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• W2091907160 abstract "X-linked spinal and bulbar muscular atrophy is a degenerative disease affecting motor neurons that is caused by polyglutamine (polyQ) expansion within the androgen receptor (AR). The polyQ-expanded form of AR is cytotoxic to cells, and proteolytic cleavage enhances cell death. The intracellular signaling pathways activated and/or required for cell death induced by the expanded form of AR (AR112) are unknown. We found that AR regulates mitogen-activated protein kinase (MAP kinase) pathways and, therefore, hypothesized that these pathway(s) may be required for AR112-induced cell death. The polyQ expansion in AR activates three MAP kinase pathways, causing increasing levels of phosphorylation of p44/42, p38, and SAPK/JNK MAP kinase. Inhibitors of either the JNK or p38 pathways had no effect on AR112-induced cell death, suggesting they are not required for polyQ-induced cell death. Strikingly, the MEK1/2 inhibitor, U0126, which selectively inhibits the p44/42 MAP kinase pathway, reduces AR112-stimulated cell death. The inhibition of the MEK1/2 pathway correlates directly with a change in phosphorylation state of the androgen receptor. Mutation of the MAP kinase consensus phosphorylation site in AR at serine 514 blocked AR-induced cell death and the generation of caspase-3-derived cleavage products. We propose a mechanism by which phosphorylation at serine 514 of AR enhances the ability of caspase-3 to cleave AR and generate cytotoxic polyQ fragments. X-linked spinal and bulbar muscular atrophy is a degenerative disease affecting motor neurons that is caused by polyglutamine (polyQ) expansion within the androgen receptor (AR). The polyQ-expanded form of AR is cytotoxic to cells, and proteolytic cleavage enhances cell death. The intracellular signaling pathways activated and/or required for cell death induced by the expanded form of AR (AR112) are unknown. We found that AR regulates mitogen-activated protein kinase (MAP kinase) pathways and, therefore, hypothesized that these pathway(s) may be required for AR112-induced cell death. The polyQ expansion in AR activates three MAP kinase pathways, causing increasing levels of phosphorylation of p44/42, p38, and SAPK/JNK MAP kinase. Inhibitors of either the JNK or p38 pathways had no effect on AR112-induced cell death, suggesting they are not required for polyQ-induced cell death. Strikingly, the MEK1/2 inhibitor, U0126, which selectively inhibits the p44/42 MAP kinase pathway, reduces AR112-stimulated cell death. The inhibition of the MEK1/2 pathway correlates directly with a change in phosphorylation state of the androgen receptor. Mutation of the MAP kinase consensus phosphorylation site in AR at serine 514 blocked AR-induced cell death and the generation of caspase-3-derived cleavage products. We propose a mechanism by which phosphorylation at serine 514 of AR enhances the ability of caspase-3 to cleave AR and generate cytotoxic polyQ fragments. Spinal and bulbar muscular atrophy (SBMA), 1The abbreviations used are: SBMA, spinal and bulbar muscular atrophy; polyQ, polyglutamine; MAPK, mitogen-activated protein kinase; MEK1, mitogen-activated protein kinase kinase; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase; AR, androgen receptor; DHT, dihydrotestosterone; SH, Src homology; MN, mouse motor neuron-neuroblastoma; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (systematic); PARP, poly(ADP-ribose) polymerase.1The abbreviations used are: SBMA, spinal and bulbar muscular atrophy; polyQ, polyglutamine; MAPK, mitogen-activated protein kinase; MEK1, mitogen-activated protein kinase kinase; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase; AR, androgen receptor; DHT, dihydrotestosterone; SH, Src homology; MN, mouse motor neuron-neuroblastoma; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol (systematic); PARP, poly(ADP-ribose) polymerase. or Kennedy's disease, is an X-linked autosomal dominant degenerative disease of the motor neurons, characterized by progressive muscle atrophy and weakness in male patients (1Fischbeck K.H. J. Inherit. Metab. Dis. 1997; 20: 152-158Crossref PubMed Scopus (113) Google Scholar). The disease is caused by a polyglutamine (polyQ) tract expansion within the transcriptional activation domain of androgen receptor (AR) (2La Spada A.R. Wilson E.M. Lubahn D.B. Harding A.E. Fischbeck K.H. Nature. 