The Focal Adhesion Protein Vinexin α Regulates the Phosphorylation and Activity of Estrogen Receptor α*

Steroid receptors are transcription factors that regulate hormone-responsive genes and whose activity is controlled by their interaction with numerous other proteins. Observations reported here reveal that estrogen receptors α and β (ERα and ERβ), androgen receptor, and glucocorticoid receptor bind in vitro to vinexin α, a multiple SH3 motif-containing protein associated with the cytoskeleton. The SH3 domains are not involved in this interaction. Furthermore, we demonstrate that vinexin α stimulates the ligand-induced transactivation function of these receptors, although it is devoid of intrinsic transcriptional activity when tethered to DNA. In addition, the ectopic coexpression of vinexin α and ERα results in a loss of ERα phosphorylation on serines and the partial redistribution of vinexin α into the nucleus, where it colocalizes with ERα. These results establish a new model of transcriptional regulation where components of the cell-cell and cell-substrate adhesion complexes can regulate the phosphorylation and activity of steroid receptors.

differentiation, and function of a wide array of target tissues, including the male and female reproductive tracts, CNS, immune system, mammary gland, and skeletal and cardiovascular systems. The modular structure of ERs into functional domains is shared with other steroid receptors and features an extremely divergent N-terminal region, a highly homologous central DNA-binding domain (DBD), and a conserved multifunctional C-terminal ligand-binding domain (LBD). Two transcription activation functions, the constitutive AF-1 and the hormone-dependent AF-2, have been allocated to the N-terminal domain and the LBD, respectively. Upon binding of ligand, steroid receptors are released from a complex with molecular chaperones and/or corepressors. Liganded receptors then bind to cognate hormone response elements, located in the promoter or enhancer regions of target genes, and stimulate transcription by transmitting signals to the transcriptional machinery through direct protein-protein interactions (8 -11). Other proteins, called coactivators, are recruited to the liganded, DNAbound steroid receptors and serve as connections with the transcription initiation complex (12,13). In addition to this so-called classic mode of action of steroid receptors, alternative ligand-independent regulation mechanisms have been identified (for review see Refs. 14 -16). These non-classic mechanisms involve phosphorylation of the receptor and cross-talk with other intracellular signaling pathways, some of which are initiated from membrane growth factor receptors.
Vinexin is a recently identified cytoskeletal protein (17), which is characterized by three SH3 domains in its C-terminal region and which exists as two subtypes, vinexin ␣or SH3containing adaptor molecule-1 (SCAM-1) and vinexin ␤. Both isoforms share a common C-terminal sequence, and the larger vinexin ␣ contains an additional N-terminal sequence (see Fig.  1). Vinexin ␣ was isolated via its interaction with vinculin, an abundant cytoskeletal protein found at cell-substrate adhesion sites (focal adhesions) and at cell-cell adhesions (intermediate junctions) where it regulates actin cytoskeletal organization. Through its association with vinculin, mediated by its first two SH3 domains, vinexin ␣ stimulates the formation of actin stress fibers (17). In addition, vinexin ␤ binds to SOS (Son of sevenless), through its C-terminal SH3 domain, and modulates EGF-induced signal transduction pathways leading to JNK/ SAPK MAP kinase activation (18). Two closely related proteins, the Arg/c-Abl-binding protein 2 (ArgBP2), which binds to the Abelson protein-tyrosine kinases (19), and the c-Cbl-associated protein CAP/ponsin/SH3P12 (20 -23), have been identified. Besides the presence of three SH3 domains at their C termini, these proteins share with vinexin ␣ a region of homology with the gut peptide sorbin (24). The sorbin homology (SoHo) domain mediates the interaction of CAP and vinexin with flotillin (a protein located at the lipid raft membrane fraction), the subsequent translocation of the c-Cbl/CAP com-plex to the lipid raft, and the propagation of the signal to guanyl nucleotide exchange proteins, resulting in the activation of intracellular signaling pathways.
In the present study, we report that vinexin ␣ also interacts with steroid receptors in vitro and stimulates their hormoneinduced transactivation potential, although it has no inherent ability to activate transcription. Furthermore, we demonstrate that ER␣ elicits the relocation of a subset of vinexin ␣ into the nucleus, where it colocalizes with ER␣. Finally, our results reveal an inverse correlation between the levels of vinexin ␣ and serine phosphorylation of ER␣. In view of its function in growth factor signaling, we propose that vinexin ␣ integrates signal transduction from the cell membrane and the regulation of steroid receptor action, further emphasizing the multifaceted role of the vinexin/ponsin/ArgBP2 family of proteins.
