Cloning and Characterization of Androgen Receptor Coactivator, ARA55, in Human Prostate

Androgen receptor (AR) is a hormone-activated transcriptional factor that can bind to androgen response elements and that regulates the transcription of target genes via a mechanism that presumably involves cofactors. We report here the cloning of a novel AR coactivator ARA55 using a yeast two-hybrid system. ARA55 consists of 444 amino acids with the predicted molecular mass of 55 kDa and its sequence shows very high homology to mouse hic5, a TGF-β1-inducible gene. Yeast and mammalian two-hybrid systems and co-immunoprecipitation assays all prove ARA55 can bind to AR in a ligand-dependent manner. Transient transfection assay in prostate cancer DU145 cells further demonstrates that ARA55 can enhance AR transcriptional activity in the presence of 1 nmdihydrotestosterone or its antagonists such as 100 nm17β-estradiol or 1 μm hydroxyflutamide. Our data also suggest the C-terminal half of ARA55, which includes three LIM motifs, is sufficient to interact with AR. Northern blot and polymerase chain reaction quantitation showed ARA55 can be expressed differently in normal prostate and prostate tumor cells. Together, our data suggests that ARA55 may play very important roles in the progression of prostate cancer by the modulation of AR transactivation. Androgen receptor (AR) is a hormone-activated transcriptional factor that can bind to androgen response elements and that regulates the transcription of target genes via a mechanism that presumably involves cofactors. We report here the cloning of a novel AR coactivator ARA55 using a yeast two-hybrid system. ARA55 consists of 444 amino acids with the predicted molecular mass of 55 kDa and its sequence shows very high homology to mouse hic5, a TGF-β1-inducible gene. Yeast and mammalian two-hybrid systems and co-immunoprecipitation assays all prove ARA55 can bind to AR in a ligand-dependent manner. Transient transfection assay in prostate cancer DU145 cells further demonstrates that ARA55 can enhance AR transcriptional activity in the presence of 1 nmdihydrotestosterone or its antagonists such as 100 nm17β-estradiol or 1 μm hydroxyflutamide. Our data also suggest the C-terminal half of ARA55, which includes three LIM motifs, is sufficient to interact with AR. Northern blot and polymerase chain reaction quantitation showed ARA55 can be expressed differently in normal prostate and prostate tumor cells. Together, our data suggests that ARA55 may play very important roles in the progression of prostate cancer by the modulation of AR transactivation. androgen receptor steroid receptor wild type AR mutant AR DNA binding domain glucocorticoid receptor progesterone receptor estrogen receptor chloramphenicol acetyltransferase transforming growth factor β1 dihydrotestosterone 17β-estradiol hydroxyflutamide amino acid reverse transcriptase-polymerase chain reaction 4-morpholinepropanesulfonic acid rapid amplification of cDNA ends base pair(s) glyceraldehyde-3-phosphate dehydrogenase synthetic dropout The androgen receptor (AR)1 is a member of the steroid receptor (SR) superfamily and plays an important role in male sexual differentiation and prostate cell proliferation (1Chang C. Saltzman A. Yeh S. Young W. Keller E. Lee H. Wang C. Mizokami A. Crit. Rev. Eukaryotic Gene Expression. 1995; 5: 97-125Crossref PubMed Scopus (245) Google Scholar). The well conserved DNA binding domain (DBD) within AR has two zinc finger structures that are involved in DNA binding. The C-terminal region of the AR, including the hinge region and the ligand-binding domain, is responsible for the functions of dimerization and androgen binding. The N-terminal region is involved in the transcriptional activation of AR. The discovery of transcriptional interference/squelching of SRs provided the concept of the existence of transcriptional cofactors that mediate SR function (2Meyer M.-E. Gronemeyer H. Turcotte B. Bocquel M.-T. Tasset D. Chambon P. Cell. 1989; 57: 433-442Abstract Full Text PDF PubMed Scopus (444) Google Scholar, 3Bocquel M.T. Kumar V. Stricker C. Chambon P. Gronemeyer H. Nucleic Acids Res. 1989; 17: 2581-2595Crossref PubMed Scopus (229) Google Scholar). Recently, several putative cofactors (either coactivators or corepressers) for SRs have been identified and characterized (4Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalski M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar, 5Jenster G. Spencer T.E. Burcin M.M. Tsai S.Y. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7879-7884Crossref PubMed Scopus (233) Google Scholar). Further studies of the interaction of SRs with these cofactors suggested that these SR-cofactor complexes play essential roles for the regulation of SRs target gene transcription by interaction with general transcription factors and the remodeling of chromatin (4Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalski M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar, 5Jenster G. Spencer T.E. Burcin M.M. Tsai S.Y. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7879-7884Crossref PubMed Scopus (233) Google Scholar). The in vivo significance of these cofactors and their relationship to diseases, however, remains unclear. Recently, an estrogen receptor coactivator, AIB1, was identified with higher expression in ovarian cancer cell lines and breast cancer cells than in other cell lines tested (6Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.-Y. Sauter G. Kallioniemi O.P. Trent J.M. Science. 1997; 277: 965-968Crossref PubMed Scopus (1432) Google Scholar), implying that increased expression of cofactors might be involved in some hormone-responsive tumors. The question whether cofactors of AR, the major promoter of prostate tumor growth, can also play vital roles for the maintenance of androgen-dependent status is thus of vital interest. Here we report for the isolation and characterization of a novel AR coactivator, ARA55, which can bind to wild type AR (wtAR) and mutant AR (mAR) in a ligand-dependent manner and enhance their transcriptional activities. The potential roles of ARA55 in prostate cancer is also discussed. A human prostate cDNA library in pACT2 yeast expression vector was a gift from Dr. S. Elledge. For construction of pAS2-wtAR or mAR, C-terminal fragments (aa 595–918) from wtAR or mAR (mART877S, point mutation threonine to serine at codon 877, from Dr. S. P. Balk (7Taplan M.-E. Bubley G.J. Shuster T.D. Frantz M.E. Spooner A.E. Ogata G.K. Keer H.N. Balk S.P. N. Engl. J. Med. 1995; 332: 1393-1398Crossref PubMed Scopus (1040) Google Scholar)), respectively, were inserted in pAS2 yeast expression vector (CLONTECH). pG5CAT reporter plasmid (CLONTECH) contains five GAL4 binding sites upstream of the E1b TATA box, linked to the CAT gene. A pACT2-prostate cDNA library that consists of the GAL4 activation domain (aa 768–881) fused with human prostate cDNA library was transformed into Y190 yeast cells with a plasmid of pAS2-mAR (mART877S) that contains GAL4DBD fused with the C-terminal domain of this mAR. Transformants were selected for growth on synthetic dropout (SD) plates with 25 mm 3-aminotriazole and 100 nm dihydrotestosterone (DHT) lacking histidine, leucine, and tryptophan. Colonies were also filter-assayed for β-galactosidase activity. DNAs from positive clones were recovered from yeast, amplified in E. coli, and confirmed by sequencing. The missing 5′-coding region was isolated by 5′-RACE-PCR according to the manufacturer's protocol of Marathon cDNA Amplification Kit (CLONTECH). The gene-specific antisense primer used for 5′-RACE-PCR was 5′-TCAGCCGAAGAGCTTCAGGAAGCAGGG-3′. The specific PCR reaction condition was 94 °C for 1 min, 5 cycles of 94 °C for 5 s → 72 °C for 3 min, 5 cycles of 94 °C for 5 s → 70 °C for 3 min, then 25 cycles of 94 °C for 5 s → 68 °C for 3 min. The PCR product was subcloned into pT7-Blue vector (Novagen) and sequenced. Lysates fromin vitro translated full-length AR and ARA55 were incubated with or without 10−8m DHT in the modified RIPA buffer (50 mm Tris-HCL, pH 7.4, 150 mmNaCl, 5 mm EDTA, 0.1% Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin, 0.25% Na-deoxycholate, 0.25% gelatin) and rocked at 4 °C for 2 h. The conjugated beads were washed four times with RIPA buffer, boiled in SDS sample buffer, and analyzed by 8% SDS-polyacrylamide gel electrophoresis and visualized by STORM 840 (Molecular Dynamics). The total RNA (25 μg) was fractionated on a 1% formaldehyde-MOPS-agarose gel, transferred onto a Hybond-N nylon membrane (Amersham Pharmacia Biotech) and prehybridized. A probe corresponding to the 900 bp C terminus of ARA55 was32P-labeled in vitro using Random Primed DNA Labeling Kit (Boehringer Mannheim) according to the manufacturer's protocol and hybridized