BAG-1L Protein Enhances Androgen Receptor Function
1998; Elsevier BV; Volume: 273; Issue: 19 Linguagem: Inglês
10.1074/jbc.273.19.11660
ISSN1083-351X
AutoresBarbara A. Froesch, Shinichi Takayama, John C. Reed,
Tópico(s)Protein Structure and Dynamics
ResumoBAG-1 is a regulator of heat shock protein (Hsp) 70/Hsc70 family proteins that interacts with steroid hormone receptors. The recently identified BAG-1 long (BAG-1L) protein, an isoform of BAG-1 that arises from translation initiation at a noncanonical CUG codon, was co-immunoprecipitated with androgen receptors (AR) from LNCaP prostate cancer cells and other cell lysates, whereas the shorter originally identified BAG-1 and BAG-1M (RAP 46) proteins were not. BAG-1L, but not BAG-1 or BAG-1M (RAP46), also markedly enhanced the ability of AR to transactivate reporter gene plasmids containing an androgen response element (ARE) in PC3 prostate cancer and other cell lines. A C-terminal region deletion mutant of BAG-1L failed to co-immunoprecipitate with AR and functioned as a trans-dominant inhibitor of BAG-1L, impairing AR-induced transactivation of ARE-containing reporter plasmids. In addition, BAG-1L significantly reduced the concentrations of 5α-dihydrotestosterone (DHT) required for AR activity but did not induce ligand-independent transactivation. BAG-1L also markedly improved the ability of AR to transactivate reporter genes when cells were cultured with DHT in combination with the anti-androgen cyproterone acetate. The effects of BAG-1L on AR could not be explained by detectable alterations in the DHT-induced translocation of AR from cytosol to nucleus, nor by BAG-1L-induced increases in the amounts of AR protein. These findings implicate BAG-1L in the regulation of AR function and may have relevance to mechanisms of prostate cancer resistance to hormone-ablative and anti-androgen therapy. BAG-1 is a regulator of heat shock protein (Hsp) 70/Hsc70 family proteins that interacts with steroid hormone receptors. The recently identified BAG-1 long (BAG-1L) protein, an isoform of BAG-1 that arises from translation initiation at a noncanonical CUG codon, was co-immunoprecipitated with androgen receptors (AR) from LNCaP prostate cancer cells and other cell lysates, whereas the shorter originally identified BAG-1 and BAG-1M (RAP 46) proteins were not. BAG-1L, but not BAG-1 or BAG-1M (RAP46), also markedly enhanced the ability of AR to transactivate reporter gene plasmids containing an androgen response element (ARE) in PC3 prostate cancer and other cell lines. A C-terminal region deletion mutant of BAG-1L failed to co-immunoprecipitate with AR and functioned as a trans-dominant inhibitor of BAG-1L, impairing AR-induced transactivation of ARE-containing reporter plasmids. In addition, BAG-1L significantly reduced the concentrations of 5α-dihydrotestosterone (DHT) required for AR activity but did not induce ligand-independent transactivation. BAG-1L also markedly improved the ability of AR to transactivate reporter genes when cells were cultured with DHT in combination with the anti-androgen cyproterone acetate. The effects of BAG-1L on AR could not be explained by detectable alterations in the DHT-induced translocation of AR from cytosol to nucleus, nor by BAG-1L-induced increases in the amounts of AR protein. These findings implicate BAG-1L in the regulation of AR function and may have relevance to mechanisms of prostate cancer resistance to hormone-ablative and anti-androgen therapy. Prostate cancer is the most common malignancy in the United States and the second leading cause of cancer-related death among men (1Parker S.L. Tong T. Bolden S. Wingo P.A. CA-Cancer J. Clin. 1997; 47: 5-27Crossref PubMed Scopus (2365) Google Scholar). The normal prostate gland contains a two-layer epithelium composed of a population of small round stem cells called basal cells, which line the basement membrane, and a population of larger differentiated epithelial cells called secretory cells, which secrete a variety of proteins and other substances into the lumen of the gland (2Bonkhoff H. Stein U. Remberger K. Prostate. 