Yeast Two-hybrid Screening and RT-PCR-The isolation of cDNAs encoding proteins that interact with ER␤ was performed in a yeast two-hybrid screening using the Gal4 MATCHMAKER system as suggested by the manufacturer (Clontech). Saccharomyces cerevisiae strain HF7c was sequentially transformed with Gal4-ER␤(AF-1) followed by a MATCHMAKER rat brain cDNA library constructed in the plasmid pGAD10. Positive clones were selected on the basis of their ability to grow on synthetic dropout plates lacking leucine, tryptophan, and histidine, in the presence of 1 mM aminotriazole. One positive clone showed homologies with a human cDNA sequence named SCAM-1 (GenBank TM accession number AF037261), which was later identified as the product of the vinexin gene (17). The full vinexin ␣ coding sequence with surrounding partial UTRs was obtained by nested PCR on human cerebellum cDNA (Clontech). A first PCR was performed on 1 l of cDNA using the ExpandLong PCR system (Roche Applied Science) with the sense primer 5Ј-CTCGCCGGGGAAGAGGACACGCAGAGGA-3Ј and the antisense primer 5Ј-GGGCAAAGCGGAGAGCTGGTCCCTGT-TAGA-3Ј for 30 cycles with a final elongation step of 10 min. Each cycle consisted of 30 s at 94°C, 30 s at 65°C, and 1 min 30 s at 68°C; an increment of 20 s was added to each elongation step for the remaining 20 cycles. 1 l of the PCR product was used as a template for the second amplification with the sense primer 5Ј-TGGCTTGCCCGGAGTCCTC-CCACCTTGAC-3Ј and the antisense primer 5Ј-TTGGGGAGTGGGGT-GGGGAGGAAATGAAAG-3Ј under the same PCR conditions. The PCR product was cloned into pGEM-T easy (Promega) and sequenced.
In Vitro Protein-Protein Interaction Assay (GST Pull-down Assay)-GST-vinexin ␣ fusion proteins were expressed in E. coli BL21(DE3)-pLysS from pGEX-4T-1-based plasmids as described previously (33) and purified in a non-denaturing extraction solution (39). All nuclear receptors that were tested in pull-down assay were transcribed in vitro and translated with [ 35 S]methionine using rabbit reticulocyte lysate (TNT-coupled in vitro system, Promega) according to the manufacturer's recommendations. Approximately 5 g of GST fusion protein bound to glutathione-Sepharose 4B beads were used in each assay. Glutathionebound GST fusion proteins were incubated with 2 l of [ 35 S]methioninelabeled protein in the presence of the appropriate ligand in Me 2 SO or ethanol at a final concentration of 1 M E2 (ERs), dexamethasone (GR), 5␣-dihydrotestosterone (AR), T3 (TR␤), 9-cis-retinoic acid (RXR␣), or with vehicle alone in a total volume of 200 l of incubation buffer (20 mM Hepes KOH (pH 7.9), 20% (v/v) glycerol, 200 mM KCl, 5 mM MgCl 2 , 0.2 mM EDTA, 0.01% Nonidet P-40, 1 mM dithiothreitol) supplemented with 1 mg/ml bovine serum albumin and a protease inhibitor mixture (Roche Applied Science). After incubation overnight at ϩ4°C with rotation, beads were sedimented by centrifugation (2000 ϫ g) and washed three times for 15 min with incubation buffer without bovine serum albumin. Washed beads were resuspended in 60 l of 1ϫ SDS sample buffer, and an aliquot was subjected to SDS-polyacrylamide gel electrophoresis. Before autoradiography, gels were stained with Coomassie Blue to control for the stability and the equal loading of the GST fusion proteins. Cell Cultures-Hepa-1c1c7, COS-7, MCF-7, and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Invitrogen). SK-N-BE cells were cultured in Roswell Park Memorial Institute medium 1640 supplemented with 10% FCS and 1% L-glutamine. HEK-293 cells were cultured in a 1:1 mixture of Ham's nutrient mixture F-12 (Invitrogen) and DMEM supplemented with 10% FCS. MCF-10-2A cells were cultured in Ham's F-12:DMEM (1:1) with 0.04 mM Ca 2ϩ , 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5% horse serum. All cultures were supplemented with 100 IU/ml penicillin and 100 mg/ml streptomycin (1% PEST). Cells were maintained in an atmosphere of 5.0% CO 2 and 37°C.
