (amino acids 360–733) and 221 nucleotides corresponding to the 3'-untranslated region of vinexin (Fig. 2A). The encoded protein contained the three SH3 domains of vinexin , which are also shared by the vinexin isoform (Fig. 2, A and B) (13).
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in which the two serines were substituted with glutamic acids, RAR(A/B)(S77E/S79E) (Fig. 1B), which mimic phosphorylated residues. This suggests that the cloned C-terminal half of vinexin would interact specifically with the non-phosphorylated A/B domain of RAR. To corroborate this hypothesis, the cloned VP16-RAR(A/B)-BP hybrid protein was expressed in the L40 yeast strain in combination with either LexA-RAR(A/B)(S77A/S79A) or LexA-RAR(A/B)(S77E/S79E). As expected, the cloned protein interacted with RAR(A/B)(S77A/S79A), as evidenced by growth of colonies in medium lacking histidine and by expression of -galactosidase (Fig. 1C). However, no interaction could be detected with RAR(A/B)(S77E/S79E) (Fig. 1C), indicating that phosphorylation of the N-terminal domain of RAR impedes the interaction with the C-terminal half of vinexin.
We also tested whether the cloned C-terminal half of vinexin was able to interact with the N-terminal domain of another RAR isotype, RAR. With this aim, the corresponding LexA-RAR(A/B) protein (either WT or S77A) (Fig. 1B) was expressed in the L40 yeast strain either alone or in combination with the cloned VP16-RAR(A/B)-BP hybrid protein. In both cases, -galactosidase was only slightly expressed (Fig. 1C), suggesting that the cloned protein interacts preferentially with RAR.
Nuclear Colocalization of Vinexin with RAR—Because our cloned RAR(A/B)-binding protein contains the three SH3 domains of vinexin , which are also shared by vinexin , one can speculate that both vinexin isoforms might be candidates for interacting with RAR. A number of previous studies (13–15) indicated that the vinexin and proteins are focal adhesion and intermediate junction proteins that play a role predominantly in cytoskeleton organization, cell spreading, and intracellular signaling. However, only vinexin (not vinexin ) could be detected also in the nucleus (13).
Thus, we investigated whether vinexin is able to colocalize with RAR in the nucleus by performing epifluorescence and confocal imaging with COS-1 cells overexpressing RAR together with FLAG-vinexin ( or ). WT RAR (Fig. 3, panels 2, 6, 10, and 14) and RAR(S77A/S79A) and RAR(S77E/S79E) (data not shown) were found exclusively in the nucleus. However, the subcellular localization of vinexin was different depending on the isoform. Indeed, FLAG-vinexin was found exclusively as dots in the cytosol of the transfected cells (Fig. 3, panels 1 and 9), whereas FLAG-vinexin was present in both the nucleus and the cytoplasm (Fig. 3, panels 5 and 13). Finally, the distribution of vinexin in the nucleus appeared to be similar to that of RAR (Fig. 3, panels 8 and 16). In contrast, vinexin and RAR were distributed in different compartments (Fig. 3, panels 4 and 12). Collectively, these results indicate that vinexin colocalizes with RAR in the nucleus and thus would be the vinexin isoform that interacts with the N-terminal domain of RAR in vivo.
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Co-immunoprecipitates with the Non-phosphorylated AF-1 Domain of RAR—To study further the data obtained by yeast two-hybrid screening and fluorescence imaging, co-immunoprecipitation experiments were performed with recombinant proteins overexpressed in COS-1 cells. FLAG-vinexin was coexpressed with full-length RAR(S77A/S79A), which is not phosphorylatable. After immunoprecipitation of the extracts with monoclonal antibodies raised against the F domain of RAR (mAb4(F)), immunoblotting with anti-FLAG antibodies showed that vinexin interacted with RAR(S77A/S79A) (Fig. 4, A, lane 3; B, lane 4). No interaction could be seen upon deletion of the N-terminal A/B domain (Fig. 4A, lane 7), in agreement with the fact that vinexin had been isolated in the two-hybrid system as a protein that interacts with this region of RAR. Similar results were obtained in the absence and presence of RA (Fig. 4A, compare lanes 3 and 4 and lanes 7 and 8), confirming that the interaction of vinexin with RAR does not involve surfaces within the AF-2 domain that are reorganized upon ligand binding (4). Finally, no interaction was seen with RAR(S77E/S79E) (Fig. 4B, lane 5), which mimics a phosphorylated receptor, in line with the observed interaction of vinexin with the non-phosphorylated A/B domain of RAR.
