Androgen Receptor Acetylation Site Mutations Cause Trafficking Defects, Misfolding, and Aggregation Similar to Expanded Glutamine Tracts

Kennedy's disease is a degenerative disorder of motor neurons caused by the expansion of a glutamine tract near the amino terminus of the androgen receptor (AR). Ligand binding to the receptor is associated with several post-translational modifications, but it is poorly understood whether these affect the toxicity of the mutant protein. Our studies now demonstrate that mutation of lysine residues in wild-type AR that are normally acetylated in a ligand-dependent manner mimics the effects of the expanded glutamine tract on receptor trafficking, misfolding, and aggregation. Mutation of lysines 630 or 632 and 633 to alanine markedly delays ligand-dependent nuclear translocation. The K632A/K633A mutant also undergoes ligand-dependent misfolding and aggregation similar to the expanded glutamine tract AR. This acetylation site mutant exhibits ligand-dependent 1C2 immunoreactivity, forms aggregates that co-localize with Hsp40, Hsp70, and the ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP), and inhibits proteasome function. Ligand-dependent nuclear translocation of the wild-type receptor and misfolding and aggregation of the K632A/K633A mutant are blocked by radicicol, an Hsp90 inhibitor. These data identify a novel role for the acetylation site as a regulator of androgen receptor subcellular distribution and folding and indicate that ligand-dependent aggregation is dependent upon intact Hsp90 function. Kennedy's disease is a degenerative disorder of motor neurons caused by the expansion of a glutamine tract near the amino terminus of the androgen receptor (AR). Ligand binding to the receptor is associated with several post-translational modifications, but it is poorly understood whether these affect the toxicity of the mutant protein. Our studies now demonstrate that mutation of lysine residues in wild-type AR that are normally acetylated in a ligand-dependent manner mimics the effects of the expanded glutamine tract on receptor trafficking, misfolding, and aggregation. Mutation of lysines 630 or 632 and 633 to alanine markedly delays ligand-dependent nuclear translocation. The K632A/K633A mutant also undergoes ligand-dependent misfolding and aggregation similar to the expanded glutamine tract AR. This acetylation site mutant exhibits ligand-dependent 1C2 immunoreactivity, forms aggregates that co-localize with Hsp40, Hsp70, and the ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP), and inhibits proteasome function. Ligand-dependent nuclear translocation of the wild-type receptor and misfolding and aggregation of the K632A/K633A mutant are blocked by radicicol, an Hsp90 inhibitor. These data identify a novel role for the acetylation site as a regulator of androgen receptor subcellular distribution and folding and indicate that ligand-dependent aggregation is dependent upon intact Hsp90 function. The androgen receptor (AR) 1The abbreviations used are: AR, androgen receptor; CHIP, carboxyl terminus of Hsc70-interacting protein; Hsp, heat shock protein; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; E3, ubiquitin-protein isopeptide ligase; DAPI, 4′,6-diamidino-2-phenylindole. is a ligand-activated transcription factor that is a pivotal regulator of sexually dimorphic traits. The receptor also plays a causative role in several human diseases including Kennedy's disease, a degenerative disorder of motor neurons caused by the expansion of a glutamine tract near the amino terminus of the receptor (1Lieberman A.P. Fischbeck K.H. Muscle Nerve. 2000; 23: 843-850Crossref PubMed Scopus (65) Google Scholar). Ligand-dependent translocation of the receptor to the nucleus is required both for normal AR function as a regulator of gene expression and for the neuronal pathology characteristic of Kennedy's disease (2Katsuno 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 (408) Google Scholar, 3Takeyama 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 (267) Google Scholar). Kennedy's disease is one of a group of inherited neurodegenerative disorders caused by the expansion of CAG/glutamine tracts in affected genes (4Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1118) Google Scholar). In all these disorders, the mutant proteins misfold and aggregate within the cytoplasm or nucleus. The presence of an expanded glutamine tract is the defining feature of these disorders and is causally related to the toxic gain-of-function conferred by the mutation. However, important differences among the polyglutamine expansion diseases exist, including the fact that distinct neuronal populations degenerate despite widespread expression of the mutant proteins. Such differences may be a consequence of altered normal function of the disease-causing protein. In Kennedy's disease, for example, the mutation causes a partial loss of AR function that may contribute to the specificity of the disease phenotype by depriving motor neurons of trophic support (5Lieberman A.P. Harmison G. Strand A.D. Olson J.M. Fischbeck K.H. Hum. Mol. Genet. 