Transactivation Assays-COS-7 cells were seeded on 6-well cell culture plates 24 h before transfection in phenol red-free DMEM containing 10% dextran-coated charcoal (DCC)-treated FCS, and 1% PEST. Transient transfection experiments utilizing the FuGENE6 TM reagent (Roche Molecular Biochemicals) were performed as described by the manufacturer, using 0.8 g of MMTV-Luc reporter plasmid, 10 ng of pCMVhGR␣, or either 10 or 100 ng of pSG5-rAR and various amounts of pSG5-VINXa in the absence or presence of ligands (as indicated in the figure legends). Transient transfection experiments utilizing the Lipofectin® reagent (Invitrogen) were carried out as instructed by the manufacturer with 0.8 g of 3xERE-TATA-Luc, 10 ng of either pSG5-ER␣, pSG5-ER␤485, or pSG5-ER␤530 and various amounts of pSG5-VINXa in the absence or presence of E2 (as indicated in the figure legends). Prior to transfection, the cell culture medium was replaced by 1 ml of phenol red-free DMEM without serum or antibiotics. Six hours after addition of the DNA⅐lipid complexes to the cells, 1 ml of phenol red-free DMEM containing 10% DCC-treated FCS, 1% PEST, 10 nM E2 or vehicle alone was added. HEK-293 cells were seeded in a 1:1 mixture of phenol red-free DMEM (Invitrogen) and F-12 containing 10% DCCtreated FCS and 1% PEST. Cells were transiently transfected using the FuGENE6 TM reagent with 0.8 g of 3xERE-TATA-Luc, 10 ng of pSG5-ER␣, and various amounts of pSG5-VINXa in the absence or presence of E2 (as indicated in the figure legends). For mammalian one-hybrid experiments, HEK-293 cells were transfected with 0.5 g of the UAStk-Luc reporter plasmid and various amounts of the different Gal4 fusion protein expression vectors (as indicated in the figure legends). After 24 h (HEK-293) or 36 -48 h (COS-7), cellular extracts were prepared and analyzed for luciferase activity using a Berthold luminometer. The luciferase detection reagents were purchased from Biothema.
Analysis of Intracellular Localization Using Confocal Microscopy-COS-7 cells were seeded onto glass coverslips in 12-well cell culture plates and grown in phenol red-free DMEM, 10% DCC-treated FBS, 1% PEST, for 24 h. The cells were transfected in OPTI-MEM I medium (Invitrogen) for 6 h with either 0.5 g of pHcRed-ER␣ or 0.3 g of pEGFP-VINXa, or both, using LipofectAMINE 2000 (Invitrogen). When necessary, the total amount of DNA was completed to 0.8 g with pSG5.
The cells were grown for 24 h after transfection in the absence or presence of 10 nM estradiol before fixing with 3% paraformaldehyde in 5% sucrose/PBS for 20 min at room temperature. The cell nuclei were stained with 5 g/ml Hoechst 33342 (Molecular Probes), and the coverslips were then mounted on glass slides using antiphotobleaching Fluorosave (Calbiochem). Subcellular localization was determined using a TCS SP Multiband confocal imaging system (Leica Corp.).
ER␣ Protein Stability-COS-7 cells were transfected using the Lipo-fectin® reagent with 0.8 g of 3xERE-TATA-Luc, 10 ng of pSG5-ER␣, and 1 g of either pSG5 or pSG5-VINXa in the absence or presence of 10 nM E2 as described above. Twenty-four hours after transfection, cells were harvested in 500 l of TEN (40 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 150 mM NaCl 2 ) and lysed in 30 l of buffer C (10 mM Hepes-KOH (pH 7.9), 10 mM dithiothreitol, 10 mM EDTA, 5% glycerol, and 1ϫ Complete TM , EDTA-free protease inhibitor mixture (Roche Applied Science)), 0.4 M NaCl, by two successive cycles of freezing on dry ice and thawing. After centrifugation for 10 min at 14,000 rpm and 4°C, the supernatant was recovered and 5 l of protein extract was used for luciferase reporter assay as described above. 10 g of proteins was subjected to SDS-PAGE and transblotted onto Hybond-P Nylon polyvinylidene difluoride membrane (Amersham Biosciences) according to the manufacturer's instructions. ER␣ was detected using the H-184 rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution in PBS, 0.1% Tween 20, 5% nonfat dried milk, and the rabbit IgG, horseradish peroxidase-linked whole antibody (Amersham Biosciences) at a 1:10000 dilution. Signals were revealed with the ECL Plus Western blotting detection system (Amersham Biosciences).
