Small Ubiquitin-like Modifier (SUMO) Modification of the Androgen Receptor Attenuates Polyglutamine-mediated Aggregation
2009; Elsevier BV; Volume: 284; Issue: 32 Linguagem: Inglês
10.1074/jbc.m109.011494
ISSN1083-351X
AutoresSarmistha Mukherjee, Monzy Thomas, Nahid Dadgar, Andrew P. Lieberman, Jorge A. Iñiguez-Lluhı́,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe neurodegenerative disorder spinal and bulbar muscular atrophy or Kennedy disease is caused by a CAG trinucleotide repeat expansion within the androgen receptor (AR) gene. The resulting expanded polyglutamine tract in the N-terminal region of the receptor renders AR prone to ligand-dependent misfolding and formation of oligomers and aggregates that are linked to neuronal toxicity. How AR misfolding is influenced by post-translational modifications, however, is poorly understood. AR is a target of SUMOylation, and this modification inhibits AR activity in a promoter context-dependent manner. SUMOylation is up-regulated in response to multiple forms of cellular stress and may therefore play an important cytoprotective role. Consistent with this view, we find that gratuitous enhancement of overall SUMOylation significantly reduced the formation of polyglutamine-expanded AR aggregates without affecting the levels of the receptor. Remarkably, this effect requires SUMOylation of AR itself because it depends on intact AR SUMOylation sites. Functional analyses, however, indicate that the protective effects of enhanced AR SUMOylation are not due to alterations in AR transcriptional activity because a branched protein structure in the appropriate context of the N-terminal region of AR is necessary to antagonize aggregation but not for inhibiting AR transactivation. Remarkably, small ubiquitin-like modifier (SUMO) attenuates AR aggregation through a unique mechanism that does not depend on critical features essential for its interaction with canonical SUMO binding motifs. Our findings therefore reveal a novel function of SUMOylation and suggest that approaches that enhance AR SUMOylation may be of clinical use in polyglutamine expansion diseases. The neurodegenerative disorder spinal and bulbar muscular atrophy or Kennedy disease is caused by a CAG trinucleotide repeat expansion within the androgen receptor (AR) gene. The resulting expanded polyglutamine tract in the N-terminal region of the receptor renders AR prone to ligand-dependent misfolding and formation of oligomers and aggregates that are linked to neuronal toxicity. How AR misfolding is influenced by post-translational modifications, however, is poorly understood. AR is a target of SUMOylation, and this modification inhibits AR activity in a promoter context-dependent manner. SUMOylation is up-regulated in response to multiple forms of cellular stress and may therefore play an important cytoprotective role. Consistent with this view, we find that gratuitous enhancement of overall SUMOylation significantly reduced the formation of polyglutamine-expanded AR aggregates without affecting the levels of the receptor. Remarkably, this effect requires SUMOylation of AR itself because it depends on intact AR SUMOylation sites. Functional analyses, however, indicate that the protective effects of enhanced AR SUMOylation are not due to alterations in AR transcriptional activity because a branched protein structure in the appropriate context of the N-terminal region of AR is necessary to antagonize aggregation but not for inhibiting AR transactivation. Remarkably, small ubiquitin-like modifier (SUMO) attenuates AR aggregation through a unique mechanism that does not depend on critical features essential for its interaction with canonical SUMO binding motifs. Our findings therefore reveal a novel function of SUMOylation and suggest that approaches that enhance AR SUMOylation may be of clinical use in polyglutamine expansion diseases. Spinal and bulbar muscular atrophy (SBMA), 2The abbreviations used are: SBMAspinal and bulbar muscular atrophyARandrogen receptorhARhuman ARAREandrogen-response elementSUMOsmall ubiquitin-like modifierSCsynergy controlSCAspinocerebellar ataxiaWTwild typeHAhemagglutininDAPI4′,6-diamidino-2-phenylindoleSTATsignal transducers and activators of transcriptionE1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligase. or Kennedy disease, is an inherited degenerative disorder of lower motor neurons (1Kennedy W.R. Alter M. Sung J.H. Neurology. 