Multiple Phosphorylation Events Control Chicken Ovalbumin Upstream Promoter Transcription Factor I Orphan Nuclear Receptor Activity
Frédérique Gay1,
Peter Baráth,
Christine Desbois-Le Péron,
Raphaël Métivier2,
Rémy Le Guével,
Darcy Birse and
Gilles Salbert
Equipe Information et Programmation Cellulaire (F.G., C.D.-L.P., R.L.G., G.S.) et Equipe Endocrinologie Moléculaire de la Reproduction (R.M.), Unité Mixte de Recherche 6026 Centre National de la Recherche Scientifique, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France; Department of Biochemistry and Biophysics (P.B., D.B.), Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm SE-106 91, Sweden
Address all correspondence and requests for reprints to: Dr. Gilles Salbert, Equipe Information et Programmation Cellulaire, Unité Mixte de Recherche 6026 Centre National de la Recherche Scientifique, Université de Rennes I, Campus de Beaulieu, 35042 Rennes cedex, France. E-mail: gilles.salbert{at}univ-rennes1.fr.
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ABSTRACT
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Chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) is an orphan member of the nuclear hormone receptor superfamily that comprises key regulators of many biological functions, such as embryonic development, metabolism, homeostasis, and reproduction. Although COUP-TFI can both actively silence gene transcription and antagonize the functions of various other nuclear receptors, the COUP-TFI orphan receptor also acts as a transcriptional activator in certain contexts. Moreover, COUP-TFI has recently been shown to serve as an accessory factor for some ligand-bound nuclear receptors, suggesting that it may modulate, both negatively and positively, a wide range of hormonal responses. In the absence of any identified cognate ligand, the mechanisms involved in the regulation of COUP-TFI activity remain unclear. The elucidation of several putative phosphorylation sites for MAPKs, PKC, and casein kinase II within the sequence of this orphan receptor led us to investigate phosphorylation events regulating the various COUP-TFI functions. After showing that COUP-TFI is phosphorylated in vivo, we provide evidence that in vivo inhibition of either MAPK or PKC signaling pathway leads to a specific and pronounced decrease in COUP-TFI-dependent transcriptional activation of the vitronectin gene promoter. Focusing on the molecular mechanisms underlying the MAPK- and PKC-mediated regulation of COUP-TFI activity, we show that COUP-TFI can be directly targeted by PKC and MAPK. These phosphorylation events differentially modulate COUP-TFI functions: PKC-mediated phosphorylation enhances COUP-TFI affinity for DNA and MAPK-mediated phosphorylation positively regulates the transactivation function of COUP-TFI, possibly through enhancing specific coactivator recruitment. These data provide evidence that COUP-TFI is likely to integrate distinct signaling pathways and raise the possibility that multiple extracellular signals influence biological processes controlled by COUP-TFI.
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INTRODUCTION
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THE NUCLEAR RECEPTOR superfamily encompasses many ligand-activated transcription factors in addition to also a large number of proteins whose ligands are unknown and which are thus termed orphan receptors (1, 2). These receptors play crucial roles both as transcriptional activators and repressors, and are regulators of a wide range of biological functions including development, metabolism and reproduction. Among nuclear orphan receptors, chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are encoded by distinct genes sharing an exceptionally high degree of sequence homology (3, 4, 5, 6, 7, 8, 9, 10). The expression patterns of COUP-TFs have been extensively studied in several divergent species and appear to be relatively similar, suggesting a conservation of COUP-TF function throughout evolution. Although each COUP-TF has its own distinct expression pattern during embryonic development and in adult mice, these patterns are often overlapping and both genes are widely expressed in the developing central nervous system (8, 11, 12, 13). These findings suggest a functional redundancy among COUP-TFs, which are classically considered to act as key factors during neurogenesis. Overexpression of COUP-TFs and gene disruption experiments performed in Drosophila, Xenopus, and mouse, define a major role of these orphan receptors in the formation of the anterior brain in tetrapods, in the patterning of the eye in the fly, and in the modulation of axogenesis and cranial neural crest cell behavior in mammals, respectively (3, 9, 14, 15, 16). The recent finding that COUP-TFI function is required for cortical layer IV cell survival through correct guidance and connection of axons from mouse thalamic neurons supports the hypothesis that COUP-TFI functions in the transcriptional control of axon guidance cues (17). Aside from neuronal expression patterns, COUP-TFs show overlapping, yet distinct, expression patterns in many tissues during development, suggesting their involvement in organogenesis (8, 13). Consistent with a role for these nuclear receptors accessory to neurogenesis, recent data show that COUP-TFII is involved in angiogenesis, vascular remodeling, and heart development (16, 18).
Initial studies identified COUP-TFs as transcriptional activators (19, 20, 21, 22); nevertheless, COUP-TFs can repress the transcriptional activation mediated by a number of ligand-bound nuclear receptors, including VDR, TRs, RXRs, and RARs (23, 24, 25, 26). A functional cross-talk between COUP-TFs and retinoid-signaling pathways during development has been proposed, based on the comprehensively documented antagonistic effects of COUP-TFs on retinoid-dependent gene expression, and the observation that COUP-TF genes are themselves targeted by retinoids (7, 8, 15, 27, 28). However, recent data indicate that the orphan receptors can also serve as accessory factors for some ligand-bound nuclear receptors such as GR, ER
, and RAR
(29, 30, 31). In some specific promoter contexts, COUP-TFs are thus able to enhance the ligand-induced transactivation function of these various receptors, suggesting that COUP-TFs may be required for effectively establishing the cues for certain genetic responses after exposure of the cell to a ligand. In this respect, it is not surprising that common phenotypes, such as similar bone fusions, are generated in COUP-TFI-/- and RAR
1-/-/RARß-/- independent knockout experiments (16, 32). The recent finding that COUP-TFI is required for the induction of growth inhibition and apoptosis by retinoic acid in cancer cells further substantiates a positive interaction between COUP-TFs and retinoid receptors in vivo (31).
With very few exceptions, most ligand-dependent nuclear receptors can be activated in a ligand-independent manner through phosphorylation events (33). As an example, ER
can activate transcription after phosphorylation, independent of supplemented E2 (33, 34, 35, 36, 37). In a similar manner, many other ligand-regulated nuclear receptors are either activated or repressed through phosphorylation events (33, 34), and these regulatory mechanisms seem relevant also to the orphan receptors such as Nur77/NGFI-B, HNF-4 or ROR
(38, 39, 40, 41). Although COUP-TFs were cloned more than 10 yr ago, a ligand has yet to be elucidated or characterized, suggesting that COUP-TFs could be true orphan nuclear receptors. The presence of several putative phosphorylation sites for MAPKs, PKC, and casein kinase II (CKII), within its sequence led us to postulate that COUP-TFI could be targeted by cellular kinases. In this report, we demonstrate that COUP-TFI can be directly phosphorylated by PKC and MAPK, these phosphorylation events differently modulating COUP-TFI functions. COUP-TFI is thus likely to be regulated by extracellular signals transduced through distinct phosphorylation pathways.
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RESULTS
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Two Nuclear Receptor Half-Binding Sites within the Vitronectin (Vn) Gene Promoter Mediate Transcriptional Activation by the Orphan Nuclear Receptor COUP-TFI
Previously, we have shown that COUP-TFI can transactivate the mouse Vn gene promoter in P19 embryonal carcinoma cells (42). Figure 1
demonstrates that the ability of COUP-TFI to activate this promoter is not restricted to P19 cells considering the observation of the similar induction in COS-7 cells (Fig. 1A
). To delineate the promoter sequences involved in this phenomenon as well as the mode of activation by COUP-TFI, we mutated individually or in combination the two consensus (TGACCC) half-binding sites for nuclear receptors present in the mouse promoter referred to as NR1 and NR2 (Fig. 1B
). Whereas the NR2 half-site could generate a direct repeat with a 3-bp spacer (DR-3) when combined to the degenerated TGACTT sequence located downstream of the NR2 site, no TGACCC-related sequence could be found in the vicinity of the NR1 sequence (Fig. 1B
). Thereby, both consensus half-sites could organize a DR-66 element stably interacting with COUP-TFI dimers. When cotransfected in COS-7 cells with a COUP-TFI expression vector, the promoter in which the NR1 site was mutated was activated at a much lower level compared with its wt counterpart (Fig. 1C
). Mutation of the NR2 site moderately reduced COUP-TFI-induced activation (Fig. 1C
). The double NR1/NR2 mutant did not show a greater reduction of COUP-TFI-induced activity compared with the single NR1 mutant (Fig. 1C
). These data strongly suggest that the NR1 site prevails over the NR2 site for COUP-TFI activation of the Vn promoter in COS-7 cells. Because COS cells are known to overproduce proteins encoded by transfected vectors, the relatively high amounts of COUP-TFI expressed in COS-7 cells could have attenuated the impact of the mutations introduced in the Vn gene promoter. Based on this observation, we transfected P19 cells with the different mutants to verify their ability to be activated by COUP-TFI (Fig. 1C
). In this cellular context, both NR1 and NR2 mutations reduced COUP-TFI activation by 50%. When combined, the NR1 and NR2 mutations resulted in a loss of COUP-TFI activation by 78% (Fig. 1C
), suggesting that binding to both sites is required for COUP-TFI transactivation in P19 cells. Consistent with the transfection data indicating that the NR1 site is involved in the Vn gene promoter regulation, we observed that COUP-TFI could effectively interact as a dimer (as suggested by the position of the protein/DNA complex) with the NR1 half-site in EMSA experiments (Fig. 1D
). Although COUP-TFI was able to interact with the NR1-containing oligonucleotide, it conferred low affinity for this element judged by the observation that cold NR1 is a poor competitor compared with the high affinity DR1 binding element (Fig. 1D
). These experiments clearly indicate that COUP-TFI could transactivate the mouse Vn gene promoter through direct binding to at least one low affinity site including the NR1 sequence, and possibly to a second site composed of the NR2 element. However, the residual activation by COUP-TFI observed with the double mutant suggests that other undetected binding-sites could be recognized by the orphan receptor, or that part of the COUP-TFI activity could be unrelated to DNA binding as previously shown in other contexts (43).

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Figure 1. The COUP-TFI Orphan Receptor Activates the Mouse Vn Gene Promoter through Direct Binding to a Consensus Half-Site
A, Results of a representative experiment run in COS-7 cells cotransfected with the wt Vn promoter linked to the Luc reporter gene and increasing amounts (6.25 to 50 ng) of COUP-TFI expression vector. Results are shown as the means ± SEM (n = 4) of Luc values corrected by ß-galactosidase activities. B, The sequence of the mouse Vn gene proximal promoter. Putative binding-sites for transcription factors are boxed. The transcription initiation site is noted +1, and the changes introduced in the promoter by site-directed mutagenesis are shown in bold letters underneath the wt sequence. C, Transient transfection analysis of the mutated promoters. COS-7 and P19 cells were cotransfected with the indicated recombinant Vn-Luc reporter plasmids and 25 ng of pcDNA3-hCOUP-TFI expression vector. The induction obtained with the wt promoter in the presence vs. in the absence of COUP-TFI expression vector was set to 100% of activation. Inductions obtained with the various mutated promoters are expressed as % of the activation obtained with the wt promoter. Results are shown as the mean ± SEM of 12 (COS-7 cells) or 6 (P19 cells) values obtained in three and two independent experiments, respectively. EMSA analysis of COUP-TFI binding to the NR1 element is shown in panel D. WCEs from COS-7 cells transfected with empty pcDNA3 (first lane) or pcDNA3-hCOUP-TFI (other lanes) were incubated with the 32P-labeled double-stranded NR1 oligonucleotide and the complexes were resolved by nondenaturing electrophoresis. The position of the specific COUP-TFI/NR1 complex, as assessed by supershifting analysis with the anti-hCOUP-TFI antibody (Ab), is indicated by an arrowhead. Nonspecific bands are indicated by asterisks. To estimate the affinity of COUP-TFI for the NR1 sequence, increasing amounts (2- to 10-fold molar excess) of NR1, DR1, and AP1 cold competitor oligonucleotides were added.
