Dependence of Selective Gene Activation on the Androgen Receptor NH2- and COOH-terminal Interaction
2002; Elsevier BV; Volume: 277; Issue: 28 Linguagem: Inglês
10.1074/jbc.m202809200
ISSN1083-351X
AutoresBin He, Lori W. Lee, John T. Minges, Elizabeth M. Wilson,
Tópico(s)Prostate Cancer Treatment and Research
ResumoThe agonist-induced androgen receptor NH2- and COOH-terminal (N/C) interaction is mediated by the FXXLF and WXXLF NH2-terminal motifs. Here we demonstrate that agonist-dependent transactivation of prostate-specific antigen (PSA) and probasin enhancer/promoter regions requires the N/C interaction, whereas the sex-limited protein gene and mouse mammary tumor virus long terminal repeat do not. Transactivation of PSA and probasin response regions also depends on activation function 1 (AF1) in the NH2-terminal region but can be increased by binding an overexpressed p160 coactivator to activation function 2 (AF2) in the ligand binding domain. The dependence of the PSA and probasin enhancer/promoters on the N/C interaction for transactivation allowed us to demonstrate that in the presence of androgen, the WXXLF motif with the sequence433WHTLF437 contributes as an inhibitor to AR transactivation. We further show that like the FXXLF and LXXLL motifs, the WXXLF motif interacts in the presence of androgen with AF2 in the ligand binding domain. Sequence comparisons among species indicate greater conservation of the FXXLF motif compared with the WXXLF motif, paralleling the functional significance of these binding motifs. The data provide evidence for promoter-specific differences in the requirement for the androgen receptor N/C interaction and in the contributions of AF1 and AF2 in androgen-induced gene regulation. The agonist-induced androgen receptor NH2- and COOH-terminal (N/C) interaction is mediated by the FXXLF and WXXLF NH2-terminal motifs. Here we demonstrate that agonist-dependent transactivation of prostate-specific antigen (PSA) and probasin enhancer/promoter regions requires the N/C interaction, whereas the sex-limited protein gene and mouse mammary tumor virus long terminal repeat do not. Transactivation of PSA and probasin response regions also depends on activation function 1 (AF1) in the NH2-terminal region but can be increased by binding an overexpressed p160 coactivator to activation function 2 (AF2) in the ligand binding domain. The dependence of the PSA and probasin enhancer/promoters on the N/C interaction for transactivation allowed us to demonstrate that in the presence of androgen, the WXXLF motif with the sequence433WHTLF437 contributes as an inhibitor to AR transactivation. We further show that like the FXXLF and LXXLL motifs, the WXXLF motif interacts in the presence of androgen with AF2 in the ligand binding domain. Sequence comparisons among species indicate greater conservation of the FXXLF motif compared with the WXXLF motif, paralleling the functional significance of these binding motifs. The data provide evidence for promoter-specific differences in the requirement for the androgen receptor N/C interaction and in the contributions of AF1 and AF2 in androgen-induced gene regulation. activation function 1 androgen receptor NH2-terminal and COOH-terminal activation function 2 transcriptional intermediary factor 2 glutathione S-transferase glucocorticoid receptor luciferase prostate-specific antigen mouse mammary tumor virus dihydrotestosterone Steroid receptors are ligand-activated transcription factors that regulate gene activation through a series of events triggered by high affinity hormone binding and mediated by receptor binding to response element DNA and coactivators. At least two domains have been identified that mediate nuclear receptor interactions with coregulators. These are activation function 1 (AF1)1in the NH2-terminal region and activation function 2 (AF2) in the ligand binding domain. The AF2 binding surface in the ligand binding domain is comprised of helices 3, 4, and 12 and forms after hormone binding. For many nuclear receptors, transactivation depends on AF2 recruitment of p160 coactivator complexes that have histone acetyl transferase activity to modify chromatin structure (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. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1070) Google Scholar). The p160 coactivators are a group of proteins that include steroid receptor coactivator 1 (SRC1), transcriptional intermediary protein 2 (TIF2, GRIP1 or SRC2), and the steroid receptor coactivator 3 subfamily (SRC3). Interaction with AF2 is mediated by the p160 coactivator LXXLL motif that forms an amphipathic α-helix and binds the AF2 hydrophobic binding surface in the nuclear receptor ligand binding domain (2Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1778) Google Scholar, 3Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (832) Google Scholar, 4Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1700) Google Scholar, 5Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar). For the androgen receptor (AR), the functional importance of AF2 recruitment of p160 coactivators is unclear, with data implicating the AR NH2-terminal AF1 region in AR-mediated gene activation. The AF2 binding site in the AR ligand binding domain was shown to mediate the agonist-induced NH2- and COOH-terminal (N/C) interaction (6Wong C.I. Zhou Z.X. Sar M. Wilson E.M. J. Biol. Chem. 1993; 268: 19004-19012Abstract Full Text PDF PubMed Google Scholar, 7Zhou Z.X. Lane M.V. Kemppainen J.A. French F.S. Wilson E.M. Mol. Endocrinol. 1995; 9: 208-218Crossref PubMed Google Scholar, 8Langley E. Zhou Z.X. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 9Langley E. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 1998; 273: 92-101Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 10He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Agonist-induced N/C interdomain interactions are also reported for the estrogen (11Kraus W.L. McInerney E.M. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12314-12318Crossref PubMed Scopus (184) Google Scholar) and progesterone receptors (12Tetel M.J. Giangrande P.H. Leonhardt S.A. McDonnell D.P. Edwards D.P. Mol. Endocrinol. 1999; 13: 910-924Crossref PubMed Google Scholar), but not for the glucocorticoid receptor (GR) (7Zhou Z.X. Lane M.V. Kemppainen J.A. French F.S. Wilson E.M. Mol. Endocrinol. 1995; 9: 208-218Crossref PubMed Google Scholar, 13He B. Bowen N.T. Minges J.T. Wilson E.M. J. Biol. Chem. 2001; 276: 42293-42301Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Two AR NH2-terminal LXXLL-like motifs that interact with the AR ligand binding domain are the FXXLF and WXXLF motifs (23FQNLF27 and433WHTLF437) (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Mutagenesis studies and mammalian two-hybrid and GST affinity matrix assays demonstrated that the FXXLF motif interacts in the presence of androgen with AF2 (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). However the site of interaction of the WXXLF motif was not determined, nor was it clear whether interaction of the WXXLF motif depends on androgen binding. In addition, previous studies made use of the MMTV luciferase reporter vector, which may direct transcription through mechanisms that differ from other androgen responsive enhancer/promoters. Here we show the functional importance of the AR N/C interaction using androgen responsive enhancer/promoter regions derived from the prostate specific antigen (PSA) and probasin genes. Use of these responsive regions allowed us to demonstrate, in addition, that the WXXLF motif inhibits recruitment of TIF2 by the AF2 region but less than does the FXXLF motif. Using a shorter NH2-terminal fragment than previously described (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), we show that the WXXLF motif interacts in the presence of androgen with the AF2 region of the ligand binding domain. The data provide evidence that the AR N/C interaction is required for AR-mediated regulation of two androgen-dependent genes. The relatively high sequence conservation of the FXXLF and WXXLF motifs among species further supports the functional importance of the AR N/C interaction. GAL-peptide fusion proteins were constructed as described (15He B. Minges J.T. Lee L.W. Wilson E.M. J. Biol. Chem. 2002; 277: 10226-10235Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) and contain GAL4 DNA binding