1968; 18: 671-680Crossref PubMed Google Scholar, 2La Spada A.R. Wilson E.M. Lubahn D.B. Harding A.E. Fischbeck K.H. Nature. 1991; 352: 77-79Crossref PubMed Scopus (2423) Google Scholar). SBMA is characterized by muscle cramps and fasciculations followed by progressive weakness and atrophy of the proximal limb and bulbar muscles (3Thomas M. Dadgar N. Aphale A. Harrell J.M. Kunkel R. Pratt W.B. Lieberman A.P. J. Biol. Chem. 2004; 279: 8389-8395Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 4Thomas M. Harrell J.M. Morishima Y. Peng H.M. Pratt W.B. Lieberman A.P. Hum. Mol. Genet. 2006; 15: 1876-1883Crossref PubMed Scopus (79) Google Scholar, 5Lieberman A.P. Fischbeck K.H. Muscle Nerve. 2000; 23: 843-850Crossref PubMed Scopus (65) Google Scholar). The causative genetic alteration is an expansion in the length of a CAG trinucleotide repeat within the coding sequence of the androgen receptor (AR) gene, leading to an expanded polyglutamine tract in the N-terminal transcriptional regulatory domain of the receptor. A similar expansion within the coding sequence of a set of additional genes is responsible for other members of the polyglutamine class of protein folding diseases (6Palazzolo I. Gliozzi A. Rusmini P. Sau D. Crippa V. Simonini F. Onesto E. Bolzoni E. Poletti A. J. Steroid Biochem. Mol. Biol. 2008; 108: 245-253Crossref PubMed Scopus (89) Google Scholar, 7Fischbeck K.H. 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The length of the CAG repeat within AR is correlated to the severity of SBMA. Although the normal repeat length is highly polymorphic and ranges between 9 and 36 copies, overt disease is associated with lengths in the range of 38–62 repeats. The presence of an expanded polyglutamine tract within AR renders the protein prone to hormone-dependent misfolding, oligomerization, and aggregation and to the formation of microscopically visible nuclear and/or cytosolic inclusions in neurons and in neuronal processes (11Cummings C.J. Zoghbi H.Y. Annu. Rev. Genomics Hum. Genet. 2000; 1: 281-328Crossref PubMed Scopus (282) Google Scholar). Although such large inclusions were initially proposed to be the proximal cause of neurodegeneration, their presence is not always correlated with the disease (12Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 13Klement I.A. Skinner P.J. Kaytor M.D. Yi H. Hersch S.M. 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Multiple mechanisms of neurotoxicity have been proposed, including alterations in transcriptional programs, mitochondrial dysfunction, and proteotoxic stress due to excessive demands placed on the ubiquitin-proteasome pathway. In addition, alterations in cellular transport mechanisms, which are critical to the function of motoneurons, have been also implicated (3Thomas M. Dadgar N. Aphale A. Harrell J.M. Kunkel R. Pratt W.B. Lieberman A.P. J. Biol. Chem. 2004; 279: 8389-8395Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 4Thomas M. Harrell J.M. Morishima Y. Peng H.M. Pratt W.B. Lieberman A.P. Hum. Mol. Genet. 2006; 15: 1876-1883Crossref PubMed Scopus (79) Google Scholar). Although the dominant inheritance of SBMA indicates the gain of a toxic function, an expanded polyglutamine tract in AR is also associated with a partial loss of function in its transcriptional properties (17Mhatre A.N. Trifiro M.A. Kaufman M. Kazemi-Esfarjani P. Figlewicz D. Rouleau G. Pinsky L. 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As in the case of other signaling processes, post-translational modifications are likely to play an important regulatory role in the adaptive responses to polyglutamine-induced aggregation. Their identity and mechanism, however, remain to be defined. In this regard, SUMOylation has been implicated in the pathogenesis of neurodegenerative diseases (21Lieberman A.P. Exp. Neurol. 2004; 185: 204-207Crossref PubMed Scopus (15) Google Scholar, 22Dorval V. Fraser P.E. Biochim. Biophys. Acta. 2007; 1773: 694-706Crossref PubMed Scopus (150) Google Scholar). Thus, enhanced neuronal immunoreactivity for SUMO1 is observed in dentatorubral-pallidoluysian atrophy, SCA-1, Machado-Joseph disease (also known as SCA-3), and Huntington disease (23Steffan J.S. Agrawal N. Pallos J. Rockabrand E. Trotman L.C. Slepko N. Illes K. Lukacsovich T. Zhu Y.Z. Cattaneo E. Pandolfi P.P. Thompson L.M. Marsh J.L. Science. 2004; 304: 100-104Crossref PubMed Scopus (561) Google Scholar). 