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in combination with a fusion protein in which the N-terminal A/B domain of RAR was fused to the DNA-binding domain of the ER. After immunoprecipitation of the extracts with monoclonal antibodies raised against the A domain of RAR (antibody mAb41(A)), immunoblotting with anti-FLAG antibodies showed that only a small amount of vinexin coimmunoprecipitated with the WT RAR A/B domain (Fig. 4C, lane 3), in agreement with our previous demonstration that a fraction of overexpressed RAR is phosphorylated in COS-1 cells (8). However, vinexin did co-immunoprecipitate with the N-terminal domain of RAR in which Ser77 and Ser79 had substituted with alanines (Fig. 4C, lane 6). Collectively, these results confirm that vinexin interacts preferentially with the non-phosphorylated N-terminal domain of RAR.
Vinexin Interacts Specifically with RAR in Vitro, but Not with RAR or RXR—The interaction of vinexin with RAR was further investigated in in vitro protein-protein interaction assays using recombinant GST-WT RAR expressed in E. coli and bound to glutathione-Sepharose beads. When expressed in E. coli, WT RAR is not phosphorylated at Ser77 and Ser79 within the A/B domain (8) and thus is more suitable than the corresponding alanine mutant to demonstrate that the interaction with vinexin occurs with the non-phosphorylated receptor. After incubation of the beads with extracts from COS-1 cells overexpressing FLAG-vinexin , we found that vinexin interacted with RAR even in the presence of high salt concentrations (Fig. 5A, lanes 3, 5, and 7). Vinexin also interacted with the isolated N-terminal A/B domain of RAR fused to GST and expressed in E. coli (Fig. 5B, lane 3), confirming that this interaction concerns this non-phosphorylated domain.
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could also interact with other retinoid receptors. Interestingly, we did not observe any binding of vinexin with RAR (Fig. 5C, lanes 3, 6, and 9), in agreement with the yeast two-hybrid experiments (Fig. 1C). Similarly, no interaction was observed with RXR (Fig. 5D, lane 3).
Overexpression of Vinexin Inhibits RAR Transcriptional Activity—Because vinexin can bind RAR, we sought to assess whether it could also regulate the transcriptional activity of this receptor. With this aim, increasing amounts of vinexin were coexpressed in COS-1 cells along with RAR (WT, S77A/S79A, or S77E/S79E), the heterodimeric partner RXR, and a CAT reporter gene under the control of a DR5 response element (Fig. 6A).
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(S77E/S79E), which behaves as the phosphorylated receptor, was almost as efficient as WT RAR in inducing the expression of the CAT reporter gene in the presence of ligand (Fig. 6A, compare bars 4 and 16). Overexpression of vinexin did not affect the transcriptional activity of this mutant in both the absence and presence of the RAR/pan-RXR agonist combination (Fig. 6A, bars 13–18), in line with the absence of any interaction between the phosphorylated form of RAR and vinexin .
In contrast, RAR(S77A/S79A), which is devoid of any phosphorylation-dependent regulation, was markedly less efficient than the WT receptor in transactivating the CAT reporter gene in response to the ligand (Fig. 6A, compare bars 4 and 10), in agreement with our previous report (8). Overexpressed vinexin further inhibited the transcriptional activity of RAR(S77A/S79A) (Fig. 6A, bars 7–12) in both the absence and presence of ligand. Consistent with the ability of vinexin to interact with this mutant, vinexin may be repressor of RAR-mediated transcription.
Overexpression of vinexin also inhibited significantly, in a dose-dependent manner, basal transcription mediated by WT RAR in the absence of ligand (Fig. 6A, bars 1–3). This is in line with the fact that, in the absence of ligand, WT RAR bound at an RA response element is not phosphorylated due to the absence of interaction of the receptor with TFIIH associated with the transcription machinery and to the absence of p38 MAPK activation (2). In response to the ligand, RAR becomes fully active subsequent to the phosphorylation of Ser79 and Ser77 by Cdk7 within TFIIH and by p38 MAPK, respectively (11, 12), as assessed by immunoblotting with antibodies specifically recognizing RAR phosphorylated at these residues (Fig. 6B, lane 2) (data not shown). Overexpression of vinexin did not prevent the increase in RAR phosphorylation that occurred in response to the ligand (Fig. 6B, lane 3). However, overexpression of vinexin inhibited WT RAR-mediated transcription in the presence of ligand (Fig. 6A, bars 4–6). Collectively, these results suggest the hypothesis that vinexin would be a repressor of RAR through its interaction with the non-liganded and non-phosphorylated form of the receptor. They also emphasize the possibility that an excess of vinexin would drive RAR to a transcriptionally inactive state even in the presence of ligand.