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Prediction of Multiple Putative Phosphorylation Sites for Distinct Signaling Pathways within the Human COUP-TFI Sequence and in Vivo Phosphorylation of COUP-TFI
As shown in Fig. 2A
, in silico analysis of the human COUP-TFI protein sequence revealed several putative phosphorylation sites targeted by distinct cellular kinases, namely CKII, PKC, and proline-directed kinases such as MAPKs. The consensus phosphorylation sites were defined as S/TXXD/E for CKII (44), S/TXR/K for PKC (45), and PX1-2S/TP for the three distinct MAPK pathways identified in mammalian cells (46). These pathways include members of the extracellular signal-regulated kinases ERK1/p44MAPK and ERK2/p42MAPK, the Jun NH2-terminal kinase and the p38 kinase (47, 48, 49). The three putative MAPK phosphorylation sites identified within the human COUP-TFI sequence were found to be highly conserved throughout evolution (8), and were clustered in the amino-terminal A/B domain (Fig. 2A
). The putative CKII and PKC phosphorylation sites, some of which were also evolutionarily conserved (8), were in contrast distributed throughout the COUP-TFI sequence (Fig. 2A
). The DNA binding domain (DBD) contained exclusively PKC target sites, whereas the A/B and the putative ligand binding domain (LBD) of the receptor displayed both CKII and PKC potential target sequences (Fig. 2A
). The presence of several putative phosphorylation sites for various cellular kinases in the human COUP-TFI sequence raises the possibility that, similar to posttranslational modifications described for several nuclear receptors, COUP-TFI could be a phosphoprotein in vivo, whose activity may be regulated by specific phosphorylation events. To test this hypothesis, we cloned the human COUP-TFI cDNA encoding residues 57 to 423 in frame with the glutathione-S-transferase (GST) coding sequence of the mammalian expression vector pBC/GST. After mutation of the PKA site present in the GST sequence, the recombinant vector was transfected in COS-7 cells. 32P-Labeled orthophosphate was then added to label phosphoproteins in vivo. Cell extracts were purified on glutathione-agarose beads and analyzed in SDS-PAGE. The positions of GST and GST::COUP-TFI were determined by western blotting with an anti-GST antibody (data not shown). Results indicate that both GST and GST::COUP-TFI had been labeled in vivo (Fig. 2B
). Although a slight incorporation of 32P was observed for GST, the GST::COUP-TFI fusion protein was approximately 90 times more radioactive than free GST, indicating that COUP-TFI is indeed a phosphoprotein in vivo.

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Figure 2. The Human COUP-TFI Sequence Contains Multiple Putative Phosphorylation Sites and Is Phosphorylated in Vivo
A, Positions of potential target sites for CKII (black arrowheads), PKC (open arrowheads), and MAPK (gray arrowheads) identified within the sequence of the human COUP-TFI receptor. B, In vivo phosphorylation of COUP-TFI. COS-7 cells were transfected with pBC/GST or pBC/GST-COUP-TFI 57423 and treated with 32P orthophosphate. Proteins were extracted and purified on glutathione-agarose beads before SDS-PAGE. a, Autoradiogram; b, Coomassie Brilliant blue staining of the gel. The positions of GST and GST::COUP-TFI were assessed by Western blot with anti-GST antibodies (data not shown). The autoradiographic signals as well as the protein levels were quantified to estimate the relative specific activity of the labeled proteins. These activities are expressed as OD arbitrary unit per picomole of protein.
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Multiple Phosphorylation Pathways Regulate COUP-TFI-Mediated Transcriptional Activation of the Vn Gene Promoter
The functional relevance of phosphorylation events for the regulation of COUP-TFI activity was first assessed by using specific inhibitors for the three above described kinases in transfection experiments (Fig. 3
). COS-7 cells were cotransfected with the COUP-TFI-dependent reporter gene Vn-luciferase (Luc) (containing the wt Vn gene promoter sequence, see Fig. 1
), in the presence or absence of increasing amounts of human COUP-TFI expression vector. Cells were treated, or not treated, with DRB (5-6-dichloro-1-ß-D ribofuranosyl benzimidazole), a CKII inhibitor (Fig. 3A
), or the p42MAPK/p44MAPK signaling pathway-specific inhibitor PD980059, or the PKC inhibitor GF 109203X (Fig. 3B
). Consistent with our previous results, cotransfection with increasing amounts of COUP-TFI expression plasmid led to a dose-dependent induction of reporter gene activity. Neither the basal nor the stimulated transcriptional activity of the reporter gene was modified by treatment of the cells with DRB (Fig. 3A
). The expression level of COUP-TFI was also unaffected by this treatment (data not shown, see Fig. 5A
). Based on these experiments defining that inhibition of the CKII pathway in vivo does not affect COUP-TFI-mediated transcriptional activation, it seems unlikely that this kinase regulates COUP-TFI function. Whereas basal activity was not altered, treatment of the cells with PD980059 led to a significant decrease (76% inhibition of the activation) in reporter gene activity observed in the presence of 50 ng of COUP-TFI expression vector (Fig. 3B
). As assessed by Western blotting, this inhibition was not due to a decrease in nuclear receptor expression (data not shown, see Fig. 5B
). The inhibitory effect of the specific MAPK inhibitor on the induction of the Vn gene promoter by COUP-TFI was not restricted to COS-7 cells as similar results were obtained in experiments utilizing P19 cells (data not shown). As shown in Fig. 3B
, treating cotransfected COS-7 cells with GF 109203X in the absence of expression vector did not influence basal activity. In contrast, GF 109203X induced a marked reduction (60% inhibition of the activation) in the reporter gene activity observed in the presence of 50 ng of COUP-TFI expression vector (Fig. 3B
). Concurrent with the previous observation, the inhibition of COUP-TFI- induced reporter gene activity was not due to a reduced expression of COUP-TFI in COS-7 cells treated with GF 109203X (data not shown, see Fig. 5C
). Thus, the blocking of either the p42MAPK/p44MAPK or the PKC signaling pathway results in a strong inhibition of the COUP-TFI-dependent transcription, suggesting that COUP-TFI function may be positively regulated by these kinases in vivo.

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Figure 3. Specific Inhibitors of MAPK and PKC Decrease COUP-TFI-Mediated Transcription
A and B, Effects of specific inhibitors of CKII (DRB, panel A), MAPK (PD980059, panel B) and PKC (GF 109203X, panel B) on the COUP-TFI-mediated transcriptional activation of the Vn gene promoter. COS-7 cells were cotransfected with the Vn-Luc reporter gene in the absence or presence of increasing amounts (6.25 to 50 ng) of the indicated pcDNA3-hCOUP-TFI (panels A and B), pcDNA3-hCOUP-TFI 57423 (panel B), or pcDNA3-hCOUP-TFI 84423 (panel B) expression vectors. Kinase inhibitors were added for 4 h before the cells were harvested. Results are expressed as fold inductions of the reporter gene activities in the presence of the various expression vectors compared with the activity in the absence of any expression vector. Results are shown as the means ± SEM of three values obtained in a representative experiment. The expression level of the various proteins in the transfected COS-7 cells was assessed by Western blotting (B, right panel). The positions of the specific band corresponding to the COUP-TFI, COUP-TFI 57423, and COUP-TFI 84423 proteins expressed in COS-7 cells are indicated by a black, gray, and open arrowhead respectively, the asterisk indicating a nonspecific band (Fig. 3B , right panel).
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Figure 5. The Selective Inhibitor of PKC Specifically Decreases COUP-TFI Binding to DNA in a Dose-Dependent Manner
The potential effects of inhibitors of CKII (DRB, panel A), MAPK (PD980059, panel B) and PKC (GF 109203X, panel C) on COUP-TFI DNA binding were assessed by EMSA. COS-7 cells were untransfected (lanes 1) or transfected with pcDNA3-hCOUP-TFI expression vector (lanes 26) and treated in the absence or presence of increasing amounts of the indicated kinase inhibitors. WCEs were then incubated with the 32P-labeled double-stranded NR1 oligonucleotide. The position of the specific COUP-TFI/NR1 complex (as assessed by supershifting analysis with the anti-hCOUP-TFI antibody, lanes 3) is indicated by an arrowhead, whereas nonspecific bands are indicated by asterisks. The expression level of COUP-TFI in the WCEs used for EMSA was controlled by Western blotting (lower panels) with the anti-HA antibody. The position of COUP-TFI receptor is indicated by an arrowhead, whereas nonspecific bands are indicated by asterisks.
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To determine the functional requirement of the phosphorylation-mediated regulation of COUP-TFI activity, similar cotransfection experiments were performed using expression vectors encoding truncated COUP-TFI receptors (Fig. 3B
). The COUP-TFI 57423 protein is truncated at the amino-terminal region of the A/B domain, lacking a putative CKII and PKC phosphorylation sequence (S7), as well as two amino-terminal (T51 and T54) MAPK phosphorylation sites. In addition to these four sites, the COUP-TFI 84423 protein, which is deleted of almost the entire A/B domain (86 first amino acids), is devoid of the third putative MAPK phosphorylation site (T63) and of a CKII phosphorylation site (T66). Western blotting showed that all the three constructs were efficiently expressed in COS-7 and displayed similar expression levels (Fig. 3B
, right panel). Independent of the amount of expression vector used, COUP-TFI 57423 was able to stimulate the reporter gene activity to a similar extent to that observed with the equivalent dose of wild-type (wt) COUP-TFI expression vector (Fig. 3B
). Moreover, both PD980059 and GF 109203X efficiently counteracted the reporter gene activation mediated by 50 ng of COUP-TFI 57423 expression vector. This observation suggests that the region spanning residues 1 to 56 of COUP-TFI, including one PKC and two MAPK potential phosphorylation sites, is dispensable for transactivation and is not involved in the regulation of COUP-TFI activity by the PKC and MAPK pathways. Cotransfection of COS-7 cells with increasing amounts of COUP-TFI 84423 expression vector resulted in a weak stimulation of the reporter gene (Fig. 3B
). Experiments run in P19 cells gave similar results (data not shown). These data suggest that residues 57 to 83 play a crucial role, directly or indirectly, in the COUP-TFI-mediated transactivation, and raise the possibility that the third (T63) MAPK phosphorylation site could be a hierarchal requisite for COUP-TFI activity and for the MAPK-mediated regulation of the nuclear receptor functions. This was, to some extent, confirmed by the observation that PD980059 is significantly less efficient in inhibiting the COUP-TFI 84423-mediated transcriptional activation than it is in decreasing COUP-TFI 57423-induced activation (Fig. 3B
). Indeed, the inhibition of COUP-TFI 84423 and COUP-TFI 57423 activity due to PD980059 was 58% and 89%, respectively. The residual effect of PD980059 on COUP-TFI 84423-mediated transactivation suggests the involvement of an additional nonconsensus MAPK phosphorylation site located between residues 84 to 423, or that MAPK indirectly modulates COUP-TFI activity. Finally, in cells cotransfected with 50 ng of COUP-TFI 84423 expression vector, treatment with GF 109203X reduced the reporter gene activity to the basal level (Fig. 3B
). This last result implies that the potential phosphorylation site(s) targeted by PKC are located outside of the A/B domain (i.e. between residues 84 and 423). All together, these findings strongly suggest that COUP-TFI activity is positively regulated by phosphorylation events in vivo, events that may be mediated by the p42MAPK/p44MAPK and PKC pathways.