ER␣ Phosphorylation-COS-7 cells were seeded onto 150-mm plates 24 h before transfection. The cells were transiently transfected using the Lipofectin® reagent with 12 g of 3xERE-TATA-Luc, 150 ng of pcDNA-FlagER␣, and 15 g of either pSG5 or pSG5-VINXa in the absence or presence of 10 nM E2. Twenty-four hours after transfection, cells were harvested in 1.5 ml of TEN and lysed as described above in 200 l of buffer C, 0.4 M NaCl, supplemented with phosphatase inhibitors: Phosphatase Inhibitor Mixture I (1:1000) (Sigma), Phosphatase Inhibitor Mixture II (1:1000) (Sigma), 1 mM NaF, 1 mM NaPP i , and 2 M fenvalerate. The luciferase reporter activity was monitored using 1 l of protein extract. FLAG-tagged ER␣ was immunoprecipitated from 500-g protein extracts using 5 g of anti-FLAG M5 antibody (Sigma) in 450 l of buffer C, 0.2 M NaCl, supplemented with phosphatase inhibitors. After 2-h incubation at 4°C under gentle agitation, 25 l of Protein G-Sepharose (Amersham Biosciences) were added, and the incubation prolonged for 2 h. After three washes with 1 ml of buffer C, 0.2 M NaCl, for 10 min and 1 wash with 1 ml of buffer C without NaCl, for 10 min, proteins retained in the immunopellets were denatured in 25 l of 2ϫ SDS loading buffer for 5 min at 100°C. Ten l of immunoprecipitates was subjected to SDS-PAGE and transblotted onto Hybond-P Nylon polyvinylidene difluoride membrane. The detection of the phosphoserine-containing proteins was achieved using the phosphoserine 1C8 mouse IgM antibody (Alexis Biochemicals) and the donkey anti-mouse IgM antibody (Jackson ImmunoResearch Laboratories), both at a 1:1000 dilution in PBS, 0.1% Tween 20, 4% bovine serum albumin. ER␣ was detected using the H-184 rabbit polyclonal antibody as described above. Signals were revealed with the ECL Plus Western blotting detection system (Amersham Biosciences).
RNA Blot Analysis-Various cell lines were grown to ϳ80% confluence, and poly(A) ϩ mRNA was isolated as previously described (40,41). Aliquots (3 g) of RNA were electrophoresed on a formaldehyde-agarose gel prior to transfer to Zeta-probe membrane (Bio-Rad Laboratories) and cross-linking. Random primed 32 P-labeled probes to a cDNA fragment corresponding to amino acids 1-159 of human vinexin ␣ and to full-length rat GAPDH cDNA were prepared using the Redi-Prime II kit (Amersham Biosciences). Human multiple tissue Northern blots (Clontech) and the cell line Northern blot were prehybridized and hybridized as recommended by the manufacturers.

RESULTS
Isolation of Vinexin ␣-We used the AF-1 region of ER␤485 (6, 42) as a bait in a screening of a rat brain cDNA library employing the Gal4-based yeast two hybrid system. We thus isolated a 1.5-kb cDNA fragment that showed high degrees of homology at the protein level with two human sequences named SCAM-1 (SH3-containing adaptor molecule-1) (Gen-Bank TM accession number AF037261) and vinexin ␤ (17) and with the mouse sequence for vinexin ␣ (17) (Fig. 1). Rat SCAM-1 cDNA contains a coding sequence starting at position 125 and ending with the end of the cloned cDNA fragment at position 1518 (data not shown). No upstream stop codon, inframe with the first translation start site, was found in the first 124 bp. Comparison of protein sequences reveals that mouse vinexin ␣ presents an extended N-terminal polypeptide that is not seen in the human or rat orthologues. The translation of the FIG. 1. Alignment of the rat SCAM-1/vinexin ␣ partial sequence with its mouse and human orthologues. The protein sequence deduced from the rat SCAM-1 partial clone was aligned against those of mouse vinexin ␣, human SCAM-1, and human vinexin ␤. To maximize the quality of the alignment, gaps (shown as dashes) were introduced in the sequences. Amino acids that are conserved between at least two sequences are shaded in gray. The sorbin homology (SoHo) is underlined; the three SH3 domains at the C terminus of SCAM-1/vinexin ␣ are delimited by black open boxes.
