The Co-repressor Hairless Protects RORα Orphan Nuclear Receptor from Proteasome-mediated Degradation
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m308152200
ISSN1083-351X
AutoresAnna N. Moraitis, Vincent Giguère,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoRORα is a constitutively active orphan nuclear receptor essential for cerebellar development and is previously shown to regulate genes involved in both myogenesis and adipogenesis. The transcriptional activity of RORα is dependent on the presence of a ubiquitous ligand and can be abolished by interaction with Hairless (Hr), a ligand-oblivious nuclear receptor co-repressor. In this study, we first demonstrate that RORα is a short-lived protein and that treatment with the MG-132 proteasome inhibitor results in the accumulation of ubiquitin-conjugated receptor and inhibition of transcription. These data show that RORα transcriptional activity and degradation are intrinsically linked. In addition, the introduction of inactivation mutations in the ligand-binding pocket and co-regulator-binding surface of RORα significantly increases protein stability, indicating that ligand and/or co-regulator binding perpetuates RORα degradation. Strikingly, expression of the co-repressor Hr results in the stabilization of RORα because of an inhibition of proteasome-mediated degradation of the receptor. Stabilization of RORα by Hr requires intact nuclear receptor recognition LXXLL motifs within Hr. Interestingly, the co-repressor nuclear receptor co-repressor (NCoR) has no effect on RORα protein turnover. This study shows that stabilization of RORα is an essential component of Hr-mediated repression and suggests a molecular mechanism to achieve transcriptional repression by a liganded receptor-co-repressor complex. RORα is a constitutively active orphan nuclear receptor essential for cerebellar development and is previously shown to regulate genes involved in both myogenesis and adipogenesis. The transcriptional activity of RORα is dependent on the presence of a ubiquitous ligand and can be abolished by interaction with Hairless (Hr), a ligand-oblivious nuclear receptor co-repressor. In this study, we first demonstrate that RORα is a short-lived protein and that treatment with the MG-132 proteasome inhibitor results in the accumulation of ubiquitin-conjugated receptor and inhibition of transcription. These data show that RORα transcriptional activity and degradation are intrinsically linked. In addition, the introduction of inactivation mutations in the ligand-binding pocket and co-regulator-binding surface of RORα significantly increases protein stability, indicating that ligand and/or co-regulator binding perpetuates RORα degradation. Strikingly, expression of the co-repressor Hr results in the stabilization of RORα because of an inhibition of proteasome-mediated degradation of the receptor. Stabilization of RORα by Hr requires intact nuclear receptor recognition LXXLL motifs within Hr. Interestingly, the co-repressor nuclear receptor co-repressor (NCoR) has no effect on RORα protein turnover. This study shows that stabilization of RORα is an essential component of Hr-mediated repression and suggests a molecular mechanism to achieve transcriptional repression by a liganded receptor-co-repressor complex. The ubiquitin-proteasome pathway is the major system employed by eukaryotes for the selective degradation of cellular proteins that play key roles in cellular processes such as cell cycle regulation, differentiation, signal transduction, transcription, and chromosomal stabilization (reviewed in Refs. 1Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1585) Google Scholar and 2Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3318) Google Scholar). Proteolytic degradation by the ubiquitin-proteasome system involves ATP-dependent covalent attachment of a macromolecular chain of ubiquitin (Ub) 1The abbreviations used are: UbubiquitinSRCsteroid receptor co-activatorSUGsuppressor of Gal4LBDligand-binding domainHrHairlessE1ubiquitin-activating enzymeE2ubiquitin-conjugating enzymeNCoRnuclear receptor co-repressorE3ubiquitin-protein isopeptide ligaseHAhemagglutinin.1The abbreviations used are: UbubiquitinSRCsteroid receptor co-activatorSUGsuppressor of Gal4LBDligand-binding domainHrHairlessE1ubiquitin-activating enzymeE2ubiquitin-conjugating enzymeNCoRnuclear receptor co-repressorE3ubiquitin-protein isopeptide ligaseHAhemagglutinin. molecules to the target protein, followed by degradation through the multicatalytic 26 S proteasome. The conjugation of Ub, a highly conserved 