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Tip110, the Human Immunodeficiency Virus Type 1 (HIV-1) Tat-interacting Protein of 110 kDa as a Negative Regulator of Androgen Receptor (AR) Transcriptional Activation

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m314321200

ISSN

1083-351X

Autores

Ying Liu, Byung Oh Kim, Chinghai Kao, Chaeyong Jung, James T. Dalton, Johnny J. He,

Tópico(s)

Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities

Resumo

Upon binding to androgen, androgen receptor (AR) can activate expression of target genes through its direct binding to the androgen-responsive elements (AREs), which are located within the target gene promoters and/or enhancers. A number of cellular proteins have been identified as co-regulators to regulate this transactivation process. One common structural feature among these co-regulators is the presence of the LXXLL motif (X, any amino acid), the so-called nuclear receptor (NR) box, through which binding of these regulatory proteins to AR occurs. We have recently shown that Tip110 functions to potentiate the transactivation activity of human immunodeficiency virus type I (HIV-1) Tat protein. In this study, we report that Tip110 is a potent AR-binding protein that can suppress AR activity. Tip110 bound to AR in an NR box-dependent manner and inhibited AREs-mediated reporter gene expression. The inhibitory effects were abolished by removal of the NR box. Moreover, knock-down of the constitutive Tip110 expression significantly augmented AR transcriptional activation. In agreement with these findings, Tip110 overexpression blocked the prostate-specific antigen (PSA) gene, a well characterized target gene of AR from expression in LNCaP cells. Further analysis revealed that Tip110 prevented the complex formation between AR and AREs. Taken together, these results indicate that Tip110 is a negative regulator of AR transcriptional activation, and may be directly involved in AR-related developmental, physiological, and pathological processes. Upon binding to androgen, androgen receptor (AR) can activate expression of target genes through its direct binding to the androgen-responsive elements (AREs), which are located within the target gene promoters and/or enhancers. A number of cellular proteins have been identified as co-regulators to regulate this transactivation process. One common structural feature among these co-regulators is the presence of the LXXLL motif (X, any amino acid), the so-called nuclear receptor (NR) box, through which binding of these regulatory proteins to AR occurs. We have recently shown that Tip110 functions to potentiate the transactivation activity of human immunodeficiency virus type I (HIV-1) Tat protein. In this study, we report that Tip110 is a potent AR-binding protein that can suppress AR activity. Tip110 bound to AR in an NR box-dependent manner and inhibited AREs-mediated reporter gene expression. The inhibitory effects were abolished by removal of the NR box. Moreover, knock-down of the constitutive Tip110 expression significantly augmented AR transcriptional activation. In agreement with these findings, Tip110 overexpression blocked the prostate-specific antigen (PSA) gene, a well characterized target gene of AR from expression in LNCaP cells. Further analysis revealed that Tip110 prevented the complex formation between AR and AREs. Taken together, these results indicate that Tip110 is a negative regulator of AR transcriptional activation, and may be directly involved in AR-related developmental, physiological, and pathological processes. The steroid hormone androgen exerts its biological functions through the androgen receptor (AR) 1The abbreviations used are: AR, androgen receptor; HIV-1, human immunodeficiency virus type 1; Tip110, HIV-1 Tat-interacting protein of 110 kDa; AREs, androgen-responsive elements; NR box, nuclear receptor box; PSA, prostate-specific antigen; DBD, DNA binding domain; LBD, ligand binding domain; AF, activation function; GST, glutathione S-transferase; NLS, nuclear localization signal; RRM, RNA recognition motifs; RT, reverse transcriptase; Luc, luciferase. (1Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. 