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Formation of an hERalpha-COUP-TFI complex enhances hERalpha AF-1 through Ser118 phosphorylation by MAPK

2002; Springer Nature; Volume: 21; Issue: 13 Linguagem: Inglês

10.1093/emboj/cdf344

1460-2075

Raphaël Métivier, Michael R. Hübner, Gilles Flouriot, Gilles Salbert, Frank Gannon, Olivier Kah, Farzad Pakdel,

RNA Research and Splicing

Article1 July 2002free access Formation of an hERα–COUP-TFI complex enhances hERα AF-1 through Ser118 phosphorylation by MAPK Raphaël Métivier Raphaël Métivier Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Present address: EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Frédérique A. Gay Frédérique A. Gay Equipe d'Information et Programmation Cellulaire (IPC), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Present address: Harvard Medical School, Department of Pathology. WAB 120, 200 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Michael R Hübner Michael R Hübner EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Gilles Flouriot Gilles Flouriot Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Search for more papers by this author Gilles Salbert Gilles Salbert Equipe d'Information et Programmation Cellulaire (IPC), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Search for more papers by this author Frank Gannon Frank Gannon EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Olivier Kah Olivier Kah Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Search for more papers by this author Farzad Pakdel Corresponding Author Farzad Pakdel Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Search for more papers by this author Raphaël Métivier Raphaël Métivier Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Present address: EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Frédérique A. Gay Frédérique A. Gay Equipe d'Information et Programmation Cellulaire (IPC), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Present address: Harvard Medical School, Department of Pathology. WAB 120, 200 Longwood Avenue, Boston, MA, 02115 USA Search for more papers by this author Michael R Hübner Michael R Hübner EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Gilles Flouriot Gilles Flouriot Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Search for more papers by this author Gilles Salbert Gilles Salbert Equipe d'Information et Programmation Cellulaire (IPC), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Search for more papers by this author Frank Gannon Frank Gannon EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Search for more papers by this author Olivier Kah Olivier Kah Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Search for more papers by this author Farzad Pakdel Corresponding Author Farzad Pakdel Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France Search for more papers by this author Author Information Raphaël Métivier1,2, Frédérique A. Gay3,4, Michael R Hübner5, Gilles Flouriot1, Gilles Salbert3, Frank Gannon5, Olivier Kah1 and Farzad Pakdel 1 1Equipe d'Endocrinologie Moléculaire de la Reproduction (EMR), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France 2Present address: EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany 3Equipe d'Information et Programmation Cellulaire (IPC), UMR CNRS 6026, Université de Rennes I, 35042 Rennes, Cedex, France 4Present address: Harvard Medical School, Department of Pathology. WAB 120, 200 Longwood Avenue, Boston, MA, 02115 USA 5EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3443-3453https://doi.org/10.1093/emboj/cdf344 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The enhancement of the human estrogen receptor α (hERα, NR3A1) activity by the orphan nuclear receptor COUP-TFI is found to depend on the establishment of a tight hERα–COUP-TFI complex. Formation of this complex seems to involve dynamic mechanisms different from those allowing hERα homodimerization. Although the hERα–COUP-TFI complex is present in all cells tested, the transcriptional cooperation between the two nuclear receptors is restricted to cell lines permissive to hERα activation