In Vitro Dephosphorylation Inhibits COUP-TFI Binding to Its DNA Target Sequences
In an attempt to identify the functions of COUP-TFI that are controlled by phosphorylation events, we first focused on DNA binding activity, and investigated whether in vitro dephosphorylation of COUP-TFI could affect its ability to bind DNA elements (Fig. 4
, A and B). In this study, COUP-TFI was expressed in COS-7 cells and whole-cell extracts (WCEs) from either untransfected or COUP-TFI-expressing cells were treated in vitro with calf intestinal phosphatase (CIP). As shown in Fig. 4A
, incubation of untreated WCEs from COUP-TFI-expressing cells with labeled double-stranded DR1 oligonucleotide led to the formation of a complex species shifted in EMSA experiments as a single band (indicated by an arrowhead). Supershifting experiments with anti-COUP-TFI antibody reduced the electrophoretic mobility of the retarded complex, confirming the presence of COUP-TFI in this complex. In vitro treatment of the WCEs prepared from COUP-TFI-expressing cells with CIP buffer alone or CIP buffer and phosphate had no effect on the intensity of the shifted band, showing that neither of the compounds affected the binding of COUP-TFI to DNA elements. In contrast, treatment of WCEs with CIP strongly decreased the intensity of the shifted band, providing evidence for a CIP-mediated decrease in the formation of the COUP-TFI/DR1 complex. The hypothesis that CIP-mediated dephosphorylation of COUP-TFI inhibited nuclear receptor binding to specific DNA elements was further substantiated by the observation that addition of phosphate to the dephosphorylation mixture abolished the effect of the CIP.

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Figure 4. Dephosphorylation of COUP-TFI Inhibits Its Ability to Bind DNA
Effects of the in vitro treatment with CIP on COUP-TFI binding to DNA were assessed by EMSA. Equivalent amounts (2 µg) of WCEs from untransfected (lanes 1) or COUP-TFI-expressing COS-7 cells (lanes 27) were incubated with CIP buffer (lanes 47), in the absence (lanes 4 and 5) or presence (lanes 6 and 7) of CIP (2.5 U) and of a competitive inhibitor of CIP (NaPO4, lanes 5 and 7). Control incubations (lanes 1 to 3) were performed in the absence of CIP buffer. The samples were then incubated with the 32P-labeled double-stranded DR1 or NR1 oligonucleotides indicated at the bottom of the gels. The position of the specific retarded complex (as assessed by supershifting analysis with the anti-hCOUP-TFI antibody, lanes 3) is indicated by an arrowhead, whereas nonspecific bands are indicated by asterisks.
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Similar gel shift experiments were performed using the most proximal half binding site for nuclear receptors (NR1) identified within the Vn gene promoter as a probe (Fig. 4B
), or other COUP-TF recognition sequences such as EREs (data not shown). Dephosphorylation of COUP-TFI-containing cell extracts systematically reduced COUP-TFI binding to these various response elements (Fig. 4B
and data not shown). The CIP-mediated inhibition of DNA binding was also observed with COUP-TFI 84423 (data not shown). These data suggest that COUP-TFI specific DNA binding activity can be directly and positively regulated by phosphorylation events in vivo.
In Vivo Inhibition of the PKC Signaling Pathway Specifically Decreases the DNA Binding Activity of COUP-TFI
The potential effects of CKII, MAPK, and PKC kinase inhibitors on COUP-TFI specific DNA binding function were assessed by EMSA experiments (Fig. 5
). COS-7 cells were transfected with COUP-TFI expression vector, and subsequently treated or untreated with increasing amounts of the different kinase inhibitors described above. WCEs from untransfected or COUP-TFI-expressing cells were then incubated with the labeled double-stranded NR1 oligonucleotide. Neither the vehicle alone nor any of the tested concentrations of DRB significantly modified the intensity of the specific shifted band (Fig. 5A
). As assessed by Western blotting, the amounts of DRB did not affect the expression of COUP-TFI in COS-7 cells (Fig. 5A
, lower panel). Similar results were obtained using the selective p42MAPK/p44MAPK inhibitor PD980059 (Fig. 5B
). Conversely, treating the transfected cells with the selective PKC inhibitor GF 109203X did influence binding of COUP-TFI to specific DNA elements (Fig. 5C
). Indeed, treatment of the cells with increasing amounts of PKC inhibitor led to a pronounced and gradual decrease in the intensity of the specific shifted band (Fig. 5C
). This reduction of COUP-TFI binding to DNA was not due to a decreased expression of the nuclear receptor in the treated cells, as revealed by Western blotting (Fig. 5C
, lower panel). Similar results were obtained with the three kinase inhibitors using the DR1 oligonucleotide as a probe (data not shown). These observations clearly indicate that the specific inhibition of the PKC pathway significantly reduces COUP-TFI binding to distinct response elements, raising the possibility that PKC can positively regulate the DNA binding activity of COUP-TFI in vivo.
PKC Phosphorylates COUP-TFI in Vitro and Enhances Its Binding Affinity for DNA
To support the previous observations, we wanted to determine if PKC could directly target COUP-TFI and subsequently regulate its binding to DNA. The ability of purified PKC to phosphorylate COUP-TFI in vitro was assessed by in vitro phosphorylation assays performed with bacterially expressed COUP-TFI (Fig. 6A
). The GST::COUP-TFI 57423 fusion protein was incubated in the absence or presence of increasing amounts of purified PKC. In all reactions,
-32P ATP was used as a donor of phosphate and PKC-mediated phosphorylation was monitored by the incorporation of 32P. As expected, no 32P incorporation was detected when GST::COUP-TFI 57423 was treated in the absence of purified kinase. In contrast, incubation of GST::COUP-TFI 57423 with increasing amounts of PKC allowed a dose-dependent incorporation of 32P, leading to the gradual appearance of a band of the expected molecular weight (indicated by an arrowhead). Because the GST moiety was not phosphorylated by purified PKC (data not shown, see Fig. 7B
), these data show that PKC directly and specifically phosphorylates COUP-TFI at one or more target sites located between residues 57 and 423 in vitro.

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Figure 7. The Modulation of COUP-TFI DNA Binding by PKC Is a Direct Mechanism and May Involve Phosphorylation of the DBD
Panel A shows SDS-PAGE analysis of the 35S methionine-labeled in vitro translated full-length COUP-TFI retained by the unphosphorylated or in vitro phosphorylated GST and GST::COUP-TFI 57423 fusion protein. The input lane (lane 1) represents half of the amount of 35S methionine-labeled COUP-TFI protein used for the pull-down assay. Glutathione-agarose beads containing 125 ng of bacterially expressed GST (lanes 2 and 3) or GST::COUP-TFI 57423 (lanes 4 and 5) fusion proteins were incubated with cold ATP, in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of 12.5 ng of purified PKC before the pull-down protocol. Panel B shows in vitro phosphorylation of bacterially expressed truncated COUP-TFI proteins by purified PKC, monitored by the incorporation of 32P. Glutathione-agarose beads containing 250 ng of bacterially expressed GST (lane 1), GST::COUP-TFI 157 (lane 2), GST::COUP-TFI 57153 (lane 3), or GST::COUP-TFI 153423 (lanes 4) fusion proteins were incubated in the presence of 12.5 ng of purified PKC and -32P ATP (5 µCi). The samples were analyzed by SDS-PAGE. The positions of the phosphorylated GST::COUP-TFI 157 and GST::COUP-TFI 57153 fusion proteins are indicated by an open and a gray arrowhead, respectively. In panel C, MALDI-MS spectra obtained after tryptic digestion of unphosphorylated (upper spectra) or PKC-phosphorylated GST::COUP-TF 57423 (lower spectra) are shown. The peaks correponding to the tryptic peptides 116-NLTYTCR-124 and 116-NLTYpTCR-124 are indicated by arrows. Note that the spectrum on the upper right does not correspond to any tryptic peptide from the unphosphorylated sample and is shown for comparison with the lower right spectrum.
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In an attempt to confirm that PKC positively regulates COUP-TFI binding to DNA, in vitro phosphorylated GST::COUP-TFI 57423 fusion protein was subsequently submitted to EMSA experiments (Fig. 6
, B and C). In the first set of experiments, increasing amounts of unphosphorylated or in vitro phosphorylated GST::COUP-TFI 57423 fusion protein were incubated with labeled double-stranded DR1 oligonucleotides (Fig. 6B
). Independent of the amount of GST::COUP-TFI 57423 fusion protein used, the intensity of the specific shifted band was consistently higher for the phosphorylated protein than for its unphosphorylated counterpart (Fig. 6B
, upper panel). The intensity of the shifted band was quantified by phosphoimaging and normalized with that of the corresponding free probe. Results were expressed in % of bound/free probe, with the binding of 20 ng of phosphorylated GST::COUP-TFI 57423 fusion protein to the DR1 oligonucleotide being normalized to 100%. This analysis confirmed that the DNA binding ability of the phosphorylated fusion protein was more than 2-fold greater than that of the unphosphorylated protein (Fig. 6B
, lower panel).
Because these data suggested that phosphorylation of COUP-TFI modulated the affinity of the receptor for DNA, we designed gel shift experiments to calculate the dissociation constant (Kd) of PKC-phosphorylated vs. unphosphorylated COUP-TFI 57423 association with DNA. Ten nanograms of recombinant protein were incubated with increasing amounts of DR-1 probe (Fig. 6C
). After electrophoresis, the bound probe and the free probe were quantified with a phosphoimager using Fujifilm FLA-300 and Image Gauge 3.45 software. The ratio of bound/free probes was plotted against the bound probe concentration, allowing the calculation of the Kd for COUP-TFI/DNA interactions. Linear transformation clearly revealed the existence of two different protein species that differed in their ability to bind DNA in the PKC-modified sample, thus generating a kink in the curve. In the specific experiment shown in Fig. 6C
, unphosphorylated COUP-TFI had a Kd value of 1.8 nM, and PKC-modified COUP-TFI had Kd values of 0.87 and 2.0 nM, suggesting that when the phosphorylated form of COUP-TF interacts with DNA, it binds with the lowest Kd value. Such experiments were replicated several times with different preparations of GST::COUP-TFI 57423 protein. The average Kd calculated from these experiments was 2.23 ± 0.33 nM (n = 6) for unphosphorylated COUP-TI and 0.724 ± 0.1 nM (n = 5) for PKC-phosphorylated COUP-TI. These data clearly demonstrate that COUP-TFI can be directly targeted by PKC in vitro, and that PKC-mediated phosphorylation enhances the affinity of the nuclear receptor for DNA by approximately 3-fold.