domain residues 1–147 and the peptide sequences indicated. pcDNA3HA- AR624–919 with wild-type AR sequence or with mutations K720A, V716R, and E897K for in vitro translation were described (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). GST·AR-(412–460) was created by amplifying the coding sequence in pCMVhAR, digesting the fragment withBamHI/EcoRI, and cloning the fragment into pGEX3X digested with the same enzymes. GST·AR-(412–460)WXXAA, where 433WHTLF437 is changed to WHTAA, was created in the same manner except by amplifying using PCR the corresponding region of the mutant pCMVhAR vector. AR-FXXAA, where 23FQNLF27 is changed to FQNAA, and AR-FXXAA/AXXAA, which in addition has433WHTLF437 changed to AHTAA, were described (13He B. Bowen N.T. Minges J.T. Wilson E.M. J. Biol. Chem. 2001; 276: 42293-42301Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 15He B. Minges J.T. Lee L.W. Wilson E.M. J. Biol. Chem. 2002; 277: 10226-10235Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). AR-AXXAA (AR-433AHTAA437) was created by amplifying using PCR the coding sequence in pCMVhAR between BstEII and HindIII using a 5′ mutant oligonucleotide primer. The fragment was inserted into pCMVhAR digested with the same enzymes. AR-FXXAA/AXXAA-K720A and AR-FXXAA/AXXAA-E897K were created by digesting pCMVhAR-K720A and pCMVhAR-E897K (10He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) withHindIII/XbaI and cloning the ligand binding domain fragments into AR-FXXAA/AXXAA digested with the same enzymes. AR-FXXAA/AXXAAΔ(142–337) was created using a double PCR mutagenesis strategy. The AR NH2-terminal region of AR-FXXAA/AXXAA was amplified using a 5′-primer preceding the BglII site, internal primers flanking the deleted region, and a primer 3′ of the BstEII site. The amplified fragments were digested withBglII/BstEII and cloned into AR-FXXAA/AXXAA digested with the same enzymes. DNA amplification by PCR was performed using Vent-polymerase (New England BioLabs). All regions of DNA that were amplified using PCR were sequenced to verify the absence of random errors. The human GR vectors GR(LXXLL)3 and GR(LXXAA)3were described (13He B. Bowen N.T. Minges J.T. Wilson E.M. J. Biol. Chem. 2001; 276: 42293-42301Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). PSA-61-luciferase (PSA61-Luc), which contains a 5.8-kb androgen responsive enhancer/promoter region was provided by Jan Trapman, Erasmus University, Rotterdam, The Netherlands. pGC′Δ9-Luc (pGCΔ9-Luc) that contains the 120-bp sex-limited protein androgen responsive enhancer was provided by Diane M. Robins, University of Michigan. BH500-Luc (probasin-Luc), which contains the 454-bp androgen responsive probasin enhancer/promoter was provided by Robert J. Matusik, Vanderbilt University. Mouse mammary tumor virus (MMTV)-Luc was provided by Ronald M. Evans, the Salk Institute for Biological Studies. The pSG5 mammalian expression vector for TIF2 was provided by Hinrich Gronemeyer, University of Louis Pasteur, Strasbourg, France. 5XGAL4Luc3 was provided by Donald P. McDonnell, Duke University. Monkey kidney CV1 cells were maintained in Dulbecco's modified Eagle's medium containing 20 mm Hepes, pH 7.2, penicillin/streptomycin, and 2 mm l-glutamine. Cells were transfected with wild-type and mutant AR expression vectors and luciferase reporter vectors using the calcium phosphate DNA precipitation method (13He B. Bowen N.T. Minges J.T. Wilson E.M. J. Biol. Chem. 2001; 276: 42293-42301Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). 50 ng of wild-type and mutant pCMVhAR vectors were cotransfected with 5 μg of MMTV-Luc or 2 μg of pGCΔ9-Luc per 6-cm dish plated the day before at 0.42 × 106 cells/dish. 100 ng of wild-type and mutant pCMVhAR vectors were expressed with 5 μg of PSA61-Luc or 5 μg probasin-Luc. Cells were incubated in serum-free, phenol red-free media for 40 h with or without hormone as indicated. Cells were harvested in 0.5 ml of 25 mm Tris phosphate, pH 7.8, 2 mm EDTA, 1% Triton X-100, and 100 μl was analyzed using an automated LumiStar Galaxy (BMG Labtechnologies) multiwell plate reader luminometer. Human epithelioid cervical carcinoma HeLa cells were maintained in Eagle's minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and 2 mm l-glutamine and penicillin/streptomycin. Cells were plated in 12-well plates at 0.1 × 106 cells/well and transfected using per well 0.05 μg of wild-type or mutant pCMVhAR, 0.15 μg of GAL-peptide, and 0.1 μg of 5XGAL4Luc3. After plating and incubating overnight, the cells were placed in 0.8 ml of fresh media containing serum and additives and transfected by the Effectine method (Qiagen) using per well: 75 μl of EC buffer, 1 μl of enhancer, 1 μl of Effectine reagent, and 400 μl of media containing 10% serum and additives. The DNA transfection mix was typically prepared for 4 wells/tube. After 24 h, cells were washed in phosphate-buffered saline, and 2 ml of serum-free media lacking phenol red was added per well. Cells were incubated for 24 h in the absence and presence of the indicated hormones and assayed for luciferase activity as described above except harvested in 0.22 ml of lysis buffer/well. GST fusion proteins were expressed in XL1-Blue Escherichia coli cells treated with 0.5 mm isopropyl β-d-thiogalactopyranoside and extracted and incubated with glutathione-agarose beads (Amersham Biosciences) as described (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). In vitro translated proteins were labeled in the presence of 25 μCi of [35S]methionine (PerkinElmer Life Sciences) using the TnT T7 Quick Coupled transcription/translation system (Promega) in the presence and absence of 1 μm dihydrotestosterone (DHT). Washed beads were boiled in SDS buffer and input lanes contained ∼10% of the binding reactions. We investigated whether androgen response regions derived from different enhancer/promoter regions have a similar requirement for the androgen-induced N/C interaction in AR-mediated gene regulation. Luciferase reporter vectors were tested that contain enhancer/promoter regions from the androgen-regulated genes, PSA (16Cleutjens K.B.J.M. van Eekelen C.C.E.M. van der Korput H.A.G.M. Brinkmann A.O. Trapman J. J. Biol. Chem. 1996; 271: 6379-6388Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 17Cleutjens K.B.J.M. van der Korput H.A.G.M. Ehren-van Eekelen C.C. Sikes R.A. Fasciana C. Chung L.W. Trapman J. Mol. Endocrinol. 1997; 11: 1256-1265Crossref PubMed Scopus (109) Google Scholar), probasin (18Rennie P.S. Bruchovsky N. Leco K.J. Sheppard P.C. McQueen S.A. Cheng H. Snoek R. Hamel A. Bock M.E. MacDonald B.S. Nickel B.E. Chang C. Liao S. Cattini P.A. Matusik R.J. Mol. Endocrinol. 1993; 7: 23-36Crossref PubMed Scopus (215) Google Scholar), sex-limited protein (pGCΔ9) (19Loreni F. Stavenhagen J. Kalff M. Robins D.M. Mol. Cell. Biol. 1988; 8: 2350-2360Crossref PubMed Scopus (38) Google Scholar, 20Scheller A. Scheinman R.I. Thompson E. Scarlett C.O. Robins D.M. Mol. Cell. Endocrinol. 1996; 121: 75-86Crossref PubMed Scopus (22) Google Scholar), and MMTV (21Chalepakis G. Postma J.P.M. Beato M. Nucleic Acids Res. 1988; 16: 10237-10247Crossref PubMed Scopus (28) Google Scholar, 22Gunzburg W.H. Salmons B. Biochem. J. 1992; 283: 625-632Crossref PubMed Scopus (77) Google Scholar, 23Cordingley M.G. Riegel A.T. Hager G.L. Cell. 1987; 48: 261-270Abstract Full Text PDF PubMed Scopus (313) Google Scholar). The role of the WXXLF and FXXLF motifs and effects of TIF2 coactivation on luciferase activity were determined using wild-type AR (AR-FXXLF/WXXLF) or AR in which FXXLF was changed to FXXAA, WXXLF was changed to AXXAA, or both mutations were created in the same protein. AR transactivation of the PSA and probasin enhancer/promoter regions (Fig. 1 A) and the pGCΔ9 and MMTV promoters (Fig. 1 B) was increased 2–3-fold by TIF2 coexpression. Mutation of the FXXLF motif (AR-FXXAA/WXXLF) decreased to low levels transactivation of the PSA and probasin luciferase reporters in the absence of TIF2 coexpression (Fig. 1 A). This decrease in activity was recovered by coexpression of TIF2. In contrast, mutation of the FXXLF motif had no major effect on transactivation of the MMTV-Luc and pGCΔ9-Luc enhancer/promoters in the absence of TIF2 coexpression (Fig. 1 B). Surprisingly, mutation of the WXXLF motif alone (AR-FXXLF/AXXAA) increased the response of the PSA and probasin enhancer/promoters, which was