function 1 (AF-1). In these cells, the physical interaction between COUP-TFI and hERα increases the affinity of hERα for ERK2/p42MAPK, resulting in an enhanced phosphorylation state of the hERα Ser118. hERα thus acquires a strengthened AF-1 activity due to its hyperphosphorylation. These data indicate an alternative interaction process between nuclear receptors and demonstrate a novel protein intercommunication pathway that modulates hERα AF-1. Introduction Estrogens (estradiol, E2) are small lipophilic steroid hormones, predominantly synthesized by ovaries. Although commonly recognized as the pivotal hormone for female reproductive tract physiology, E2 is also involved in homeostasis of other tissues, and also performs important roles in males (Lombardi et al., 2001; Nilsson et al., 2001). Critical roles of E2 involve a control of the cell proliferation/differentiation balance (Feigelson and Henderson, 1996). Most of the physiological functions of E2 are exerted by specific nuclear receptors, termed estrogen receptors (ERα and ERβ; NR3A1 and NR3A2; Nuclear Receptor Committee, 1999). These proteins involved in controlling endocrine and reproductive functions (Couse and Korach, 1999) are evolutionarily widely distributed from chordates to mammals, and belong to the transcription factor superfamily of nuclear receptors (NRs; Laudet, 1997). This superfamily includes ligand-inducible transcription factors, such as the steroid, retinoid and thyroid hormone receptors, as well as orphan receptors for which no ligand has been identified so far (Giguere, 1999). ERs bind as dimers to DNA, on specific estrogen response elements (EREs; Parker, 1995). ERs activate transcription of target genes through an N-terminal activation function (AF-1) in the B domain, and a C-terminal AF-2 region (Parker, 1995; see Figure 1A). These AFs exhibit distinct properties, which depend on both cell and promoter contexts (Metzger et al., 1992). AF-2-mediated activation requires ligand-induced conformational changes, which allow interaction with co-activators that possess histone acetyltransferase activity and multiprotein complexes that interact with the general transcription machinery (Freedman, 1999). Extracellular signals influence ER functions by modulating the receptor phosphorylation state (Weigel, 1996). ER actions are also influenced by other transcription factors (Gaub et al., 1990; Salvatori et al., 2000), and orphan receptors such as SF-1 (NR5A1; Le Dréan et al., 1996). Figure 1.Physical and transcriptional interrelationships between hERα and COUP-TFI. (A) Schematic illustration of the hERα sequence: domains and associated functions. (B) Transfection experiments in CHO-K1 cells were performed using the 0.2 basic, ERE-TK or chicken vitellogenin (Vg) reporters, pCH110 internal control and 25 ng of each expression vector (pCMV5/hERα and pCDNA/COUP-TFI). Cells were treated with ethanol (EtOH) or 10−8 M estradiol (E2). Values are shown as the fold induction of normalized luciferase reporter activity (mean ± SEM). (C) Co-immunoprecipitation of the endogenous hERα–COUP-TFI complex in MCF-7 cells using anti-COUP-TFI- specific antibody. A goat pre-immune serum (PreI) was the control. The presence of each component of the complex was checked by western blots. (D) GST pull-down experiments performed using 250 ng of the GST–COUP-TFI 57–423 fusion protein or GST as a control, with in vitro labeled hERα. Input is 40% of the labeled protein used in the assay. Download figure Download PowerPoint COUP-TFs are orphan NRs that are highly expressed in the developing nervous system, where they exert crucial roles in specific cell fates (Qiu et al., 1997). In addition, COUP-TFs are expressed in a wide variety of tissues such as liver, uterus and mammary gland, where they may regulate vital biological functions and correct organogenesis (Pereira et al., 1995). Several of these tissues also express ERs, and cross-talk between both NRs was suggested in the context of genes expressed in estrogen-sensitive tissues such as the uterus. In agreement with a role for COUP-TFs as transcriptional repressors (Cooney et al., 