Enhancement of COUP-TFI DNA Binding Activity by PKC Is a Direct Mechanism that Is Likely to Be Mediated by Phosphorylation within the DBD
Having demonstrated that COUP-TFI 57423 is a substrate for PKC in vitro and that phosphorylation enhances the binding of the nuclear receptor to DNA, it was of interest to determine whether the PKC-mediated phosphorylation events affected DNA binding function directly or indirectly (i.e. via the dimerization function). The potential effects of PKC-mediated phosphorylation on the ability of the GST::COUP-TFI 57423 fusion protein to retain 35S methionine-labeled in vitro translated COUP-TFI were determined by GST pull-down assays (Fig. 7A
). Bacterially expressed GST and GST::COUP-TFI 57423 fusion proteins were treated in vitro in the absence or in the presence of purified PKC before incubation with 35S methionine-labeled COUP-TFI-programmed lysate. As depicted in Fig. 7A
, the unphosphorylated GST::COUP-TFI 57423 fusion protein retained a significant amount of 35S methionine-labeled COUP-TFI when compared with the input lane, showing that phosphorylation is not required for dimerization of the nuclear receptor. In vitro phosphorylation of the GST::COUP-TFI 57423 fusion protein by purified PKC did not affect the amount of labeled COUP-TFI retained by the fusion protein bound to glutathione-agarose beads (Fig. 7A
). The PKC-induced enhancement of DNA binding is therefore unlikely to result from an increase in the ability of COUP-TFI to homodimerize. To further delineate the mechanism of this PKC-mediated regulation, we aimed to map the functional domain(s) of COUP-TFI that were targeted by PKC in vitro. COUP-TFI functional domain mapping was assessed by using various truncated bacterially expressed GST::COUP-TFI fusion proteins (Fig. 7B
). Glutathione-agarose beads containing either GST, GST::COUP-TFI 157, GST::COUP-TFI 57153 or GST::COUP-TFI 153423 fusion proteins were incubated with purified PKC in the presence of
-32P ATP (Fig. 7B
). The GST moiety and the GST::COUP-TFI 153423 fusion protein were not phosphorylated by the purified PKC. In contrast, incorporation of 32P was observed when the GST::COUP-TFI 157 and the GST::COUP-TFI 57153 fusion proteins were incubated with the purified PKC, but not when the kinase was omitted in the reaction mixture (data not shown). The positions of the specific bands corresponding to the phosphorylated GST::COUP-TFI 157 and GST::COUP-TFI 57153 were assessed by Coomassie Brilliant blue staining (data not shown) and are indicated by an open and gray arrowhead, respectively (Fig. 7B
). We can therefore conclude that multiple functional domains of COUP-TFI are selectively phosphorylated by PKC in vitro. Consistent with the result that PKC positively regulates the DNA binding activity of COUP-TFI without affecting its dimerization function, we provide evidence that the DBD of COUP-TFI, but not its LBD, is directly targeted by PKC. Mass spectrometry confirmed the effect of PKC on the phosphorylation status of the DBD and allowed to specifically identify the modified amino acid residues (Fig. 7C
). Phosphorylation of S94 or S113 could not be detected in these experiments, but mass spectrometry data showed definitively that T121 was phosphorylated by PKC. Mass spectrometry data indicated a posttranslational modified peptide at position mass 927.44 (116-NLTYTCR-124). The spectra showed an additional peak +80 Da at 1007.5 indicating a phosphorylation modification at the T121 position. The presence of both peaks at m/z = 927.44 and 1007.4 indicates that both unphosphorylated and phosphorylated COUP-TFI are present after PKC action, which is consistent with what we observed in gel-shift experiments. These data demonstrate that T121 is a target for PKC in vitro.
MAPK Directly Targets Distinct Phosphorylation Sites within COUP-TFI in Vitro
Previous results strongly suggested that the p42MAPK/p44MAPK signaling pathway could also be involved in the regulation of COUP-TFI activity in vivo, possibly through phosphorylation events occurring in the A/B domain. To investigate this hypothesis further, we performed in vitro phosphorylation assays to determine which functional domain(s) of COUP-TFI could be targeted by the purified MAPK (ERK2/p42MAPK) in vitro. As shown in Fig. 8
, the GST::COUP-TFI 157 (open arrowhead) and GST::COUP-TFI 57153 (gray arrowhead) fusion proteins were labeled, whereas the GST::COUP-TFI 153423 protein did not incorporate 32P in the presence of purified MAPK. These data clearly show that purified ERK2/p42MAPK directly targets COUP-TFI in vitro, through at least two distinct phosphorylation sites located between residues 157 and 57153, respectively. In light of our previous results, it was particularly tempting to speculate that the third putative MAPK phosphorylation site (T63) identified within the C-terminal region of the A/B domain of COUP-TFI could be involved in the regulation of COUP-TFI activity by the MAPK (p42MAPK/p44MAPK) signaling pathway in vivo.

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Figure 8. MAPK Directly Targets COUP-TFI in Vitro at Multiple Phosphorylation Sites
The ability of purified MAPK to phosphorylate bacterially expressed truncated COUP-TFI proteins in vitro was monitored by incorporation of 32P. Glutathione-agarose beads containing 250 ng of bacterially expressed GST (lane 1), GST::COUP-TFI 157 (lane 2), GST::COUP-TFI 57153 (lane 3), or GST::COUP-TFI 153423 (lane 4) fusion proteins were incubated in the presence of 25 ng of purified MAPK and -32P ATP (5 µCi). The samples were analyzed by SDS-PAGE, the positions of the phosphorylated GST::COUP-TFI 157 and GST::COUP-TFI 57153 fusion proteins being indicated by an open and a gray arrowhead, respectively.
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Cell-Specific Enhancement of COUP-TFI Transactivation Function through Phosphorylation of Its Amino-Terminal T63 Residue by the MAPK Pathway
Considering that treatment with a specific MAPK inhibitor resulted in a pronounced decrease in the COUP-TFI-mediated transcriptional activation of the Vn gene promoter in vivo (see Fig. 3B
), various experiments were completed to delineate the function(s) of COUP-TFI that could be targeted by the MAPK-mediated phosphorylation events. Transfection experiments performed in COS-7 cells revealed that neither the deletion of the A/B domain of COUP-TFI (i.e. deletion of residues 184, encompassing the three conserved putative MAPK target sites), nor treatment of cells expressing the full-length COUP-TFI with the selective MAPK inhibitor modified the subcellular distribution of the nuclear receptor (data not shown). Distinct sets of experiments demonstrated that in vivo inhibition of the MAPK pathway (see Fig. 5B
) and in vitro phosphorylation by purified MAPK (data not shown) did not affect the DNA binding activity of COUP-TFI, which is specifically targeted by PKC. Our next objective was to determine whether MAPK-mediated phosphorylation could directly regulate the transactivation function of COUP-TFI. Based on the results obtained using COS-7 cells (Fig. 3B
) and P19 cells (data not shown) showing that COUP-TFI-dependent transcription was inhibited by deletion of the A/B domain (i.e. deletion of residues 184) while remaining unaffected by deletion of the N-terminal region spanning amino acids 157, we postulated that the third consensus phosphorylation site (T63) identified within the sequence of COUP-TFI could be targeted by the MAPK pathway in vivo. This putative MAPK phosphorylation site was therefore inactivated through replacement of the conserved threonine residue to a nonphosphorylatable alanine residue. The ability of the mutated COUP-TFI receptor to activate the Vn gene promoter was assessed by cotransfection experiments performed in COS-7 cells (Fig. 9A
) and P19 cells (Fig. 9B
). Cotransfection of increasing amounts of wtCOUP-TFI expression vector led to a dose-dependent increase in the Vn-Luc reporter gene activity in COS-7 cells (Fig. 9A
) and P19 cells (Fig. 9B
). Moreover, in both cellular contexts, treatment with PD980059 significantly inhibited COUP-TFI-dependent transcription (Fig. 9
, A and B). As shown in Fig. 9A
, mutation of the third putative MAPK phosphorylation site has no effect on the transcriptional activation of the Vn-Luc reporter gene mediated by COUP-TFI in COS-7 cells (Fig. 9A
). Furthermore, mutating the third putative MAPK phosphorylation site did not prevent the inhibitory effect of the MAPK inhibitor (Fig. 9A
). This was in sharp contrast to the results obtained in P19 cells. Indeed, in this cellular context, mutation of the third MAPK phosphorylation site resulted in a significant decrease in the transactivation of the Vn gene promoter by COUP-TFI (Fig. 9B
). Furthermore, consistent with the involvement of the third MAPK phosphorylation site in the positive regulation of COUP-TFI transactivation function by the MAPK pathway in P19 cells, mutation of the conserved T63 residue completely abolished the effect of the selective p42MAPK/p44MAPK pathway inhibitor on the COUP-TFI-dependent transcription (Fig. 9B
). All together, these data demonstrate that the MAPK pathway positively regulates COUP-TFI transactivation function in the context of the Vn gene promoter in a cell-specific manner. While this regulation is likely to be mediated through direct phosphorylation of the third evolutionarily conserved MAPK target site of COUP-TFI by the p42MAPK/p44MAPK signaling pathway in P19 cells, this phosphorylation site does not seem to be involved in the modulation of COUP-TFI transcriptional activity in COS-7 cells.

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Figure 9. Mutation of the Third Putative Target Site for MAPK (T63) Located in the N-Terminal Region of COUP-TFI Inhibits Its Transactivation Function in a Cell-Specific Manner
The evolutionarily conserved threonine residue (T63) of the third consensus MAPK phosphorylation site identified within the N-terminal A/B domain of hCOUP-TFI was mutated in an unphosphorylatable alanine residue (T63 A). COS-7 cells (A) or P19 cells (B) were cotransfected with the Vn-Luc reporter gene in the absence or presence of increasing amounts (6.25 to 50 ng) of the indicated pcDNA3-hCOUP-TFI or pcNDA3-hCOUP-TFI T63 A expression vectors. Transfected cells were treated or not with the selective p42MAPK/p44MAPK pathway inhibitor (PD980059, 50 µM). Results are expressed as fold inductions of the reporter gene activities in the presence of the various expression vectors compared with the activity in the absence of any expression vector. Results are shown as the means ± SEM of three values obtained in a representative experiment.
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DISCUSSION
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Among all posttranslational modifications, phosphorylation is a frequently occurring and a potent mechanism for the rapid modulation of transcription factor activity in response to environmental conditions and hormonal signals (50). Most, if not all, nuclear receptors studied to date are phosphoproteins whose functions are regulated by phosphorylation. Indeed, apart from their participation in conventional hormone-dependent activation process, phosphorylation-mediated ligand-independent regulation of nuclear receptors activity has been well documented (33, 34). Despite these findings, such a mechanism may not be ubiquitous; indeed, the GR appears to be refractory to activation in the absence of its cognate ligand. This mode of action seems to be relevant for most other steroid/thyroid hormone and retinoid receptors as well as for various orphan receptors (33, 38, 39, 40, 41). The identification of several putative phosphorylation sites for CKII, PKC, and MAPK, led us to study the potential involvement of these signaling pathways in the regulation of COUP-TFI function. Our data first show that COUP-TFI is a phosphoprotein in vivo. Despite the observation that human COUP-TFI sequence contains eight putative target sites for CKII, our data strongly suggest that this kinase is unlikely to regulate the nuclear receptor activity, in the cellular and promoter contexts considered. In contrast, we provide evidence for the involvement of both PKC and MAPK (p42MAPK/p44MAPK) transduction pathways in the modulation of COUP-TFI functions in vivo.