1994; 24: 114-118Crossref PubMed Scopus (220) Google Scholar, 3Bonkhoff H. Stein U. Remberger K. Hum. Pathol. 1994; 25: 42-46Crossref PubMed Scopus (201) Google Scholar). Although both basal and secretory cells contain androgen receptors (AR) 1The abbreviations used are: AR, androgen receptor(s); ARE, androgen response element; DHT, 5α-dihydrotestosterone; CAT, chloramphenicol acetyltransferase; Hsp, heat shock protein; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; CPA, cyproterone acetate; PBS, phosphate-buffered saline; CMV, cytomegalovirus. (4Soeffing W.J. Timms B.G. J. Androl. 1995; 16: 197-208PubMed Google Scholar, 5Evans G. Chandler J. Prostate. 1987; 11: 339-351Crossref PubMed Scopus (132) Google Scholar), only the luminal secretory epithelial cells are dependent on steroid hormone for their function, growth, and survival (4Soeffing W.J. Timms B.G. J. Androl. 1995; 16: 197-208PubMed Google Scholar). In the absence of testosterone or related androgens, which can serve as ligands for AR, the secretory cells undergo rapid programmed cell death (6Kyprianou N. Isaacs J.T. Endocrinology. 1988; 122: 552-562Crossref PubMed Scopus (648) Google Scholar). Current treatment for metastatic adenocarcinoma of the prostate is predicated on the cell death-inducing effects of anti-androgens and hormone-ablative measures, which reduce endogenous production of androgens. However, nearly all hormone-dependent prostate cancers eventually relapse as fatal hormone-independent disease (7Crawford E.D. Eisenberger M.A. McLeod D.C. Spaulding J. Benson R. Dorr F.A. Blumenstein B.A. Davis M.A. Goodman P.J. N. Engl. J. Med. 1989; 321: 419-424Crossref PubMed Scopus (1364) Google Scholar). Multiple, still largely unidentified mechanisms may account for the complete independence or reduced dependence of prostate cancers on androgens (reviewed in Refs. 8Marcelli M. Tilley W.D. Zoppi S. Griffin J.E. Wilson J.D. McPhaul M.J. J. Endocrinol. Invest. 1992; 15: 149-159Crossref PubMed Scopus (13) Google Scholar, 9McPhaul M.J. Marcelli M. Zoppi S. Griffin J.E. Wilson J.D. J. Clin. Endocrinol. Metab. 1993; 76: 17-23Crossref PubMed Scopus (139) Google Scholar, 10Coetzee G.A. Ross R.K. J. Natl. Cancer Inst. 1994; 86: 872-873Crossref PubMed Scopus (129) Google Scholar). AR gene deletion or sequestration of the AR from the nucleus to the cytoplasm have been described in some hormone-independent tumors, implying that genetic alterations associated with tumor progression can abrogate the necessity for AR in some cases. However, many tumors may rely on other strategies that allow cancer cells to grow in low concentrations of androgens, including AR gene amplification or overexpression (11Koivisto P. Kononen J. Palmberg C. Tammela T. Hyytinen E. Isola J. Trapman J. Cleutjens K. Noordzij A. Visakorpi T. Kallioniemi O.-P. Cancer Res. 1997; 57: 314-319PubMed Google Scholar, 12Visakorpi T. Hyytinen E. Koivisto P. Tanner M. Keinanen R. Palmberg C. Palotie A. Tammela T. Isola J. Kallioniemi O.P. Nat. Genet. 1995; 9: 401-406Crossref PubMed Scopus (1270) Google Scholar) and AR mutations that permit transactivation of target genes with little or no requirement for steroid hormones (9McPhaul M.J. Marcelli M. Zoppi S. Griffin J.E. Wilson J.D. J. Clin. Endocrinol. Metab. 