1993), these orphan receptors were shown to repress the estrogen-regulated expression of the oxytocin and hepatocyte growth factor (HGF) genes (Jiang et al., 1997; Chu et al., 1998). On the other hand, a positive transcriptional effect of COUP-TFI on ERα activity was demonstrated for the first time on the rainbow trout ERα (rtERα) promoter (Lazennec et al., 1997). The aim of this current study was to investigate the mechanisms underlying the positive action of COUP-TFI on ERα activity. First, we extend this positive interplay to other promoters and to the human ERα (hERα). Then, we describe a new pathway where the formation of a tight ERα–COUP-TFI intermediate complex leads to an increased recruitment of the ERK2/p42MAPK, phosphorylation of the hERα on Ser118 and enhancement of its transcriptional activity. Results Transcriptional cooperation and physical interaction between hERα and COUP-TFI Although previously described as a repressive factor, COUP-TFI enhances the hERα activity on the rtERα promoter gene, an ERE-TK synthetic promoter, the chicken vitellogenin (Vg) promoter in CHO-K1 cells (Figure 1B) and, hence, presumably on other target genes in vivo. One way that COUP-TFI could influence hERα activity is by a direct interaction between the two proteins, the resulting complex exhibiting properties that differ from those of each NR. The occurrence of an interaction between both endogenous proteins was examined in MCF-7 cells that express these NRs (Figure 1C). A co-immunoprecipitation experiment performed with a specific antibody directed against COUP-TFI revealed that endogenous hERα and COUP-TFI are found in the same protein complex, with or without E2 treatment. To demonstrate a physical interaction between the two NRs, we used a GST fusion protein with the longest COUP-TFI sequence possible [residues 57–423, encompassing half of the N-terminal region, the DNA-binding domain (DBD) and the ligand-binding domain (LBD)]. Subsequent in vitro GST pull-down assays confirm the occurrence of a ligand-independent interaction between COUP-TFI and in vitro labeled hERα (Figure 1D). Molecular dissection of the physical interaction between hERα and COUP-TFI To map the domains of both NRs involved in their interaction, we performed two-hybrid assays, whose accuracy was tested by fusing the full-length COUP-TFI and hERα to the activation domain (AD) or DBD of the yeast transcriptional factor Gal4. Subsequent assays shown in Figure 2A confirm the ligand independence of the interaction between the two NRs. A dominant-negative form of COUP-TFI (Adam et al., 2000), harboring a C141S point mutation within its DBD, is unable to interact with hERα (Figure 2A). On the contrary, this mutant maintains its ability to interact with wild-type COUP-TFI. This indicates that the mechanisms underlying the interaction between COUP-TFI and hERα are probably different from those involved in COUP-TFI homodimerization. Figure 2.The hERα–COUP-TFI complex is required for transcriptional cooperation but is destabilized by DNA. (A) Yeast two-hybrid assays using full-length hERα, wild-type and dominant-negative COUP-TFI (C141S point mutation) fused to the Gal4AD or Gal4DBD. Yeast transformants were grown in media including ethanol (EtOH) or 10−8 M estradiol (E2). Liquid β-galactosidase assays were then performed. (B and C) The different domains of COUP-TFI were fused to the Gal4AD or Gal4DBD for two-hybrid assays, which were performed using as bait the complementary fusion proteins indicated at the top of each panel. Results are expressed as the mean ± SD from three independent experiments. (D) Ability of COUP-TFI mutants to enhance hERα activity on the ERE-TK reporter gene, in CHO-K1 cells treated with EtOH or 10−8 E2. Results (mean ± SEM) are expressed as the reporter fold stimulation in the presence of E2. (E) Pull-down experiments were performed with in vitro labeled hERα and COUP-TFI using the GST–COUP-TFI 57–423 or GST–hERα DF proteins. During the incubation, increasing amounts of DR-1, ERE (25–200 ng) and AP-1 as non-specific control (50–200 ng) oligonucleotides were added. Download figure Download