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The identity of the SUMO interacting factors responsible for the effects of SC motifs, however, remains to be fully defined. In light of the established role of SUMO modification in regulating the transcriptional activity of AR, coupled with the potential for this modification to influence polyglutamine diseases, we investigate, in the present study, the role of SUMO modification in the aggregation behavior of polyglutamine-expanded AR. Our analysis reveals that SUMOylation significantly reduces AR aggregation. Notably, the mechanisms responsible are clearly distinct from those involved in regulating the transcriptional activity of AR and thus reveal a novel mode of action for this versatile post-translational modification. Expression vectors for AR forms are derivatives of the p5HB hAR cytomegalovirus-driven expression vector for WT human AR bearing a Gln24 tract. The SC motif mutants (p5HB hAR K385R/K518R) and (p5HB hAR I384N/V517N) were generated using the QuikChange Multi site-directed mutagenesis approach using p5HB hAR WT Gln24 as a template. To generate deletions of the first and second SC motifs (p5HB hAR ΔSC), 7 amino acids (Δ 382–388 in the first motif and Δ 515–521 in the second motif) were removed by seamless cloning through PCR and the type II restriction enzyme EarI, which cleaves outside its recognition site. The expanded (Gln113) glutamine tract (3Thomas M. Dadgar N. Aphale A. Harrell J.M. Kunkel R. Pratt W.B. Lieberman A.P. J. Biol. Chem. 2004; 279: 8389-8395Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) was transferred to p5HB hAR Gln24 as an EagI/AflII fragment to generate p5HB hAR Gln113. The mutant SC motif forms were transferred as RsrII/HindIII fragments from the Gln24 forms and ligated to the same sites of p5HB hAR Gln113. To generate the expression vector for the non-cleavable fusion of HA-SUMO3 at the N terminus of AR, p5HB hAR Gln113 K385R/K518R was first modified by site-directed mutagenesis to remove the second BamHI site located downstream of the AR coding sequence and to introduce an NheI site upstream of the AR coding sequence. This yielded p5HB NB hAR Gln113 AR KRKR. The HA-SUMO portion from pcDNA3 HA-SUMO3 (−Gly) Gal4 (36Holmstrom S. Van Antwerp M.E. Iñiguez-Lluhí J.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 15758-15763Crossref PubMed Scopus (130) Google Scholar, 38Chupreta S. Brevig H. Bai L. Merchant J.L. Iñiguez Lluhí J.A. J. Biol. Chem. 2007; 282: 36155-36166Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) was then excised as an NheI/BamHI fragment and ligated into the same sites of p5HB NB hAR Gln113 AR KRKR. The HA-tagged version of the preprocessed protease-resistant form of SUMO3 (HA-SUMO3-Q89P-GGstop) and the surface mutant (HA-SUMO3-Q89P-K33E/K42E-GGstop) were generated by site-directed mutagenesis using as a template the pCMV-driven (pcDNA3) expression vector for HA-tagged SUMO3 described previously (36Holmstrom S. Van Antwerp M.E. Iñiguez-Lluhí J.A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 15758-15763Crossref PubMed Scopus (130) Google Scholar). The reporter plasmid pΔ(TAT)4-Luc harbors four copies of a minimal response element from the tyrosine aminotransferase (TAT) gene upstream of a minimal Drosophila distal alcohol dehydrogenase promoter (−33 to +55) driving the luciferase gene (40Iñiguez-Lluhí J.A. Pearce D. Mol. Cell Biol. 2000; 20: 6040-6050Crossref PubMed Scopus (179) Google Scholar, 59Iñiguez-Lluhí J.A. Lou D.Y. Yamamoto K.R. J. Biol. Chem. 1997; 272: 4149-4156Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The FKBP5 luciferase reporter, which harbors a 500-bp genomic region centered on the AR binding region in the first intron of the human FKBP5 gene, was a kind gift of Dr Keith Yamamoto (60So A.Y. Chaivorapol C. Bolton E.C. Li H. Yamamoto K.R. PLoS Genet. 2007; 3: e94Crossref PubMed Scopus (245) Google Scholar). The pCMV Β-galactosidase and pRSV Β-galactosidase, which are cytomegalovirus- and Rous sarcoma virus-driven Β-galactosidase expression vectors, respectively, were used to correct for transfection efficiency. HeLa and human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10 or 5% charcoal-stripped fetal bovine serum, respectively. In all transfection experiments, cells received equimolar amounts of each type of expression plasmid to control for promoter dosage effects. HeLa cells (7.5 × 104/well) were seeded onto 6-well plates and co-transfected 24 h later using FuGENE-6® transfection reagent with 1 μg of expression vectors for expression of the Gln113 AR with WT or mutant SC motifs together with 0.3 μg of the