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Phosphorylation by p38MAPK and recruitment of SUG-1 are required for RA-induced RARgamma degradation and transactivation

2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês

10.1093/emboj/cdf374

1460-2075

Maurizio Gianni’,

Ubiquitin and proteasome pathways

Article15 July 2002free access Phosphorylation by p38MAPK and recruitment of SUG-1 are required for RA-induced RARγ degradation and transactivation Maurizio Giannì Maurizio Giannì Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France Present address: Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy Search for more papers by this author Annie Bauer Annie Bauer Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France Search for more papers by this author Enrico Garattini Enrico Garattini Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy Search for more papers by this author Pierre Chambon Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France Search for more papers by this author Cécile Rochette-Egly Corresponding Author Cécile Rochette-Egly Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France Search for more papers by this author Maurizio Giannì Maurizio Giannì Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France Present address: Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy Search for more papers by this author Annie Bauer Annie Bauer Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France Search for more papers by this author Enrico Garattini Enrico Garattini Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy Search for more papers by this author Pierre Chambon Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France Search for more papers by this author Cécile Rochette-Egly Corresponding Author Cécile Rochette-Egly Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France Search for more papers by this author Author Information Maurizio Giannì1,2, Annie Bauer1, Enrico Garattini3, Pierre Chambon1 and Cécile Rochette-Egly 1 1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 163, 67404 Illkirch, cedex, France 2Present address: Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy 3Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3760-3769https://doi.org/10.1093/emboj/cdf374 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The nuclear retinoic acid receptor RARγ2 undergoes proteasome-dependent degradation upon ligand binding. Here we provide evidence that the domains that signal proteasome-mediated degradation overlap with those that activate transcription, i.e. the activation domains AF-1 and AF-2. The AF-1 domain signals RARγ2 degradation through its phosphorylation by p38MAPK in response to RA. The AF-2 domain acts via the recruitment of SUG-1, which belongs to the 19S regulatory subunit of the 26S proteasome. Blocking RARγ2 degradation through inhibition of either the p38MAPK pathway or the 26S proteasome function impairs its RA-induced transactivation activity. Thus, the turnover of RARγ2 is linked to transactivation. Introduction The highly pleiotropic effects of retinoic acid (RA), the physiologically active derivative of vitamin A, are mediated by two families of ligand-dependent transcriptional regulators, the RA nuclear receptors (RARs) and the retinoid X nuclear receptors (RXRs) that function as RAR/RXR heterodimers both in vitro and in vivo (Kastner et al., 1995; Mangelsdorf and Evans, 1995; Chambon, 1996; Chiba et al., 1997; Mark et al., 1999). There are three RARs (α, β and γ) and three RXRs (;α, β and γ), and for each isotype there are at least two main isoforms differing only in their N-terminal region. RARs bind both all-trans and 9-cis RA, while RXRs bind 9-cis RA only (Chambon, 1996). As with most nuclear receptors, RARs and RXRs contain two transactivation functions (AFs) (Chambon, 1996). The AF-1 domain, located at the N-terminal end (A/B region) of the receptor, is ligand-independent, while the ligand-activated AF-2 domain that overlaps the ligand binding domain (LBD) is located in the C-terminal region E. AF-2 requires the