1993; 76: 17-23Crossref PubMed Scopus (139) Google Scholar, 13McPhaul M.J. Marcelli M. Tilley W.D. Griffin J.E. Wilson J.D. FASEB J. 1991; 5: 2910-2915Crossref PubMed Scopus (83) Google Scholar). Since most hormone-insensitive prostate cancers still retain a wild-type AR, presumably alterations in the factors that control the levels of AR and its function appear to play a major role in resistance to anti-androgen and hormone-ablative therapies. Thus, a need exists to understand more about the molecular mechanisms that govern the activity of AR. Steroid hormones mediate their effects by binding to specific intracellular receptors that act as hormone-dependent transcription factors. Upon binding steroid ligands, the AR undergo a conformational change, translocate to the nucleus, and bind to specific DNA sequences located near or in promoter regions of target genes. After binding DNA, the receptor interacts with components of the basal transcription machinery and sometimes sequence-specific transcription factors, resulting in positive or negative effects on gene transcription (14Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2727) Google Scholar, 15Anzick S. Kononen J. Walker R. Azorsa D. Tanner M. Guan X. Sauter G. Kallioniemi O. Trent J. Meltzer P. Science. 1997; 277: 965-968Crossref PubMed Scopus (1447) Google Scholar). A number of proteins have been identified that associate with the inactive or hormone-bound hormone-receptor complexes, including several heat shock family proteins and various types of transcription co-activators (reviewed in Refs. 16Pratt W.B. Welsh M.J. Cell Biol. 1994; 5: 83-93Google Scholar and 17Shibata H. Spencer T.E. Onate S.A. Jenster G. Tsai S.Y. Tsai M.J. O'Malley B.W. Recent Prog. Horm. Res. 1997; 52: 141-164PubMed Google Scholar). However, many details remain unclear as to the molecular mechanisms by which these proteins modulate the activities of steroid hormone receptors, and even less is known about whether alterations in their expression or function might contribute to the deregulation of steroid hormone responses in cancers. Recently, an isoform of the human BAG-1 protein (known as RAP46 (see below)) (18Takayama S. Sato T. Krajewski S. Kochel K. Irie S. Millan J.A. Reed J.C. Cell. 1995; 80: 279-284Abstract Full Text PDF PubMed Scopus (810) Google Scholar, 19Zeiner M. Gehring U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11465-11469Crossref PubMed Scopus (164) Google Scholar) has been reported to bind several steroid hormone receptors in vitro, including AR (19Zeiner M. Gehring U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11465-11469Crossref PubMed Scopus (164) Google Scholar). It is unknown, however, what effect if any, BAG-1 has on the functions of these steroid-dependent transcription factors. Interestingly, BAG-1 and its alternative isoform RAP46 were recently shown to bind tightly to heat shock protein (Hsp) 70/Hsc70 family proteins and modulate their chaperone activity in vitro (20Takayama S. Bimston D.N. Matsuzawa S. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.J. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (441) Google Scholar, 21Zeiner M. Gebauer M. Gehring U. EMBO J. 1997; 16: 5483-5490Crossref PubMed Scopus (148) Google Scholar, 22Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (341) Google Scholar). In this regard, BAG-1 appears to function analogously to bacterial GrpE, stimulating the exchange of ADP for ATP on Hsc70 (22Höhfeld J. Jentsch S. EMBO J. 1997; 16: 6209-6216Crossref PubMed Scopus (341) Google Scholar). It seems plausible therefore that BAG-1 could alter the bioactivity of AR and other steroid hormone receptors, given that many steroid hormone receptors are constitutively bound to heat shock proteins and that their hormone binding affinity and DNA binding activity can be increased in the presence of Hsp90 and Hsp70, respectively, under some circumstances (23Landel C.C. Kushner P.J. Greene G.L. Mol. Endocrinol. 