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• W2055330198 abstract "The p160 coactivators, such as GRIP1, bind nuclear receptors and help to mediate transcriptional activation. β-Catenin binds to and serves as a coactivator for the nuclear receptor, androgen receptor (AR), and the Lymphoid Enhancer Factor/T Cell Factor family member, Lef1. Here we report that GRIP1 and β-catenin can bind strongly to each other through the AD2 domain of GRIP1. Furthermore, GRIP1 and β-catenin can synergistically enhance the activity of both AR and Lef1, and both coactivators are recruited specifically to AR-driven and Lef1-driven promoters. However, the mechanism of β-catenin-GRIP1 coactivator function and synergy is different with AR and Lef1. While β-catenin can bind directly to both AR and Lef1, GRIP1 can only bind directly to AR; the ability of GRIP1 to associate with and function as a coactivator for Lef1 is entirely dependent on the presence of β-catenin. Thus, whereas GRIP1 coactivator function involves direct binding to nuclear receptors and most other classes of DNA-binding transcriptional activator proteins, the coactivator function of GRIP1 with Lef1 follows a novel paradigm where GRIP1 is recruited indirectly to Lef1 through their mutual association with β-catenin. The β-catenin-GRIP1 interaction represents another potential point of cross-talk between the AR and Wnt signaling pathways. The p160 coactivators, such as GRIP1, bind nuclear receptors and help to mediate transcriptional activation. β-Catenin binds to and serves as a coactivator for the nuclear receptor, androgen receptor (AR), and the Lymphoid Enhancer Factor/T Cell Factor family member, Lef1. Here we report that GRIP1 and β-catenin can bind strongly to each other through the AD2 domain of GRIP1. Furthermore, GRIP1 and β-catenin can synergistically enhance the activity of both AR and Lef1, and both coactivators are recruited specifically to AR-driven and Lef1-driven promoters. However, the mechanism of β-catenin-GRIP1 coactivator function and synergy is different with AR and Lef1. While β-catenin can bind directly to both AR and Lef1, GRIP1 can only bind directly to AR; the ability of GRIP1 to associate with and function as a coactivator for Lef1 is entirely dependent on the presence of β-catenin. Thus, whereas GRIP1 coactivator function involves direct binding to nuclear receptors and most other classes of DNA-binding transcriptional activator proteins, the coactivator function of GRIP1 with Lef1 follows a novel paradigm where GRIP1 is recruited indirectly to Lef1 through their mutual association with β-catenin. The β-catenin-GRIP1 interaction represents another potential point of cross-talk between the AR and Wnt signaling pathways. The androgen receptor (AR) 1The abbreviations used are: ARandrogen receptorHAhemagglutininGSTglutathione S-transferaseChIPchromatin immunoprecipitation assayDHTdihydrotestosteronePSAprostate-specific antigenco-IPco-immunoprecipitationADactivation domainMMTVmouse mammary tumor virusLEF/TCFlymphoid enhancer factor/T cell factor.1The abbreviations used are: ARandrogen receptorHAhemagglutininGSTglutathione S-transferaseChIPchromatin immunoprecipitation assayDHTdihydrotestosteronePSAprostate-specific antigenco-IPco-immunoprecipitationADactivation domainMMTVmouse mammary tumor virusLEF/TCFlymphoid enhancer factor/T cell factor. belongs to the nuclear receptor family of DNA-binding transcriptional activator proteins (1Jenster G. Semin. Oncol. 1999; 26: 407-421PubMed Google Scholar). Binding of the natural steroid hormones testosterone or dihydrotestosterone (DHT) to AR causes the protein to translocate from cytoplasm to nucleus where it binds to the promoters of androgen target genes and recruits specific coactivators to activate transcription. Among the many coactivators that contribute to the transcriptional activation process (2Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 3McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar, 4Heinlein C.A. Chang C. Endocr. Rev. 2002; 23: 175-200Crossref PubMed Scopus (631) Google Scholar), the steroid receptor coactivator/nuclear receptor