integrity of a highly conserved amphipatic α-helix, the AF-2 AD core that corresponds to the LBD helix 12. Binding of an agonistic ligand results in a transconformation of the LBD that involves helix 12 and creates a new surface for binding of coactivators (Moras and Gronemeyer, 1998; Chen, 2000; Glass and Rosenfeld, 2000). The AF-1 domain contains conserved serine residues which belong to consensus phosphorylation sites for proline-dependent protein kinases such as cyclin-dependent kinases (CDKs) and the mitogen-activated protein kinases (MAPKs) (Morgan 1997; Pearson et al. 2001; and references therein). Unliganded RARα1 and RARγ2 are constitutively phosphorylated at these sites (Rochette-Egly et al., 1997; Bastien et al., 2000) by cdk7/cyclin H associated to TFIIH, a general transcription factor also involved in DNA repair (Egly, 2001). Phosphorylation of these sites appears to be required for RAR transactivation (Keriel et al., 2002). Nuclear hormone receptors have been recently reported to be degraded by the ubiquitin–proteasome pathway upon ligand binding (Hauser et al., 2000; Lange et al., 2000; Lonard et al., 2000). In the case of ER and PR, this ligand-dependent degradation has been correlated to transcriptional activity (Lonard et al., 2000; Shen et al., 2001). In addition, phosphorylation of the N-terminal A/B region by MAPKs has been shown to play an essential role in the degradation of PR, whereas the integrity of the AF-2 domain is required for ER degradation. It has also been suggested that the proteasomal SUG-1 subunit could be recruited by liganded nuclear receptors to selectively specify their own degradation (vom Baur et al., 1996). In the presence of retinoic acid, RARα1 and RARγ2 are also degraded by the ubiquitin–proteasome pathway (Kopf et al., 2000). How these receptors are targeted for degradation, and whether their degradation is linked to their ability to activate transcription is however unknown. In the present study, we have investigated whether the targeted degradation of RARγ2 might be linked to its transactivation capacity by examining whether its activation functions are also involved in its downregulation. We show that the activation domains AF-1 and AF-2 cooperate in the RA-induced RARγ2 degradation. The RA-liganded AF-2 domain acts through the recruitment of the proteasomal SUG-1 subunit while the AF-1 domain requires to be phosphorylated. In that respect, we show that the phosphorylation of the serine residues located in the AF-1 domain is markedly increased in response to RA through a RA-induced activation of p38MAPK. Finally, we show that blocking RARγ2 degradation in the presence of ubiquitin proteasome inhibitors, p38MAPK inhibitors, or through competition of endogenous proteasomal SUG-1, impairs RA-induced transcriptional activation by RARγ2. Results Proteasome-dependent degradation is required for activation of transcription by RA-liganded RARγ2 To address the role of proteasome-mediated degradation in RARγ2-activated transcription, COS-1 cells cotransfected with a RARγ2 expression vector and a CAT reporter gene controlled by a DR5 RA-responsive element (DR5-tk-CAT) were treated with RA and the proteasome inhibitor MG132, or their respective vehicles. RA treatment resulted in a significant decrease in the amount of RARγ2 that was maximal at 48 h (Figure 1A, lane 2; data not shown). Cotransfection of RXRα increased the degradation of liganded RARγ2 (Figure 1A, lane 6), as well as its ability to induce CAT activity (Figure 1B, compare lanes 2 and 3), in agreement with the known heterodimer requirement for both processes (Kopf et al., 2000). Figure 1.RA-induced RARγ2 degradation and transactivation are reversed by the proteasome inhibitor MG132 and require engagement of the receptor at a RARE. (A) COS-1 cells cotransfected with the DR5-tk-CAT reporter construct and the expression vectors for mRARγ2 (WT or ΔC) and RXRα, were treated for 48 h with vehicle or 1 × 10−6 M RA. When mentioned, MG132 was added 15 h before harvesting. In lanes 15 and 16 the DR5 element of the CAT reporter