1994; 8: 1407-1419PubMed Google Scholar, 24Fang Y. Fliss A.E. Robins D.M. Caplan A.J. J. Biol. Chem. 1996; 271: 28697-28702Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 25Srinivasan G. Patel N.T. Thompson E.B. Mol. Endocrinol. 1994; 8: 189-196Crossref PubMed Scopus (35) Google Scholar). The human and murine BAG-1 proteins are predicted to be amino acids 230 and 219 base pairs in length, respectively, based on cDNA cloning (18Takayama S. Sato T. Krajewski S. Kochel K. Irie S. Millan J.A. Reed J.C. Cell. 1995; 80: 279-284Abstract Full Text PDF PubMed Scopus (810) Google Scholar, 19Zeiner M. Gehring U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11465-11469Crossref PubMed Scopus (164) Google Scholar, 26Takayama S. Kochel K. Irie S. Inazawa J. Abe T. Sato T. Druck T. Huebner K. Reed J.C. Genomics. 1996; 35: 494-498Crossref PubMed Scopus (44) Google Scholar, 27Packham G. Brimmell M. Cleveland J.L. Biochem. J. 1997; 328: 807-813Crossref PubMed Scopus (167) Google Scholar). However, recently longer isoforms of the human and mouse BAG-1 proteins have been identified that can arise by translation initiation from noncanonical CUG codons located upstream and in frame with the originally described BAG-1 open reading frames (27Packham G. Brimmell M. Cleveland J.L. Biochem. J. 1997; 328: 807-813Crossref PubMed Scopus (167) Google Scholar, 28Glass C.K. Rose D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1997; 9: 222-232Crossref PubMed Scopus (604) Google Scholar). This longer isoform of BAG-1 contains a basic motif resembling nuclear localization sequences and preferentially targets to nuclei. The human BAG-1 and BAG-1 long (BAG-1L) proteins migrate as ∼36-kDa and 57–58-kDa proteins, respectively in SDS-PAGE experiments. In addition, a less abundant isoform of BAG-1 that migrates at ∼46–53 kDa has been described and termed either BAG-1M or RAP46. The BAG-1M (RAP46) protein arises from translation initiation at an AUG codon located upstream of the usual start site in the BAG-1 mRNA (27Packham G. Brimmell M. Cleveland J.L. Biochem. J. 1997; 328: 807-813Crossref PubMed Scopus (167) Google Scholar). 2S. Takayama, S. Krajewski, M. Krajewska, S. Kitada, J. Zapata, K. Kochel, D. Knee, D. Scudiero, G. Tudor, G. J. Miller, M. Yamada, T. Miyashita, and J. Reed, submitted for publication. BAG-1M (RAP46) is produced in human, but not mouse, cells.2 Like BAG-1, the BAG-1L and BAG-1M proteins also bind to Hsp70 and Hsc70.2 BAG-1 is ubiquitously expressed, whereas BAG-1L is found preferentially in steroid hormone-dependent tissues such as testis, ovary, breast, and prostate.2 Although little is known about the expression of BAG-1 and BAG-1L in cancers, both proteins were detected by immunoblotting in 9 of 9 prostate cancer cell lines tested.2 In this report, we present evidence that the BAG-1L protein may play an important role in the AR signaling pathway, in that it can form complexes with AR and enhance the androgen-dependent transactivation function of this steroid hormone receptor. The plasmids pcDNA3-hu-BAG-1L and pcDNA3-hu-BAG-1 were generated as described previously (26Takayama S. Kochel K. Irie S. Inazawa J. Abe T. Sato T. Druck T. Huebner K. Reed J.C. Genomics. 