coactivator (SRC/NcoA/p160) family, encoding proteins of ∼160 kDa, play central roles by serving as scaffold proteins or primary coactivators that bind directly to the DNA-bound nuclear receptors and recruit secondary coactivators, including histone-modifying enzymes. The three members of the p160 family are SRC-1, GRIP1/TIF-2, and p/CIP/AIB1/ACTR/RAC3/TRAM1 (2Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 3McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar, 4Heinlein C.A. Chang C. Endocr. Rev. 2002; 23: 175-200Crossref PubMed Scopus (631) Google Scholar). All have a highly conserved N terminus, three nuclear receptor interaction motifs (LXXLL, where L is leucine and X is any amino acid) in the middle of the polypeptide chain, and two activation domains (AD1 and AD2) near the C terminus. AD1 and AD2 transmit the activating signal to the transcription machinery by binding to secondary coactivators: AD1 binds the histone acetyltransferases CBP and p300, while AD2 recruits the arginine-specific histone methyltransferase CARM1 (5Stallcup M.R. Kim J.H. Teyssier C. Lee Y.H. Ma H. Chen D. J. Steroid Biochem. Mol. Biol. 2003; 85: 139-145Crossref PubMed Scopus (94) Google Scholar). The multifunctional protein CBP/p300 serves as a coactivator for numerous transcriptional activators, including nuclear receptors such as hormone-activated AR (6Lee Y.H. Koh S.S. Zhang X. Cheng X. Stallcup M.R. Mol. Cell. Biol. 2002; 22: 3621-3632Crossref PubMed Scopus (151) Google Scholar) and the lymphoid enhancer factor/T cell factor (LEF/TCF) family proteins (7Sun Y. Kolligs F.T. Hottiger M.O. Mosavin R. Fearon E.R. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12613-12618Crossref PubMed Scopus (102) Google Scholar) through its intrinsic histone acetyltransferase activity and also perhaps through its ability to interact with basal transcription factors associated with RNA polymerase II (8Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar, 9Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 10Korzus E. Torchia J. Rose D.W. Xu L. Kurokawa R. McInerney E.M. Mullen T.-M. Glass C.K. Rosenfeld M.G. Science. 1998; 279: 703-707Crossref PubMed Scopus (556) Google Scholar). CARM1 contributes to transcriptional activation by methylation of specific arginine residues of histone H3 (11Ma H. Baumann C.T. Li H. Strahl B.D. Rice R. Jelinek M.A. Aswad D.W. Allis C.D. Hager G.L. Stallcup M.R. Curr. Biol. 2001; 11: 1981-1985Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) and possibly by methylation of or protein-protein interactions with other components of the transcription machinery (12Xu W. Chen H. Du K. Asahara H. Tini M. Emerson B.M. Montminy M. Evans R.M. Science. 2001; 294: 2507-2511Crossref PubMed Scopus (334) Google Scholar, 13Teyssier C. Chen D. Stallcup M.R. J. Biol. Chem. 2002; 277: 46066-46072Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). androgen receptor hemagglutinin glutathione S-transferase chromatin immunoprecipitation assay dihydrotestosterone prostate-specific antigen co-immunoprecipitation activation domain mouse mammary tumor virus lymphoid enhancer factor/T cell factor. androgen receptor hemagglutinin glutathione S-transferase chromatin immunoprecipitation assay dihydrotestosterone prostate-specific antigen co-immunoprecipitation activation domain mouse mammary tumor virus lymphoid enhancer factor/T cell factor. The AR signaling pathway is critical in the progression of prostate cancers, even in the androgen-independent stage in which androgen deprivation is no longer effective in blocking the transcription of AR target genes such as PSA (1Jenster G. Semin. Oncol. 1999; 26: 407-421PubMed Google Scholar, 14Ross R.K. Pike M.C. Coetzee G.A. Reichardt J.K. Yu M.C. Feigelson H. Stanczyk F.Z. Kolonel L.N. Henderson B.E. Cancer Res. 1998; 58: 4497-4504PubMed Google Scholar). Androgen independence can be caused by amplification of the AR gene, the emergence of AR mutants with a broadened spectrum of ligand agonists, or overexpression of p160 coactivators (1Jenster G. Semin. Oncol. 