gene was mutated and in lanes 17 and 18 the DR5 element was deleted. WCEs were immunoblotted with RPγ(F) (upper panels) or actin antibodies (lower panels). (B) COS-1 cells were cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2, in the absence (−) or presence (+) of RXRα as indicated. In lane 5 the DR5 responsive element was mutated. The cells were treated as in (A) and analyzed for CAT activity. The results are the mean ± SD of three independent experiments. Download figure Download PowerPoint MG132 prevented RARγ2 degradation (Figure 1A, lanes 4 and 8), confirming that proteasome mediates the degradation of the receptor (Kopf et al., 2000). MG132 did not alter RARγ2 levels in the absence of RA (Figure 1A, compare lanes 1 and 3, and 5 and 7), nor β-actin levels (Figure 1A, lower panel). Interestingly, MG132 also abrogated the induction of CAT activity (Figure 1B). The basal level of CAT activity observed in the absence of RARγ2 and/or in the absence of RA was unaltered by the proteasome inhibitor (Figure 1B, lane 1). Similarly, the activity of the β-galactosidase gene, which was added to allow normalization of the results according to transfection efficiency, was not affected (data not shown). Note that similar results were obtained with lactacystin, a specific proteasome inhibitor (data not shown). Importantly, RARγ2 was not significantly degraded when cotransfected with a reporter gene in which the DR5 RA responsive element had been deleted (the tk-CAT reporter gene; Figure 1A, lanes 17 and 18) or mutated (the DR5 mut-tk-CAT reporter gene; Figure 1A, lanes 15 and 16), even in the presence of cotransfected RXRα. Moreover, a transcriptionally inactive deletion mutant of RARγ2 lacking the DNA binding domain (RARγΔC) was also resistant to the RA-induced degradation (Figure 1A, lanes 9–12). Altogether, these data demonstrate that RARγ2 degradation and transactivation processes are coupled, and indicate that degradation of the receptor requires its engagement in transcription. The effects of proteasome inhibitors were also tested in F9 embryocarcinoma cells, on both degradation of endogenous RARγ2 and RA-induced RARγ2-mediated transcription of endogenous RA-target genes. (for a review, see Rochette-Egly and Chambon, 2001). In these cells, maximal degradation of RARγ2 occurs at 48 h of RA treatment (Kopf et al., 2000; see also Figure 2A, lane 2), when the transactivation of RA-target genes (collagen IV, laminin B1, Stra4, HNF1β and HNF3α) is maximal and reaches a plateau (Figure 2B). Note that the heterodimerization partner RXRα was not degraded (Figure 2A, lane 2). However, RA-dependent degradation of RARγ2 requires the presence of RXRα (Kopf et al., 2000), and accordingly RXRα−/− F9 cells (Clifford et al., 1996) did not show any evidence of RARγ2 degradation in response to RA (Figure 2C). In F9 cells, MG132 and lactacystin abrogated the RA-induced decrease in RARγ2 levels (Figure 2A, compare lanes 2 and 4; data not shown), but did not alter RARγ2 levels in the absence of RA (Figure 2A, compare lanes 1 and 3), nor β-actin levels (Figure 2A, lower panel). MG132 also abrogated the RA-induced expression of RA target genes (Figure 2B). This lack of induction did not result from a general block in transcription, as MG132 did not affect the basal level of expression of the tested genes and the expression of RA unresponsive genes (RARγ2, 36B4, Nedd4, Pin-1 and many others; Figure 2B; data not shown). Collectively these results indicate that proteasome activity is also required for RA-induced RARγ2-mediated transcriptional activation through endogenous promoters. Figure 2.RA-induced RARγ2 degradation and transcription are also reversed by MG132 in F9 cells. (A) F9 WT cells were treated for 48 h with vehicle or 1 × 10−7 M RA. When mentioned, MG132 was added 15 h before harvesting. Whole cell extracts (WCEs) were resolved by SDS–10% PAGE and immunoblotted with RPγ(F), RPRXα(A), or actin antibodies. (B) F9 WT cells were treated for 48 h as in (A), as indicated. Transcripts for collagen IV, laminin B1, Stra4, HNF1β, HNF3α