PowerPoint To obtain further information on the specific mechanisms of interaction between the two NRs, we identified their contacting surfaces. The different domains of COUP-TFI were fused to the Gal4AD, while the C–F (CF) or D–F (DF) domains of hERα fused to the Gal4DBD were used as bait proteins (Figure 2B, left). These hERα proteins exhibit no intrinsic transcriptional activity that might have interfered in these assays. This property allowed us to show the ligand independence of all detected interactions (data not shown). The hERα DBD + LBD bait (hERα CF/Gal4DBD) associates with COUP-TFI N-terminal (amino acids 1–57), C-terminal (amino acids 153–423) and DBD (amino acids 57–153) regions. Performing the two-hybrid assays with the hERα LBD alone (hERα DF/Gal4DBD) showed that it associates with COUP-TFI N- and C-terminal regions (Figure 2B, right). The absence of the DBD in this hERα DF/Gal4DBD bait and its failure to bind the COUP-TFI DBD demonstrate that the DBDs of the two NRs form direct contacts (Figure 2B, right). We next wanted to see if the hERα N-terminal domain was able to contact COUP-TFI. When fused to the Gal4DBD, the hERα N-terminal domain activates transcription in yeast through its AF-1 (Métivier et al., 2001). Since such a protein is not informative in two-hybrid assays, we therefore conducted inverse assays. hERα AB domains were fused to the Gal4AD, and COUP-TFI domains to the Gal4DBD. These assays demonstrate that the hERα N-terminal region associates with the C-terminal part of COUP-TFI (Figure 2C). In conclusion, three surfaces of interaction are identified within the two NRs: one mediated by their DBD, one by their C-terminal region and one involving their N- and C-terminal regions. In vitro pull-down assays confirmed the yeast two-hybrid data (see below), indicating that the interaction surfaces used are specific. Correlation between physical interaction and transcriptional cooperation between hERα and COUP-TFI We next identified the domains of COUP-TFI involved in the enhancement of hERα transcriptional activity to look for a relationship between physical interaction and transcriptional cooperation. This direct relationship is suggested first by the fact that the COUP-TFI C141S mutant is not able to enhance hERα activity (Figure 2D). The partial or total deletion of the COUP-TFI N-terminal region (57–423 and 87–423 constructs) does not affect the increase in hERα activity (Figure 2D). In contrast, deleting the COUP-TFI C-terminal region (57–153 protein) reduces the transcriptional cooperation by 60%. This points to the involvement of the COUP-TFI DBD and LBD in the transcriptional cooperation with hERα. This is demonstrated further by the hERα activity enhancement by the COUP-TFI DBD alone (residues 57–153) with hERα. In the case of the COUP-TFI LBD, we had to fuse the COUP-TFI 153–423 region to the SV40 nuclear localization signal (NLS; Figure 2D) to obtain a correct localization of the protein, as revealed by green fluorescent protein (GFP; not shown). Expression of this GFP–NLS/COUP-TFI 153–423 protein increases hERα-mediated transcription on the ERE-TK-Luc reporter by 2-fold. In conclusion, both an intact DBD and LBD are required for COUP-TFI to interact physically with hERα and to enhance its activity. Binding to DNA response elements specific for both NRs destabilizes the hERα–COUP-TFI complex The above results indicate that the transcriptional cooperation between hERα and COUP-TFI reflects their physical interaction. COUP-TFI can bind EREs (Klinge et al., 1997). One can thus postulate that the hERα–COUP-TFI complex might possess a higher affinity for EREs, compared with hERα homodimers, hence explaining the transcriptional cooperation. Electro phoretic mobility shift assays (EMSAs) were performed using various types of response elements, but in all cases we failed to detect a complex other than each of the NRs homodimers on DNA (data not shown). On the other hand, adding DNA-binding sites for COUP-TFI (DR-1) or ER (ERE), but not unspecific (AP1) sequence, destabilizes the hERα–COUP-TFI complex. As a control, the COUP-TFI–COUP-TFI 57–423 