overnight. After washing, the blot was exposed and quantified by PhosphorImager (Molecular Dynamics). GAPDH was used to monitor the amount of total RNA in each lane. DU145 cells and PC3 cells were grown in Dulbecco's minimal essential medium (DMEM) containing 5% fetal calf serum (FCS). One hour before transfection, the medium was changed to DMEM containing 5% charcoal-stripped fetal calf serum. Phenol red-free medium was used with the E2 experiments. Cells were transfected using the modified calcium phosphate technique for 24 h, the medium was changed, and cells were treated with either steroid hormones or hydroxyflutamide (HF) for another 24 h. The cells were harvested, and cell lysates were normalized by β–galactosidase internal control and assayed for chloramphenicol acetyltransferase (CAT) activity. CAT activity was quantified by PhosphorImager. For RT-PCR, the total RNA (2 μg) from each sample was reverse-transcribed using SuperScript Preamplification System (Life Technologies, Inc.) in a total reaction volume of 20 μl. cDNA (1 μl) was amplified by PCR with AmpliTaq Gold (Perkin-Elmer). The following sense and antisense primers were used: sense primer, 5′-GCACTTCGTTTGCGGAGGC-3′; antisense primer, 5′-CCGAAGAGCTTCAGGAAGC-3′. This combination of primers amplifies 633 bp of the C-terminal region of ARA55. After an initial denaturation at 95 °C for 9 min, 33 cycles of amplification (denaturation at 94 °C for 9 min, annealing at 63 °C for 1 min, and extension at 72 °C for 1 min) were followed by a terminal extension at 72 °C for 1 min. PCR products were visualized on a 1% agarose gel containing ethidium bromide. The 600-bp fragment of GAPDH, which was amplified with 30 amplification cycles using the sense and antisense primers for human GAPDH (Stratagene), was used to demonstrate comparable RNA amounts and quality among samples. Loss of androgen specificity in mAR may contribute to the development of prostate cancer from an androgen-dependent to androgen-independent state (7Taplan M.-E. Bubley G.J. Shuster T.D. Frantz M.E. Spooner A.E. Ogata G.K. Keer H.N. Balk S.P. N. Engl. J. Med. 1995; 332: 1393-1398Crossref PubMed Scopus (1040) Google Scholar, 8Gaddipati J.P. McLeod D.G. Heidenberg H.B. Sesterhenn I.A. Finger M.J. Moul J.W. Srivastava S. Cancer Res. 1994; 54: 2861-2864PubMed Google Scholar). We were interested to know if wtAR and mAR might exert their functions by recruiting additional cofactors. A yeast two-hybrid system with mART877S as a bait was used to screen the human prostate cDNA library. As a result, six clones that interacted with mART877S were isolated, and one of them, named ARA55, was further characterized because its DNA sequence was highly homologous to the C terminus of mouse hic5 (a hydrogen peroxide-inducible clone) (9Shibanuma M. Mashimo J. Kuroki T. Nose K. J. Biol. Chem. 1994; 269: 26767-26774Abstract Full Text PDF PubMed Google Scholar). Northern blot analysis indicated that ARA55 mRNA, with a size near 2 kilobases, could be detected in HeLa and prostate PC-3 cells but not in other cell lines such as HepG2, H1299, MCF7, CHO, PC12, P19, and DU145 (data not shown). The 5′-RACE-PCR technique was then used to clone the full-length ARA55 from HeLa cells. Sequence analysis determined that the open reading frame between the first ATG and terminal TGA encoded 444 aa for human ARA55 with the predicted molecular mass of 55 kDa (Fig.1 A). Amino acid sequence analysis indicated that human ARA55 shares 90.5% homology with mouse Hic5 (9Shibanuma M. Mashimo J. Kuroki T. Nose K. J. Biol. Chem. 1994; 269: 26767-26774Abstract Full Text PDF PubMed Google Scholar). Another interesting finding from this deduced aa sequence was the existence of four LIM motifs in the C-terminal regions (Fig. 1 B). The LIM motif is a cysteine-rich zinc-binding motif with the consensus sequence: CX2CX16–23HX2CX2CX2CX16–21CX2(C, H, D) (10Sadler I. Crowford A.W. Michelsen J.W. Beckerle M.C. J. Cell Biol. 1992; 119: 1573-1587Crossref PubMed Scopus (295) Google Scholar). Although the function of the LIM motif has not been fully defined, some data suggest that it might be involved in the protein-protein interaction (11Schmeichel K.L. Beckerle M.C. Cell. 1994; 79: 211-219Abstract Full Text PDF PubMed Scopus (408) Google Scholar). Among all identified SR-associated proteins, only the thyroid hormone interacting protein 6 (Trip 6) has these similar LIM motifs (12Lee J.W. Choi H.