1999; 26: 407-421PubMed Google Scholar, 14Ross R.K. Pike M.C. Coetzee G.A. Reichardt J.K. Yu M.C. Feigelson H. Stanczyk F.Z. Kolonel L.N. Henderson B.E. Cancer Res. 1998; 58: 4497-4504PubMed Google Scholar, 15Gregory C.W. He B. Johnson R.T. Ford O.H. Mohler J.L. French F.S. Wilson E.M. Cancer Res. 2001; 61: 4315-4319PubMed Google Scholar). In addition, altered regulation of other signaling pathways can contribute to androgen-independent proliferation of prostate cancer; for example, mutant β-catenin was found in one study in 5% of primary prostate cancers and thus may also contribute to unregulated cell growth in prostate cancer (16Chesire D.R. Ewing C.M. Sauvageot J. Bova G.S. Isaacs W.B. Prostate. 2000; 45: 323-334Crossref PubMed Scopus (155) Google Scholar). β-Catenin is a key component of the Wnt signaling pathway, in which it serves as a coactivator for LEF/TCF transcriptional activator proteins (17Barker N. Clevers H. Bioessays. 2000; 22: 961-965Crossref PubMed Scopus (207) Google Scholar), but β-catenin also binds to and serves as a coactivator for AR (18Truica C.I. Byers S. Gelmann E.P. Cancer Res. 2000; 60: 4709-4713PubMed Google Scholar, 19Song L.N. Herrell R. Byers S. Shah S. Wilson E.M. Gelmann E.P. Mol. Cell. Biol. 2003; 23: 1674-1687Crossref PubMed Scopus (142) Google Scholar, 20Pawlowski J.E. Ertel J.R. Allen M.P. Xu M. Butler C. Wilson E.M. Wierman M.E. J. Biol. Chem. 2002; 277: 20702-20710Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 21Mulholland D.J. Cheng H. Reid K. Rennie P.S. Nelson C.C. J. Biol. Chem. 2002; 277: 17933-17943Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 22Yang F. Li X. Sharma M. Sasaki C.Y. Longo D.L. Lim B. Sun Z. J. Biol. Chem. 2002; 277: 11336-11344Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). β-Catenin is an armadillo motif-containing protein that is involved in regulation of cell-cell adhesion and transcriptional activation (17Barker N. Clevers H. Bioessays. 2000; 22: 961-965Crossref PubMed Scopus (207) Google Scholar). β-Catenin protein levels are usually maintained at a low level through active degradation mediated by the glycogen synthase kinase (GSK) complex that contains adenomatous polyposis coli (APC) and axin. Upon the activation of the Wnt signaling pathway, GSK kinase activity is inhibited as the GSK complex is dissociated; this allows β-catenin to bypass the destruction pathway. Increased accumulation allows some β-catenin to translocate to the nucleus where it binds to LEF/TCF proteins and enhances transcriptional activation of Wnt target genes, including c-Myc, cyclin D1, and ITF-2. Similarly, nuclear β-catenin also interacts with hormone activated AR (18Truica C.I. Byers S. Gelmann E.P. Cancer Res. 2000; 60: 4709-4713PubMed Google Scholar) and retinoic acid receptor (23Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar) and activates the transcription of their specific target genes. Binding of β-catenin to LEF/TCF causes displacement of corepressor complexes containing histone deacetylases (24Billin A.N. Thirlwell H. Ayer D.E. Mol. Cell. Biol. 2000; 20: 6882-6890Crossref PubMed Scopus (189) Google Scholar), and recruitment of CBP and p300, which act synergistically with β-catenin as coactivators (7Sun Y. Kolligs F.T. Hottiger M.O. Mosavin R. Fearon E.R. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12613-12618Crossref PubMed Scopus (102) Google Scholar, 25Hecht A. Vleminckx K. Stemmler M.P. van Roy F. Kemler R. EMBO J. 2000; 19: 1839-1850Crossref PubMed Google Scholar, 26Miyagishi M. Fujii R. Hatta M. Yoshida E. Araya N. Nagafuchi A. Ishihara S. Nakajima T. Fukamizu A. J. Biol. Chem. 2000; 275: 35170-35175Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In addition, β-catenin can associate directly or indirectly with the TATA box-binding protein (27Bauer A. Chauvet S. Huber O. Usseglio F. Rothbacher U. Aragnol D. Kemler R. Pradel J. EMBO J. 2000; 19: 6121-6130Crossref PubMed Google Scholar) and the chromatin-remodeling protein Brg-1 (28Barker N. Hurlstone A. Musisi H. Miles A. Bienz M. Clevers H. EMBO J. 2001; 20: 4935-4943Crossref PubMed Scopus (354) Google Scholar), suggesting that β-catenin may help to recruit these transcription components to the promoter. Recently, it