heterodimer remains unaffected by these DNA sequences (Figure 2E). These results suggest that the mechanisms that allow COUP-TFI to enhance hERα activity occur before hERα interacts with its target genes. Physical interaction with COUP-TFI increases hERα AF-1 activity and phosphorylation of the hERα N-terminal region In yeast cells, the hERα N-terminal AF-1 is required for the enhancement of hERα transcriptional activity by COUP-TFI (Petit et al., 1999). Moreover, deleting the hERα N-terminal region (hERα CF) abrogates the transcriptional cooperation between the two NRs in CHO-K1 cells (Figure 3A). We thus hypothesized that the actions of COUP-TFI might target specifically hERα AF-1. To test this assumption, the effect of COUP-TFI on hERα activity was checked in different cell lines, as it has been shown that the relative involvement of AF-1 and AF-2 in hERα transcriptional activity depends upon the cell type (Metzger et al., 1992). Cell contexts can thus be defined as AF-1 or AF-2 permissive, depending upon which AF is involved principally in hERα activity. The relative sensitivity of different cell lines to AF-1 and AF-2 was evaluated by comparing the transcriptional activity of full-length hERα with that of a mutant lacking AF-1 (hERα CF). A similar transcriptional activity of the two proteins would indicate AF-2-permissive cell contexts; no activity of the mutant would indicate AF-1-permissive contexts; and a reduced activity would indicate a mixed context permissive to both AF-1 and AF-2. Based on this classification, data obtained demonstrate that COUP-TFI increases hERα activity in AF-1-permissive cells (mixed and exclusive; Figure 3B). This was not due to cell-specific differences in COUP-TFI or hERα expression, as detected by western blots (see below). Figure 3.Cell-specific enhancement of hERα AF-1 by COUP-TFI. (A) Transfection of CHO-K1 cells with 0.2 basic reporter, internal control pCH110 and expression plasmids for COUP-TFI, full-length or N-terminally truncated hERα (hERα CF). (B) Nine cell lines exhibiting a differential sensitivity to ERα AFs were transfected with vectors encoding hERα alone or with COUP-TFI. Cells were treated with ethanol (EtOH) or 10−8 M estradiol (E2). Values are the fold induction of normalized luciferase reporter activity (mean ± SEM). Download figure Download PowerPoint A cell-specific formation of the hERα–COUP-TFI complex might be one basis for the cell-dependent action of COUP-TFI on hERα activity. However, co-immunoprecipitation experiments detect a protein complex containing the two NRs even in cells restricted to AF-2 activation (see below). Thus, physical interactions alone do not account for the effect of COUP-TFI on hERα activity in certain cells. AF-1-mediated transcription stimulation is strongly modulated by cell-specific phosphorylation events (Ali et al., 1993; Le Goff et al., 1994). We therefore postulated that phosphorylation could mediate the cell-specific effect of COUP-TFI on hERα AF-1. To obtain information about a direct impact of COUP-TFI interaction on the hERα phosphorylation state, we developed an in vitro assay in which proteins are first subjected to a GST pull-down procedure. This step is followed by in vitro phosphorylation using whole cell extracts (WCEs) as a donor of kinases. As shown above, two regions of COUP-TFI allow a transcriptional cooperation with hERα: the LBD (amino acids 153–423) and the DBD (amino acids 57–153). These regions were used in the phosphorylation assays, in conjunction with either GST–hERα B or GST–hERα BD domain fusion proteins, with which they can interact (Figure 4A). Data shown in Figure 4B illustrate a typical result obtained after in vitro phosphorylation of the protein complexes. We have shown previously that one of the consequences of the direct interaction between the hERα N- and C-terminal domains is an increase in hERα B domain phosphorylation (Métivier et al., 2001). This provided a positive control for COUP-TFI actions. The quantification of these assays performed with several WCEs as donors of kinases are shown in Figure 4C and D, and demonstrate that (i) COUP-TFI