1991; 352: 77-79Crossref PubMed Scopus (2380) Google Scholar). The normal number of CAG repeats is 11–36, whereas the mutant gene has up to 68 repeats. Presumably SBMA is caused by a gain-of-function mutation because severe testicular feminization patients where AR is fully inactivated do not have motor neuron disease (3Brown T.R. Lubahn D.B. Wilson E.M. Joseph D.R. French F.S. Migeon C.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8151-8155Crossref PubMed Scopus (174) Google Scholar, 4Yong E.L. Ng S.C. Roy A.C. Yun G. Ratnam S.S. Lancet. 1994; 344: 826-827Abstract PubMed Scopus (4) Google Scholar). However, male patients with SBMA exhibit some symptoms of partial androgen insensitivity, including gynecomastia, small testicles, and feminization, suggesting some impairment of AR function (5Pinsky L. Trifiro M. Kaufman M. Beitel L.K. Mhatre A. Kazemi-Esfarjani P. Sabbaghian N. Lumbroso R. Alvarado C. Vasiliou M. Gottlieb B. Clin. Invest. Med. 1992; 15: 456-472PubMed Google Scholar). SBMA is one of a growing number of polyQ-repeat diseases, including Huntington's disease and the spinocerebellar ataxias, that are characterized by the death of specific neuronal subsets (6Ross C.A. Neuron. 1995; 15: 493-496Abstract Full Text PDF PubMed Scopus (228) Google Scholar). It is particularly attractive to study the effect of a polyQ expansion in the context of AR because, unlike the other polyQ disease proteins, it has a well characterized function. AR is a transcription factor and a member of a large family of steroid hormone receptors (7Beato M. Herrlich P. Schutz G. Cell. 1995; 83: 851-857Abstract Full Text PDF PubMed Scopus (1630) Google Scholar, 8Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6028) Google Scholar).Almost all the polyQ proteins identified to date are substrates for cell death proteases or caspases (9Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (205) Google Scholar, 10Wellington C.L. Ellerby L.M. Hackam A.S. Margolis R.L. Trifiro M.A. Singaraja R. McCutcheon K. Salvesen G.S. Propp S.S. Bromm M. Rowland K.J. Zhang T. Rasper D. Roy S. Thornberry N. Pinsky L. Kakizuka A. Ross C.A. Nicholson D.W. Bredesen D.E. Hayden M.R. J. Biol. Chem. 1998; 273: 9158-9167Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 11Goldberg Y.P. Nicholson D.W. Rasper D.M. Kalchman M.A. Koide H.B. Graham R.K. Bromm M. Kazemi-Esfarjani P. Thornberry N.A. Vaillancourt J.P. Hayden M.R. Nat. Genet. 1996; 13: 442-449Crossref PubMed Scopus (501) Google Scholar, 12Miyashita T. Okamura-Oho Y. Mito Y. Nagafuchi S. Yamada M. J. Biol. Chem. 1997; 272: 29238-29242Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Ellerby L.M. Andrusiak R.L. Wellington C.L. Hackam A.S. Propp S.S. Wood J.D. Sharp A.H. Margolis R.L. Ross C.A. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Biol. Chem. 1999; 274: 8730-8736Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The expanded form of AR is toxic to cells in both cell culture systems and transgenic animal models (9Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (205) Google Scholar, 14Abel A. Walcott J. Woods J. Duda J. Merry D.E. Hum. Mol. Genet. 2001; 10: 107-116Crossref PubMed Scopus (111) Google Scholar). Recent studies both in Drosophila and in transgenic mouse models of SBMA, expressing full-length mutant protein, demonstrate that the addition of ligand accelerates the phenotypes in these models and truncated fragments are found in the affected tissue as well as postmortem SBMA brain tissue (15Katsuno M. Adachi H. Kume A. Li M. Nakagomi Y. Niwa H. Sang C. Kobayashi Y. Doyu M. Sobue G. Neuron. 