and RARγ2 were analyzed by quantitative RT–PCR. The presented values are the mean ± SD of three individual experiments and correspond to the fold-induction relative to the amount of transcripts present in vehicle-treated cells which was given an arbitrary value of 1. (C) RXRα−/− F9 cells were treated as in (A) and WCEs were immunoblotted with RPγ(F) or actin antibodies. (D) F9 cells that were either WT (lanes 1–4) or expressing RARγΔH12 (lanes 5–8) or RARγS66/68A (lanes 9–12) were treated as in (A) and WCEs were immunoblotted with RPγ(F) or actin antibodies. Download figure Download PowerPoint Contribution of the activation domains AF-1 and AF-2 to RA-induced RARγ2 degradation To determine which receptor domain(s) (see Figure 3A) contribute to the proteasome-dependent degradation of RARγ2, expression vectors for mutant forms of the receptor were cotransfected with the DR5-tk-CAT reporter gene in COS-1 cells. After treatment with RA, in the presence or absence of MG132, the degradation (Figure 3B) and the transcriptional activity (Figure 3C) of each mutant were analyzed. Figure 3.Both the AF-1 and AF-2 activation domains contribute to the RA-induced degradation of RARγ2. (A) Schematic representation (not to scale) of mRARγ2 with the DBD and the functional AF-1 and AF-2 domains, which lie in the A/B and E regions, respectively. The target sequence for phosphorylation by proline-directed kinases in the B region is shown and the corresponding serine residues, which have been mutated to alanine (S66 and S68), are indicated. (B) COS-1 cells were cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2 either WT, ΔF, ΔH12, ΔAB, ΔA, ΔB or S66/68A and treated with vehicle or 1 × 10−6 M RA as indicated. When mentioned, MG132 was added 15 h before harvesting. Equal amounts of WCEs, as estimated by immunoblotting with actin antibodies (data not shown) were resolved by SDS–10% PAGE and immunoblotted with RPγ(F) or Ab5γ(D) in the case of RARγ2ΔF. (C) Cells transfected as in (B) were analyzed for CAT activity. The results are the mean ± SD of three independent experiments. Download figure Download PowerPoint When compared with the wild-type receptor, deletion of the C-terminal F region (RAR2γΔF) did not affect the RA-induced degradation and transcriptional activity of the receptor, nor the inhibition by MG132. Thus, the F region appears to be dispensible for RA-dependent degradation of RARγ2. The E region contains the transactivation function AF-2 whose activity is dependent on the integrity of the AF-2 AD core, which corresponds to helix 12 of the C-terminal end of the ligand-binding domain. Deletion of helix 12 in RARγ2 (RARγ2ΔH12) resulted in abrogation of both RA-induced RARγ2 transcriptional activity and degradation (Figure 3B and C). Note that RARγ2ΔH12 was in fact stabilized by RA, most likely because the ligand induced a conformational change protecting the receptor from proteolytic digestion (Wijayaratne and McDonnell, 2001). RARγ2, lacking the entire N-terminal A/B region which contains the ligand-independent transactivation function AF-1 (RARγΔAB), was transcriptionally impaired and was not degraded in response to RA (Figure 3B and C). In contrast, deletion of the A region (RARγΔA) had no effect on RA-induced receptor degradation and transactivation. On the other hand, elimination of the B region (RARγΔB) resulted in a receptor with the same characteristics as RARγΔAB. Interestingly, mutation of the two phosphorylation sites, serines 66 and 68 (see Figure 3A) present in the B region of RARγ2 (RARγS66/68A) (Bastien et al., 2000) decreased RA-induced transactivation and abrogated RA-induced degradation. Note that the addition of MG132 suppressed the residual amount of RA-induced transactivation and further protected this mutant against degradation. Similar results were obtained in RARγ−/− F9 embryocarcinoma cells expressing RARγ2ΔH12 or RARγ2S66/68A. These cells are known to be deficient for RA-induced expression of RA-target genes (Taneja et al., 1997; Plassat et al., 2000). In both cell lines, RARγ2 levels were not affected