LBD and DBD enhance the phosphorylation of the hERα B or BD domains by 2- to 3.5-fold in a ligand-independent manner; and (ii) this increase occurs with WCEs from CHO-K1 and HepG2 cells (AF-1-permissive) but not from HeLa cells (AF-2-permissive). These actions of COUP-TFI on hERα B domain phosphorylation are specific, since (i) as previously reported, the interaction of hERα LBD with its own B domain requires E2 to affect AF-1 phosphorylation (Métivier et al., 2001); and (ii) hERα DBD interaction with the hERα BD domains does not reproduce the phosphorylation enhancement observed with COUP-TFI DBD. Figure 4.Cell-specific enhancement of hERα phosphorylation upon interaction with COUP-TFI. (A) GST pull-down experiments performed with 250 ng of the GST–hERα B or 150 ng of the GST–hERα BD with in vitro labeled COUP-TFI and hERα regions. Controls were made using 250 ng of GST alone, and inputs (I) are 20% of the labeled protein used. (B) In vitro phosphorylation assays were performed using similar amounts of the fusion proteins as in (A), with retained D–F (DF; 153–423) or C–D (DBD; 57–153) domains of hERα or COUP-TFI, in the presence of 5 × 10−6 M estradiol (E2) or ethanol (EtOH). Controls were made using an in vitro translation performed with empty pSG5 vector (Unpg Lys). Phosphorylation was ensured using 10 μCi of [γ-32P]ATP and 10 or 20 μg of CHO-K1 and HepG2 or HeLa WCEs, respectively. (C and D) Phosphoimager quantification of the in vitro phosphorylation assays performed using the same conditions as above. The values shown, normalized by the basal 32P incorporation detected with Unpg Lys, were reproducible (± 1%). Download figure Download PowerPoint COUP-TFI targets hERα Ser118 phosphorylation by MAPK To identify the amino acids within the hERα B domain targeted by this phosphorylation process, we conducted transfection experiments with hERα mutated at the known phosphorylated serines (Ali et al., 1993; Arnold et al., 1994; Le Goff et al., 1994; Rogatsky et al., 1999). COUP-TFI enhanced the activity of the receptors mutated at residues S167, S154, S104 and S106, but not that of S118A. Further, placing a negative charge at the 118 position (S118E) abrogated COUP-TFI action, but also enhanced the activity of the receptor to the same extent that COUP-TFI does on wild-type hERα (Figure 5A). These differences in transactivation are not due to an altered interaction of COUP-TFI with the two S118 mutants (Figure 5B). COUP-TFI is also unable to enhance the phosphorylation state of hERα domains mutated at residue S118 in vitro (Figure 5C). These results suggest that the basis of the transcriptional cooperation mechanism between hERα and COUP-TFI is the provision of a negative charge at the hERα S118 position, which is an essential residue for the hERα AF-1 activity. Figure 5.COUP-TFI interaction with hERα targets Ser118 phosphorylation. (A) CHO-K1 cells were transfected with the ERE-TK reporter, pCH110 and plasmids expressing hERα point mutated on each of the residues indicated. (B) GST pull-down assays using in vitro labeled wild-type or point-mutated hERα with 250 ng of each of the COUP-TFI fusion proteins or GST alone. Inputs are 20% of the labeled protein used. (C) Coupled GST pull-down/phosphorylation assays performed with GST fused to point-mutated hERα domains. Experiments used 10 μg of CHO-K1 WCE and in vitro translated hERα or COUP-TFI domains. Quantification of the radioactivity incorporation is shown as in Figure 4. Download figure Download PowerPoint S118 is a target of the mitogen-activated protein kinase (MAPK) pathway (Kato et al., 1995). We thus performed transfection experiments in the presence or absence of an inhibitor of this pathway, PD098059. Inhibiting the MAPK pathway markedly decreases COUP-TFI DBD- and LBD-mediated enhancement of hERα activity (Figure 6A). Western blots using an antibody specifically targeting the phosphorylated S118 were conducted to test the in vivo relevance of the phosphorylation processes (Figure 6B). In transfected HepG2 cells (AF-1-permissive), the amounts of hERα phosphorylated at S118 are increased after E2 treatment, as