2002; 35: 843-854Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 16Takeyama K. Ito S. Yamamoto A. Tanimoto H. Furutani T. Kanuka H. Miura M. Tabata T. Kato S. Neuron. 2002; 35: 855-864Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). We have shown previously that AR is a substrate for caspases, the disease form of AR is toxic to cells, and mutation of the caspase cleavage site in AR attenuates cell death (9Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (205) Google Scholar). These observations along with other recent studies suggest that proteolytic cleavage by caspases may be important in SBMA and other polyQ diseases (17DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2284) Google Scholar, 18Wellington C.L. Ellerby L.M. Gutekunst C.A. Rogers D. Warby S. Graham R.K. Loubser O. van Raamsdonk J. Singaraja R. Yang Y.Z. Gafni J. Bredesen D. Hersch S.M. Leavitt B.R. Roy S. Nicholson D.W. Hayden M.R. J. Neurosci. 2002; 22: 7862-7872Crossref PubMed Google Scholar).Because AR and other steroid receptors are highly dependent upon phosphorylation for function as transcription factors (19Brinkmann A.O. Trapman J. Adv. Pharmacol. 2000; 47: 317-341Crossref PubMed Scopus (36) Google Scholar, 20Lin H.K. Yeh S. Kang H.Y. Chang C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7200-7205Crossref PubMed Scopus (337) Google Scholar), we reasoned that a critical step in the production of proteolytic fragments might require phosphorylation. Further, the MAP kinase signal transduction pathway is involved in AR function and phosphorylation (21Yeh S. Lin H.K. Kang H.Y. Thin T.H. Lin M.F. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5458-5463Crossref PubMed Scopus (498) Google Scholar, 22Kyriakis, J. M. (2000) Science's STKE http://STKE.sciencemag.org/cgi/content/full/sigtrans;2000/48/pe1.Google Scholar, 23Migliaccio A. Castoria G. Di Domenico M. de Falco A. Bilancio A. Lombardi M. Barone M.V. Ametrano D. Zannini M.S. Abbondanza C. Auricchio F. EMBO J. 2000; 19: 5406-5417Crossref PubMed Google Scholar, 24Ueda T. Bruchovsky N. Sadar M.D. J. Biol. Chem. 2002; 277: 7076-7085Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Therefore, we tested whether the polyQ expansion in AR activates three major MAP kinase pathways (p44/42, p38, and SAPK/JNK MAP kinases), whether these pathways were required for AR112-mediated cell death, and whether phosphorylation of AR112 was required for AR112-mediated cell death. We found the p44/42 MAP kinase pathway is required for AR112 cell death and mutation of the MAP kinase consensus phosphorylation site in AR at serine 514 blocked AR-induced cell death and the generation of caspase-3-derived cleavage products. The results demonstrate the proteolytic cleavage of mutant AR is modulated by phosphorylation and demonstrate the MEK1/2 inhibitor, U0126, can be used to block AR112-induced cellular toxicity.EXPERIMENTAL PROCEDURESCell Culture and Transfections—HEK 293T cells were cultured as described previously (9Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (205) Google Scholar). Transient transfections were performed using Superfect reagent (Qiagen) according to the manufacturer's instructions. Briefly, cells were plated at an initial density of 1 × 106 cells for 10-cm plates and 2 × 105 for 6-well plates. pcDNA3, pcDNA3-AR12, pcDNA3-AR50, pcDNA3-AR112, pcDNA3-AR112 (S28A), pcDNA3-AR112 (T438A), or pcDNA3-AR112 (S514A) (10 or 2 μg) were transfected into 293T cells. Dihydrotestosterone (DHT) (Sigma) treatment was carried out in Dulbecco's modified Eagle's medium containing charcoal-stripped fetal bovine serum. MAP kinase inhibitors U0126 (Cell Signaling), SB203580 (Calbiochem), and SP-600125 (Biomol) were solubilized in Me2SO (Sigma), and treatments were carried out 16 h after transfection for 48 h. Cells were treated with Me2SO as controls. Cells were harvested 48 h after transfection by scraping and centrifugation at 500 × g.Site-directed Mutagenesis—Site-directed mutagenesis was performed using the QuikChange kit (Stratagene) on template pcDNA3-AR112 using the following