was also shown that CARM1 interacts with β-catenin in vivo, and can function in synergy with β-catenin as a coactivator for AR and LEF/TCF (29Koh S.S. Li H. Lee Y.H. Widelitz R.B. Chuong C.M. Stallcup M.R. J. Biol. Chem. 2002; 277: 26031-26035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Whether there are other downstream coactivators that mediate the coactivator activity of β-catenin, and whether there are specific coactivator requirements for different enhancer element-binding transcriptional activators are important questions that remain to be addressed. β-Catenin binds in an agonist-dependent manner to the AF-2 activation function within the hormone binding domain of AR; the AF-2 region of AR also contains another distinct binding site for GRIP1 (19Song L.N. Herrell R. Byers S. Shah S. Wilson E.M. Gelmann E.P. Mol. Cell. Biol. 2003; 23: 1674-1687Crossref PubMed Scopus (142) Google Scholar). VP16-fused β-catenin was shown to modulate the ability of GRIP1 to enhance transcriptional activation by GAL4-DBD fused to the AR hormone binding domain (19Song L.N. Herrell R. Byers S. Shah S. Wilson E.M. Gelmann E.P. Mol. Cell. Biol. 2003; 23: 1674-1687Crossref PubMed Scopus (142) Google Scholar). However, it remains unknown whether the intrinsic activation function of β-catenin cooperates with p160 coactivators in AR-mediated transcriptional activation. Here we demonstrate that the p160 coactivator GRIP1 binds strongly to β-catenin in vitro and in vivo, independent of AR, through the GRIP1 AD2 domain. We also show that the β-catenin-GRIP1 binding is important for their synergistic coactivator function with AR and Lef1. However, the manner in which these two coactivators are recruited to the AR and Lef1 transcription initiation complexes is different, since GRIP1 does not interact directly with Lef1. The β-catenin-GRIP1 interaction represents another potential point of cross-talk between the AR and Wnt signaling pathways. Cell Culture—CV-1 and COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen), and LNCaP cells were cultured in RPMI1640. Both media were supplemented with penicillin and streptomycin and 10% fetal bovine serum. Plasmids—Plasmids pSG5.HA-GRIP1, pSG5.HA-GRIP1(1–1121), pVP16-GRIP1, pVP16-GRIP1(1122–1462), pGEX-4T1.GRIP1(5–765), pGEX1-GRIP1(730–1121), pGEX-4T1-GRIP1(1122–1462), and pSV40 AR were previously described (6Lee Y.H. Koh S.S. Zhang X. Cheng X. Stallcup M.R. Mol. Cell. Biol. 2002; 22: 3621-3632Crossref PubMed Scopus (151) Google Scholar, 30Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (214) Google Scholar, 31Chen D. Ma H. Hong H. Koh S.S. Huang S.M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2177Crossref PubMed Scopus (983) Google Scholar, 32Hong H. Kohli K. Trivedi A. Johnson D.L. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4948-4952Crossref PubMed Scopus (610) Google Scholar, 33Hong H. Kohli K. Garabedian M.J. Stallcup M.R. Mol. Cell. Biol. 1997; 17: 2735-2744Crossref PubMed Scopus (493) Google Scholar). The following luciferase reporter gene plasmids were described previously: for AR, MMTV-LUC (30Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (214) Google Scholar); for Lef1, pGL3OT containing three wild type Lef1 binding elements and pGL3OF containing mutant Lef1 binding sites (29Koh S.S. Li H. Lee Y.H. Widelitz R.B. Chuong C.M. Stallcup M.R. J. Biol. Chem. 2002; 277: 26031-26035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar); for Gal4 fusion proteins, GK1-LUC (30Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (214) Google Scholar). Lef1 and Lef1ΔN67 (24Billin A.N. Thirlwell H. Ayer D.E. Mol. Cell. Biol. 2000; 20: 6882-6890Crossref PubMed Scopus (189) Google Scholar) were encoded in expression vector pME18. For expression of Lef1 and β-catenin in mammalian cells, the following mammalian expression vectors were used: pSG5.HA for expressing N-terminal hemagglutinin (HA)-tagged proteins; pM (Clontech) for expressing N-terminal Gal4 DNA binding domain (DBD) fusion proteins. The PCR-amplified Lef1 coding region from pM18Lef1 was digested with EcoRI and BamHI and inserted into EcoRI and BglII sites of pSG5.HA