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Cell Lines and Cell Transfection—293T cells and LNCaP cells were purchased from American Tissue Culture Collection (ATCC, Manassas, VA) and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) at 37 °C with 5% CO2. For luciferase reporter gene assay and detection of prostate-specific antigen (PSA) expression, LNCaP cells were maintained in DMEM supplemented with 10% dextran charcoal-stripped fetal bovine serum at 37 °C with 5% CO2. 293T cells were transfected by the standard calcium phosphate precipitation method as previously described (38Liu Y. Li J. Kim B.O. Pace B.S. He J.J. J. Biol. Chem. 2002; 277: 23854-23863Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). LNCaP cells were transfected using the LipofectAMINE 2000 according to the manufacturer's instructions (Invitrogen). Transfection medium was replaced with fresh medium containing 100 nm R1881 (Sigma) 10–12 h after transfection unless otherwise stated, and cells were harvested for protein analysis by Western blot or immunoprecipitation followed by Western blot, or for assay of the luciferase reporter gene activity, or for isolation of total RNAs and RNA analysis by reverse transcription-PCR (RT-PCR) or Northern blot. In all transfections, pcDNA3 was used to equalize the amount of DNA transfected, and pTKβgal was included to normalize the variations in transfection efficiency. Plasmids—Plasmid pTip110.His, the deletion mutants ΔCT, ΔNLS, ΔRRM, and ΔNLSΔRRM, pAs-Tip110, and pTKβgal are described elsewhere (38Liu Y. Li J. Kim B.O. Pace B.S. He J.J. J. Biol. Chem. 2002; 277: 23854-23863Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Tip110 mutants; ΔNR deleted for the NR box, L118A (leucine at position 118 mutated to alanine), L121A (leucine at position 121 mutated to alanine), and L122A (leucine at position 122 mutated to alanine); were constructed similarly in the context of the pTip110.His backbone using the ExSite PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, CA). The oligonucleotides for each mutant are: ΔNR, 5′-GTC CAC ATG GCA GTT GTA GTC ATA-3′ and 5-AGG CTC GAG GGG GAG CTT ACC AAG GTG-3′; L118A, 5′-CAG GTG TAC CGT CAA CAT CAG TAT CTG C-3′ and 5′-CGG CTA GTC TGA CGA GTC CGA CCT TCC CC-3′; L121A, 5′-AGA CTA GTT CAG GTG TAC CGT CAA CAT C-3′ and 5′-CGG GAG TCC GAC CTT CCC CTC GAA TGG-3′; L122A, 5′-GTC AGA CTA GTT CAG GTG TAC CGT C-3′ and 5′-CGG TCC GAC CTT CCC CTC GAA TGG TTC C-3′. The pGEX-Tip110 plasmid expressing Tip110 as the glutathione S-transferase (GST) fusion protein was constructed in the context of the pGEX4T-3 backbone purchased from Amersham Biosciences (Piscataway, NJ), using the standard PCR cloning technique with oligonucleotides 5′-GGA ATT CAC CAT GGC GAC TGC GGC CGA A-3′ (EcoRI site underlined) and 5′-CCC GCT CGA GTC AAT GAT GAT GAT GAT GAT GCT TTC TCA GAA ACA GCT TGG C-3′ (XhoI site underlined and His tag in italics). pGL3.TATA-Luc containing the synthetic adenovirus E1b TATA sequence (TATATAAT) and p4GRE.TATA-Luc containing the TATA and 4 tandem copies of GREs (TGTACAGGATGTTCT) from the murine mammary tumor virus promoter were constructed in the context of the pGL3 backbone (Promega, Madison, WI). pAR.FLAG expressing the human AR tagged with the FLAG epitope at the COOH terminus is described elsewhere (39Wang C. Yeung F. Liu P.C. Attar R.M. Geng J. Chung L.W. Gottardis M. Kao C. J. Biol. Chem. 2003; 278: 32423-32430Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Baculovirus transfer vector pBlueBacHis2A-hAR expressing the full-length human AR is described elsewhere (40Zhu Z. Bulgakov O.V. Scott S.S. Dalton J.T. Biochem. Biophys. Res. Commun. 