Note that no effect of vinexin overexpression could be detected when the DR5 response element was mutated and unable to bind the RAR/RXR heterodimers (data not shown). Moreover, vinexin did not affect the transcriptional activity of RXR homodimers on a DR1-tk-CAT reporter gene (Fig. 6C), in accordance with the absence of any significant interaction of vinexin with this receptor (Fig. 5D). This also indicates that the observed inhibition of RAR activity does not reflect a general inhibition of transcription.
In F9 Cells, Expression of RA Target Genes Requires Phosphorylation of the N-terminal Domain of RAR, and Vinexin Colocalizes with RAR in the Nucleus—Having demonstrated the importance of vinexin in the transcriptional activity of RAR overexpressed in COS-1 cells, we asked whether the same conclusions could be made in vivo in mouse embryo carcinoma cells (F9 cell line), which constitute a well established cell autonomous model system for investigating RA signaling (29). In F9 cells, RA induced the expression of several target genes within 24 h as assessed by quantitative RT-PCR (Fig. 7A). The RA-induced expression of these genes involves the activation of RAR/RXR heterodimers, as it was reduced in F9 RAR–/– cells (Fig. 7B) (data not shown). Most interestingly, the activation of the RA target genes was less efficiently restored upon re-expression of RAR mutated at the phosphorylation sites than upon re-expression of WT RAR (Fig. 7B) (data not shown), indicating that phosphorylation of the AF-1 domain of RAR plays a crucial role in RAR-mediated transcription.
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is present in the nucleus of F9 cells, in addition to RAR. With this aim, we generated mouse monoclonal antibodies against vinexin. As shown in Fig. 7C, these antibodies efficiently recognized recombinant FLAG-vinexin ( and ) overexpressed in COS-1 cells. Nuclear and cytoplasmic extracts were prepared from F9 cells and immunoblotted with these anti-vinexin antibodies. A protein species with an apparent molecular mass corresponding to that of vinexin (82 kDa) was detected only in the cytosol (Fig. 7D, middle panel, lane 1). In contrast, vinexin (37 kDa) was detected not only in the cytosolic compartment, but also in the nucleus of F9 cells (Fig. 7D, lower panel). Thus, in F9 cells, vinexin appeared to colocalize with RAR (Fig. 7D, upper panel). Collectively, these results indicate that F9 cells would constitute a good model to determine whether vinexin modulates the expression of RAR target genes.
Vinexin Is an Inhibitor of Several RAR Target Genes in F9 Cells—To determine the role of vinexin in RAR-mediated transcription, we analyzed the consequences of siRNA-mediated knockdown of vinexin in F9 cells and investigated whether reduction in the level of vinexin would influence the expression of the RA target genes. Transfection into F9 cells of the siRNA targeting vinexin reduced the expression levels of both vinexins and at both the mRNA and protein levels as shown by quantitative RT-PCR and immunoblotting, respectively (Fig. 8, A, bars 1–3; and B). In contrast, the expression of RAR was not affected (Fig. 8, A, bars 4–6; B, lower panel). When vinexin was reduced by RNA interference, the RA-induced expression of all tested RA target genes such as CYP26, Hoxb-1, CRABPII, Stra4, Hoxa-1, HNF3, and HNF1 (Fig. 8C) (data not shown), which depend on RAR phosphorylation (see above), was significantly up-regulated. The expression of RAR was not affected (Fig. 8C), indicating that our results do not reflect an increase in RAR levels. Thus, decreasing vinexin levels appears to improve the efficiency of transcription of RAR target genes, consistent with an inhibitory role of vinexin.
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in the expression of RAR target genes, a stable cell line overexpressing vinexin , the F9 V(5) cell line, was established from F9 WT cells. In this cell line, vinexin mRNA levels were significantly increased as assessed by quantitative RT-PCR (Fig. 9A, bar 2). The corresponding protein was also increased in both the cytosolic and nuclear compartments (Fig. 9B) as assessed by immunoblotting. We investigated whether the expression of the RA target genes was affected in this cell line compared with the WT counterpart. Our results clearly show that, in the F9 V(5) cell line, the RA-induced expression of some RA target genes such as CYP26, Hoxb-1, and CRABPII (Fig. 9C) was significantly lower than in the parental F9 WT cell line, confirming that vinexin is a repressor of RAR transcriptional activity. The expression of RAR was not affected (Fig. 9C), indicating that the observed results do not reflect an inhibition of RAR levels. However, the expression of some other RA target genes such as Stra4, Hoxa-1, HNF3, and HNF1 (Fig. 9C) (data not shown) was not affected, although it was up-regulated in the siRNA vinexin knockdown cells (Fig. 8C). This suggests that some genes may be differentially regulated by overexpressed vinexin , probably due to different conformations of the transcriptional complexes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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as a novel cofactor of the RAR isotype. Vinexin was originally identified as a focal adhesion and intermediate junction protein that plays a role in cytoskeleton organization and cell spreading as well as in intracellular signaling (13–15). In addition to these membrane-associated functions, we present evidence that vinexin is present in the nucleus, interacts with the non-phosphorylated form of RAR, and negatively controls its transcriptional activity.