PCR primers; S28A, 5′-GGAGCTTTCCAGAATCTGTTCCAGGCCGTGCGCGAAGTGATCCAG-3′; T438A, 5′-TCC-TCATCCTGGCACACTCTCTTCGCAGCCGAAGAAGGCCAGTTG-3′; S514A, 5′-GTGAGCAGAGTGCCCTATCCCGCTCCCACTTGTGTCAAAAGCGAAATGGGC-3′. Primers were purchased from IDT.Immunoprecipitation and Western Blot Analysis—Cells (10-cm plate) were washed in phosphate-buffered saline and lysed in Nonidet P-40 buffer (50 mm HEPES, 250 mm NaCl, 5 mm EDTA, 0.1% Nonidet P-40) with protease inhibitor Minicomplete (Roche Applied Science) for 10 min and centrifuged at 15,000 × g. Lysate (200 μg) was precleared with anti-mouse IgG agarose (30 μl; Sigma) for 45 min. AR was pulled down with anti-AR-441 monoclonal antibody (5 μg) for 18 h at 4 °C and washed in Nonidet P-40 lysis buffer (5×). Proteins were resolved on Nupage BisTris 4–12% precast gels (Invitrogen). Proteins were transferred to polyvinylidene difluoride (BioRad) for 1 h at 25 mA. Membranes were blocked in 5% bovine serum albumin (Sigma) (for serine phospho-specific antibodies) or milk in TBST and probed with primary antibody (1:1000) for 2 h at room temperature or 18 h at 4 °C. Antibodies utilized were: phospho-p44/42 MAPK, phospho-SAPK/JNK, phospho-p38 MAPK, phospho-ATF-2 monoclonal antibodies (Cell Signaling); AR N-20, AR-441 polyclonal antibodies (Santa Cruz Biotechnology); and phosphoserine monoclonal antibody 16B4 (Calbiochem). Monoclonal β-tubulin T-4026 (1:500; Sigma) was used to confirm equal loading of samples. Secondary anti-rabbit or anti-mouse antibody (1:3000; Amersham Biosciences) was applied for 45 min at room temperature, and ECL (Amersham Biosciences) was used for detection.Cytotoxicity Assay—Cells were transiently transfected in 6-well dishes. After 48 h, cells were harvested by centrifugation at 500 × g for 10 min. Cells were lysed (Apoalert kit lysis buffer, Clontech) for 10 min and assayed according to the manufacturer's instructions. Lysates (20 μg) were resolved by SDS-PAGE, and Western blotting was performed using the AR N-20 and tubulin antibody to confirm equal expression. Lactate dehydrogenase assays were performed according to Ref. 25Koh J.Y. Choi D.W. J. Neurosci. Methods. 1987; 20: 83-90Crossref PubMed Scopus (1234) Google Scholar.Subcellular Fractionation—293T cells were transiently transfected and harvested at 48 h by scraping, and subcellular fractionation was carried out as described previously (26Lewis J.M. Baskaran R. Taagepera S. Schwartz M.A. Wang J.Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15174-15179Crossref PubMed Scopus (268) Google Scholar). Nuclear fraction was verified by immunoblotting with anti-PARP antibody (1:500; Biomol).In vitro Translation and Caspase-3 Cleavage—AR constructs were in vitro translated (IVT) as previously described (9Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (205) Google Scholar). IVT reaction (5 μl) was incubated with various concentrations of caspase-3 (gift from Guy Salvesen) for 4 h at 37 °C. Cleavage products were resolved using 4–12% BisTris precast gels (Invitrogen). Gels were dried and autoradiographed, and densitometry was performed using ChemiImager software (Alpha Innotech).RESULTSExpression of the PolyQ-expanded Form of AR Activates MAPK Pathways in 293T Cells—The MAP kinase pathways are known to relay, integrate, and amplify signals from a number of physiological responses, including differentiation, proliferation, inflammation, and apoptosis. Recently, using gene chip analysis, we have found that AR regulates a number of genes involved in the MAP kinase pathway and the polyQ-expanded form of AR differentially regulates these genes. 