5Ј-UTR in rat SCAM-1/vinexin ␣ showed that the sequence conservation between rat and mouse is restricted to 19 codons directly upstream from the translation start site in the rat sequence (data not shown). Strikingly, no such similarity is observed in the more distal region of the rat 5Ј-UTR, in contrast to the overall high degree of conservation between the rat and the mouse sequences. We suggest that the conservation of the sequence upstream of the first ATG results from the presence of regulatory sequences necessary for the initiation of translation. Overall, the rat SCAM-1 partial protein sequence shares 96% identity with mouse vinexin ␣ and 80% identity with human SCAM-1, in their overlapping regions (Fig. 1). A similar degree of conservation is found between the full-length mouse and human sequences. In particular, functional domains that have been characterized so far in mouse vinexin ␣ present very little divergence between rodents and human: at the N terminus, the SoHo domain, and in the C-terminal half, the three SH3 domains. To investigate whether vinexin ␣ affects signaling from ERs and other nuclear receptors, we isolated the full coding sequence of human SCAM-1 by RT-PCR on cerebellum cDNAs. For clarity, we will refer to human SCAM-1 as vinexin ␣ herein.
Vinexin ␣ Is Widely Distributed in Adult Tissues with a High Predominance of the Testis-The distribution of vinexin ␣ in human tissues was investigated by Northern blotting with a probe corresponding to amino acids 1-159, which discriminates between the two vinexin isoforms. In most tissues we observed the presence of a single mRNA species with an apparent size of 3.0 kb ( Fig. 2A), in agreement with the length of the reported sequence in the GenBank TM data base. Interestingly, the expression of vinexin ␣ was highly prevalent in the testis, where a second shorter (2.8 kb) minor mRNA species was also detected. In contrast, the expression of vinexin ␣ was extremely weak or undetectable in the kidney, the liver, peripheral blood leukocytes, and the thymus. RT-PCR experiments on multiple human tissue cDNA panels imparted similar conclusions on the wide distribution of vinexin ␣ in human tissues and on its foremost expression in the testis (data not shown). In contrast, expression of vinexin ␣ appears to be restricted to few cell lines among those we tested. A noticeable signal was only observed in the human breast adenocarcinoma MCF-7 cells and neuroblastoma SK-N-BE cells (Fig. 2B).
Vinexin ␣ Interacts in Vitro with Steroid Receptors Independently of the SH3 Domains-The partial rat SCAM-1 clone was originally isolated via its interaction with ER␤ in the yeast two-hybrid system. To assess whether human vinexin ␣ could also interact with other NRs, we set up an in vitro proteinprotein interaction assay with different regions of human vinexin ␣ (Fig. 3A) and various NRs. The translation of ER␤530 in vitro produced two major protein species of apparent masses of 59 and 55 kDa, respectively (Fig. 3B; lane 1), most likely corresponding to alternate usage of two translation start sites (Met at positions 1 and 46) leading to the expression of the two isoforms ER␤530 and ER␤485, respectively. Both isoforms interacted with the N terminus (N; aa 1-201) and the middle region (M; aa 202-386) of vinexin ␣ (Fig. 3B). This interaction was independent of the presence of ligand and was not modified by the addition of E2. In contrast, no binding (above the GST control) to the C-terminal region (C; aa 387-671) was detected. Similarly, we observed a binding between vinexin ␣ and three other steroid receptors, ER␣, AR, and GR (Fig. 3C)  extremely weak interaction with two other ligand-inducible NRs, the retinoid receptor RXR␣ and the thyroid hormone receptor TR␤ (Fig. 3C), or with the orphan nuclear receptors Dax-1 and SF-1 (Fig. 3D). As seen with ER␤, the interaction between vinexin ␣ and other steroid receptors was not affected by the presence of ligand and was independent of the SH3 domains (data not shown).
Vinexin ␣ Is an Activator of the Transcriptional Function of Steroid Receptors-Considering that vinexin ␣ could bind to steroid receptors in vitro, we sought to assess whether it could also regulate the transcriptional activity of ER␣, ER␤, AR, or GR, ectopically expressed in different cells lines. Experiments conducted in COS-7 cells showed that vinexin ␣ could increase the activity of both ER␣ (Fig. 4A) and ER␤ (Fig. 4, C and D) in the presence of E2. No striking difference in the amplitude of the coactivation of these receptors was observed. It is notable that the extension of the N terminus in the ER␤530 isoform did not alter the coactivation of ER␤ (Fig. 4, C and D), indicating that this additional sequence does not play any role in the activation of the liganded ER␤ by vinexin ␣. The coactivation of ER by vinexin ␣ is not restricted to a particular cellular context, because we detected a similar effect of vinexin ␣ on the activity of ER␣ in HEK-293 cells (Fig. 4B) and on that of the different ER isoforms in HeLa cells (data not shown). Although both ER␣ and ER␤ are targets for vinexin ␣ signaling, they nonetheless present a significant disparity in their mode of activation. We observed that the activity of the non-liganded ER␣ was increased by vinexin ␣ in a dose-dependent manner ( Fig. 4, A and B), whereas non-liganded ER␤ was not responsive to vinexin ␣ at any dose tested (Fig. 4, C and D).