2001; 284: 828-835Crossref PubMed Scopus (7) Google Scholar). Preparation of Whole Cell Lysates, Immunoprecipitation, and Western Blot—Cells were washed twice with ice-cold phosphate-buffered saline before they were harvested for preparing whole cell lysates according to the methods previously described (38Liu Y. Li J. Kim B.O. Pace B.S. He J.J. J. Biol. Chem. 2002; 277: 23854-23863Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Briefly, cell pellets were suspended in two volumes of the whole cell lysis buffer containing 10 mm NaHPO4, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 0.2% sodium azide, 0.5% sodium deoxycholate, 0.004% sodium fluoride, and 1 mm sodium orthovanadate, and incubated on ice for 10 min. Whole cell lysates were obtained by centrifugation and removal of the cell debris. For Western blots, cell lysates (25 μg of protein) or immunoprecipitates (500 μg of protein) were electrophoretically separated on 10% SDS-PAGE and analyzed by immunoblotting using anti-His (Qiagen, Valencia, CA), anti-FLAG (Sigma), anti-PSA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-AR antibodies (Santa Cruz Biotechnology), or anti-β-actin antibody (Sigma) at dilutions recommended by the manufacturers, followed by appropriate horseradish peroxidase-conjugated secondary antibodies, and then visualized with the ECL system (Amersham Biosciences). RNA Analysis by RT-PCR and Northern Blot—Total RNA was extracted from LNCaP cells using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. 0.2 μg of total RNA was reverse-transcribed and PCR-amplified with PSA-specific 5′-GGCAGGTGCTTGTAGCCTCTC-3′ and 5′-CACCCGAGCAGGTGCTTTTGC-3′) using the Titan One Tube RT-PCR system (Roche Diagnostics, Indianapolis, IN). The PCR program consisted of 2 min of denaturation at 94 °C, 10 cycles of amplifications with 45 s of elongation time, followed by 25 cycles of amplifications with 45 s of elongation time plus 5 s for each cycle. The expected PCR DNA products were about 522 bp in length. The human β-actin gene was included in the RT-PCR as an internal control, with primers 5′-CAGCACTGTGTTGGCGTACAGGTC-3′ and 5′-CAGGTCATCACCATTGGCAATCAG-3′, and the expected PCR DNA was 139 bp in length. For Northern blots, total RNA was extracted from LNCaP cells as described above, and 25 μg of RNA was fractionated on a 1.2% denaturing agarose gel. The gel was stained with ethidium bromide for 18 and 28 S rRNAs, and the PSA mRNA was detected by hybridization with the 32P-labeled 522-bp PSA probe (the RT-PCR product). The probe was prepared using the random-primed DNA labeling kit (Roche Diagnostics). Recombinant Protein Purification—GST-Tip110 and GST proteins were expressed in Escherichia coli BL21 strain. Bacteria harboring the pGST-Tip110 or pGEX-4T-3 plasmid were grown in LB medium containing 50 μg/ml ampicillin at 26 °C overnight, diluted by 1:20 with fresh LB media, and allowed to grow until the OD600 of the culture reached 0.6. To induce the fusion protein expression, isopropyl-1-thio-β-galactoside was added to the culture at a final concentration of 0.1 mm, and the culture was continued for 3 h. The bacteria were then lysed in a lysis buffer containing 100 mm Tris-HCl, pH 8.0, 5 mm EDTA, 5 mm dithiothreitol, 5 mm benzamidine, and 200 μg/ml lysozyme and sonicated. The supernatants were collected by centrifugation and allowed to bind to immobilized glutathione-Sepharose beads (Pierce Biotechnology). Bound Tip110-GST fusion protein or GST protein was eluted by 0.4 m reduced glutathione in phosphate-buffered saline (PBS), and the glutathione was removed by extensive dialysis against PBS. For the full-length recombinant AR protein, preparation of baculovirus stock, infection of SF9 cells, and AR purification were performed exactly as previously described (40Zhu Z. Bulgakov O.V. Scott S.S. Dalton J.T. Biochem. Biophys. Res. Commun. 2001; 284: 828-835Crossref PubMed Scopus (7) Google Scholar). The purity of GST, Tip110-GST, and AR protein was determined to be higher than 95% by SDS-PAGE and Coomassie staining. Electrophoretic Mobility Shift Assay (EMSA)—The double-stranded AR DNA binding fragment ARE was made by annealing two complementary oligonucleotides: 5′-GAAGTCTGGTACAGGGTGTTCTTTTTG-3′ and 5′-CAAAAAGAACACCTGTACCAGACTTC-3′. The ARE DNA was radiolabeled using [α-32P]dATP and Klenow enzyme to a specific activity of 5 × 108 cpm/μg, removing the free [α-32P]dATP. The AR-ARE complex formation was carried out in a buffer containing 10 mm HEPES, pH 7.9, 100 mm KCl, 5 mm MgCl2, 10% glycerol, 1 mm dithiothreitol, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 100 μg/ml poly(dI:dC), and in the presence of 100 mm