8.6-kDa protein, to its target protein is mediated by the serial action of three enzymes: E1, the Ub-activating enzyme, activates Ub in an ATP-dependent manner; E2, the Ub-conjugating enzyme, catalyzes the attachment of Ub to the substrate protein; and E3, the Ub-ligases, serves as a scaffold between E2 and the substrate and provides recognition specificity of the substrate. Ubiquitinylation of a substrate is reversible, and Ub moieties can be cleaved from a target protein by deubiquitinating enzymes. These enzymes assure that the cell is not depleted of a Ub pool. A protein tagged with a polyubiquitin chain is recognized and degraded by the 26 S proteasome complex. This complex is composed of a 19 S regulatory subcomplex, consisting of a "lid" subunit and a "base" subunit, the latter containing the six ATPases required for the degradation executed by the 20 S catalytic subcomplex (2Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3318) Google Scholar). ubiquitin steroid receptor co-activator suppressor of Gal4 ligand-binding domain Hairless ubiquitin-activating enzyme ubiquitin-conjugating enzyme nuclear receptor co-repressor ubiquitin-protein isopeptide ligase hemagglutinin. ubiquitin steroid receptor co-activator suppressor of Gal4 ligand-binding domain Hairless ubiquitin-activating enzyme ubiquitin-conjugating enzyme nuclear receptor co-repressor ubiquitin-protein isopeptide ligase hemagglutinin. The Ub-proteasome pathway has recently emerged as a key regulator of transcription controlling the level, location, and activity of transcription factors and associated co-factors (3Muratani M. Tansey W.P. Nat. Rev. Mol. Cell Biol. 2003; 4: 192-201Crossref PubMed Scopus (670) Google Scholar). Nuclear receptors are short-lived transcription factors whose turnover is mediated by the Ub-proteasome complex (reviewed in Ref. 4Dennis A.P. Haq R.U. Nawaz Z. Front. 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Proteasomal inhibition is thus unfavorable to RORα transcriptional activity. Taken together, these results show that protection of RORα from the activity of the Ub-proteasome pathway by Hr is an integral component of RORα-regulated transcription. Plasmids—pCMX-hRORα1 wild type and ligand-binding domain mutants L361F, V364G, K357A, and E509K as well as pCMX-FLAG-hRORα1 have been previously described (32Moraitis A.N. Giguère V. Thompson C.C. Mol. Cell Biol. 2002; 22: 6831-6841Crossref PubMed Scopus (81) Google Scholar). N-terminal deletion mutants of hRORα1, RORαΔN12, RORαΔN25, and RORαΔN35 have been described elsewhere (42Giguère V. McBroom L.D.B. Flock G. Mol. Cell Biol. 1995; 15: 2517-2526Crossref PubMed Google Scholar). pCMV-HA-Ub consists of a octameric Ub construct; each Ub is preceded at its N terminus by an HA tag as described in (43Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (846) Google Scholar). PRK5-Myc-rHr and LXXLL mutants (Hrm1, Hrm2, and Hrm3) have been previously described (32Moraitis A.N. Giguère V. Thompson C.C. Mol. Cell Biol. 2002; 22: 6831-6841Crossref PubMed Scopus (81) Google Scholar). The pCMX-hNCoR was a gift from G. Rosenfeld (La Jolla, CA) and was described in Ref. 44Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamel Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1699) Google Scholar. Cell Culture and Transient Transfection—Cos-1 cells obtained from the American Type Culture Collection were cultured in Dulbecco's minimal essential medium containing penicillin (25 units/ml), streptomycin (25 units/ml), and 10% fetal calf serum at 37 °C with 5% CO2. Twenty-four hours prior to transfection, the cells were split and seeded in 12-well plates. The cells were transfected with FuGENE 6 Transfection Reagent (Roche Applied Science), following the protocol supplied by the manufacturer. A total of 1 μg of DNA/well was transfected including 0.05 μg of pCMX-hRORα1 or mutant derivatives, 0.5 μg of pCMX-hNCoR or pRK5-Myc-rHR, 0.5 μg of reporter plasmid, and 0.25 μg of internal control pCMVβGal. The cells were treated with ethanol (vehicle) or 0.1, 0.5, or 1.0 μm MG-132 for 6–24 h, as specified in the figure legends. The cells were harvested and assayed for luciferase and β-galactosidase activities. The normalized values are expressed in terms of relative luciferase units. The error bars represent the standard deviations between duplicate samples. Each graph is one representative experiment of a total of three independent experiments. Co-immunoprecipitation and Immunoblotting Assays—Cos-1 cells in 10-cm dishes were transiently transfected as described above with 10 μg of FLAG-RORα and HA-Ub