In parallel, we observed that vinexin ␣ is able to stimulate the transcriptional activity of two other steroid receptors, AR and GR, in the presence of their cognate ligands (Fig. 5). On the very robust dexamethasone-induced activation (270-fold) of GR, vinexin ␣ further increased the transcription from the MMTV promoter 4-fold (Fig. 5A). On the same promoter, AR was less responsive to DHT than GR was to dexamethasone; a maximal 7-fold activation of the transcription was reached when a dose of 100 ng of pSG5-ratAR was used (Fig. 5B). The addition of vinexin ␣ further enhanced the transcriptional activity of AR 4-fold. Remarkably, at a lower dose of AR expression plasmid (10 ng), where the receptor was almost nonresponsive to DHT, vinexin ␣ was more potent to stimulate AR activity (Fig. 5C). In addition, vinexin ␣ could also enhance the basal activity of AR and GR in the absence of ligand (data not shown), as already observed with ER␣.
To investigate whether vinexin ␣ holds an intrinsic transcriptional function, its complete coding sequence was fused to the Gal4 DNA-binding domain (DBD), and its transcriptional activation potential was analyzed in a mammalian one-hybrid assay. As shown in Fig. 6, vinexin ␣ does not elicit any increase of the reporter gene activity above the basal level, in contrast to a fragment of the activation domain of RAP250 (33), which showed a very robust activation of transcription. Thus, we conclude that vinexin ␣ cannot exert any transcriptional activity on its own. Altogether, our observations from functional assays in mammalian cells indicate that vinexin ␣ can stimulate the activity of steroid receptors, although it is unable, on its own, to transmit any positive signal to the basal transcription machinery, unlike RAP250 and classic steroid receptor coactivators.
Vinexin ␣ Regulates ER␣ Phosphorylation-ER transcriptional activity can be influenced by different parameters, including protein stability (43,44) and phosphorylation (45)(46)(47). To understand the molecular mechanisms underlying ER activation by vinexin ␣, we investigated whether ER␣ protein stability and/or phosphorylation would be altered in the presence of vinexin ␣. Transient transfection experiments revealed that the levels of ectopically expressed ER␣ in COS-7 cells are only marginally modified in the presence of vinexin ␣ (Fig. 7, A  and B). An increase of the ER␣ protein levels by 25% was  (B and C)). Forty-eight hours after transfection, luciferase reporter activity was measured. The results represent the -fold activation by the ligand and were normalized to the activity of each receptor in the absence of vinexin ␣, which was set as 1. The results represent the mean of at least two independent values from a typical experiment. detected when vinexin ␣ was expressed, both in the presence or absence of E2.
The strategy we developed for the study of ER␣ phosphoryl-ation relied on the characterization of phosphorylation on serine residues by Western blot, using the 1C8 antibody directed against phosphoserine, following the immunopurification of FLAG-tagged ER␣ from transiently transfected COS-7 cells. Under these conditions, we were able to detect a specific signal of the right size in cells transfected with FLAG-ER␣ in the absence of vinexin ␣ and E2 (Fig. 7C). Either treatment with 10 nM E2 for 20 h or coexpression of vinexin ␣ almost completely abolished this signal. The quantification of the signals revealed that phosphorylation levels of ER␣ in the presence of E2 or vinexin ␣ were reduced to 5-10% of those in their absence (Fig.  7D). The combination of both E2 treatment and vinexin ␣ expression resulted in the total loss of ER␣ phosphorylation as detected by the 1C8 antibody (Fig. 7, C and D). Overall, our results indicate a critical role for vinexin ␣ in the regulation of ER␣ phosphorylation that occurs along with the transcriptional activation of the receptor. The target of this regulation is one or a few yet unidentified serines, the phosphorylation of which is also influenced by the interaction with ligand.