2002; 11: 1967-1976Crossref PubMed Scopus (125) Google Scholar). Recent data also indicate that sequences outside the glutamine repeat may modulate toxicity of the mutant protein and contribute to selective neuronal vulnerability. In SCA1, another polyglutamine neurodegenerative disease, a phosphorylation site near the carboxyl terminus of ataxin-1 affects aggregation and toxicity of the mutant protein (6Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 7Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). The identification of similar sequences that influence misfolding and toxicity of other disease-causing proteins could provide further insight into the pathogenic mechanisms underlying these diseases. Among the CAG repeat disorders, the mutant protein with one of the best understood normal functions is AR. Much progress has been made in defining ligand-dependent post-translational modifications of the receptor that are required for regulated gene expression; however, far less is known about whether these modifications impact the toxicity of the mutant protein. Ligand binding to the receptor is accompanied by alterations in the tertiary structure of the receptor and by several post-translational modifications. Among these ligand-dependent modifications is the acetylation of lysine residues 630, 632, and 633 (5Lieberman A.P. Harmison G. Strand A.D. Olson J.M. Fischbeck K.H. Hum. Mol. Genet. 2002; 11: 1967-1976Crossref PubMed Scopus (125) Google Scholar, 8Fu M. Wang C. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). This acetylation may be mediated by the transcriptional co-activators p300/CBP-associated factor and p300 and was shown previously to affect ligand-regulated activation of hormone-responsive promoters. These lysine residues that are modified by acetylation are adjacent to the bipartite nuclear localization sequence of the receptor, which is encoded by amino acids 608-625 (9Jenster G. Trapman J. Brinkmann A.O. Biochem. J. 1993; 293: 761-768Crossref PubMed Scopus (219) Google Scholar), and are in a region shown previously to affect nuclear trafficking (10Poukka H. Karvonen U. Yoshikawa N. Tanaka H. Palvimo J.J. Janne O.A. J. Cell Sci. 2000; 113: 2991-3001Crossref PubMed Google Scholar). Since the expanded glutamine tract is known to delay the ligand-dependent nuclear translocation of the receptor (11Becker M. Martin E. Schneikert J. Krug H.F. Cato A.C. J. Cell Biol. 2000; 149: 255-262Crossref PubMed Scopus (35) Google Scholar) and to inhibit the activity of acetyltransferases (12McCampbell A. Taylor J.P. Taye A.A. Robitschek J. Li M. Walcott J. Merry D. Chai Y. Paulson H. Sobue G. Fischbeck K.H. Hum. Mol. Genet. 2000; 9: 2197-2202Crossref PubMed Scopus (487) Google Scholar, 13McCampbell A. Taye A.A. Whitty L. Penney E. Steffan J.S. Fischbeck K.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15179-15184Crossref PubMed Scopus (223) Google Scholar, 14Taylor J.P. Taye A.A. Campbell C. Kazemi-Esfarjani P. Fischbeck K.H. Min K.T. Genes Dev. 2003; 17: 1463-1468Crossref PubMed Scopus (127) Google Scholar), we sought to determine the affect of acetylation site mutations on AR. We now demonstrate that these lysine residues play a critical role in the regulation of the subcellular localization and folding of the receptor. Mutation of these amino acids impairs ligand-dependent cytoplasmic to nuclear translocation and can lead to protein misfolding and aggregation similar to that which occurs in Kennedy's disease. Materials—MN-1 cells and plasmids encoding Q24 or Q112 AR were from Dr. K. Fischbeck. K630A and K632A/K633A mutants (numbering based on a Q19 AR) were described previously (8Fu M. Wang C. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), and GFPu was from Dr. R. Kopito. Antibodies against AR (N-2O), Hsp90, and β-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Hsp40 was from Stressgen (Victoria, British Columbia, Canada), and anti-Hsp70 was from Upstate Biotechnology (Lake Placid, NY). CHIP antibody, a gift from Dr. C. Patterson, was described previously (15Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (766) Google Scholar). Anti-polyglutamine antibody (1C2) was from Chemicon (Tamecula, CA). FITC and Texas red-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). R1881 was from PerkinElmer Life Sciences. Hydroxyflutamide was from Schering-Plough. Cell Transfection—HeLa cells were grown in phenol red-free Dulbecco's modified Eagle's medium supplemented with 10% charcoal and dextran-stripped fetal bovine serum. Cells were transfected with FuGENE 6 transfection reagent using 3 μl of FuGENE 6 and 1 μg of DNA. MN-1 cells stably expressing AR were isolated after co-transfection with pTracer EF/Bsd (Invitrogen) and indicated AR expression constructs. Cells were selected in medium supplemented with 7 μg/ml blasticidin. Clonal lines expressing equivalent levels of AR protein by Western blot were chosen for further analysis. Western Blot Analysis—Cells were harvested, washed with phosphate-buffered saline, and lysed in radioimmune precipitation buffer buffer. Protein samples were electrophoresed through a 10% SDS-polyacrylamide gel and transferred to Immunobilon-P membranes using a semidry transfer apparatus. Immunoreactive proteins were detected by chemiluminescence. Quantification of AR Localization and Aggregation—Following fixation and staining, cells were visualized using a Zeiss Axioplan 2 imaging system. AR localization was scored as described previously (16Galigniana M.D. Radanyi C. Renoir J.M. Housley P.R. Pratt W.B. J. Biol. Chem. 2001; 276: 14884-14889Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) using the following scale: 4 = nuclear fluorescence much greater than cytoplasmic fluorescence, 3 = nuclear fluorescence greater than cytoplasmic fluorescence, 2 = nuclear fluorescence equal to cytoplasmic fluorescence, 1 = nuclear fluorescence less than cytoplasmic fluorescence, and 0 = nuclear fluorescence much less than cytoplasmic fluorescence. AR aggregation was scored by determining the percentage of transfected cells with visible protein aggregates. For each experiment and each condition, >100 transfected cells were scored in a blind manner. Immunofluorescence Microscopy—Cells cultured on chambered slides were fixed at -20 °C with methanol and stained. Confocal images were captured using a Zeiss LSM 510 microscope and a ×63 water immersion objective. For quantification, cells were examined using a Zeiss Axiopan 2 imaging system. Electron Microscopy—Cell were fixed in suspension with 4% glutaraldehyde in 0.1 m calcodylate buffer, pH 7.3, overnight at 4 °C, and then post-fixed in 2% osmium tetraoxide. After dehydration with ethanol and propylene oxide, cells were embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and observed on a Philips 400T transmission electron microscope. Flow Cytometry—Cells were harvested by trypsinization, washed, and resuspended in phosphate-buffered saline. Cytometric analysis was performed using a Coulter Epics XL flow cytometer. Data analysis was performed using Winlist for Win 32 software. The cell population was gated to exclude necrotic cells and cell debris. 20,000 events within the gated region were analyzed per sample. Specific fluorescence of GFPu-expressing cells was determined by subtracting the background fluorescence of mock-transfected cells. We first observed that point mutations in AR at lysine residues normally acetylated in a ligand-dependent manner disrupted the nuclear translocation of the receptor (Fig. 1). These initial studies utilized AR containing either 19 or 24 glutamines, both of which fall within the normal range of glutamine tract length in humans. Mutation of lysine residues 632 and 633 to alanine (K632A/K633A) shifted the distribution of the receptor toward the cytoplasm in the absence of ligand and delayed nuclear translocation of the receptor upon addition of ligand (Fig. 1A). Altered ligand-dependent translocation was observed in cells both transiently and stably expressing the mutant receptor. Clonal lines stably expressing wild-type or K632A/K633A mutant AR were established using MN-1 motor neuron-neuroblastoma hybrid cells. Treatment with ligand for 24 h revealed near complete nuclear translocation of the wild-type but not the mutant receptor (Fig. 1B). Similarly, lysine to alanine substitution of residue 630 (K630A) completely blocked ligand-dependent nuclear translocation of the receptor, even up to 24 h following the addition of ligand (Fig. 1C). Subcelluar localization of the androgen receptor was determined by indirect immunofluorescence and scored in >100 transfected cells expressing the K630A mutant following with or ligand for 24 This analysis revealed translocation of for cells with R1881 and 0.1 for cells with These data indicated that lysine residues that are modified by acetylation are for normal ligand-dependent nuclear translocation of AR. ligand-dependent nuclear may contribute to the previously loss of receptor function conferred by these mutations (8Fu M. Wang C. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, M. Wang C. Wang J. T. C. T. E. Palvimo J.J. Janne O.A. Pestell R.G. Mol. Cell Biol. 2002; PubMed Scopus Google Scholar). The K632A/K633A mutant both delayed nuclear and ligand-dependent misfolding and aggregation (Fig. first as as after ligand addition and in of transfected cells following ligand for 24 h (Fig. in cells expressing AR with an expanded tract of aggregates of the K632A/K633A mutant in the cytoplasm and were not within as (Fig. and Ligand-dependent aggregation of the K632A/K633A mutant AR misfolding similar to that caused by the presence of an expanded glutamine the K632A/K633A mutant with 24 and AR with an expanded tract of were detected by the antibody 1C2 (Fig. that an by proteins with glutamine 1C2 of the K632A/K633A mutant was detected on Western blot after the addition of ligand (Fig. and with the of 1C2 protein aggregates by immunofluorescence (Fig. 1C2 of the K632A/K633A mutant was detected in the absence of ligand by immunofluorescence not In the K630A mutant that to the nucleus in to ligand not ligand-dependent aggregates (Fig. it 1C2 on Western blot (Fig. Data from cell and of CAG repeat disorders indicate that proteins with expanded glutamine tracts with and inhibit proteasome function (4Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1118) Google Scholar, H.T. Genes Dev. 2001; PubMed Scopus Google Scholar). We sought to determine whether the Q24 K632A/K633A mutant with and affected proteasome function in a ligand-dependent manner. and with aggregates by the mutant receptor (Fig. as with aggregates by proteins with expanded glutamine The ubiquitin ligase CHIP P. C.A. J. Wu Y. Thompson L.J. J. Patterson C. Cell Biol. 2001; PubMed Scopus Google Scholar) and for by the proteasome 2001; PubMed Scopus Google Scholar) also with aggregates by the K632A/K633A mutant (Fig. In GFPu was in the cytoplasm of cells with wild-type Q24 AR not of Hsp90 to aggregates was not detected (Fig. determine whether ligand-dependent aggregation of the K632A/K633A mutant proteasome cells were with GFPu wild-type AR, the K632A/K633A or the Q112 receptor. with or ligand for 24 fluorescence was determined by flow (Fig. Ligand-dependent in fluorescence, ligand-dependent of proteasome function 2001; PubMed Scopus Google Scholar), was greater in cells with the K632A/K633A mutant than in cells with the wild-type receptor. Q112 AR also fluorescence in a ligand-dependent manner. In with the androgen caused aggregation not proteasome (Fig. of proteasome function by the expanded glutamine AR may contribute to the of ligand-dependent in Kennedy's disease. nuclear and ligand-dependent aggregation were of the K632A/K633A of nuclear translocation the K630A In both these point mutations caused either partial or near complete of the that normally ligand-dependent nuclear translocation. This which has been for the receptor, is based upon of the receptor to the motor protein with W.B. Biol. 2003; PubMed Scopus Google Scholar). similar nuclear translocation of AR V. C. 2002; PubMed Scopus Google Scholar). that ligand-dependent nuclear translocation of wild-type AR was cells at 4 °C were first with ligand to the receptor and then with the Hsp90 AR subcellular localization was determined following a at °C (Fig. Treatment with ligand-dependent nuclear of wild-type AR, in a distribution near These data indicated that translocation was an We sought to determine whether ligand-dependent aggregation of the K632A/K633A mutant was dependent upon Cells expressing the mutant receptor were at 4 °C with ligand ligand then radicicol, or ligand then the proteasome AR aggregation was after a at °C (Fig. and ligand-dependent misfolding and aggregation of the mutant receptor, as both by of cells containing aggregates and by the of 1C2 AR on Western the of 1C2 was less after a ligand than after a and the was chosen for these to toxicity associated with to The of on misfolding and aggregation was not to AR by the proteasome a similar was observed in the presence of these data indicated that ligand-dependent nuclear translocation of AR was dependent upon Mutation of lysine residues 632 and 633 and caused ligand-dependent misfolding and aggregation of the receptor that required intact Hsp90 function. Recent studies shown that AR undergoes ligand-dependent acetylation at lysine residues in a adjacent to the (8Fu M. Wang C. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). at these has been to play an important role in AR activation by with transcriptional M. Wang C. Wang J. T. C. T. E. Palvimo J.J. Janne O.A. Pestell R.G. Mol. Cell Biol. 2002; PubMed Scopus Google Scholar). Our data now demonstrate that mutation of the acetylation site also impairs ligand-dependent trafficking of the receptor from the cytoplasm to the nucleus and may receptor misfolding and aggregation. These studies identify another by which mutation of the AR acetylation site receptor function as a ligand-dependent transcriptional Ligand-dependent misfolding and aggregation of the K632A/K633A mutant is of ligand-dependent aggregation of the expanded glutamine AR that causes motor in Kennedy's disease. the K632A/K633A mutant and the Q112 receptor ligand-dependent aggregates in cell that are The presence of cytoplasmic aggregates or may contribute to the of by the expanded glutamine AR G. A. M. K. M. Neuron. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, P. S. P. U. G. L. R. A. J. 2002; PubMed Scopus Google Scholar). the K632A/K633A mutant and Q112 AR are by the antibody aggregate with and may inhibit proteasome function. and Hsp70, the ubiquitin ligase CHIP, and for by the proteasome co-localize with aggregates by the K632A/K633A that these of protein In Hsp90 is required for ligand-dependent of AR but not to the aggregates. and aggregation of the K632A/K633A mutant are associated with proteasome activity in cells with a similar was used previously to altered proteasome function in intact cells 2001; PubMed Scopus Google Scholar), it is also that other mechanisms contribute to the observed fluorescence. acetylation of and activity of the protein in several of CAG repeat disorders, including and of Kennedy's disease (12McCampbell A. Taylor J.P. Taye A.A. Robitschek J. Li M. Walcott J. Merry D. Chai Y. Paulson H. Sobue G. Fischbeck K.H. Hum. Mol. Genet. 2000; 9: 2197-2202Crossref PubMed Scopus (487) Google Scholar, 13McCampbell A. Taye A.A. Whitty L. Penney E. Steffan J.S. Fischbeck K.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15179-15184Crossref PubMed Scopus (223) Google Scholar, 14Taylor J.P. Taye A.A. Campbell C. Kazemi-Esfarjani P. Fischbeck K.H. Min K.T. Genes Dev. 2003; 17: 1463-1468Crossref PubMed Scopus (127) Google Scholar). Our data that acetylation of the expanded glutamine AR may contribute to to ligand-dependent protein aggregates by with cytoplasmic to nuclear The of on ligand-dependent misfolding and aggregation may the established role of Hsp90 in protein folding and of proteins. However, Hsp90 also plays a critical role in ligand-dependent of the androgen receptor and other W.B. Biol. 2003; PubMed Scopus Google Scholar). Our data indicate that is required for misfolding and aggregation of the K632A/K633A mutant receptor that occurs in the presence of with is the that the K630A mutant not ligand-dependent it ligand-dependent misfolding and aggregation. misfolding and delayed ligand-dependent nuclear is a feature by both the K632A/K633A acetylation site mutant and the expanded glutamine receptor (11Becker M. Martin E. Schneikert J. Krug H.F. Cato A.C. J. Cell Biol. 2000; 149: 255-262Crossref PubMed Scopus (35) Google Scholar). Such receptor trafficking may contribute to the partial loss of AR function that occurs in Kennedy's disease. Recent that sequences outside the glutamine tract may influence aggregation and toxicity of other proteins with glutamine In data the role of phosphorylation of ataxin-1 as a of aggregation and toxicity (6Emamian E.S. Kaytor M.D. Duvick L.A. Zu T. Tousey S.K. Zoghbi H.Y. Clark H.B. Orr H.T. Neuron. 2003; 38: 375-387Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 7Chen H.K. Fernandez-Funez P. Acevedo S.F. Lam Y.C. Kaytor M.D. Fernandez M.H. Aitken A. Skoulakis E.M. Orr H.T. Botas J. Zoghbi H.Y. Cell. 2003; 113: 457-468Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). AR is to ligand-dependent acetylation mediated by the co-activators p300/CBP-associated or (8Fu M. Wang C. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, L. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is in which AR is acetylated and whether acetyltransferases may also of ligand-dependent AR acetylation in motor neurons may contribute to the neuronal that Kennedy's disease. of the other proteins in the polyglutamine expansion diseases are known to regulated that the influence of the acetylation site on folding and aggregation may be to AR. be to which ligand-dependent acetylation of AR in motor neurons and to determine whether the activity of these is altered in Kennedy's disease. We Dr. for on Dr. for the GFPu expression Dr. Fischbeck for Q24 and Q112 AR, Dr. Patterson for the antibody, and Pestell and for K630A and K632A/K633A constructs.

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