Focusing on the PKC pathway, we demonstrated that selective inhibition of this pathway in vivo actively decreases COUP-TFI-dependent activation of the Vn gene promoter. In this study, we provide data that clearly show that PKC directly targets COUP-TFI in vitro and enhances its binding to various response elements. Precedents for both positive and negative regulations of DNA binding by phosphorylation exist for numerous nuclear receptors, including ER
, GR, RAR, TR
2, TRß1, Nur77/NGFI-B, and HNF-4. These regulatory mechanisms are not yet fully understood and highly complex, as they involve a wide range of protein kinases with target sites located within, nearby, or far from the DBD. While phosphorylation-mediated by PKA enhances GR binding to DNA (51), this kinase has been shown to inhibit binding of HNF-4 in vitro and in vivo (40). Consistent with the involvement of the A-box in the high affinity DNA binding of HNF-4 (52), phosphorylation at a consensus PKA-dependent site located within this region results in a modification of the receptor binding to DNA (40). Similarly, phosphorylation of NGFI-B within its A-box also decreases DNA binding (38, 39). In both cases, the reduction of DNA binding of the receptor is likely to reflect a local effect: phosphorylations occurring nearby to the DBD could either induce a local conformational change impairing DNA recognition, inhibit protein/DNA contacts by steric hindrance, or electrostatically hinder interactions between the phosphate groups of the protein and the similarly charged phosphate backbone of the DNA. In contrast, phosphorylation of TR
2, a variant of the thyroid hormone receptor, within a unique carboxy-terminal region mediated by CKII decreases its binding to DNA via a mechanism that is likely to involve a phosphorylation-induced change in the overall conformation of the protein (53). A similar mechanism, which is likely to underlie the positive regulation exerted by CKII on the binding of ER
to its response element, involves phosphorylation events occurring at an amino acid residue located within the amino-terminal A/B domain of the receptor, resulting in intramolecular interactions affecting the DNA binding domain and its function (54, 55). In addition to these direct mechanisms, indirect phosphorylation-mediated regulations of DNA binding have also been reported; these are often due to a modulation of the dimerization interface of the nuclear receptors by phosphorylation events. For example, phosphorylation of TRß1 enhances TRß1/RXR heterodimerization, leading to an increase in DNA binding (56, 57). Such an indirect regulation of DNA binding is also relevant for RAR/RXR heterodimers where PKC-mediated phosphorylation event at a serine residue located in the T-box of RAR prevents heterodimerization (58, 59). Based on the finding that the T-box of RAR engages dimerization contacts with the DBD of RXR (60), phosphorylation occurring within this region may introduce strong conformational constraints on the dimerization interface, thus impairing heterodimerization and subsequent DNA binding (58, 59). While PKC has been shown to down-regulate DNA binding for members of the nuclear receptor superfamily, both through direct and indirect mechanisms, there is, to our knowledge, no evidence for PKC-mediated positive effects on the DNA binding of any member of this superfamily. In this report, we demonstrate that phosphorylation of COUP-TFI by PKC increases the affinity of the orphan receptor for specific DNA elements. Moreover, we show that the enhancement of DNA binding does not result from a modification of the ability of COUP-TFI to homodimerize, but rather appears to be a direct mechanism. Consistent with this idea, the DBD of COUP-TFI is targeted by PKC in vitro, whereas the LBD, which contains the major homodimerization surface (61), is not. Mass spectrometry analysis allowed us to ascertain that T121 is a target for PKC, whereas no other modifications were detected. Although the region spanning amino acids 1 to 57 also contains at least one target site for PKC, our data demonstrate that the amino-terminal A/B domain of the nuclear receptor is dispensable for DNA binding and its phosphorylation-dependent regulation. We propose that COUP-TFI binding to DNA is positively and directly regulated by PKC-mediated phosphorylation targeting T121 within the DNA binding domain of the receptor.
COUP-TFI may integrate multiple signaling pathways considering our finding that the MAPK pathway specifically regulates COUP-TFI transactivation function. While having no effect on nuclear translocation or DNA binding function, inhibition of the p42MAPK/p44MAPK pathway in vivo strongly inhibits the COUP-TFI-mediated transcriptional activation of the Vn gene promoter. Moreover, we showed that ERK2/p42MAPK directly targets COUP-TFI at distinct phosphorylation sites in vitro. While the LBD is not phosphorylated by MAPK, at least one target site is present in the amino-terminal region of the A/B domain (i.e. amino acids 157) of COUP-TFI. The functional relevance of this phosphorylation event in vivo remains unclear because the deletion of the amino-terminal region to residue 57 does not affect the subcellular distribution, DNA binding activity and transactivation function of the receptor, or the MAPK-mediated regulation of the COUP-TFI-dependent transcription. Other phosphorylation sites, located between residues 57 and 153, are directly targeted by ERK2/p42MAPK in vitro, and the ERK2-mediated phosphorylation event that might specifically target T63 in vivo is shown here to play an active role in cell-specific transcriptional activation by COUP-TFI. Such a modulation by MAPK-mediated phosphorylation could result from modifications in coactivator recruitment as previously reported for various nuclear receptors. Indeed, ERß can be activated by the Ras-dependent MAPK pathway in the absence of its cognate ligand via direct phosphorylation within the A/B domain (62, 63). Recent studies have shown that phosphorylation of MAPK target sites located in the AF-1 region stimulates ligand- and AF-2-independent interaction between ERß and SRC-1, thus allowing for ligand-independent activation of ERß (63). In addition to ERß, several authors have reported observations of functional cross-talk between growth factors and ER
signaling pathways, both in the absence and presence of ligand (64, 65, 66, 67, 68). Consistent with the requirement of the AF-1 region for the activation of the apo-form ER
in response to growth factors, one serine residue S118 located in the A/B domain of ER
is directly targeted by the MAPK in response to EGF, and these phosphorylation events increase the AF-1 activity (66, 67, 68). The mechanism underlying this enhancement has recently been elucidated and involves the MAPK-dependent recruitment of the coactivator p68 by the AF-1 region of ER
(69). In addition to these local effects, the MAPK pathway can, by intramolecular communication between different nuclear receptor domains, positively or negatively regulate coactivator binding by domains that are distinct from the ones targeted by specific phosphorylation events (70, 71). For example, phosphorylation of a serine residue located within the hinge region of SF-1 is required for maximal interaction of its LBD with the coactivator GRIP-1, results in the ligand-independent activation of SF-1 (71). Similarly, MAPK-mediated phosphorylation of a single amino acid residue in the A/B domain of PPAR
alters the overall conformation of the apo-form receptor in a way that impairs ligand binding, thereby indirectly preventing the recruitment of coactivators such as SRC-1 (70). Thus, interdomain communication allows phosphorylation events targeting the amino-terminal A/B domain of PPAR
to regulate independent properties of the LBD. This regulatory mechanism is involved in the MAPK-mediated inhibition of PPAR
ligand-dependent transactivation function in response to various growth factors and cytokines, and has important repercussions on PPAR
activity in vivo (70, 72, 73). Taken together, these results show that MAPK-mediated phosphorylation events play crucial roles in the modulation of nuclear receptor/coactivator interactions, a regulatory mechanism that, according to recent data, may also be relevant to nuclear receptor/corepressor interactions (74, 75). Further experiments will tell if ERK2 can modify COUP-TFI/coactivator interactions.
Integration of multiple signaling pathways by the human COUP-TFI nuclear receptor would undoubtedly have repercussions on a wide range of biological processes that involve COUP-TFI, such as cellular growth, differentiation, apoptosis as well as embryonic development (8, 11, 13, 16, 17, 31, 76). Results described in this study indicate that there is a possibility that a variety of extracellular stimuli, transduced by MAPK and PKC signaling pathways, participate in the multifactorial regulation of the diverse biological processes that are controlled by COUP-TFI.
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MATERIALS AND METHODS
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Chemicals and Materials
The affinity-purified rabbit polyclonal anti-hemagglutinin (HA) antibody (HA-probe; Y-11) and goat polyclonal anti-human (h) COUP-TFI antibody (COUP-TFI; N-19) were from TEBU (Le Perray-en-Yvelines, France). Klenow fragment of Escherichia coli DNA polymerase I, CIP, T4 DNA polymerase, T4 DNA ligase, and the fugene transfection reagent were from Roche Molecular Biochemicals (Mannheim, Germany). The TNT-coupled reticulocyte lysate and purified PKC were from Promega Corp. (Madison, WI). Recombinant MAPK, phosphatidyl serine, DRB, reduced glutathione, glutathione-agarose beads, protease inhibitors and antibiotics were from Sigma (St. Louis, MO). PD980059 (2'-Amino-3'-methoxyflavone) and GF 109203X (Bisindolylmaleimide) were purchased from Calbiochem (La Jolla, CA). T4 polynucleotide kinase, culture media, and FCS were from Life Technologies, Inc. (Eragny, France). The phosphatase inhibitors okadaic acid and sodium orthovanadate (Na3VO4),
-32P ATP,
-32P deoxy-CTP (dCTP), 32P orthophosphate, and 35S methionine were from ICN Pharmaceuticals, Inc. (Irvine, CA). The Luc assay system was from Promega Corp., and Luc activity was measured on a Dynatech Corp. luminometer (Guyancourt, France). The chemiluminescent substrate protein detection kit and the Hybond C nitrocellulose membrane were from Amersham Pharmacia Biotech (Orsay, France).
Oligonucleotides
Oligonucleotides were synthesized by Genset Oligos (Paris, France). Double-stranded oligonucleotides were used for EMSA, whereas site-directed mutagenesis (SDM) was performed with single-stranded oligonucleotides, with the exception of the PKA site in pBC/GST, which was mutated by PCR. The oligonucleotides used were as follows: DR1 (EMSA), 5'-TCGATGACCTCTGACCCA-3'; AP1 (EMSA), 5'-TCGACGCTT GATGACTCAGCCGGAA-3'; NR1 (EMSA), 5'-TCGAGGGGTTCTGGGAATGTGACCCTTTGC-3'; mutant (m)NR1 (SDM), 5'-TGCAGCAAAGTTTCACATTCCCAG-3'; mNR2 (SDM), 5'-AAAGTCAGTAGTTTCACGGGCAGGA-3'; COUP-3m (SDM), 5'-GTGCCGGGGGCGGCGGGCGCT-3'; pBC/GST-PKAm up (SDM), 5'-GGTCGTGGGATCAGAAGAGCATGCGTGCATATGGATATC-3'. The double-stranded oligonucleotides used for gel shift assays were end-labeled with
-32P dCTP by Klenow fill-in and purified on a Sephadex G-50 column. The single-stranded oligonucleotides used for SDM were phosphorylated with T4 polynucleotide kinase and ATP before annealing to the single-stranded DNA templates.