1996; 35: 494-498Crossref PubMed Scopus (44) Google Scholar).2 Translation of the longer form (BAG-1L) was forced by mutation of the noncanonical in frame first CTG codon of the cDNA to ATG.2 pcDNA3-BAG-1/BAG-1M lacks the upstream CTG-containing region of the cDNA and encodes both the originally described ∼36-kDa form of BAG-1 and ∼the 46–53-kDa BAG-1M (RAP46) proteins. The plasmid pcDNA3-hu-BAG-1L (ΔC) (lacking the last 47 amino acids of the human BAG-1 protein) was generated by polymerase chain reaction using pcDNA3-BAG-1L as a template and the EcoRI-containing forward primer 5′-GGGAATTCAGTGCGGGCATGGCTC-3′ together with the XhoI containing reverse primer 5′-CCCTCGAGTTATGGCAGGATCAGTGTGTG-3′. After digestion of the polymerase chain reaction product at theEcoRI and XhoI sites, the resulting ∼0.8-kilobase pair fragments were subcloned intoEcoRI/XhoI-digested pcDNA3. pSG5-AR contains the cDNA for the wild-type AR (29Lee H.J. Kokontis J. Wang K.C. Chang C. Biochem. Biophys. Res. Commun. 1993; 194: 97-103Crossref PubMed Scopus (16) Google Scholar). The reporter pLCI plasmid contains the full-length mouse mammary tumor virus long terminal repeat sequence linked with the chloramphenicol acetyltransferase (CAT) gene (29Lee H.J. Kokontis J. Wang K.C. Chang C. Biochem. Biophys. Res. Commun. 1993; 194: 97-103Crossref PubMed Scopus (16) Google Scholar, 30Mowszowicz I. Lee H.J. Chen H.T. Mestayer C. Portois M.C. Cabrol S. Mauvais-Jarvis P. Chang C. Mol. Endocrinol. 1993; 7: 861-869PubMed Google Scholar). pCMV-p53wt expression vector, MYH101–81 containing the p53 response element and the TATA box from the BAX promoter, and pUCSV3-CAT containing a SV40 early region promoter have been described (31Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar, 32Miyashita T. Harigai M. Hanada M. Reed J.C. Cancer Res. 1994; 54: 3131-3135PubMed Google Scholar). The human prostate cancer cell lines LN-CaP and PC3, the transformed human embryonal kidney 293, and the monkey kidney COS7 cell lines were obtained from the American Type Culture Collection (Rockville, MD). The ALVA31 human prostate cancer cell line was generously provided by Dr. G. Miller (University of Colorado, Denver, CO). Cells were maintained in a humidified atmosphere with 5% CO2 in RPMI 1640 or Dulbecco's modified Eagle's medium (293 and COS7) supplemented with 10% FCS, 3 mm glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (Life Technologies, Inc.). Two days prior to experiments, cells were transferred into CT-FCS to reduce background levels of steroids. 5α-Dihydro-testosterone (DHT) (Sigma) and cyproterone acetate (CPA) (Sigma) were dissolved in dimethyl sulfoxide and added to the cultures at a minimum dilutions of 0.0001% (v/v). Control cells received an equivalent amount of solvent only. COS7, PC3, and 293T cells at 60% confluency were transfected by a standard calcium phosphate precipitate method (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The medium was replaced with fresh charcoal-treated fetal calf serum/Dulbecco's modified Eagle's medium 1 h before transfection. The total amount of plasmid DNA used was normalized to 2.5 μg/well and 8 μg/plate for transfection in 12-well and 6-cm2 plates, respectively, by the addition of empty plasmid. For reporter gene assays, 0.2 μg of a β-galactosidase expression plasmid pCMV-βgal was co-transfected with the CAT reporter gene to normalize the transfection efficiency. Cells were exposed to the precipitate for 5 h at 37 °C. For COS7 and PC3 cells, a glycerol shock was applied. Cells were exposed to 15% glycerol in HBS buffer (25 mm HEPES pH 7.05, 0.75 mm Na2HPO4, 140 mmNaCl) for 4 min. The glycerol was removed by washing three times with PBS and replacement with fresh charcoal-treated fetal calf serum medium. For 293 cells, the medium was replaced without applying a shock. ALVA31 cells were transfected by a lipofection method. Briefly, 1.3 μg of DNA was diluted into 50 μl of Opti-MEM medium (Life Technologies) and combined with 3.3 μl of Lipofectamine (Life Technologies) in 50 μl of Opti-MEM. After incubation for 20 min, 0.35 ml of Opti-MEM was added, and the mixtures were overlaid onto monolayers of cells. After culturing at 37 °C and 5% CO2 for 6 h, 0.45 ml of Opti-MEM containing 20% charcoal-stripped fetal bovine serum was added to the cultures. At 32–36 h after transfection, cells were stimulated with 0.001–10 nm DHT or 0.1 nm R1881 (ALVA31). Cell extracts were prepared 48 h after transfection. For reporter gene experiments, cells lysates were made as described in Ref. 34Nielsen D.A. Chang T.C. Shapiro D.J. Anal. Biochem. 