by RA addition (Figure 2D, lanes 6 and 10). Thus, taken together, the above results indicate that the integrity of the AF-2 domain and of the phosphorylation sites located in the B region are essential for both RA-induced degradation of, and transactivation by, RARγ2. Ubiquitylation is required for RA-induced RARγ2 degradation and transactivation Polyubiquitylation is a prerequisite for the degradation of proteins through the proteasome pathway (Laney and Hochstrasser, 1999). The ts85 mouse mammary carcinoma cell line, which harbors a temperature-sensitive mutation of the ubiquitin activating (UBA) enzyme, is defective for protein ubiquitylation (Finley et al., 1984). These cells transiently transfected with the RARγ2 expression vector and the DR5-tk-CAT reporter gene, were treated with RA and incubated at the permissive (30°C) or the restrictive (37°C) temperatures. Under these conditions, RARγ2 was degraded at the permissive, but not at the restrictive temperature (Figure 4A, compare lanes 2 and 4). Moreover, incubation at the restrictive temperature decreased RA-induced RARγ2-mediated transcription (Figure 4B, compare lanes 2 and 4). Incubation of ts85 cells at the restrictive temperature had no influence on the basal level of CAT activity observed in the absence of RARγ2 and/or in the absence of RA (Figure 4B, lanes 1 and 3; data not shown). Moreover, it did not affect the expression of β-actin (Figure 4A, lower panel) or the levels of β-galactosidase activity expressed from the cotransfected control plasmid pCH110 (data not shown), indicating specificity of the RA-induced degradation of RARγ2 by the ubiquitin–proteasome pathway. Altogether, these results indicate that ubiquitylation is essential for both RA-induced RARγ2 degradation by the proteasome pathway and transactivation. Figure 4.Disruption of the ubiquitin-activating enzyme (UBA) function abrogates RA-induced RARγ2 degradation and transactivation. The temperature-sensitive UBA mutant ts85 cell line was transfected with the DR5-tk-CAT reporter gene and the expression vector for mRARγ2, treated with vehicle or 1 × 10−6 M RA and incubated at permissive (30°C) or restrictive (37°C) temperature for 24 h before harvesting. Extracts were immunoblotted with RPγ(F) and actin antibodies (A) and analyzed for CAT activity (B). The results, which correspond to the fold-induction relative to the CAT activity in vehicle-treated cells, are the mean ± SD of three independent experiments. Download figure Download PowerPoint RA induces phosphorylation of RARγ2 in its AF-1 domain through activation of p38MAPK Serine 66 and/or serine 68 in the AF-1 domain of RARγ2 are constitutively phosphorylated by cdk7 within TFIIH (Bastien et al., 2000). As these residues are also substrates for MAPKs and SAPKs in vitro (Bastien et al., 2000; our unpublished data), we investigated whether RARγ2 degradation might be linked to phosphorylation of the receptor by MAPKs in response to RA. RARγ2-transfected COS-1 cells were treated with RA and labelled with [32P]orthophosphate. Interestingly, the phosphorylation of RARγ2 was increased after 24 h of RA treatment (Figure 5A, lane 2), thus before its degradation could be seen (data not shown). This increase was not detectable up to 16 h of RA treatment (Bastien et al., 2000; data not shown). Figure 5.RA increases the amount of RARγ2 phosphorylated in its AF-1 domain, subsequent to the activation of p38MAPK. (A) COS-1 cells plated in 10 cm Petri dishes and cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2 either WT (lanes 1–4) or S66/68A (lanes 5 and 6) were treated with vehicle (lane 1) or 1 × 10−6 M RA (lane 2). In lanes 3 and 4, RA was combined with SB203580 (10 μM) or PD98059 (5 μM), respectively. Lanes 7 and 8 correspond to F9 WT cells, treated or not with RA (1 × 10−7 M). Cells were labelled with [32P]orthophosphate and WCEs were immunoprecipitated with mAb2γ(mF). Immunoprecipitates containing equal amounts of RARγ2 were resolved by SDS–10% PAGE, electrotransferred onto nitrocellulose (NC) filters, autoradiographed [32P] and