expected (Kato et al., 1995). Co-expressing COUP-TFI also induces this process, but in a ligand-independent manner. This COUP- TFI-mediated effect needs the formation of the hERα–COUP-TFI complex. Indeed, the COUP-TFI C141S mutant, which cannot interact with hERα (Figure 2), is unable to increase in vivo the S118 phosphorylation above the level found for ER in the absence of COUP-TFI. Treatment with PD098059 abolishes this COUP- TFI-mediated effect, as does S118 point mutation. In AF-2-permissive HeLa cells, the hERα S118 is not phosphorylated, and COUP-TFI has no effect. The results obtained using the PD098059 inhibitor indicated that the MAPKs are involved, either directly or indirectly, in the COUP-TFI-mediated processes that affect hERα phosphorylation and activity. This was analyzed further using a purified constitutively active ERK2/p42MAPK (Sigma) instead of WCE in the pull-down/in vitro phosphorylation assay. Phosphorylation of the hERα N-terminal region by ERK2 is not enhanced after interaction with any hERα domains in the presence of estradiol (Figure 6C). In contrast, COUP-TFI 153–423 and 57–153 regions increase the phosphorylation state of the hERα B and BD domains by 6- or 14-fold, respectively, in a ligand-independent manner. Together with previous results indicating a requirement for an AF-1-permissive cell context, these data demonstrate that a phosphorylation process involving MAPK pathways is the key for the cell-specific enhancement of hERα transcriptional activity by COUP-TFI. Figure 6.In vivo enhancement of hERα activity by COUP-TFI involves the MAPK pathway. (A) hERα was expressed alone or in combination with COUP-TFI constructs in CHO-K1 cells, which were treated subsequently with 10 μM of the ERK inhibitor PD098059. Normalized luciferase activities are shown as the mean ± SEM. (B) WCEs from HepG2 or HeLa transfected cells were subjected to western blots using the antibodies indicated on the left. (C) A 25 ng aliquot of purified MAPK was used in the GST pull-down/in vitro phosphorylation assays using 250 or 150 ng of the GST–hERα B or BD fusion proteins, respectively, with either the DF or CD domains of hERα or COUP-TFI. 32P incorporation was measured and expressed as in Figure 4. Download figure Download PowerPoint hERα hyperphosphorylation induced by COUP-TFI involves an enhanced recruitment of MAPK The connection between the enhanced phosphorylation state and an increased targeting of ERK2 to hERα through interaction with COUP-TFI subsequently was tested. As illustrated in Figure 7A, a His-tagged ERK2 protein binds to hERα and COUP-TFI in a ligand-independent manner. This interaction requires the in vitro phosphorylation of the bacterially expressed ERK2 by CHO-K1 WCE. In contrast, phosphorylation by HeLa WCE allows a 10- to 20-fold less efficient association of ERK2 with both NRs, although ERK2 has been phosphorylated to the same extent by both WCEs (Figure 7A, right). Co-immunoprecipitation experiments (Figure 7B) demonstrate this enhanced recruitment in vivo. COUP-TFI does not enhance the level of phosphorylated ERK2, as revealed by western blot using a specific antibody against the phosphorylated form of ERK2. COUP-TFI increases the amounts of activated ERK2 present in the hERα immunoprecipitated complex from HepG2 but not HeLa cells. This requires the physical interaction between the two NRs, since the COUP-TFI C141S mutant does not allow this increase in ERK2 recruitment. Altogether, these data demonstrate that the molecular basis of the positive hERα–COUP-TFI interplay is an enhanced recruitment of ERK2. Figure 7.Interaction with COUP-TFI enhances ERK2/p42MAPK recruitment by hERα. (A) In vitro pull-down experiments were performed using 2 μl of the in vitro labeled proteins indicated and 500 ng of the His-tagged ERK2 previously phosphorylated by 10 or 20 μg of CHO-K1 or HeLa WCE, respectively. Controls (C) were run using ion resin previously incubated with bacterial extract expressing no fusion proteins. Inputs (I) are 25% of the proteins used. A radioactive in vitro phosphorylation assay is shown on the right, revealing similar activation