R1881, unlabeled ARE DNA, GST, or GST-Tip110 protein. Complex formation was determined on a pre-run 4% native polyacrylamide gel (29:1) containing 2.5% glycerol in 0.5× Tris borate/EDTA buffer (45 mm Tris, pH 8.0, 45 mm boric acid, and 1 mm EDTA). The gel was dried and autoradiographed. Tip110 Binding to AR—We have recently identified a new protein Tip110 as a co-transactivator of human immunodeficiency virus type I (HIV-1) Tat protein (38Liu Y. Li J. Kim B.O. Pace B.S. He J.J. J. Biol. Chem. 2002; 277: 23854-23863Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Further examination of the Tip110 primary amino acid sequence revealed an LXXLL motif between amino acid positions118 and 122. Various androgen receptor co-regulators contain this LXXLL motif, which is recognized by and recruited through the activation function 2 (AF2) domain of AR. To investigate whether Tip110 binds to AR, 293T cells were transfected with Tip110 expression plasmid pTip110.His, AR expression plasmid pAR.FLAG, or both. His and FLAG epitope tags were used to facilitate detection of Tip110 and AR, respectively. pcDNA3 was included to normalize the amount of DNAs among transfections. Expression of Tip110 or AR was determined by Western blot of cell lysates, and the binding by immunoprecipitation of cell lysates with anti-FLAG antibody, followed by Western blot against anti-His antibody. Tip110 and AR were expressed at the expected molecular sizes and comparable levels when they were transfected separately or together (Fig. 1, top and middle panels). Tip110 was detected in immunoprecipitates of cell lysates prepared from co-transfection of Tip110 and AR (Fig. 1, lane 4, bottom panel), but not in immunoprecipitates of cell lysates from AR expression plasmid transfection alone (lane 3) or transfections without AR expression plasmids (lanes 1 and 2). Complex formation of Tip110 and AR was also detected in immunoprecipitates against anti-His antibody followed by Western blot against the FLAG antibody in the cell lysates of Tip110 and AR co-transfections (data not shown). These results suggest that Tip110 was able to bind to AR. NR-dependent Tip110 Binding to AR—Besides the NR box, Tip110 has been found to contain a nuclear localization signal (NLS, amino acids 600–670) and RNA recognition motifs (RRM, amino acids 740–850) (38Liu Y. Li J. Kim B.O. Pace B.S. He J.J. J. Biol. Chem. 2002; 277: 23854-23863Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). To determine the specificity of Tip110 binding to AR and the role of the NR box of Tip110 in its binding to AR, we constructed a series of Tip110 deletion mutants including the mutant deleted for the NR box (Fig. 2A). Similarly, we transfected 293T cells with the AR expression plasmid in combination with each of Tip110 mutants and determined its binding to each of the Tip110 mutants by immunoprecipitation and Western blot. All mutants except the Tip110ΔRRM mutant were expressed at the expected molecular sizes and at comparable levels (Fig. 2B, top panel). Like the wild-type Tip110, deletion of the nuclear localization signal (ΔNLS), the RNA recognition motifs (ΔRRM), or the COOH terminus domain (ΔCT, amino acids 670–963) retained the ability of Tip110 to bind to AR (Fig. 2B, bottom panel). In contrast, NR box deletion (ΔNR) abolished Tip110 binding to AR (Fig. 2B, lane 3). Interestingly, the mutant deleted for both NLS and RRM (ΔNLSΔRRM) rendered Tip110 unable to bind to AR (Fig. 2B, lane 7), suggesting that in addition to the NR box, intramolecular interaction or appropriate conformation of Tip110 may also be required for Tip110 interaction with AR. To ascertain whether the NR box is directly involved in Tip110 binding to AR, we sequentially mutated each of the leucine amino residues at amino acid positions 118, 121, and 122 to alanine within the NR box. Similarly, we co-expressed these site-directed Tip110 mutants with AR and analyzed their interaction. The results showed no complex formation detected between AR and any of these three Tip110 mutants, although they were expressed at comparable levels (Fig. 2C, lanes 4–6). In addition, we also co-expressed the wild-type Tip110 with AR in 293T