Vinexin Interacts with the Non-phosphorylated AF-1 Domain of RAR—During the last decade, a number of co-regulators, including the p160 family of coactivators, have been identified as nuclear receptor-binding proteins that are recruited by the AF-2 domain subsequent to ligand-induced conformational changes (30–33). In contrast, only a few proteins have been reported to interact with the N-terminal domain of nuclear receptors (34–38). This study is the first reporting the identification of a protein that interacts with the AF-1 domain of an RAR. It is also the first reporting the interaction of an RAR with an adaptor protein (vinexin ) known to regulate cytoskeleton organization and signal transduction.
The main characteristic of the vinexin protein is the presence of three SH3 domains (13) that are known to interact with proline-rich sequences containing the PXXP core (16). Such proline-rich motifs are relatively rigid and exposed and thus are favorable for interactions with SH3, WW, and many other protein interaction domains (16, 39). The interactions with these proline-rich motifs are not as tight as those with globular domains, but they present the advantage of being modulated rapidly upon covalent modifications such as phosphorylation. Indeed, phosphorylation of serine residues flanking proline motifs has the ability to positively or negatively regulate the binding of SH3 domains (16, 40).
Interestingly, the AF-1 domain of RARs is characterized by the presence of a conserved proline-rich motif (Fig. 1A). In RAR, this proline-rich motif contains two serine residues that can be phosphorylated by two types of kinases, Cdk7 within TFIIH and p38 MAPK (2, 11). Here, we have demonstrated that vinexin interacts with the non-phosphorylated form of the N-terminal AF-1 domain of RAR and that its phosphorylation prevents the interaction with vinexin . These results challenge the hypothesis (see below) that the phosphorylationdependent modulation of the interaction with vinexin is, at least in part, one of the mechanisms involved in the control of RAR activity.
According to our data, the interaction with vinexin appears to concern only RAR and not the other RAR isotypes or RXR. However, it must be noted that other nuclear receptors have been recently found to interact with proteins of the same vinexin family (41). Indeed, the progesterone receptor interacts with c-Cbl-associated protein/ponsin through a proline-rich motif in its N-terminal domain (42). The ER has also been recently reported to interact with the isoform of vinexin through its N-terminal domain (43). Based on these observations, it appears that this family of proteins, which can be present not only in the cytoplasm, but also in the nucleus, would be novel regulators of nuclear receptors.
Vinexin Is an Inhibitor of RAR-mediated Transcription— Although the potential importance of phosphorylation of the N-terminal domain of RAR has been established, the underlying mechanism for the phosphorylation-dependent transcriptional activity of RAR has not been elucidated yet. Here, we propose the hypothesis that non-phosphorylated RAR is transcriptionally inactive due, at least in part, to the interaction of its AF-1 domain with vinexin . Consequently, phosphorylation of the AF-1 domain of RAR would control RAR-mediated transcription through triggering the dissociation of vinexin . To address this hypothesis, we used not only transfected COS-1 cells, but also F9 cells, which constitute a well established cell autonomous model system for investigating RA signaling (29). Most interestingly, in these cells, the functional RAR isotype, RAR, controls most RA-induced events through its phosphorylation and colocalizes with vinexin in the nucleus. Vinexin overexpression and siRNA knockdown experiments led us to propose the following model.
The traditional view is that, in the absence of ligand, RAR resides in the nucleus, binds to response elements of target genes, and interacts with corepressors associated with large complexes with histone deacetylase activity, resulting in chromatin compaction and transcriptional repression (30, 44, 45). In this context, due to the absence of recruitment of the general transcription factor TFIIH by the promoter and to the absence of p38 MAPK activation, RAR is not phosphorylated (2). Thus, from our data, one can suggest that, at that stage, vinexin , which colocalizes with RAR in the nucleus, interacts with the non-phosphorylated N-terminal AF-1 domain of RAR and participates with corepressors in transcriptional repression. In support of this hypothesis is the recent description of a direct interaction between vinexin and SAFB2, a novel nuclear receptor corepressor (46). It is also consistent with our observation that, in transfected COS-1 cells, overexpression of vinexin completely blocked the activity of RAR(S77A/S79A) (Fig. 6A), which mimics a non-phosphorylated receptor and is devoid of any phosphorylation-dependent regulation.