2M. LaFevre-Bernt, E. Hermel, J. P. Taylor, M. W. Eschoo, and L. M. Ellerby, submitted for publication. We hypothesized that expression of the expanded form of AR, which induces cell death, would result in alterations in the MAP kinase pathway(s). To evaluate the effect of expression of AR12 or AR112 (12 or 112 glutamines, respectively) on the activation of the MAP kinase pathways, 293T cells were transiently transfected with AR constructs. In our cell culture model, expression of AR112 alone is sufficient to induce caspase activation and subsequent cell death. MAP kinase activation was determined by Western blot analysis with phospho-specific antibodies that recognize only the phosphorylated forms of the MAP kinases (Fig. 1, A–C). AR112 leads to increased phosphorylation of p44/42 MAPK (Fig. 1A), SAPK/JNK MAPK (Fig. 1B), and the p38 MAPK (Fig. 1C) when compared with AR12 or vector control. Reprobing the Western blot confirmed equal expression of AR, total p44/42 MAPK, and β-tubulin (Fig. 1, D and E). The lower band in the AR112 lane is a proteolytic fragment. These results suggest that activation of a MAPK pathway might play a role in expanded AR-induced cell death.MEK1/2 Pathway Is Required for AR-induced Cytotoxicity— To investigate whether the cytotoxicity of AR112 is mediated by MAPK pathways, we treated 293T cells that had been transiently transfected with AR112 with MAP kinase inhibitors: the MEK1/2 inhibitor, U0126, the SAPK/JNK inhibitor, SP-600125, or the p38 MAPK inhibitor, SB 203580. Forty-eight hours after treatment, cytotoxicity was evaluated by monitoring caspase-3 activity (Fig. 2). Addition of the MEK1/2 inhibitor, U0126, dramatically reduced AR112-induced cytotoxicity, reducing it to levels of vector alone (Fig. 2A). Treatment with either a JNK/SAPK inhibitor (SP-600125) or a p38 MAPK inhibitor (SB 203580) did not reduce the toxicity of AR112 (Fig. 2A). Similar results were observed using a cell viability (WST) assay (data not shown). Interestingly, MEK1/2 inhibitor treatment also inhibits the cleavage of AR112 (Fig. 2B). Cleavage of AR has been correlated with cellular toxicity (9Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (205) Google Scholar). As a control for kinase inhibition, Western blot analysis demonstrated that the inhibitor doses utilized led to a decrease in phosphorylation of the kinases themselves and of ATF-2, a downstream target of the JNK pathway (Fig. 2C). Inhibitor treatments did not affect the expression level of AR. It is possible that the SAPK/JNK and p38 MAPK (stress-activated) pathways are activated in a protective response to AR112 cytotoxicity; this explains why the inhibitors of these pathways do not decrease toxicity. Indeed, if these pathways are activated as a protective mechanism in response to cellular stress, inhibition would be expected to increase toxicity, as is observed here (Fig. 2A). Our results suggest that the MEK1/2 pathway is required for AR112-induced cell death.Fig. 2MEK1/2 inhibitor (U0126), but not JNK or p38 MAP kinase inhibitors (SP-600125 and SB203580), decreases AR112-induced cell death in 293T cells. 293T-transfected cells were treated with U0126 (10 μm) or SP-600125 and SB203580 (30 μm) 16 h after transfection for 48 h in the absence of DHT. A, caspase-3 activity was measured using the substrate N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC). **, p <0.01, *, p <0.05, compared with untreated (t test). B, Western blot analysis of cell lysates expressing AR112 treated with U0126. C, Western blot analysis of cell lysates with phospho-specific antibodies confirms effective kinase inhibitor doses were utilized. Phospho-ATF-2 (Thr-71) is a downstream target of SAP/JNK MAPK.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Serine Phosphorylation of AR is PolyQ Repeat Length-dependent—Given the increased activation of the MAP kinases, which are serine/threonine kinases, we examined whether AR might be a downstream target of serine phosphorylation. We immunoprecipitated AR12, AR50, or AR112 from transiently transfected 293T cells and performed Western blot analysis. An antibody that recognizes phosphorylated serine with a proline immediately C-terminal (phospho-Ser-Pro) (Fig. 3A, right) was used to evaluate AR phosphorylation because only six serine-proline sites are present in AR. Equivalent amounts of AR were pulled down in each immunoprecipitation (Fig. 3A, left panel). Increasing amounts of phosphorylated serine