1989; 179: 19-23Crossref PubMed Scopus (102) Google Scholar and assayed for CAT and β-galactosidase activity. All transfection experiments were carried out in triplicate, repeated at least three times, and normalized for β-galactosidase activity. For gene expression experiments, cells were washed two times in PBS and lysed in radioimmune precipitation buffer (35Reed J.C. Meister L. Tanaka S. Cuddy M. Yum S. Geyer C. Pleasure D. Cancer Res. 1991; 51: 6529-6538PubMed Google Scholar) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 0.28 trypsin inhibitory units/ml aprotinin, 50 μg/ml leupeptin, 1 μmbenzamidine, 0.7 μg/ml pepstatin). For protein localization experiments, nuclear and nonnuclear fractions were prepared according to the method of Schreiber et al. (36Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6418Crossref PubMed Scopus (4013) Google Scholar). Briefly, cells were collected and washed two times with ice-cold PBS. Cell pellets were resuspended in buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 2.5 mm dithiothreitol, protease inhibitors) and left on ice for 15 min prior to the addition of Nonidet P-40 to 0.6% (v/v) final concentration. After centrifugation, supernatants (cytoplasmic fractions) were collected, and the nuclear pellets were washed twice in the same buffer. Pellets were finally resuspended in buffer B (20 mm HEPES, pH 7.9, 400 mm NaCl, 25% glycerol, 0.1 mm EDTA, 0.1 mm EGTA, 2.5 mmdithiothreitol, protease inhibitors) and vigorously shaken for 10 min, and the postnuclear supernatants were cleared by centrifugation. Fractions were normalized based on the bicinchoninic acid method (Pierce) prior to SDS-PAGE/immunoblot assay. Aliquots containing 25 μg of protein were subjected to SDS-PAGE using 10% gels, followed by electrotransfer to Immobilon-P transfer membranes (Millipore Corp., Bedford, MA). Immunodetection was accomplished using 1:1000 (v/v) of anti-BAG-1 monoclonal antibody ascites (26Takayama S. Kochel K. Irie S. Inazawa J. Abe T. Sato T. Druck T. Huebner K. Reed J.C. Genomics. 1996; 35: 494-498Crossref PubMed Scopus (44) Google Scholar, 37Krajewski S. Zapata J.M. Reed J.C. Anal. Biochem. 1996; 236: 221-228Crossref PubMed Scopus (88) Google Scholar)2 or polyclonal rabbit AR antiserum (Clone AR N20, Santa Cruz Biotechnology, Inc., Santa Barbara, CA), followed by horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech). Detection was performed using an enhanced chemiluminescence detection method (ECL; Amersham Pharmacia Biotech) or the Vector SG substrate (Vector Laboratories, Burlingame, CA). LN-CaP cells (2 × 107) were collected at 70% confluency and lysed in HKMEN buffer (10 mm HEPES, pH 7.2, 142 mm KCl, 5 mm MgCl2, 2 mm EGTA, 0.2% Nonidet P-40, protease inhibitors). Cell lysates were passaged several times through a 30½-gauge needle to disrupt the nuclei. Altnernatively, COS7 cells were transiently transfected with AR and BAG-1 expression plasmids, washed several times in PBS, and treated with 1 mm dimethyl-3,3′-dithiobispropionimadate (Pierce) in PBS for 30 min on ice. After extensive washing in ice-cold PBS, cells were lysed in radioimmune precipitation buffer containing protease inhibitors. Immunoprecipitations were performed in HKMEN either using the IgG1 anti-BAG-1 monoclonal KS6C8 (26Takayama S. Kochel K. Irie S. Inazawa J. Abe T. Sato T. Druck T. Huebner K. Reed J.C. Genomics. 