Then, upon ligand binding, subsequent to the combinatorial recruitment of chromatin-modifying and chromatin-remodeling complexes, repressive chromatin is decompacted, allowing the positioning of the transcription machinery at the promoter and the interaction of the general transcription factor TFIIH with RAR (2, 7, 45). This makes the Cdk7 subunit of TFIIH able to phosphorylate one serine residue located in the proline-rich motif of the AF-1 domain of RAR (2, 8). In the same time slot, p38 MAPK becomes activated and phosphorylates the second nearby serine residue (2, 11). Our data suggest that phosphorylation would induce the dissociation of vinexin , therefore allowing transcription to proceed. Such a model is supported by other studies showing that phosphorylation processes also promote the dissociation of vinexin from other partners (14, 15). Whether vinexin dissociates subsequent to RAR phosphorylation by TFIIH and/or by p38 MAPK is a current matter of investigation in our laboratory. It also remains to be determined whether vinexin dissociation would allow the subsequent recruitment by the phosphorylated AF-1 domain of other complexes participating in the initiation of transcription. The characterization of these complexes is presently under investigation.
According to this model, vinexin appears to be a repressor of RAR, which is recruited through the non-phosphorylated N-terminal domain of the receptor. This is substantiated by our results showing that siRNA knockdown of vinexin potentiates RA-induced activation of most RAR target genes. It is also corroborated by our vinexin overexpression experiments in COS-1 and F9 cells showing a significant decrease in the RA-induced expression of several RA target genes. This implies that an increased ratio of vinexin drives the equilibrium to an inactive state, even though the RA-induced phosphorylation of the RAR AF-1 domain is not compatible with vinexin interaction. Note that a similar dynamic inhibition of nuclear receptor activation has been recently reported upon corepressor overexpression even in the presence of ligand (47, 48). Regardless of the detailed mechanism involved, our results strongly emphasize the possibility that, when present at an increased ratio, vinexin does not impede RAR phosphorylation, but would function as a scaffolding protein that blocks RAR in an inactive state, unable to recruit, through its phosphorylated AF-1 domain, the co-regulators necessary to achieve transactivation. Note, however, that this mechanism depends on the promoter context, as the expression of some genes was not affected upon vinexin overexpression. The reason why genes are differentially regulated by vinexin is not yet known. Nevertheless, one can suggest that, depending on the promoter context, the different RAR-interacting protein complexes are endowed with different and specific conformations that dictate the consequences of vinexin overexpression.
In conclusion, we have identified vinexin as a novel repressor of RAR that is recruited through the non-phosphorylated N-terminal domain of the receptor. We propose that the dissociation of vinexin upon phosphorylation of the AF-1 domain of RAR would be a physiologically relevant part of the phosphorylation-dependent transcriptional activation process of RAR. However, it must be noted that vinexin can also be phosphorylated, raising the hypothesis that the association/dissociation of vinexin and RAR might be controlled by the phosphorylation of both partners. Therefore, we can conclude, along with others, that vinexin functions as a scaffolding protein that controls RAR function through its association/dissociation in response to phosphorylation processes.
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FOOTNOTES |
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Supported by INSERM and the Region Alsace.
To whom correspondence should be addressed: Dept. of Cell Biology and Signal Transduction, IGBMC, BP 10142, 67404 Illkirch Cedex, Communauté Urbaine de Strasbourg, France. Tel.: 33-3-8865-3459; Fax: 33-3-8865-3201; E-mail: cegly{at}igbmc.u-strasbg.fr.
1 The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; TF, transcription factor; MAPK, mitogen-activated protein kinase; SH3, Src homology 3; SoHo, sorbin homology; h, human; m, mouse; ER, estrogen receptor; GST, glutathione S-transferase; WT, wild-type; mAb, monoclonal antibody; CAT, chloramphenicol acetyltransferase; RT, reverse transcription; CRAB-PII, cellular retinoic acid-binding protein II; HNF, hepatocyte nuclear factor; siRNA, small interfering RNA; BP, binding protein.
2 The oligonucleotide sequences are available upon request.
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ACKNOWLEDGMENTS |
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