Plasmids
The GST::COUP-TFI 157, GST::COUP-TFI 57153, GST::COUP-TFI 153423, and GST::COUP-TFI 57423 fusion proteins were expressed in E. coli under isopropyl-1-thio-ß-D-galactopyranoside (IPTG) induction by using the pGEX-2T plasmid (Amersham Pharmacia Biotech). The pGEX-2T-COUP-TFI 157, pGEX-2T-COUP-TFI 57153, pGEX-2T-COUP-TFI 153423, and pGEX-2T-COUP-TFI 57423 recombinant plasmids contain the human COUP-TFI coding sequence, spanning amino acids 157, 57153, 153423, and 57423, respectively. The pcDNA3-hCOUP-TFI, pcDNA3-hCOUP-TFI 57423, and pcDNA3-hCOUP-TFI 84423 expression vectors encode either the full length or various truncated human COUP-TFI constructs as fusion proteins with the HA epitope tag peptide. The corresponding fragments of the COUP-TFI coding sequence, amplified by PCR or generated by enzymatic cleavage, were first inserted in the pACT2 vector (CLONTECH Laboratories, Inc., Palo Alto, CA) downstream of the HA epitope coding sequence before being introduced into pcDNA3 expression vector (Invitrogen). The pcDNA3-hCOUP-TFI recombinant plasmid was used to generate the pcDNA3-hCOUP-TFI T63
A expression vector by site-directed mutagenesis. For mammalian GST fusion protein expression, the PKA site of pBC/GST (77) was first mutated before the human COUP-TFI cDNA (lacking the coding region corresponding to amino acid residues 1 to 56) was inserted in HpaI-linearized vector. The p19-Vn-Luc (Vn-Luc) reporter plasmid (78) contains the 5' flanking region (bp -528 to +47) of the mouse Vn gene, inserted in the p19-Luc plasmid (79). For SDM of each or both of the two putative half binding sites for nuclear receptors NR1 (bp -89 to -84) and NR2 (bp -162 to -157) identified within the Vn gene promoter, the Vn gene promoter insert was transferred into the Bluescript vector (Stratagene, La Jolla, CA), which contains the f1 origin of replication and therefore allows production of single-stranded DNA. The mutated promoter sequences were then reinserted in the p19-Luc plasmid to generate the mNR1-Vn-Luc, mNR2-Vn-Luc, and mNR1/mNR2-Vn-Luc recombinant reporter genes.
SDM
SDM was performed as previously described (30) by using the mNR1 and mNR2 oligonucleotides on the Bluescript-Vn construct, containing the 5'-flanking region (bp -528 to +47) of the murine Vn gene, to mutate one or both of the two putative nuclear receptors half-binding sites (NR1 and NR2: TGACCC
TGAAAC) identified within this promoter. Similarly, the COUP-3m oligonucleotide was used on the pcDNA3-hCOUP-TFI expression vector, encoding the full-length human COUP-TFI receptor fused to the HA epitope, to inactivate the third consensus phosphorylation site for MAPK located in the N-terminal A/B domain of COUP-TFI. This was achieved through replacing the putative targeted threonine residue (T63) by a nonphosphorylatable alanine residue. For all SDM reactions, 1 µg of single-stranded uracil-containing DNA templates were prepared and annealed to each corresponding mutagenic primer (10 pmol). The annealed primer was then elongated by T4 DNA polymerase, and after ligation, the reaction mixture was used to transform E. coli DH5
competent cells. All resulting point mutations were confirmed by sequencing. The two mutated Vn gene promoter inserts were then transferred into p19-Luc reporter plasmid for transfection experiments. The QuikChange site-directed mutagenesis kit (Stratagene) was used to mutate the PKA site (S to C substitution) of pBC/GST (77).
Generation of Proteins
The TNT-coupled reticulocyte lysate allowed us to produce the 35S methionine-labeled COUP-TFI protein used for the GST pull-down assay. Translation efficiencies of the 35S-labeled proteins were checked by SDS-PAGE analysis of 2 µl of translation products. The GST and GST::COUP-TFI fusion proteins were generated using the pGEX-2T vector, which allows bacterial expression under IPTG induction. After IPTG induction, cells were collected by centrifugation at 4000 rpm for 20 min at 4 C. The supernatant was discarded; the pellet was resuspended in NETN buffer [20 mM Tris-HCl, pH 8.0; 100 mM NaCl; 1 mM EDTA; 0.5% Nonidet P-40; 1 mM phenylmethylsulfonyl fluoride (PMSF); 10 µg/ml aprotinin; 10 µg/ml leupeptin; 10 µg/ml pepstatin], and sonicated twice for 30 sec. The suspension was then centrifuged for 15 min at 12,000 rpm and the soluble fraction, containing the GST fusion proteins, was incubated overnight at 4 C with 100 µl of glutathione-agarose beads prepared and stored in NETN buffer. The beads were washed twice with NETN buffer and a fraction of each sample was loaded onto an SDS-10% polyacrylamide gel and analyzed by Coomassie Brilliant blue staining. The bead-bound GST fusion proteins were collected by centrifugation and directly used for the in vitro phosphorylation and pull-down assays. For EMSA, the fusion proteins were eluted from the beads by competition with reduced glutathione. Briefly, the GST fusion proteins bound to beads were incubated in 10 µl of elution buffer (Tris-HCl 100 mM, pH 8; NaCl 120 mM; 50 mM reduced glutathione) at 4 C for 30 min. After centrifugation, a fraction of the eluted proteins was loaded onto an SDS-10% polyacrylamide gel and analyzed by Coomassie Brilliant blue staining, while remaining eluted proteins were phosphorylated in vitro and submitted to EMSA.
Preparation of WCEs
Transfected COS-7 cells were harvested 36 h after transfection in PBS and collected by centrifugation at 2000 rpm, at 4 C for 15 min. Cells were then resuspended in WCE buffer [20 mM HEPES, pH 7.9; 2 mM dithiothreitol (DTT); 20% glycerol]-0.4 M KCl, containing 5 µg/ml aprotinin, leupeptin, pepstatin and 0.1 mM PMSF. After three cycles freezing (-80 C) and thawing (37 C), the cellular debris were pelleted by centrifugation at 12,000 rpm for 15 min at 4 C. The protein concentration of the supernatant was assessed by Bradford assay and ranged from 26 µg/µl. These WCEs were used for EMSA and Western blot analysis.
In Vitro Phosphorylation of hCOUP-TFI
For in vitro phosphorylation with PKC purified from rat brain, 125500 ng of bacterially expressed GST and GST::COUP-TFI fusion proteins bound to beads were incubated with 5 µCi of
-32P ATP in 50 µl of phosphorylation buffer (20 mM HEPES, pH 7.4; 10 mM MgCl2; 1.7 mM CaCl2; 600 µg/ml phosphatidyl serine; 1 mM DTT; 50 µM ATP), in the absence or presence of increasing amounts of PKC (3.125 to 25 ng). For in vitro phosphorylation with activated recombinant MAPK, the fusion proteins were incubated with 5 µCi of
-32P ATP in 50 µl of phosphorylation buffer (50 mM Tris-HCl, pH 8; 0.5 mM EDTA; 25 mM MgCl2; 1 mM DTT; 50 µM ATP; 10% glycerol), in the absence or presence of 25 ng of MAPK. All phosphorylation reactions were carried out at 30 C for 30 min, in the presence of proteases and phosphatases inhibitors (1 µM okadaic acid; 200 µM Na3VO4). The glutathione-agarose beads were then washed twice with washing buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.02% Tween 20; 1 mM PMSF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; 10 µg/ml pepstatin), solublized in 1x SDS loading buffer and heated at 100 C for 5 min. The in vitro phosphorylated proteins were resolved in a SDS-10% polyacrylamide gel, which was dried and exposed to a radiographic film. For the GST pull-down assay and EMSA, cold ATP (250 µM) was added instead of
-32P ATP as a donor of phosphate.
Mass Spectrometry
Purified GST::COUP-TFI was phosphorylated by PKC (Promega Corp.) according to manufacturers instructions and subjected to SDS-PAGE. Samples were prepared according to Shevchenko et al. (80). After tryptic digest of phosphorylated and control samples, peptides were extracted and analyzed by matrix-assisted laser desorption/ionization mass spectrometry using Voyager Biospectrometry Workstation with Delayed Extraction Technology (PerSeptive Biosystems, Inc., Foster City, CA). Obtained data were analyzed using Moverz software (Proteometrics, LLC, Winnipeg, Canada).
GST Pull-Down Assay
For in vitro protein-protein interactions, 125 or 250 ng of unphosphorylated or in vitro phosphorylated GST::COUP-TFI fusion proteins bound to beads were incubated for 4 h at 4 C with 35S methionine-labeled in vitro-translated hCOUP-TFI in 400 µl of binding buffer (50 mM Tris-HCl, pH 8.0; 50 mM NaCl; 0.02% Tween 20; 0.02% BSA; 1 mM PMSF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; 10 µg/ml pepstatin). The beads were then washed with washing buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.02% Tween 20; 1 mM PMSF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; 10 µg/ml pepstatin) and the proteins were solubilized in 1x SDS loading buffer, heated at 100 C for 5 min and resolved in a SDS-polyacrylamide gel.
In Vitro Dephosphorylation of hCOUP-TFI
To study the potential effect of the in vitro dephosphorylation of COUP-TFI on its ability to bind DNA, human COUP-TFI expressed in transfected COS-7 cells was treated in vitro with CIP before the EMSA protocol. Two micrograms of WCEs were incubated with 2.5 units of CIP in 10 µl of CIP buffer (10 mM Tris-acetate, pH 8; 10 mM magnesium-acetate; 50 mM potassium-acetate), control reactions being performed without CIP buffer, or with CIP buffer but in the absence of enzyme. Some incubations were also performed in the absence or in the presence of NaPO4 (100 mM) to inhibit the CIP activity. All dephosphorylation reactions were carried out at 25 C for 1 h, the dephosphorylation mixture being then submitted to the EMSA protocol.
EMSA
EMSA experiments were performed with WCEs prepared from transfected COS-7 cells or bacterially expressed COUP-TFI proteins. When WCEs from transfected cells treated with specific kinase inhibitors were used, the expression levels of hCOUP-TFI receptor were controlled by Western blot analysis before EMSA. Two micrograms of WCEs from COUP-TFI-expressing COS-7 cells or increasing amounts (0.62520 ng) of glutathione-agarose beads-purified GST::COUPTFI fusion proteins were incubated for 20 min at room temperature with 1 µg of poly (deoxyinosine-deoxycytidine) in 20 µl of binding buffer (20 mM HEPES, pH 7.9; 1 mM DTT; 50 mM KCl; 2.5 mM MgCl2; 10% glycerol), before incubation of the samples with 0.2 ng of double-stranded oligonucleotide, labeled with
-32P dCTP (20,000 cpm). After 20 min at room temperature, the protein-DNA complexes were resolved on a 4% polyacrylamide gel, in 0.5x TBE (45 mM Tris base; 45 mM boric acid; 1 mM EDTA). For Kd calculation, 10 ng of GST::COUP-TFI 57423 were incubated with increasing amounts of 32P-labeled DR1 double-stranded oligonucleotide. The gels were dried before autoradiography, and the intensity of the specific retarded complex was assessed by phosphoimaging. In most of the experiments, the presence of hCOUP-TFI in the specific shifted complex was controlled by supershifting analysis, performed with 2 µg of anti-hCOUP-TFI antibody.
Western Blot Analysis
WCEs (1030 µg) from transfected COS-7 cells were fractioned by SDS-PAGE and electrotransferred to a nitrocellulose membrane. The membrane was stained with Ponceau S to locate the molecular weight markers and washed in PBS containing 0.1% Tween 20. All washes and incubations were performed at room temperature. The nonspecific sites on the membrane were then blocked with PBS containing 0.1% Tween 20, 1% BSA and 5% nonfat milk powder for 2 h. Because the human COUP-TFI protein expressed in transfected cells was fused to an eleven amino acid peptide derived from the influenza HA protein, the membrane was probed overnight with 0.5 µg/ml anti-HA in PBS containing 0.1% Tween 20, 1% BSA. Immunodetection was then carried out with horseradish peroxidase-conjugated goat antirabbit antibodies, revealed with the electrochemiluminescence protein detection kit according to the manufacturers protocol.