indicated HA-SUMO3 expression vectors. Cultures were supplemented with 10 nm R1881 or vehicle (0.1% ethanol) 24 h after transfection and harvested 20 h later. Cells were lysed for 15 min on ice with 300 μl of high salt lysis buffer (20 mm Hepes, pH 7.5, 400 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1% Nonidet P-40) containing 20 mm N-ethylmaleimide and one tablet per 10 ml of CompleteTM protease inhibitors (Roche Diagnostics). After 5 min, N-ethylmaleimide was quenched by the addition of dithiothreitol to 40 mm final concentration and a further 2-min incubation on ice. A fraction (5%) of each sample was reserved for analysis, and the remaining cleared cell lysates were immunoprecipitated with 4 μl of rabbit polyclonal AR-N20 antibody (Santa Cruz Biotechnology) at 4 °C for 2 h. Complexes were recovered with 60 μl of 50% protein A-agarose (Invitrogen) at 4 °C for 2 h. The immunoprecipitates were washed three times in low salt lysis buffer (200 mm NaCl) and eluted in 4× Laemmli sample buffer. Samples were resolved by 7.5% SDS-PAGE and processed for immunoblotting as described below. For cell fractionation studies, quenched lysates were centrifuged at 15,000 × g for 15 min at 4 °C. Pellets were resuspended in a volume equal to the supernatant using low salt lysis buffer. Equal amounts of supernatant and pellet fractions were resolved by 7.5% SDS-PAGE and processed for immunoblotting as described below. As indicated, the supernatants were further fractionated by ultracentrifugation at 100,000 × g for 30 min at 4 °C. The supernatants were resolved by 4–20% gradient SDS-PAGE and processed for immunoblotting as described below. For the in vivo SUMOylation experiments, immunoblots were probed with primary rabbit polyclonal AR-N20 (Santa Cruz Biotechnology), mouse monoclonal HA-11 (Covance), or rabbit polyclonal SUMO2/3 (Abcam) antibodies followed by goat anti-rabbit or mouse IgG peroxidase-conjugated (Bio-Rad) secondary antibodies. AR expression levels were confirmed by Western blotting. Cells were transfected as described for the functional assays and lysed in 4× SDS-PAGE sample buffer resolved by 7.5% SDS-PAGE, transferred to Immobilon-P membranes (Millipore) using a wet transfer apparatus, and processed for immunoblotting. Immunoreactive proteins were detected by chemiluminescence. Images were captured in a Kodak Image Station 440 CF using SuperSignal West Femto substrates (Pierce). All the experiments were performed at least thrice with similar results. HeLa cells were transfected as described above for the in vivo SUMOylation experiments and seeded onto chambered slides 18 h after transfection. Cultures were supplemented with 10 nm R1881 or vehicle (0.1% ethanol) 6 h later and incubated a further 20 h. Cells were then fixed at −20 °C with methanol for 10 min, washed in phosphate-buffered saline, and blocked in 5% goat serum for 60 min at room temperature. Slides were incubated with primary rabbit polyclonal (AR-N20, 1:250) or mouse monoclonal (HA-11, 1:100) antibodies for 2 h at room temperature. Donkey anti-rabbit IgG conjugated to fluorescein and donkey anti-mouse IgG conjugated to Texas Red (from Jackson ImmunoResearch Laboratories) were used as secondary antibodies at 1:500 and 1:250 dilutions, respectively. After a 45-min incubation, slides were washed, and nuclei were counterstained with DAPI. Confocal images were captured using a Zeiss LSM 510 microscope and a ×63 water immersion objective. For quantitation, cells were examined using a Zeiss Axioplan 2 imaging system under a ×40 objective. AR aggregation was scored by determining the percentage of transfected cells with visible protein aggregates. For each experiment, transfections were performed in triplicate, and between 100 and 150 cells were scored in a blinded manner for each sample. Each experimental condition was then repeated in separate occasions between three and seven times. The data are expressed as the average ± S.E. Statistical analysis was carried out using the two-tailed Student's t test. Differences remained significant even after arcsine square root transformation of the proportions or when tested with the non-parametric χ2 test. For functional assays in HeLa cells (see Fig. 3), cells were seeded onto 24-well plates (1.8 × 104/well) and co-transfected 24 h later with 1 ng of AR expression vector, the indicated amounts of expression vector for HA-SUMO3Q89P, 100 ng of reporter plasmid, and 50 n
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