and EcoRI and BamHI sites of pM; for bacterial expression, the EcoRI-NotI Lef1 cDNA fragment was inserted into homologous restriction sites of pGEX-5X1 (Amersham Biosciences). A PCR-amplified cDNA fragment encoding β-catenin was inserted as an EcoRI-BamHI fragment into homologous restriction sites of pSG5.HA and pM vectors; for bacterial expression, an EcoRI-XhoI β-catenin cDNA fragment was inserted into the homologous sites of pGEX-4T1. Transfection—For co-immunoprecipitation assay, COS-7 cells were transiently transfected in 100-mm dishes as described previously (34Li H. Park S. Kilburn B. Jelinek M.A. Henschen-Edman A. Aswad D.W. Stallcup M.R. Laird-Offringa I.A. J. Biol. Chem. 2002; 277: 44623-44630Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). For reporter co-immunoprecipitation assays, COS-7 cells were transfected with 1 μg of each protein expression vector and 2 μg of reporter plasmids. After 48 h, a soluble chromatin fraction was prepared according to chromatin immunoprecipitation assay protocol (see below). For reporter gene assays, CV-1 cells were transiently transfected in 12-well dishes as previously described (35Kim J. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Protein-Protein Interaction in Vitro—Glutathione S-transferase (GST) pull-down assays were performed as described previously (29Koh S.S. Li H. Lee Y.H. Widelitz R.B. Chuong C.M. Stallcup M.R. J. Biol. Chem. 2002; 277: 26031-26035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), except that incubation was overnight in a total volume of 0.5 ml. Co-immunoprecipitation and Immunoblot—Cell lysates were cleared with protein A/G beads (Santa Cruz Biotechnology) for 1 h at 4 °C. 1 μg rabbit anti-GRIP1 (Bethyl Laboratories) or normal rabbit IgG (Santa Cruz Biotechnology) was added to the cell lysates and incubated overnight at 4 °C on a rotator. 30 μl protein A/G beads were added and incubated for another 3 h. Beads were washed three times with radioimmune precipitation assay buffer and subjected to SDS-PAGE. Blots were probed with rabbit anti-β-catenin (Upstate Biotechnology) at 1 μg/20 ml blocking buffer (5% nonfat milk in TBST: 150 mm NaCl, 10 mm Tris-HCl, pH 8.0, and 0.1% Tween-20). The blot was stripped with stripping buffer (100 mm 2-mercaptoethanol, 2% SDS, and 62.5 mm Tris-HCl, pH 6.7) and re-probed with anti-GRIP1 antibody at 1 μg/20 ml blocking buffer to check the efficiency of immunoprecipitation. 5% of cell extracts were also subjected to SDS-PAGE as input and probed with either rat monoclonal anti-HA antibody (Roche Applied Science) or anti-β-catenin (Santa Cruz Biotechnology) at 1 μg/20 ml blocking buffer. HRP-conjugated secondary antibodies (Santa Cruz) were used at 1 μg/10 ml blocking buffer and ECL reagents (Amersham Biosciences) were used for detection. Chromatin Immunoprecipitation (ChIP) and Reporter Co-immunoprecipitation (Reporter Co-IP)—ChIP assays were performed largely as described previously (11Ma H. Baumann C.T. Li H. Strahl B.D. Rice R. Jelinek M.A. Aswad D.W. Allis C.D. Hager G.L. Stallcup M.R. Curr. Biol. 2001; 11: 1981-1985Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 36Ma H. Shang Y. Lee D.Y. Stallcup M.R. Methods Enzymol. 2003; 364: 284-296Crossref PubMed Scopus (23) Google Scholar). LNCaP cells were grown in phenol red-free Dulbecco's modified Eagle's medium supplemented with 5% dextran charcoal-stripped serum in 150 mm dishes for 3 days. Cells were then treated with or without 100 nm DHT for 45 min. Immunoprecipitation was performed with 1 μg of anti-AR (Santa Cruz Biotechnology), anti-GRIP1, anti-Lef1 (Santa Cruz Biotechnology), or anti-β-catenin antibodies. PCR amplification was performed with DNA extracted from the immunoprecipitates, using 35–42 cycles and primers flanking the PSA promoter (–170/+19) (37Kang Z. Pirskanen A. Janne O.A. Palvimo J.J. J. Biol. Chem. 2002; 277: 48366-48371Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). PCR products were run on 2% agarose gels and visualized by ethidium bromide staining. For all ChIP and reporter co-IP experiments, varying amounts of input and precipitated DNA samples were tested in PCR reactions