were found in AR with 50 or 112 repeats (Fig. 3A, right panel), although AR12 was not phosphorylated at this site. The nonspecific band that is recognized by the phosphoserine antibody (denoted by *) is not AR, because the AR antibody does not recognize this band in the vector control lane and this band runs at a molecular weight that is between the molecular weight of the AR12 and AR50 bands. Therefore, the expanded form of androgen receptor has increased serine phosphorylation, correlating with the increase in MAP kinase activation.Fig. 3Serine phosphorylation is polyQ repeat length-dependent in 293T cells. A, AR was immunoprecipitated and Western blot analysis was carried out with either AR N-terminal antibody (left) or phosphoserine-specific antibody 16B4 (proline consensus phosphoserine site, phospho-Ser-Pro, right). * indicates a nonspecific immunoreactive band. B, possible serine phosphorylation sites in AR transcriptional activation domain (TAD). C, the MAP kinase consensus sequence is conserved in human (h), mouse (m), and rat (r) AR. D, Western blot of immunoprecipitated AR112 treated with U0126 or 1 μm dihydrotestosterone (DHT) probed with phosphoserine-specific antibody 16B4. E, mutation of serine phosphorylation sites in AR results in reduced isoforms when compared with AR112. Western blot was probed with AR N-terminal antibody. F, mutation of serine phosphorylation site 514 in AR results in the elimination of one isoform of AR112 immunoreactive to phosphoserine-specific antibody 16B4. AR112 was expressed in 293T cells in the absence of testosterone unless otherwise indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Phosphorylation of Serine 514 Is Required for AR112-induced Cytotoxicity—Several serine residues, including Ser-19, -210, -514, and -790, are phosphorylated in AR. Phosphorylation at these sites can lead to an increase, no change, or a decrease in AR transactivation activity (20Lin H.K. Yeh S. Kang H.Y. Chang C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7200-7205Crossref PubMed Scopus (337) Google Scholar, 21Yeh S. Lin H.K. Kang H.Y. Thin T.H. Lin M.F. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5458-5463Crossref PubMed Scopus (498) Google Scholar, 27Gioeli D. Ficarro S.B. Kwiek J.J. Aaronson D. Hancock M. Catling A.D. White F.M. Christian R.E. Settlage R.E. Shabanowitz J. Hunt D.F. Weber M.J. J. Biol. Chem. 2002; 277: 29304-29314Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Particularly of interest is Ser-514, in the transcriptional activation domain (Fig. 3B), which is phosphorylated by MEK1/2 in a prostate cancer cell line (21Yeh S. Lin H.K. Kang H.Y. Thin T.H. Lin M.F. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5458-5463Crossref PubMed Scopus (498) Google Scholar), is in a MEK1/2 consensus sequence that has a proline at the +1 position (Fig. 3C), and would be recognized by the serine phospho-specific antibody utilized in Fig. 3A. Treatment of AR112-expressing cells with the MEK1/2 inhibitor U0126 reduced the phosphorylated form of the AR that is immunoreactive to the phospho-Ser-Pro antibody (Fig. 3D, upper panel). Treatment with DHT, which often modulates phosphorylation of AR, did not alter phosphorylation of AR immunoreactive to the phospho-Ser-Pro antibody (Fig. 3D, lower panel). Inhibition of phosphorylation of AR by U0126 suggests the serine phospho-specific antibody recognizes a site in AR phosphorylated by MEK1/2 or downstream targets of this pathway. Ser-28 and Thr-438 also lie in the transcriptional activation domain (Fig. 3B) and are adjacent to FXXLF motifs, which mediate interactions of AR with its transcriptional co-activators. These sites are consensus sites for p90RSK, a kinase that phosphorylates transcription factors and is a downstream target of MEK1/2 (28Sampt E.R. Fernandez G.A. Lehman J.A. Corey S.J. Huang C.K. Gomez-Cambronero J. J. Biochem. Biophys. Methods. 