Nuclear Colocalization of Vinexin ␣ with ER␣-A previous study highlighted the role of vinexin proteins at focal adhesions (17). In particular, the observation of the subcellular localization of vinexin by GFP fluorescence revealed that both vinexin isoforms, exogenously expressed in NIH 3T3 fibroblasts, are predominantly localized at focal adhesions with vinculin. In contrast to vinexin ␤, which showed cytoplasmic and nuclear localization, vinexin ␣ was only detected in the cytoplasm of transfected cells, where it formed vinculin-free aggregates. In the present report, we show that vinexin ␣ can bind in vitro to ER␣ (Fig. 3C); to investigate whether these two proteins can indeed interact in the cell, we performed confocal microscopy studies in COS-7 cells transfected with GFP-vinexin ␣ and HcRed-ER␣. In agreement with the established view of a predominant nuclear subcellular localization of ER␣, both in the absence or presence of E2, we detected the HcRed-ER␣ fusion protein in the nucleus of transfected cells (Fig. 8, A and B). A decrease in the overall intensity of the red fluorescent signal was observed in E2-treated cells (data not shown), suggesting a higher turnover of ER␣ in the presence of E2. In the absence of ER␣, GFP-vinexin ␣ formed numerous aggregates in the cytoplasm of COS-7 cells (Fig. 8C). A very large aggregate could also be observed in the perinuclear region of many cells, likely resulting from very high levels of expression of the exogenous protein. Furthermore, some of the small aggregates were present in the nucleus. The coexpression of ER␣, irrespective of the ligand status, resulted in the relocation of a fraction of the total GFP-vinexin ␣ into the nucleus of a subset of cells (Fig. 8, D and  G), where it colocalized with ER␣ (Fig. 8, F and I). No change in the subcellular distribution of ER␣ could be detected in the presence of vinexin ␣ (Fig. 8, E and H). Our observations establish that ER␣ and vinexin ␣ colocalize in the nucleus of transfected cells, where the possible interaction of the two proteins would result in a modification of ER␣ phosphorylation and transcriptional activity. DISCUSSION Vinexin ␣ and vinexin ␤ are newly identified focal adhesion and intermediate junction proteins that play a role in cytoskel-etal organization and cell spreading (17) as well as intracellular signaling (18,48). In addition to its membrane-associated functions, we present here evidence that vinexin ␣ exerts a role in the nucleus, where it stimulates the transactivation function of the steroid receptors, ER␣, ER␤, AR, and GR. With the notable exception of ER␤, vinexin ␣ enhances the transcriptional activity of steroid receptors in their unliganded state; a further and higher increase of activity occurs in the presence of the cognate ligands. We show that, in the absence of hormone, vinexin ␣ can indeed associate with steroid receptors in vitro and colocalize with ER␣ in the nucleus. Whereas vinexin binds to vinculin and SOS at focal adhesions through its SH3 domains (17,18), the interaction surface with steroid receptors is restricted to the N-terminal region of vinexin ␣ and does not include any of the three SH3 domains. The interaction domain on the steroid receptors remains to be delineated. The isolation of vinexin ␣ in a yeast two-hybrid screening with the AF-1 region of ER␤ suggests that this domain might be involved; however, this region shows no homology between steroid receptors, and we can suspect that another more conserved domain, i.e. the DBD or the LBD, might also contribute to the binding to vinexin ␣. AF-2-directed coactivators contain conserved leucine-rich motifs with the consensus sequence LXXLL (L, leucine; X, any amino acid), called NR boxes, which form an ␣-helix and bind to a hydrophobic groove on the ligand-occupied LBD of nuclear receptors (49). Whereas vinexin ␣ contains the NR box-like sequence IEVLL (aa 271-275), and motifs with the sequence IXXLL can function as AF-2 interacting interfaces, 2 the steroid receptor binding domain of vinexin ␣ extends far over the putative NR-box and the region between amino acids 1 and 201 is sufficient for this interaction. Furthermore, the binding of steroid receptors with vinexin ␣, and their subsequent activation, occurs irrespective of ligand binding in contrast to the AF-2-NR box interaction. Finally, vinexin ␣ does not exhibit any intrinsic transcriptional activity and can be distinguished from classic NR coactivators in this aspect too.