to determine the linear range of the reaction, and the data shown are from PCR reactions performed with a DNA amount from the linear range. In the reporter co-IP assay, to test the recruitment of proteins to transiently transfected plasmids with AR or Lef1 response elements, COS-7 cells were transfected with reporter plasmids and expression vectors as indicated. The soluble chromatin fraction was prepared and immunoprecipitated. PCR was performed on the extracted DNA from the immunoprecipitates as described above: for AR, primers spanning the nucleosome B region of the mouse mammary tumor virus (MMTV) promoter were used, 5′-ATTAGCCTTTATTTGCCCAACCTTG-3′ (forward) and 5′-CAGCACTCTTTTATATTATGGTTTAC-3′ (reverse); for Lef1, primers representing backbone elements of reporter plasmids pGL3OT and pGL3SV40 and spanning the promoter regions were used, 5′-CAAGTGCAGGTGCCAGAACA-3′ (forward) and 5′-ACCAGGGCGTATCTCTTCAT-3′ (reverse). GRIP1 Binds β-Catenin, and Both Act Synergistically as Coactivators for AR—β-Catenin and GRIP1 bind to distinct sites in the AR AF-2 activation domain in a ligand-dependent manner (19Song L.N. Herrell R. Byers S. Shah S. Wilson E.M. Gelmann E.P. Mol. Cell. Biol. 2003; 23: 1674-1687Crossref PubMed Scopus (142) Google Scholar). Moreover, each protein can enhance transcriptional activation by AR (18Truica C.I. Byers S. Gelmann E.P. Cancer Res. 2000; 60: 4709-4713PubMed Google Scholar, 30Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (214) Google Scholar). We therefore tested whether GRIP1 and β-catenin can act synergistically as coactivators for AR in transient transfection assays with a luciferase reporter plasmid controlled by the MMTV promoter. β-Catenin enhanced DHT-activated AR activity 2.5-fold, and GRIP1 increased activity 4.4-fold in CV-1 cells (Fig. 1). When these two coactivators were co-expressed, a synergistic 25-fold activation was observed. The synergy suggests that the functions of GRIP1 and β-catenin are not redundant, and thus that they have different downstream targets or contribute to transcriptional activation in different ways. Since GRIP1 and β-catenin were shown to bind to distinct sites in the AR AF-2 domain, it was previously assumed that the association between GRIP1 and β-catenin was through the androgen receptor (19Song L.N. Herrell R. Byers S. Shah S. Wilson E.M. Gelmann E.P. Mol. Cell. Biol. 2003; 23: 1674-1687Crossref PubMed Scopus (142) Google Scholar). However, we tested whether GRIP1 and β-catenin might bind to each other even in the absence of AR. To test for binding in vitro GRIP1 was translated in vitro in the presence of [35S]methionine (Fig. 2A, lane 1) and incubated with agarose beads containing bound GST or GST-βcatenin. GRIP1 bound strongly to GST-β-catenin (lane 3), but only background binding was observed with GST (lane 2). Since no AR or DHT was present, these results suggest a direct interaction between GRIP1 and β-catenin. β-Catenin also bound to the two other members of the p160 family, SRC-1 and ACTR, in similar GST pull-down assays (data not shown), indicating that binding to β-catenin is a conserved function of p160 coactivators. We next tested for binding in vivo by co-expressing GRIP1 and β-catenin proteins in COS-7 cells, monkey kidney cells with no endogenous AR. Both HA-tagged GRIP1 and HA-tagged β-catenin were expressed well in COS-7 cells (Fig. 2B, top panel), and anti-GRIP1 antibodies effectively precipitated HA-GRIP1 (bottom panel). In the absence of exogenously expressed HA-GRIP1, anti-GRIP1 antibody co-immunoprecipited a low level of β-catenin (middle panel, lane 2), which might be mediated by endogenous GRIP1. No β-catenin was detected in the immunoprecipitates when the β-catenin expression vector was omitted from the transfection (middle panel, lane 1). However, a strong co-immunoprecipitated β-catenin band was observed when both proteins were overexpressed (middle panel, lane 3), which indicates a β-catenin-GRIP1 complex that is independent of AR and DHT. Binding of endogenous GRIP1 and β-catenin was examined in LNCaP prostate cancer cells, which also express endogenous AR. While no GRIP1 or