immunoprobed with RPγ(F) by western blotting (WB). (B) Two-dimensional tryptic phosphopeptide mapping of 32P-labelled immunoprecipitated RARγ2WT (panels 1–3) and RARγ2S66/68A (panel 4). (C) RA activates p38MAPK. Transfected COS-1 cells (lanes 1 and 2) and F9 WT cells (lanes 3 and 4), were treated for 24 h with vehicle or RA as indicated. Then the cells were lysed and immunoprecipitated with a p38MAPK rabbit polyclonal antibody immobilized on Protein A–Sepharose beads. The immunoprecipitates were immunoblotted with antibodies recognizing specifically p38MAPK or its phosphorylated form, P-p38MAPK. (D) Phosphorylation of ATF-2 upon activation of p38MAPK. F9 WT cells were treated for 48 h with vehicle (lane 1), 1 × 10−7 M RA (lane 2), 10 μM SB203580 (lane 3), or 5 μM PD98058 (lane 5). In lanes 4 and 6, RA was combined with SB203580 or PD98058. WCEs were immunoprecipitated with a Phospho-p38MAPK rabbit polyclonal antibody immobilized on Protein A–Sepharose beads, washed and processed for phosphorylation of the 40 kDa ATF-2 fusion protein (5 μg) in kinase buffer (25 mM HEPES, 25 mM MgCl2, 25 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3V04 and 20 μM ATP) for 30 min at room temperature. The reaction was terminated upon addition of the SDS sample buffer, and the phospho (P-ATF-2) and non-phospho forms of ATF-2 were detected by immunoblotting with specific antibodies. Download figure Download PowerPoint In the absence of RA treatment, tryptic phosphopeptide mapping of RARγ2 yields four phosphorylated peptides (Figure 5B, panel 1). Phosphopeptides a and b contain the phosphorylated residues of the AF-1 domain (spot a is diphosphorylated at serines 66 and 68, while spot b is monophosphorylated at serine 68; see Bastien et al., 2000). The other phosphopeptides (d and e) map to region F (Bastien et al., 2000). RA markedly increased the phosphorylation of peptide a, and to a lesser extent that of peptide b (Figure 5B, panel 2). Thus, RA treatment increased selectively the phosphorylation of both serines 66 and 68. Accordingly, in COS-1 cells overexpressing a RARγ2 with the two serine residues mutated into alanine (RARγS66/68A), no increase in the amount of phosphorylated receptor was observed upon RA treatment (Figure 5A, lanes 5 and 6) and spots a and b were absent (Figure 5B, panel 4). F9 WT cells also responded to RA by an increase in the amount of phosphorylated RARγ2 (Figure 5A, lanes 7 and 8). No such increase could be seen in F9 cells expressing RARγS66/68A in a RARγ-null background (Taneja et al., 1997; data not shown). To determine whether MAPKs are involved in RA-induced phosphorylation of RARγ2, transfected COS-1 cells were treated with RA in the absence or presence of MAPK inhibitors before labeling with 32P. SB203580 (10 μM), a highly specific inhibitor of p38MAPK (Cohen, 1996), abrogated the RA-dependent increase in phosphorylated RARγ2 (Figure 5A, lane 3) and in phosphopeptides a and b (Figure 5B, panel 3). In contrast, the MEK1 inhibitor PD98059 (5 μM; Cohen, 1996) had no significant effect (Figure 5A, lane 4). Altogether, these results indicate that RA increases the amount of phosphorylated RARγ2, through activation of p38MAPK, but not of Erks. Thus, we determined the state of phosphorylation and activation of p38MAPK in RA-treated transfected COS-1 cells and F9 WT cells. In both cell systems, the phosphorylation of p38MAPK was induced after 24 h of RA treatment (Figure 5C), and was maximal at 48 h (data not shown), as assessed by western blotting analysis with specific antibodies recognizing the phosphorylated form of the kinase (P-p38). RA had no effect at earlier times (data not shown). Under these conditions, p42/p44 Erks were not activated (data not shown). That RA activates p38MAPK was confirmed by the observation that p38MAPK phosphorylates ATF-2 (a well known substrate of the kinase) more efficiently when immunoprecipitated from RA-treated F9 cells rather than from control untreated cells. (Figure 5D, lane 2). As expected, this phosphorylation of ATF-2 was completely abolished upon incubation of RA-treated