cells and cultured the transfected cells in medium containing dextran charcoal-stripped fetal bovine serum. The results showed no complex formation between Tip110 and AR in lysates prepared from these cells (Fig. 2C, lane 7). Taken together, these results demonstrated that the NR box was directly involved in Tip110 binding to AR and further support that Tip110 bound to AR in a specific manner. Inhibition of AR Transcriptional Activation by Tip110 —A number of AR co-regulators have been identified to interact with AR and function as either AR co-activators or co-repressors in AR-mediated gene expression. Our results showed complex formation between Tip110 and AR. Thus, we decided to investigate the effect of Tip110 on AR transcriptional activation. We took advantage of an AR reporter gene assay, which involves use of an AR-responsive DNA element-driven fruit fly luciferase reporter gene p4GRE.TATA-Luc containing 4 tandem repeats of AR-responsive DNA elements (39Wang C. Yeung F. Liu P.C. Attar R.M. Geng J. Chung L.W. Gottardis M. Kao C. J. Biol. Chem. 2003; 278: 32423-32430Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) and human prostate LNCaP cancer cells, constitutively expressing AR. LNCaP cells were transfected with p4GRE.TATA-Luc plasmid alone or in combination with Tip110 expression plasmid, and the transfected cells were allowed to grow in the absence or presence of a synthetic androgen R1881. As expected, addition of 100 nm R1881 increased AR-mediated Luc expression by about 3.5-fold (Fig. 3A). Expression of Tip110 resulted in a considerable reduction of AR-mediated Luc expression in the presence of R1881, and the inhibition appeared to be correlated with the amount of Tip110 DNA transfected (Fig. 3A). Nevertheless, Tip110 expression in the absence of R1881 showed little effect on the basal level of AR-mediated Luc expression (Fig. 3A). Moreover, the inhibitory effects of Tip110 were not caused by decreased levels of AR expression (Fig. 3B). Similar results were obtained in non-prostate cancer cells such as 293T cells by co-transfection of p4GRE.TATA-Luc, AR, and Tip110 (data not shown). These results suggest that Tip110 functioned as a repressor of ligand-dependent AR transcriptional activation. Ablation of Tip110 Inhibition of AR Transcriptional Activation by Removal of the NR Box—Although the NR box has been shown to be important for AR binding to its co-regulators, its direct function on AR transcriptional activation remains largely inconsistent. For example, the NR box in Tip60 is directly involved in Tip60-induced AR transcriptional activation, but the NR box in p160 has no effect on AR transcriptional activation. Thus, we next determined whether NR-mediated Tip110 binding to AR is attributable to the inhibitory effect of Tip110 on AR transcriptional activation activity. LNCaP cells were transfected with p4GRE.TATA-Luc in combination with each of the Tip110 mutants, and the AR-mediated Luc expression was determined in the presence of R1881. Tip110 mutants ΔCT, ΔNLS, and ΔRRM exhibited Luc expression at a level similar to that of the wild-type Tip110, whereas both mutants ΔNR and ΔNLSΔRRM showed no inhibitory effect on Luc expression (Fig. 4). These effects were in agreement with the abilities of Tip110 mutants to bind to AR. These results suggest that direct binding of Tip110 with AR is a prerequisite for Tip110-induced inhibition of AR transcriptional activation. Enhancement of AR Transcriptional Activation by Tip110 Knock-down—Our previous studies have shown ubiquitous expression of Tip110 mRNA in a variety of human tissues and cell lines (41Liu Y. Wang Z. Rana T.M. J. Biol. Chem. 1996; 271: 10391-10396Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). To further characterize the potential roles of Tip110 in AR transcriptional activation, Tip110 expression was determined in LNCaP cells. Western blot analysis showed that Tip110 protein was expressed in LNCaP cells (Fig. 5A, lanes 1 and 2), which raised the possibility that Tip110 may be a constitutive repressor of AR. To test this possibility, we down-modulated constitutive Ti

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