Cell Culture and Transient Transfection
COS-7 and P19 cells were routinely grown at 37 C in DMEM containing 10% FCS, and 100 U/ml penicillin, 100 µg/ml streptomycin and 25 µg/ml Amphotericin. Cells were transfected in 24-well plates by using the calcium phosphate/DNA method. Briefly, 300 ng of recombinant Vn-Luc reporter plasmids, containing either the wt (wtVn) or the mutated (mNR1-Vn, mNR2-Vn, and mNR1/mNR2-Vn) gene promoter sequences (bp -528 to +47) were cotransfected with 150 ng of pCH110 control plasmid (a ß-galactosidase expression vector). Various amounts (6.2550 ng per well) of pcDNA3 expression vectors (pcDNA3-hCOUP-TFI, pcDNA3-hCOUP-TFI T63
A, pcDNA3-hCOUP-TFI 57423, and pcDNA3-hCOUP-TFI 84423) were cotransfected, the expression levels of the proteins being checked by Western blot analysis. In all wells, the total amount of CMV promoter (100 ng per well) and the total amount of DNA (1 µg per well) were maintained constant through addition of pcDNA3 and of Bluescript plasmids respectively. After 18 h, cells were washed with PBS and fresh medium was added, the cells being harvested 36 h after the calcium phosphate/DNA precipitate was removed. In some experiments, specific kinase inhibitors were incubated with transfected COS-7 cells during the last 4 h of culture: MEK1/2 inhibitor (PD98059 in Me2SO), PKC inhibitor (GF109203X in Me2SO) and CK-II inhibitor (DRB in ethanol) were added to the medium to give a final concentration of 50 µM, 5 µM and 50 µM, respectively. The control incubations were performed with dimethyl sulfoxide (Me2SO) or ethanol instead of the corresponding protein kinase inhibitor, the final concentration of vehicle in any experiment never exceeding 0.1% (vol/vol). After harvesting the cells, 10% of the cellular extract was used to measure the Luc activity; half of the remaining extract was taken to perform the ß-galactosidase assay. Luc activities were normalized for transfection efficiency with the ß-galactosidase activities. For COUP-TFI expression, COS-7 cells were plated in 5-cm diameter petri dishes and transfected with 5 µg of expression plasmid by the calcium phosphate/DNA coprecipitation technique. After 18 h, cells were rinsed with PBS and medium was renewed. In some experiments, treatment with various specific kinase inhibitors was performed during the last 4 h of culture, under the same conditions as those described above. To prepare WCEs, cells were harvested 36 h after transfection in PBS.
In Vivo Metabolic Labeling
COS-7 cells were transfected in 10 cm dishes by the fugene reagent with pBC/GST or pBC/GST-COUP-TFI 57423. After 36 h, cells were transferred in phosphate-free DMEM. One hour later, medium was replaced by phosphate-free DMEM containing 75 µCi/ml of 32P-labeled orthophosphate. This medium was left on the cells for 4 h. Cells were then washed and scraped before being lysed in 20 mM Tris-HCl, pH 8.0; 0.3 M NaCl; 1 mM EDTA; 2 mM DTT; 0.5% NP-40; 20% glycerol, containing protease inhibitors (10 µg/ml aprotinin, leupeptin, pepstatin; 0.1 mM PMSF). The cell lysate was incubated overnight at 4 C with 100 µl of a 50% glutathione-agarose bead suspension in binding buffer (50 mM Tris-HCl, pH 8.0; 0.4 M NaCl; 0.5% NP-40, and protease inhibitors). The beads were washed twice in Washing buffer I (50 mM Tris-HCl, pH 8.0; 0.4 M NaCl; 0.5% NP-40; protease inhibitors) and twice in Washing buffer II (50 mM Tris-HCl, pH 8.0; 0.8 M NaCl; 1% NP-40; protease inhibitors). The proteins bound to the beads were resuspended in 1x SDS loading buffer, heated at 100 C for 5 min and resolved in an SDS-polyacrylamide gel. After Coomassie Brilliant blue staining and drying of the gel, the incorporation of 32P was monitored by autoradiography. The resulting signal was quantified with Scion Image software (Scion Corp., Frederick, MD), and normalized with the corresponding amount of purified GST and GST::COUP-TFI 53423 fusion proteins.
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ACKNOWLEDGMENTS
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We thank M. Pfahl (Sidney Kimmel Cancer Center, La Jolla, CA) for providing us with the hCOUP-TFI expression vector, and D. Loskutoff (Scripps Research Institute, La Jolla, CA) for the gift of the p19-Vn-Luc reporter construct.
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FOOTNOTES
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This work was supported by grants from the Ministère de la Recherche et de lEnseignement, and the Centre National de la Recherche Scientifique. F.G. and R.M. were supported by fellowships, and G.S. by a research grant (ARC No. 5982), from the Association pour la Recherche sur le Cancer. F.G., C.D.-L.P., R.M., R.L.G., and G.S. also thank the Fondation Langlois for financial support. P.B. is supported by a fellowship, and G.S. and D.B. are supported by a research grant from the European Union 5th Framework Programme (NORTh No. QLG1-CT-2001-01513).
1 Present address: Harvard Medical School, Department of Pathology, WAB 120, 200 Longwood Avenue, Boston, Massachusetts 02115. 
2 Present Address: European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfasch 102209, Heidelberg D-69012, Germany. 
Abbreviations: CIP, Calf intestinal phosphatase; CKII, casein kinase II; COUP-TFI, chicken ovalbumin upstream promoter transcription factor I; DBD, DNA binding domain; DRB, 5-6-dichloro-1-ß-D ribofuranosyl benzimidazole; dCTP, deoxy-CTP; DTT, dithiothreitol; E2, estradiol; GST, glutathione-S-transferase; h, human; HA, hemagglutinin; IPTG, isopropyl-1-thio-ß-D-galactopyranoside; Kd, dissociation constant; LBD, ligand binding domain; Luc, luciferase; m, mutant; PMSF, phenylmethylsulfonyl fluoride; NR1 or 2, nuclear receptor half-binding site 1 or 2; SDM, site-directed mutagenesis; Vn, vitronectin; WCE, whole-cell extracts; wt, wild-type.
Received for publication April 20, 2001.
Accepted for publication January 31, 2002.
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REFERENCES
|
---|
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Giguère V 1999 Orphan nuclear receptors: from gene to function. Endocr Rev 20:689725[Abstract/Free Full Text]
-
Mlodzik M, Hiromi Y, Weber U, Goodman CS, Rubin GM 1990 The Drosophila seven-up gene a member of the steroid receptor gene superfamily controls photoreceptor cell fates. Cell 60:211224[Medline]
-
Ritchie HH, Wang LH, Tsai SY, OMalley BW Tsai MJ 1990 COUP-TF gene: a structure unique for the steroid/thyroid receptor superfamily. Nucleic Acids Res 18:68576862[Abstract]
-
Chan SM, Xu N, Niemeyer CC, Bone JR, Flytzanis CN 1992 SpCOUP-TF: a sea urchin member of the steroid/thyroid hormone receptor family. Proc Natl Acad Sci USA 89:1056810572[Abstract]
-
Matharu PJ, Sweeney GE 1992 Cloning and sequencing of a COUP transcription factor gene expressed in xenopus embryos. Biochim Biophys Acta 1129:331334[Medline]
-
Fjose A, Nornes S, Weber U, Mlodzik M 1993 Functional conservation of vertebrate seven-up related genes in neurogenesis and eye development. EMBO J 12:14031414[Abstract]
-
Jonk LJ, de Jonge ME, Pals CE, Wissink S, Vervaart JM, Schoorlemmer J, Kruijer W 1994 Cloning and expression during development of three murine members of the COUP family of nuclear orphan receptors. Mech Dev 47:8197[CrossRef][Medline]
-
Fjose A, Weber U, Mlodzik M 1995 A novel vertebrate svp-related nuclear receptor is expressed as a step gradient in developing rhombomeres and is affected by retinoic acid. Mech Dev 52:233246[CrossRef][Medline]
-
Qiu Y, Krishnan V, Zeng Z, Gilbert DJ, Copeland NG, Gibson L, Yang-Feng T, Jenkins NA, Tsai MJ, Tsai SY 1995 Isolation characterization and chromosomal localization of mouse and human COUP-TF I and II genes. Genomics 29:240246[CrossRef][Medline]
-
Qiu Y, Cooney AJ, Kuratani S, DeMayo FJ, Tsai SY, Tsai MJ 1994 Spatiotemporal expression patterns of chicken ovalbumin upstream promoter-transcription factors in the developing mouse central nervous system: evidence for a role in segmental patterning of the diencephalon. Proc Natl Acad Sci USA 91:44514455[Abstract]
-
Lopes da Silva S, Cox JJ, Jonk LJ, Kruijer W, Burbach JP 1995 Localization of transcripts of the related nuclear orphan receptors COUP-TF I and ARP-1 in the adult mouse brain. Brain Res Mol Brain Res 30:131136[CrossRef][Medline]
-
Pereira FA, Qiu Y, Tsai MJ, Tsai SY 1995 Chicken ovalbumin upstream promoter transcription factor (COUP-TF): expression during mouse embryogenesis. J Steroid Biochem Mol Biol 53:503508[CrossRef][Medline]
-
Hiromi Y, Mlodzik M, West SR, Rubin GM, Goodman CS 1993 Ectopic expression of seven-up causes cell fate changes during ommatidial assembly. Development 118:11231135[Abstract/Free Full Text]
-
Schuh TJ, Kimelman D 1995 COUP-TFI is a potential regulator of retinoic acid-modulated development in Xenopus embryos. Mech Dev 51:3949[CrossRef][Medline]
-
Qiu Y, Pereira FA, DeMayo FJ, Lydon JP, Tsai SY, Tsai MJ 1997 Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion axonal projection and arborization. Genes Dev 11:19251937[Abstract/Free Full Text]
-
Zhou C, Qiu Y, Pereira FA, Crair MC, Tsai SY, Tsai MJ 1999 The nuclear orphan receptor COUP-TFI is required for differentiation of subplate neurons and guidance of thalamocortical axons. Neuron 24:847859[Medline]
-
Pereira FA, Qiu Y, Zhou G, Tsai MJ, Tsai SY 1999 The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development. Genes Dev 13:10371049[Abstract/Free Full Text]
-
Pastorcic M, Wang H, Elbrecht A, Tsai SY, Tsai MJ, OMalley BW 1986 Control of transcription initiation in vitro requires binding of a transcription factor to the distal promoter of the ovalbumin gene. Mol Cell Biol 6:27842791[Medline]
-
Sagami I, Tsai SY, Wang H, Tsai MJ, OMalley BW 1986 Identification of two factors required for transcription of the ovalbumin gene. Mol Cell Biol 6:42594267[Medline]
-
Wang LH, Tsai SY, Cook RG, Beattie WG, Tsai MJ, OMalley BW 1989 COUP transcription factor is a member of the steroid receptor superfamily. Nature 340:163166[CrossRef][Medline]
-
Ladias JA, Karathanasis SK 1991 Regulation of the apolipoprotein AI gene by ARP-1 a novel member of the steroid receptor superfamily. Science 251:561565[Medline]
-
Cooney AJ, Tsai SY, OMalley BW, Tsai MJ 1992 Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements allowing COUP-TF to repress hormonal induction of the vitamin D3 thyroid hormone and retinoic acid receptors. Mol Cell Biol 12:41534163[Abstract]
-