F9 cells with the p38MAPK inhibitor SB203580, but not with the MEK1 inhibitor PD98059 (Figure 5D, lanes 4 and 6). RA-induced phosphorylation of RARγ2 is required for RARγ2 degradation and transactivation As RARγS66/68A is resistant to RA-induced degradation (Figure 3A and Figure 2D, lanes 9–12) and impaired in transactivation (Rochette-Egly et al., 1997; Taneja et al., 1997; Figure 3C), we investigated whether the RA-induced phosphorylation of RARγ2 affects its degradation and transactivation activity. To this end we inhibited the RA-dependent RARγ2 phosphorylation. SB203580 treatment of transfected COS-1 cells abrogated RA-induced degradation of RARγ2 (Figure 6A, lanes 3 and 4), whereas PD98059 was ineffective (Figure 6A, lanes 5 and 6). SB203580 also decreased RA-induced degradation of RARγ2 in F9 cells (data not shown) and blocked the induced expression of laminin, HNF3α and HNF1β, which was observed after 48 h of RA treatment (Figure 6B), while PD98059 had no effect (Figure 6B). Figure 6.Phosphorylation by p38MAPK is required for RA-induced RARγ2 degradation and transactivation. (A) COS-1 cells cotransfected with the DR5-tk-CAT reporter construct and the expression vector for mRARγ2 were treated for 48 h with vehicle, RA, SB203580 or PD98058, either alone or in combination, as indicated. WCEs were resolved by SDS–10% PAGE and immunoblotted with RPγ(F) (upper panels) or actin antibodies (lower panels). (B) F9 WT cells were treated for 48 h with vehicle, 1 × 10−7 M RA, 10 μM SB203580 or 5 μM PD98059 either alone or in association as indicated. Transcripts for laminin B1, HNF3α, HNF1β and RARγ2 were analyzed by quantitative RT–PCR. The presented results are an average of at least three independent experiments which agreed within ±15%. The values correspond to the fold-induction relative to the amount of transcripts present in vehicle-treated cells. Download figure Download PowerPoint Collectively, these results indicate that the RA-induced phosphorylation of RARγ2 by p38 MAPK is required for both RARγ2 degradation and RARγ2-mediated activation of transcription. Overexpression of SUG-1, a subunit of the 26S proteasome complex, reverses RA-induced degradation of RARγ2 As the proteasome activity is required for RA-induced degradation of RARγ2, we investigated whether SUG-1 could be involved in this process. SUG-1 is a nuclear receptor-interacting protein which belongs to the 19S regulatory complex of the 26S proteasome (Rubin et al., 1996; Glickman et al., 1998; DeMartino and Slaughter, 1999). The RA-dependent interaction of SUG-1 with RARs requires the integrity of the receptor AF2-AD core (helix 12) (vom Baur et al., 1996) and of the SUG-1 AAA module (ATPases Associated with a variety of cellular Activities), which contains a putative ATP binding site consensus motif (Fraser et al., 1997). To investigate whether proteasomal SUG-1 is recruited by liganded RARγ2 to selectively specify its degradation, competition experiments were performed by transfecting COS-1 cells with increasing concentrations of an expression vector for SUG-1, either WT or mutated at its AAA module (K196H mutation in SUG-1m) (Fraser et al., 1997), along with RARγ2 and the DR5-tk-CAT reporter gene. Interestingly, overexpressed SUG-1WT abrogated the RA-induced degradation of RARγ2 (Figure 7A, lanes 3–5) and inhibited its transactivation activity (Figure 7B, lanes 4–6). In contrast, the SUG-1 K196H mutant which cannot bind nuclear receptors (vom Baur et al., 1996) did not affect RARγ2 degradation (Figure 7A, lanes 8–10) and transactivation activity (Figure 7B, lanes 7–9). These effects of SUG-1 were specific for RARγ2, as β-actin levels (Figure 7A, lower panels) and the basal CAT activity observed in the absence of RARγ2 (Figure 7B, lanes 1 and 2) were unaffected. In contrast, overexpression of the coactivators TIF2 and SRC-1 did not exert any significant effect on the RA-induced degradation of RARγ2 (Figure 7C, compare lanes 1–4). Note that overexpressed SUG-1, TIF2 and SRC-1 did not undergo any degradation upon addition of RA (Figure 7A; data n