β-catenin was immunoprecipitated by normal rabbit IgG (Fig. 2C, lane 2), anti-GRIP1 antibody coimmunoprecipitated β-catenin with GRIP1 from LNCaP cells that were grown in charcoal-stripped serum without DHT (Fig. 2C, lane 3). When similar experiments were performed using lysates from LNCaP cells treated with DHT, no enhancement of the β-catenin-GRIP1 interaction was observed (data not shown). Taken together, these results indicate that there is a physiological, AR-independent interaction between GRIP1 and β-catenin. The AD2 Domain of GRIP1 Is Required For Its Interaction with β-Catenin and For Their Synergistic Activation of AR— Although GRIP1 and β-catenin can each bind directly to AR, we asked whether their synergistic effect on AR function requires the binding between GRIP1 and β-catenin. We first defined the region of GRIP1 that binds β-catenin, by testing the ability of in vitro-translated β-catenin to bind to each of three different GRIP1 fragments fused to GST. Concentrations of these GST fusion proteins were checked by Coomassie Blue staining (data not shown), and similar amounts of each protein bound to beads were mixed with 35S-labeled β-catenin. While GST, GST-GRIP1-(5–765), and GST-GRIP1-(730–1121) did not bind to β-catenin (Fig. 3A, lanes 2–4), a strong interaction between the C-terminal GRIP1 AD2 domain (amino acids 1122–1462) and β-catenin was observed (lane 5). To test whether the β-catenin-GRIP1AD2 interaction also occurs in vivo, a mammalian two-hybrid assay was used. Gal4DBD-β-catenin activated a transiently transfected reporter plasmid containing Gal4 response elements, confirming the transcriptional activation function of β-catenin (Fig. 3B, lanes 2 and 5). This activity was enhanced by co-expression of VP16 fused to either full-length GRIP1 or GRIP1-(1122–1462) (AD2) (lanes 6 and 7), but not by VP16 fused to GRIP1 AD1 (data not shown), suggesting that the interaction between GRIP1 and β-catenin is specifically through the AD2 region in GRIP1. Based on these results, we then tested whether the GRIP1 AD2 region is important for synergistic coactivator function of GRIP1 and β-catenin in AR-mediated transcriptional activation. GRIP1 enhanced AR activity by 5-fold, β-catenin enhanced AR activity by 3-fold, and together they caused a synergistic 15-fold enhancement (Fig. 3C, lanes 3–6). GRIP1ΔAD2 (lacking AD2) alone enhanced AR activity 3-fold, but the effect of GRIP1ΔAD2 and β-catenin together was 7-fold, which is additive rather than synergistic (lanes 7 and 8). Thus, while GRIP1ΔAD2 was still able to bind AR (30Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (214) Google Scholar) and enhance AR function, its inability to function synergistically with β-catenin as a coactivator correlated with its inability to bind β-catenin. These data suggest that the binding of β-catenin to GRIP1 through the GRIP1 AD2 domain is required for the synergistic coactivator function between GRIP1 and β-catenin. β-Catenin Is Recruited by AR to Promoters of Transiently Transfected MMTV-LUC Reporter Gene and Endogenous PSA Gene in a Hormone-dependent Manner—To test whether the coactivator function of β-catenin involves recruitment of β-catenin to the promoter by AR, we employed a reporter co-immunoprecipitation (reporter co-IP) assay. AR and β-catenin were co-expressed in COS-7 cells in the presence of MMTV-LUC reporter gene. Chromatin immunoprecipitation assays were then performed with untreated or DHT-treated cells. Primers flanking the hormone response elements (AR binding sites) in the MMTV promoter were used in PCR reactions to detect this promoter in the chromatin and reporter gene fragments immunoprecipitated by antibodies against AR or β-catenin. Both AR (used as a positive control) and β-catenin were found to be associated with the transiently transfected MMTV promoter in a DHT-dependent manner (Fig. 4A). In contrast, equal, low background levels of PC" @default.
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• W2055330198 title "Synergistic Effects of Coactivators GRIP1 and β-Catenin on Gene Activation" @default.
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