and treated with ethanol (vehicle) or 1 μm MG-132 for 24 h. The cells were lysed in IP buffer (1% Nonidet P-40, 10% glycerol, 150 mm NaCl, 50 mm Tris-HCl, pH 7.5) supplemented with protease inhibitor mixture (Complete Mini EDTA-free; Roche Applied Science). The lysates (containing a total of 250 μg of protein) were incubated with 5 μg of FLAG antibody (Sigma) overnight at 4 °C with gentle rotation. The proteins were collected on protein G-Sepharose for 2 h at 4 °C with mild rotation and then washed three times with ice-cold low salt buffer (1% Nonidet P-40, 50 mm Tris-HCl, pH 8.0). The immunoprecipitates were resolved by SDS-PAGE, transferred to a hydrophobic polyvinylidene difluoride membrane (Amersham Biosciences), and immunoblotted with FLAG antibody or HA antibody (HA.11; Berkeley Antibody Company). The proteins were visualized with the POD chemiluminescence kit following the manufacturer's instructions (Roche Applied Science). Immunoblotting for detection of RORα wild type and mutants, Myc-Hr, SRC-1, hNCoR, or actin was similarly done using anti-RORα antibody (C-16; Santa Cruz Biotechnology), anti-c-Myc antibody (Roche Applied Science), anti-SRC-1 antibody (M-341; Santa Cruz Biotechnology), anti-hNCoR antibody (H-303; Santa Cruz Biotechnology), and anti-actin antibody (I-19; Santa Cruz Biotechnology), respectively. The lysates were prepared from transiently transfected Cos-1 harvested in modified RIPA buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mm Na3VO4, 1mm NaF), resolved by SDS-PAGE, transferred, and immunoblotted as described above. In Vitro Degradation Assay—The cell extract was prepared from Cos-1 cells harvested in modified RIPA buffer. 5 μlof in vitro translated [35S]methionine-labeled RORα, using TnT rabbit reticulocyte lysate (Promega, Madison, WI), was incubated with 50 μg of cell extract, ethanol (vehicle), or 50 μm MG-132, 20 μm lactacystin, 50 μg/ml expressed sequence tag, 2 mm phenylmethylsulfonyl fluoride in a final volume of 50 μl of degradation buffer (20 mm Tris-HCl, pH 7.4, 50 mm NaCl, 0.2 mm dithiothreitol) for 2 h at 37 °C. The samples were resolved by SDS-PAGE. The gels were fixed, treated with the fluorographic reagent Amplify (Amersham Biosciences), dried, and exposed. Pulse-Chase Assay—Cos-1 cells in 10-cm dishes were transiently transfected as described above, with 5 μg of pCMX-hRORα1 and pRK5-Myc-rHr or pCMX-hSRC-1 for 24 h as specified in figure legends. The cells were washed carefully with 1× phosphate-buffered saline, and the medium was replaced with Dulbecco's modified Eagle's medium (-methionine/-cysteine) for 2 h, followed by the addition of [35S]methionine (100 μCi/ml) for an additional 1 h. The cells were washed with 1× phosphate-buffered saline and chased with Dulbecco's modified Eagle's medium for the times indicated in the figure legends. The cells were harvested and lysed in IP buffer. 400 μg of lysate was immunoprecipitated with 5 μl of anti-RORα antibody (C-16; Santa Cruz Biotechnology) for 30 min at 4 °C with gentle rotation, followed by incubation with 50% slurry of protein G-Sepharose for an additional 30 min. The beads were then washed with low salt buffer (1% Nonidet P-40, 50 mm Tris-HCl, pH 8.0), followed by a wash in high salt buffer (500 mm NaCl, 1% Nonidet P-40, 50 mm Tris-HCl, pH 8.0), and resuspended in 2× SDS sample buffer. The samples were boiled for 3 min and resolved by SDS-PAGE. The gels were fixed and treated with fluorographic reagent, dried, and exposed. Quantification was performed using the Typhoon 8600 PhosphorImager (Amersham Biosciences). RORα, a Target of the Ub-Proteasome Complex—Nuclear receptors are short-lived proteins that are rapidly turned over by the Ub-proteasome complex. Using pulse-chase analysis, we first determined that the half-life of RORα in transiently transfected Cos-1 cells is ∼1.3 h (Fig. 1A). This rapid turnover is reminiscent of liganded nuclear receptors, which have a shorter half-life than their unliganded counterparts. We next investigated whether RORα degradation is mediated by the Ub-proteasome complex using pharmacological inhibitors. Peptide aldehydes (MG-132) or natural products (lactacystin) act as pseudosubstrates that become covalently linked to the 26 S proteasome and inactivate its chymotryptic and tryptic-like activities (45Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1236) Google Scholar). As shown in Fig. 1B, RORα is not expressed endogenously in Cos-1 cells, although RORα expressed through transient transfection is detected by immunoblotting with anti-RORα antibody. Blocking of the 26 S proteosome