-S. Gyuris J. Brent R. Moore D.D. Mol. Endocrinol. 1995; 9: 243-254Crossref PubMed Google Scholar). We first tested whether the interaction between ARA55 and AR is ligand-dependent in the yeast and mammalian two-hybrid systems. Y190 yeast cells were transformed with GAL4AD fused with ARA55251–444 and GAL4DBD fused with the C-terminal region (aa 595–918) of mART877S or wtAR. Transformants were selected by their growth on plates with DHT, testosterone, E2, progesterone, dexamethasone, or vehicle (ethanol) only. Colonies were also assayed for β–galactosidase activity. As shown in Fig.2 A, DHT and testosterone promoted the interaction between ARA55 and wtAR or mAR at concentrations of greater than 1 nm. E2 and progesterone promoted the interaction only at much higher concentrations (1 μm for E2 and 0.1 μm for progesterone). Dexamethasone and vehicle did not promote this interaction. There were no differences between wtAR and mAR in their interactions with ARA55 (data not shown). Together, these data suggested that ARA55 can interact with not only mAR, but also wtAR in the presence of DHT, testosterone, and higher concentrations of E2 or progesterone and that the presence of only the C-terminal region of ARA55 is the minimal requirement for interaction. Next, we tested this interaction in a mammalian two-hybrid system. DU145 cells were with a plasmid the hormone binding domain of wtAR fused to the GAL4 and with a plasmid full-length ARA55 fused to the activation domain of was by the of CAT activity from the reporter plasmid (Fig. 2 B). A combination of vector and did not CAT activity (Fig. 2 A combination of and vector showed a amount of CAT activity and a of CAT activity was by the of and only in the presence of nm DHT (Fig. 2 3 that ARA55 can interact with AR and that this interaction is further prove that the interaction between AR and ARA55 is the a co-immunoprecipitation assay was using an in vitro which expressed high of AR and ARA55. A was for and the complexes were to SDS-polyacrylamide gel As shown in 2 the AR can only be by with ARA55 that is fused with and the of 10−8m DHT the specific interaction between AR and ARA55 (Fig. 2 3 yeast and mammalian two-hybrid systems and all indicated that ARA55 can interact with AR in a we were then interested in if such an interaction can further AR transcriptional activity. As shown in the AR only minimal reporter activity with or without the ARA55, and ARA55 also showed only minimal reporter activity (Fig. of nm DHT in a of AR transcriptional activity and ARA55 further increased this by to in a manner (Fig. The activity a at the of to A similar could also be we DU145 cells with PC-3 or with a reporter plasmid that a promoter region of gene containing androgen regions (data not shown). We then the of ARA55 on two in the presence of concentrations of DHT, E2, and One mAR, was identified in cells and in prostate with a point mutation at codon to J.P. McLeod D.G. Heidenberg H.B. Sesterhenn I.A. Finger M.J. Moul J.W. Srivastava S. Cancer Res. 1994; 54: 2861-2864PubMed Google J. C. G. C. E. J. E. Res. PubMed Scopus Google Scholar). The other mAR, has a point mutation at codon to This was isolated by a yeast with of to and E2 as with wtAR The Androgen and on the of Androgen of Scholar). As shown in wtAR and these well to DHT at to and ARA55 their by the presence of E2 or wtAR was at high concentrations for E2 and for of wtAR with ARA55 at a however, increased AR transcriptional activity in the presence of for E2 or to for with much to E2 and and ARA55 could further enhance its transcriptional activity. could well to DHT and ARA55 could also further enhance its transcriptional showed very response to E2 and at the high concentrations and ARA55 did not have on its transactivation. data suggest that at AR may play important roles for the AR transcriptional ARA55 may be for the or or AR transcriptional activity. As of the identified cofactors for SRs are general and not specific to only one receptor H. Spencer T. G. Tsai S.Y. Tsai M.-J. O'Malley B.W. Res. 1997; Google Scholar), we were interested in if ARA55 was a specific coactivator for AR. the we tested the of ARA55 on by glucocorticoid receptor progesterone receptor and estrogen receptor in DU145 cells. As shown in ARA55 to be specific to AR, it also and to a and has only a on we not know if the we used in the prostate DU145 cells were for and our data did that ARA55 could and to a greater with another AR coactivator, S. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). showed much higher specificity to AR (Fig. which may suggest ARA55 has to interact with other The and were identified in prostate cancer and could to or estrogen as well as ARA55 could enhance the of these as well as we were interested in whether the prostate cancer cells or the prostate could the ARA55. Northern blot analysis that DU145 and cells did but PC-3 cells did ARA55 A). Next, we ARA55 expression in the prostate and the cell using (Fig. 6 because these were by and the amount of the RNA was not sufficient for Northern As the Northern PC-3 cells expressed ARA55 mRNA, but DU145 and cells did prostate including and expressed ARA55 mRNA, but were some differences in its expression among the samples. This of ARA55 expression might be one of the factors that the of prostate The sequence of ARA55 this AR coactivator the of SR coactivators that Tsai S.Y. Tsai M.-J. O'Malley B.W. Science. 1995; PubMed Scopus Google Scholar), C. Chambon P. Gronemeyer H. J. PubMed Scopus Google Scholar, H. K. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and (4Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalski M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar, S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.-Y. Sauter G. Kallioniemi O.P. Trent J.M. Science. 1997; 277: 965-968Crossref PubMed Scopus (1432) Google Scholar, H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). For ARA55 some motifs as domain and that are by SR ARA55 have three LIM motifs in the interaction domain of the C-terminal The LIM motif is a cysteine-rich motif that is in several with functions and The and the function of the LIM motifs have not been fully defined, and it has been suggested that their function is in regulation (10Sadler I. Crowford A.W. Michelsen J.W. Beckerle M.C. J. Cell Biol. 1992; 119: 1573-1587Crossref PubMed Scopus (295) Google A.W. Beckerle M.C. J. Cell Biol. 1994; PubMed Scopus Google Scholar). and Beckerle that LIM motifs might be involved in protein-protein interaction (11Schmeichel K.L. Beckerle M.C. Cell. 1994; 79: 211-219Abstract Full Text PDF PubMed Scopus (408) Google Scholar). the LIM motifs in the C-terminal region of ARA55 may contribute to its interaction with AR. There is also no homology between ARA55 and the first identified AR coactivator, S. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. H. C. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. H. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, H. Yeh S. G. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, H. Yeh S. H. E. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. H. K. H. J. P. Wang C. C. C. Res. PubMed Scopus Google Scholar). Although AR coactivators enhance AR transcriptional activity in DU145 ARA55 is general to SRs is specific to ARA55 has a than on in the presence of E2 and and ARA55 is a gene. The role of these two cofactors may the on prostate cells. One of the very interesting of ARA55 sequence is its high homology to mouse hic5, a well TGF-β1-inducible gene (9Shibanuma M. Mashimo J. Kuroki T. Nose K. J. Biol. Chem. 1994; 269: 26767-26774Abstract Full Text PDF PubMed Google Scholar). studies suggested that Hic5 may play important roles in the with M. E. R. Mashimo J. N. S. T. M. Nose K. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar), the of hic5 to steroid hormones and their has not been Our here may a potential to to AR transcriptional activity via of ARA55 in cancer is the tumor and the of cancer in T. S. J. 1995; PubMed Scopus Google Scholar). the only for prostate cancer is that or with of such as or Although is very and of the to this the of may within J. 1992; 70 PubMed Scopus Google Scholar). The by which prostate cancer cells to unclear. One to prostate can from androgen to androgen is that in AR may this to other steroid hormones or such as E2 and As ARA55 was to transcriptional activity of wtAR and mAR in the presence of DHT, E2, and and ARA55 was expressed differently in prostate tumor ARA55 may play some important roles for the progression of prostate cancer and its to Further studies of the function of ARA55 a or that can with the interaction of AR and may a novel for the development of of the prostate We S. S. P. and D. for and and K. for