1996; 35: 494-498Crossref PubMed Scopus (44) Google Scholar)2 or a polyclonal rabbit AR antiserum (clone AR PA1–110 ABR, Inc.) conjugated to protein G-agarose (Zymed, San Francisco, CA). Control immunoprecipitations were performed using IgG1 or rabbit preimmune serum. Immune complexes were analyzed by SDS-PAGE/immunoblot assay using anti-BAG-1 monoclonal antibody with an enhanced chemiluminescence detection method. The human BAG-1M (RAP46) protein had been shown to bind to AR in vitro (19Zeiner M. Gehring U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11465-11469Crossref PubMed Scopus (164) Google Scholar). We therefore asked whether BAG-1 family proteins can influence the transcriptional activity of this steroid hormone receptor. For these experiments, three different cell lines were transiently co-transfected with plasmids encoding various BAG-1 isoforms and AR, together with a ARE-containing CAT reporter plasmid. The cells were then cultured in the presence or absence of DHT. In the presence of hormone, BAG-1 family proteins increased the transcriptional activity of AR in a concentration-dependent manner, with the plasmid producing the BAG-1L protein displaying far more effect than the plasmid encoding for both BAG-1 and BAG-1M (Fig. 1). The extent of BAG-1L-mediated up-regulation of AR-induced transactivation varied among cell lines, with COS7 and PC3 demonstrating as much as ∼ 5-fold increases when transfected with BAG-1L but 293T cells exhibiting only a modest effect. Immunoblot analysis confirmed the production of the BAG-1, BAG-1M, BAG-1L, and AR proteins in the transfected cells and demonstrated production of similar amounts of BAG-1 and BAG-1M compared with BAG-1L (see below for examples). Thus, differences in the relative amounts of BAG-1, BAG-1M, and BAG-1L proteins produced could not account for the greater potency of BAG-1L. The transcription-potentiating effect of BAG-1L was dependent on the addition of androgen to cultures. As shown in Fig.2, AR-mediated transactivation of the ARE-CAT reporter plasmid remained at background levels when cells were co-transfected with plasmids encoding BAG-1 family proteins but cultured in the absence of DHT. However, cells transfected with the BAG-1L-producing plasmid displayed greater sensitivity to androgen compared with control transfected cells or cell over-expressing BAG-1/BAG-1M. The BAG-1L-mediated increases in AR-induced transactivation of the ARE-CAT reporter gene were detected at concentrations as low as 0.01 nm DHT and were substantially higher than control cells or BAG-1/BAG-1M-expressing cells over a broad range of hormone concentrations (0.01- 10 nm). The effects of BAG-1L were dependent on AR, since co-transfections lacking the AR-encoding plasmid failed to result in ARE-CAT plasmid reporter gene transactivation above background levels (not shown). To further examine the specificity of BAG-1L-mediated enhancement of AR transcriptional activity, the effects of BAG-1L on expression of other reporter genes were evaluated. The tumor suppressor p53 was chosen because, by analogy to steroid receptors, p53 is often associated with Hsp90 and Hsp70 in the cytoplasm and must translocate from cytosol to nucleus to exert its transcriptional regulatory action (38Selkirk J.K. Merrick B.A. Stackhouse B.L. He C. Appl. Theor. Electrophor. 