2001; 48: 219-237Crossref PubMed Scopus (2) Google Scholar).We used site-directed mutagenesis to eliminate these phosphorylation sites. Mutation of any of these sites eliminates the doublet that is seen in cells expressing AR112, suggesting that these sites affect post-translational modification of AR112 (Fig. 3E). Two isoforms of AR112 are reactive to the phospho-Ser-Pro antibody (Fig. 3F). Immunoreactivity of one of the isoforms of AR112 to the phospho-Ser-Pro antibody is eliminated by mutation of Ser-514 to Ala (Fig. 3F).To investigate the effect of phosphorylation on AR112-induced cytotoxicity, we expressed AR112, AR112 (S28A), AR112 (T438A), and AR112 (S514A) in 293T cells and performed cytotoxicity assays. Transient expression of AR112 (S28A) and AR112 (T438A) increased (1.5-fold) caspase-3 activity (Fig. 4A). However, expression of AR112 (S514A) decreased cytotoxicity (2-fold) in 293T cells. Expression levels of AR112 and the mutants in lysates used for these assays were equivalent (Fig. 3E). Similar results were obtained using a lactate dehydrogenase assay to measure cell death (data not shown). Interestingly, both AR112 and AR112 S514A activate p44/42 MAP kinase pathway (data not shown), suggesting the polyQ expansion activates this pathway but kinase activation is not sufficient for cell death.Fig. 4Phosphorylation of Ser-514 is required for AR112 cytotoxicity in 293T cells and in mouse motor neuron hybrid cell line. A, 293T cells were transfected with AR112, AR112 (S28A), AR112 (T438A), and AR112 (S514A) for 48 h in the absence of DHT. Caspase-3 activity was measured using the substrate DEVD-AFC. Equivalent expression of AR112 mutants was confirmed by Western blot analysis with the AR N-terminal antibody (see Fig. 3E). B, mouse motor neuron (MN) hybrid cells were transfected with pcDNA3, AR12, AR112, AR112 (S514A), and AR112 (D154N) in the presence and absence of 1 μm DHT. Equivalent expression of AR112 mutants was confirmed by Western blot analysis with the AR N-terminal antibody. Lactate dehydrogenase activity was measured. ***, p <0.005; **, p <0.01; *, p <0.05, compared with AR112 (t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further evaluate whether the phosphorylation status of androgen receptor affected cellular cytotoxicity, we transiently transfected mouse motor neuron-neuroblastoma (MN) hybrid cells, a neuronal cell line directly relevant to SBMA with AR112 and AR112 (S514A). We found, by LDH assay (Fig. 4B) and by caspase activation assay (data not shown), that AR112 (S514A) was less toxic than AR112. Because we have previously reported that the caspase-resistant form of the polyQ-expanded form of AR has reduced cellular toxicity, we compared AR112 (S514A) to AR112 (D154N). Interestingly, the decreased cytotoxicity of the AR112 (S514A) was similar to that observed for AR112 (D154N), a caspase-resistant form of AR that cannot generate N-terminal polyQ-containing caspasemediated cleavage products (Fig. 4B). Next we evaluated whether addition of DHT, which results in nuclear localization and transcriptional activation of AR, modulated cellular toxicity. Cellular toxicity of AR112 expressed in MN hybrid cells was enhanced (Fig. 4B, 1.3-fold, **, p <0.01). DHT did not modulate the toxicity of cells expressing AR112 (S514A) or AR112 (D154N). Expression levels of AR112 and the mutants in lysates used for these assays were equivalent (Fig. 4B). The effect of the Ser-514 mutant is consistent with the decrease in cytotoxicity seen upon treatment of cells with the MEK1/2 inhibitor and further suggests that the MEK1/2 pathway, and specifically MAPK phosphorylation of AR112, is required for cytotoxicity in our cell culture model. Therefore, we focused on the AR112 (S514A) mutant for further characterization.Phosphorylation of Ser-514 Does Not Alter Localization of AR112—In many polyQ diseases, nuclear localization correlates with enhanced cell dysfunction or cell death (29Klement I.A. Skinner P.J. Kaytor M.D. Yi H. Hersch S.M. Clark H.B. Zoghbi H.Y. Orr H.T. Cell. 1998; 95: 41-53Abstract Full Text Full Tex" @default.
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