Because vinexin ␣ is present at focal adhesions and intermediate junctions (17), and interacts with the lipid raft protein flotillin (24), its involvement in intracellular signaling pathways has been suggested. This hypothesis has been further strengthened by the observation of the role of the shorter vinexin ␤ in EGF signal transduction to JNK/SAPK MAP kinase (18). Yet, no evidence exists to establish a similar role for vinexin ␣. The cross-talk from membrane-associated peptide growth factors and cytosolic signaling pathways to intracellular steroid receptors has been clearly demonstrated for ER (for review see Ref. 7) and AR (50 -53). In many instances, it has been established that the activation of steroid receptor function occurs via phosphorylation. Our study reveals that vinexin ␣ down-regulates ER␣ phosphorylation on serine, at a site that remains to be identified. Interestingly, a similar effect on ER␣ phosphorylation was observed after E2 binding. Arnold et al. (54,55) reported that a single site of ER␣ is dephosphorylated after treatment of MCF-7 cells with E2. Of potential significance in this context, a single serine residue in the ER␣ DBD (Ser-236) has been identified as a site for phosphorylation by protein kinase A (PKA), leading to a decrease in dimerization and DNA-binding capabilities of ER␣ (56). Ser-236 is immediately adjacent to a basic amino acid and could thus form an epitope for the phosphoserine antibody that we used, suggesting that Ser-236 could be the target for regulation by vinexin ␣. Sequence homology analyses show that vinexin ␣ does not contain any known catalytic domain, suggesting that external enzymatic activity would be required. Whether vinexin ␣ exerts 2 E. Treuter, personal communication. its action on ER␣ phosphorylation through the inhibition of PKA, or other kinases, or via the recruitment and activation of yet unidentified phosphatases remains to be elucidated. A recent report demonstrated that ER␣ undeniably exists in complexes with phosphatases as well as kinases (57). Protein phosphatase 2A directly interacts with ER␣, dephosphorylates serine 118 of the receptor, and thus prevents the formation of an ER␣-activated MAP kinase complex and subsequent transcriptional activity. Conversely, a direct interaction of AR or ER␣ with the p21-activated kinase PAK6, an effector of Cdc42 and Rac, leads to the inhibition of their transcriptional activity (58,59). In that respect, Suwa et al. (48) reported in the Gen-Bank TM data base that vinexin ␣ binds to PAK and, more recently, the functional interaction between vinexin ␤ and PKA-PAK signaling has been established for the anchorage-dependent activation of MAP kinase.
Human genome sequences available in the GenBank TM data base reveal that vinexin ␣ and ␤ are encoded by 21 and 10 exons, respectively, nine of which are common at the 3Ј terminus, indicating that the two vinexin transcripts result from alternative promoter usage. Hence, vinexin ␣ and ␤ exhibit different expression patterns in human adult tissues. Kioka et al. (17) reported that, whereas the expression of vinexin ␤ is fairly ubiquitous, vinexin ␣ displays a more restricted distribution with higher expression in the adult skeletal muscle. In the present report, we show that vinexin ␣ is more widely distributed that previously stated, although expression levels are low in most tissues. We actually observed higher amounts of vinexin ␣ mRNA in the skeletal muscle than in other tissues. We present here evidence, for the first time, that vinexin ␣ expression is predominant in the adult testis. In line with these findings, a high expression of vinexin ␣ in the mouse developing testis has also been reported (60), suggesting an important role for vinexin ␣ in testicular ontogeny and function. Because steroid hormones and their receptors are key regulators of the development and function of the testis, our results raise the question of the significance of the cross-talk between vinexin ␣ and steroid receptors in this context. Of particular interest is the high expression of PAK6 that is observed in the testis (58). To get further insights into the function of vinexin ␣ in the testis, the presence of vinexin ␣, PAK 6, AR, or ERs in the same cells should be investigated. The possible colocalization of these proteins might suggest a role for the cross-talk between vinexin ␣-PAK signaling and regulation of testicular function by steroid receptors. However, other functions that vinexin ␣ might exert in the testis independently of steroid receptor signaling should be considered, in particular with respect to cytoskeletal organization, maintenance of cellular structure and tissue integrity, as well as EGF signal transduction. Further experiments will be required to fully comprehend the importance of the cross-talk between vinexin ␣ and steroid receptors in the modulation of the testicular function.
In conclusion, we have identified vinexin ␣ as a novel activator of steroid receptor transcriptional function, in addition to its established roles in the regulation of cytoskeleton/cell spreading and in the transduction of the EGF signal along the MAP kinase pathways. We propose that vinexin ␣ exerts its action on steroid receptors via the regulation of their phosphorylation. The recent description of a direct interaction between vinexin and SAFB2, a novel ER corepressor (61), further emphasizes the possibility of a cross-talk between cell-cell and cell-substrate adhesion complexes and nuclear receptors. We propose, along with others, that vinexin ␣ functions as a scaffolding protein that integrates signals from different pathways.