Kliewer SA, Umesono K, Heyman RA, Mangelsdorf DJ, Dyck JA, Evans RM 1992 Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling. Proc Natl Acad Sci USA 89:14481452[Abstract]
-
Tran P, Zhang XK, Salbert G, Hermann T, Lehmann JM, Pfahl M 1992 COUP orphan receptors are negative regulators of retinoic acid response pathways. Mol Cell Biol 12:46664676[Abstract]
-
Widom RL, Ladias JA, Kouidou S, Karathanasis SK 1991 Synergistic interactions between transcription factors control expression of the apolipoprotein AI gene in liver cells. Mol Cell Biol 11:677687[Medline]
-
Neuman K, Soosaar A, Nornes HO, Neuman T 1995 Orphan receptor COUP-TF I antagonizes retinoic acid-induced neuronal differentiation. J Neurosci Res 41:3948[Medline]
-
Van der Wees J, Matharu PJ, de Roos K, Destree OH, Godsave SF, Durston AJ, Sweeney GE 1996 Developmental expression and differential regulation by retinoic acid of xenopus COUP-TF-A and COUP-TF-B. Mech Dev 54:173184[CrossRef][Medline]
-
Hall RK, Sladek FM, Granner DK 1995 The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids. Proc Natl Acad Sci USA 92:412416[Abstract]
-
Lazennec G, Kern L, Valotaire Y, Salbert G 1997 The nuclear orphan receptors COUP-TF and ARP-1 positively regulate the trout estrogen receptor gene through enhancing autoregulation. Mol Cell Biol 17:50535066[Abstract]
-
Lin B, Chen GQ, Xiao D, Kolluri SK, Cao X, Su H, Zhang XK 2000 Orphan receptor COUP-TF is required for induction of retinoic acid receptor ß growth inhibition and apoptosis by retinoic acid in cancer cells. Mol Cell Biol 20:957970[Abstract/Free Full Text]
-
Luo J, Sucov HM, Bader JA, Evans RM, Giguère V 1996 Compound mutants for retinoic acid receptor (RAR) ßand RAR
1 reveal developmental functions for multiple RAR ß isoforms. Mech Dev 55:3344[CrossRef][Medline]
-
Weigel NL, Zhang Y 1998 Ligand-independent activation of steroid hormone receptors. J Mol Med 76:469479[CrossRef][Medline]
-
Weigel NL 1996 Steroid hormone receptors and their regulation by phosphorylation. Biochem J 319: 657667
-
Chen D, Pace PE, Coombes RC, Ali S 1999 Phosphorylation of human estrogen receptor
by protein kinase A regulates dimerization. Mol Cell Biol 19:10021015[Abstract/Free Full Text]
-
Rogatsky I, Trowbridge JM, Garabedian MJ 1999 Potentiation of human estrogen receptor alpha transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A-CDK2 complex. J Biol Chem 274:2229622302[Abstract/Free Full Text]
-
Chen D, Riedl T, Washbrook E, Pace PE, Coombes RC, Egly JM, Ali S 2000 Activation of estrogen receptor
by S118 phosphorylation involves a ligand-dependent interaction with TFIIH and participation of CDK7. Mol Cell 6:127137[Medline]
-
Hirata Y, Kiuchi K, Chen HC, Milbrandt J, Guroff G 1993 The phosphorylation and DNA binding of the DNA-binding domain of the orphan nuclear receptor NGFI-B. J Biol Chem 268:2480824812[Abstract/Free Full Text]
-
Li Y, Lau LF 1997 Adrenocorticotropic hormone regulates the activities of the orphan nuclear receptor Nur77 through modulation of phosphorylation. Endocrinology 138:41384146[Abstract/Free Full Text]
-
Viollet B, Kahn A, Raymondjean M 1997 Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4. Mol Cell Biol 17:42084219[Abstract]
-
Kane CD, Means AR 2000 Activation of orphan receptor-mediated transcription by Ca(2+)/calmodulin-dependent protein kinase IV. EMBO J 19:691701[Abstract/Free Full Text]
-
Adam F, Sourisseau T, Métivier R, Le Page Y, Desbois C, Michel D, Salbert G 2000 COUP-TFI (chicken ovalbumin upstream promoter transcription factor I) regulates cell migration and axogenesis in differentiating P19 embryonal carcinoma cells. Mol Endocrinol 14:19181933[Abstract/Free Full Text]
-
Power SC, Cereghini S 1996 Positive regulation of the vHNF1 promoter by the orphan receptors COUP-TFI/Ear3 and COUP-TFII/Arp1. Mol Cell Biol 16:778791[Abstract]
-
Pinna LA 1990 Casein kinase 2: an "eminence grise" in cellular regulation. Biochim Biophys Acta 1054:267284[Medline]
-
Kishimoto A, Nishiyama K, Nakanishi H, Uratsuji Y, Nomura H, Takeyama Y, Nishizuka Y 1985 Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem 260:1249212499[Abstract/Free Full Text]
-
Gonzalez FA, Raden DL, Davis RJ 1991 Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases. J Biol Chem 266:2215922163[Abstract/Free Full Text]
-
Davis RJ 1994 MAPKs: new JNK expands the group. Trends Biochem Sci 19:470473[CrossRef][Medline]
-
Marshall CJ 1995 Specificity of receptor tyrosine kinase signaling: transient vs. sustained extracellular signal-regulated kinase activation. Cell 80:179185[Medline]
-
Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ 1995 Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270:74207426[Abstract/Free Full Text]
-
Hunter T, Karin M 1992 The regulation of transcription by phosphorylation. Cell 70:375387[Medline]
-
Rangarajan PN, Umesono K, Evans RM 1992 Modulation of glucocorticoid receptor function by protein kinase A. Mol Endocrinol 6:14511457[Abstract]
-
Jiang G, Sladek FM 1997 The DNA binding domain of hepatocyte nuclear factor 4 mediates cooperative specific binding to DNA and heterodimerization with the retinoid X receptor
. J Biol Chem 272:12181225[Abstract/Free Full Text]
-
Katz D, Reginato MJ, Lazar MA 1995 Functional regulation of thyroid hormone receptor variant TR
2 by phosphorylation. Mol Cell Biol 15:23412348[Abstract]
-
Arnold SF, Obourn JD, Jaffe H, Notides AC 1995 Phosphorylation of the human estrogen receptor by mitogen-activated protein kinase and casein kinase II: consequence on DNA binding. J Steroid Biochem Mol Biol 55:163172[CrossRef][Medline]
-
Castano E, Vorojeikina DP, Notides AC 1997 Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation. Biochem J 326:149157[Medline]
-
Lin KH, Ashizawa K, Cheng SY 1992 Phosphorylation stimulates the transcriptional activity of the human ß 1 thyroid hormone nuclear receptor. Proc Natl Acad Sci USA 89:77377741[Abstract]
-
Bhat MK, Ashizawa K, Cheng SY 1994 Phosphorylation enhances the target gene sequence-dependent dimerization of thyroid hormone receptor with retinoid X receptor. Proc Natl Acad Sci USA 91:79277931[Abstract]
-
Tahayato A, Lefebvre P, Formstecher P, Dautrevaux M 1993 A protein kinase C-dependent activity modulates retinoic acid-induced transcription. Mol Endocrinol 7:16421653[Abstract]
-
Delmotte MH, Tahayato A, Formstecher P, Lefebvre P 1999 Serine 157 a retinoic acid receptor
residue phosphorylated by protein kinase C in vitro is involved in RXR.RAR
heterodimerization and transcriptional activity. J Biol Chem 274:3822538231[Abstract/Free Full Text]
-
Rastinejad F, Perlmann T, Evans RM, Sigler PB 1995 Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature 375:203211[CrossRef][Medline]
-
Perlmann T, Umesono K, Rangarajan PN, Forman BM, Evans RM 1996 Two distinct dimerization interfaces differentially modulate target gene specificity of nuclear hormone receptors. Mol Endocrinol 10:958966[Abstract]
-
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguère V 1997 Cloning chromosomal localization and functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353365[Abstract/Free Full Text]
-
Tremblay A, Tremblay GB, Labrie F, Giguère V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513519[Medline]
-
Ali S, Metzger D, Bornert JM, Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO J 12:11531160[Abstract]
-
Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:14911494[Abstract]
-
Bunone G, Briand PA, Miksicek RJ, Picard D 1996 Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15:21742183[Abstract]
-
El-Tanani MK, Green CD 1997 Two separate mechanisms for ligand-independent activation of the estrogen receptor. Mol Endocrinol 11:928937[Abstract/Free Full Text]
-
Smith CL 1998 Cross-talk between peptide growth factor and estrogen receptor signaling pathways. Biol Reprod 58:627632[Abstract]
-
Endoh H, Maruyama K, Masuhiro Y, Kobayashi Y, Goto M, Tai H, Yanagisawa J, Metzger D, Hashimoto S, Kato S 1999 Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor
. Mol Cell Biol 19:53635372[Abstract/Free Full Text]
-
Shao D, Rangwala SM, Bailey ST, Krakow SL, Reginato MJ, Lazar MA 1998 Interdomain communication regulating ligand binding by PPAR-
. Nature 396:377380[CrossRef][Medline]
-
Hammer GD, Krylova I, Zhang Y, Darimont BD, Simpson K, Weigel NL, Ingraham HA 1999 Phosphorylation of the nuclear receptor SF-1 modulates cofactor recruitment: integration of hormone signaling in reproduction and stress. Mol Cell 3:521526[Medline]
-
Hu E, Kim JB, Sarraf P, Spiegelman BM 1996 Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPAR
. Science 274:21002103[Abstract/Free Full Text]
-
Adams M, Reginato MJ, Shao D, Lazar MA, Chatterjee VK 1997 Transcriptional activation by peroxisome proliferator-activated receptor
is inhibited by phosphorylation at a consensus mitogen-activated protein kinase site. J Biol Chem 272:51285132[Abstract/Free Full Text]
-
Lavinsky RM, Jepsen K, Heinzel T, Torchia J, Mullen TM, Schiff R, Del-Rio AL, Ricote M, Ngo S, Gemsch J, Hilsenbeck SG, Osborne CK, Glass SK, Rosenfeld MG, Rose DW 1998 Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc Natl Acad Sci USA 95:29202925[Abstract/Free Full Text]
-
Juge-Aubry CE, Hammar E, Siegrist-Kaiser C, Pernin A, Takeshita A, Chin WW, Burger AG, Meier CA 1999 Regulation of the transcriptional activity of the peroxisome proliferator-activated receptor
by phosphorylation of a ligand-independent trans-activating domain. J Biol Chem 274:1050510510[Abstract/Free Full Text]
-
Lu XP, Salbert G, Pfahl M 1994 An evolutionary conserved COUP-TF binding element in a neural-specific gene and COUP-TF expression patterns support a major role for COUP-TF in neural development. Mol Endocrinol 8:17741788[Abstract]
-
Chatton B, Bahr A, Acker J, Kedinger C 1995 Eukaryotic GST fusion vector for the study of protein-protein associations in vivo: application to interaction of ATFa with Jun and Fos. BioTechniques 18:142145[Medline]
-
Seiffert D, Curriden SA, Jenne D, Binder BR, Loskutoff DJ 1996 Differential regulation of vitronectin in mice and humans in vitro. J Biol Chem 271:54745480[Abstract/Free Full Text]
-
Van Zonneveld AJ, Curriden SA, Loskutoff DJ 1988 Type 1 plasminogen activator inhibitor gene: functional analysis and glucocorticoid regulation of its promoter. Proc Natl Acad Sci USA 85:55255529[Abstract]
-
Shevchenko A, Chernushevich I, Wilm M, Mann M 2000 De Novo peptide sequencing by nanoelectrospray tandem mass spectrometry using triple quadrupole and quadrupole/time-of-flight instruments. Methods Mol Biol 146:116[Medline]