with MG-132 leads to a substantial increase of RORα protein, suggesting that RORα is a likely substrate of the Ub-proteasome complex (Fig. 1B). We next used Cos-1 extract as a source of Ub-proteasome complex in an in vitro degradation assay to determine whether in vitro translated and labeled RORα is proteolytically degraded. Loss of labeled RORα was observed upon incubation with Cos-1 extracts, a process that was not observed with extracts obtained from Cos-1 cells previously treated with the inhibitor MG-132, indicating that RORα is degraded by the 26 S proteasome in vitro (Fig. 2A). In addition to MG-132, treatment with lactacystin, an irreversible specific inhibitor of the 20 S proteasome, also blocked RORα degradation as demonstrated by a marked increase in protein expression in comparison with the control sample (Fig. 2B). In contrast, the lysosomal-specific cysteine protease inhibitor expressed sequence tag, as well as the nonspecific serine protease inhibitor phenylmethylsulfonyl fluoride, failed to stabilize RORα protein levels, providing further evidence that RORα is specifically degraded by the 26 S proteasome. Substrates destined for proteasomal degradation are tagged by covalent attachment of a macromolecular Ub chain. Co-immunoprecipitation of RORα and Ub resulted in the appearance of high molecular weight Ub-conjugated RORα complexes in cells treated with MG-132 (Fig. 2C). Given the absence of Ub-RORα complexes in untreated cells, Ub-tagged RORα is likely rapidly degraded by the 26 S proteasome under normal conditions (Fig. 2C).Fig. 2RORα is ubiquitinylated and degraded by the 26 S proteasome.A, in vitro degradation assay of in vitro translated and labeled RORα incubated with Cos-1 cell extracts treated with ethanol (-) or 50 μm MG-132 (+). The input (i) represents labeled RORα not subjected to the 37 °C incubation required for the degradation reaction. B, in vitro degradation assay of in vitro translated and labeled RORα incubated with Cos-1 extract in the presence of vehicle (ethanol), MG-132, lactacystin, expressed sequence tag (EST), or phenylmethylsulfonyl fluoride (PMSF) inhibitors as specified under "Experimental Procedures." C, Cos-1 cells transiently transfected with HA-tagged Ub (HAUb) and FLAG-tagged RORα (Flag-RORα) treated with ethanol (-) or 1 μm MG-132 (+) for 16 h. The lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody and immunoblotted (Blot) with anti-FLAG or anti-HA antibodies as specified under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) A Putative PEST Motif Is Not Involved in Degradation— Proteins targeted for degradation by the Ub-proteasome complex often contain a short hydrophilic stretch of at least 12 amino acids termed a PEST motif. A PEST region serves as a proteolytic signal leading to rapid destruction of the protein (46Rogers S. Wells R. Rechsteiner M. Science. 1986; 234: 364-368Crossref PubMed Scopus (1949) Google Scholar, 47Rechsteiner M. Rogers S.W. Trends Biochem. Sci. 1996; 21: 267-271Abstract Full Text PDF PubMed Scopus (1398) Google Scholar). We first used a PESTfind program () to identify putative PEST sequences in RORα. This algorithmic program scores the hydrophilicity in a range of -50 to +50, and scores above +5.0 are considered more probable PEST motif candidates. A putative PEST motif with a score of +6.88 was located in the N-terminal region of the protein (Fig. 3A). To determine the involvement of this putative PEST motif in signaling RORα degradation, we tested three N-terminal deletion mutants referred to as RORαΔ12, RORαΔ25, and RORαΔ35 (Fig. 3B). These constructs were transiently transfected in Cos-1 cells, and their transactivation potential was assessed on a RORE-driven reporter. All deletion proteins displayed potent transcriptional activity, which was inhibited by treatment of the cells with the MG-132 (Fig. 3B). Given that protein expression of RORαΔ25 is stabilized upon treatment with MG-132 (Fig. 3C), we have to conclude that deletion of the N-terminal PEST motif does not affect RORα degradation. Proteasomal Degradation Is an Integral Part of RORα Transactivation Potential—Ub-mediated degradation of nuclear receptors and other transcription factors is tightly coupled to their transactivation potential, providing the cell with a mechanism that protects it against possible deleterious prolonged periods of transcription at specific genes. In particular, it has been demonstrated that this pathway is imperative for a functional hormone-mediated transcriptional response of the estroge
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