1994; 4: 11-18PubMed Google Scholar). Co-transfection of BAG-1L-encoding expression plasmid into PC3 cells with a p53-producing vector and a p53-RE-CAT reporter gene demonstrated that BAG-1L does not influence p53-mediated transactivation (Fig.3). Similarly, BAG-1L had no effect on the constitutive expression of either a SV40 early region promoter-driven CAT or the CMV immediate early region lacZreporter gene plasmid used for normalizing transfection efficiencies (Fig. 3 and data not shown). These viral promoter/enhancers contain Sp1 binding sites, thus suggesting that BAG-1L does not nonspecifically modulate this family of transcription factors. The observation that BAG-1L increased the sensitivity of AR to its ligand DHT (Fig. 2) prompted us to explore the effects of BAG-1L on the suppression of AR transactivity by the anti-androgen cyproterone acetate. For these experiments, AR and ARE-CAT were transfected into COS7 cells with either pcDNA3 control DNA or an equivalent amount of pcDNA3BAG-1L. The cells were treated ∼1.5 days later with 1 nm DHT alone or in combination with various concentrations of CPA. Relative CAT activity was then measured 12–14 h later. As shown in Fig. 4, CPA reduced in a concentration-dependent manner the DHT-induced transactivation of the ARE-CAT reporter gene plasmid in both control and BAG-1L-transfected COS7 cells. However, because AR-mediated reporter gene transactivation started at higher levels in BAG-1L transfectants, approximately 2 log higher concentrations of CPA androgens were generally required to reduce reporter gene activity to levels comparable with control-transfected cells (Fig. 4). Although BAG-1M (RAP46) has been reported to bind AR in vitro, the interaction of these proteins has not been demonstrated previously in cells. Co-immunoprecipitation assays were therefore performed using lysates prepared from untransfected LN-CaP cells, which constitutively express high levels of the BAG-1, BAG-1M, BAG-1L, and AR proteins (39Veldscholte J. Berrevoets C.A. Mulder E. J. Steroid Biochem. Mol. Biol. 1994; 49: 341-346Crossref PubMed Scopus (62) Google Scholar).2 A polyclonal anti-AR antiserum or a preimmune control serum was employed for immunoprecipitations, and the resulting immune complexes were subjected to SDS-PAGE/immunoblot analysis using the anti-BAG-1 monoclonal antibody KS6C8. As a control, BAG-1 proteins were also immunoprecipitated using the same anti-BAG-1 monoclonal antibody. Alternatively, an IgG1 control antibody was employed to confirm specificity. As shown in Fig. 5, the BAG-1L protein was readily detected in association with anti-AR immune complexes (lane 5). In contrast, the BAG-1 and BAG-1M proteins did not co-immunoprecipitate with AR but were found in anti-BAG-1 immune complexes, confirming their presence in LN-CaP cells under these conditions. The specificity of these results was confirmed by the absence of BAG-1 family and AR proteins in immune complexes prepared using IgG1 control monoclonal antibody or the preimmune control serum. Although BAG-1L could be detected in AR-containing immune complexes, the reciprocal experiment involving the use of anti-BAG-1 antibody in attempts to co-immunoprecipitate AR proved unsuccessful. Additional experiments suggested that this was due to antibody-induced disruption of BAG-1L interactions with AR (data not shown). Attempts to determine whether BAG-1L can associate with AR in the absence of steroid hormone have been hampered by the rapid turnover of unliganded AR, resulting in lower levels of AR and making quantitative comparisons difficult. However, thus far, we have detected association of BAG-1L with AR only when androgens have been present. Previously, we showed that the last 47 amino acids of the BAG-1 protein are required for binding to the ATPase domain of Hsc70 (20Takayama S. Bimston D.N. Matsuzawa S. Freeman B.C. Aime-Sempe C. Xie Z. Morimoto R.J. Reed J.C. EMBO J. 1997; 16: 4887-4896Crossref PubMed Scopus (441) Google Scholar
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