increased further by TIF2 coexpression (Fig. 1 A). In contrast, mutation of WXXLF had relatively little effect on the response of MMTV-Luc and pGCΔ9-Luc (Fig. 1 B). Mutating both binding motifs (AR-FXXAA/AXXAA) decreased transactivation of the PSA and probasin enhancer/promoters, which was rescued by coexpression of TIF2 (Fig. 1 A), again with relatively little effect on the pGCΔ9 and MMTV promoters. The results indicate that in contrast to the pGCΔ9 and MMTV enhancer/promoters, androgen regulation of the PSA and probasin enhancer/promoter regions depends on the AR N/C interaction mediated by the FXXLF and WXXLF motifs in the presence of androgen. The increase in transactivation by mutating WXXLF alone suggests an inhibitory role of this motif in AR activity, whereas a decrease in transactivation by the FXXAA and FXXAA/AXXAA mutants indicates a strong requirement for the N/C interaction. The results are in agreement with the FXXLF motif primarily mediating the N/C interaction, whereas mutation of the WXXLF motif alone does not abolish the N/C interaction (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The detrimental effect on transactivation of losing the N/C interaction was recovered by TIF2 overexpression. The results support previous evidence from androgen insensitivity syndrome mutations (9Langley E. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 1998; 273: 92-101Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) where the N/C interaction is critical for AR-mediated transactivation of androgen-dependent genes in vivo. We investigated further the inhibitory effect of the WXXLF motif in AR-mediated transactivation of the PSA enhancer/promoter by mutating the WXXLF and FXXLF binding motifs in an AR mutant in which the NH2-terminal AF1 residues 142–337 were deleted. Deletion of AF1 resulted in nearly background levels of activity in the absence or presence of TIF2 coexpression (Fig.2), indicating an important role for AF1 in AR-mediated transactivation. Introducing mutations into the FXXLF (AR-FXXAAΔ142–337) and FXXLF plus WXXLF motifs (AR-FXXAA/AXXAAΔ142–337) increased AR-mediated transactivation of the PSA61-Luc reporter in the absence and presence of TIF2 coexpression. When the WXXLF motif alone was mutated in the AF1 deletion mutant (AR-FXXLF/AXXAAΔ142–337, Fig.2), there was only a small increase in transactivation by TIF2. Essentially identical results were obtained using the probasin-Luc reporter (data not shown). We conclude that in the absence of AF1, both FXXLF and WXXLF motifs have a small inhibitory effect on AR transactivation in the absence of TIF2 coexpression. There was a synergistic inhibitory effect by both motifs, possibly reflecting inhibition of endogenous TIF2 coactivation through AF2 in the ligand binding domain. The greater level of TIF2-stimulated luciferase activity observed with the FXXLF mutant compared with the WXXLF mutant indicates that FXXLF more effectively inhibits AR-mediated gene activation by TIF2 than does WXXLF. The TIF2-stimulated increase in luciferase activity of the double mutant provides further evidence that the WXXLF motif contributes to inhibiting coactivation by TIF2 even though mutagenesis of the WXXLF motif itself did not effectively increase transactivation by TIF2. Enhancement of PSA and probasin luciferase activity observed with the WXXLF mutant alone (Fig. 1 A) appeared to depend on AF1 because the increase in activity was less apparent when the WXXLF mutant was combined in the AF1 deletion mutant. To evaluate further the requirement for the N/C interaction in transactivation of the PSA enhancer/promoter regionversus the MMTV promoter, we made use of a previously described GR chimera GR(LXXLL)3 in which an artificial N/C interaction was introduced (13He B. Bowen N.T. Minges J.T. Wilson E.M. J. Biol. Chem. 2001; 276: 42293-42301Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). GR(LXXLL)3 contains the 3 LXXLL motif region of TIF2 that interacts in the presence of dexamethasone with the GR ligand binding domain AF2 region and results in a 5-fold slower dissociation half-time of [3H]dexamethasone compared with wild-type GR. In the presence of 1 or 10 nm dexamethasone, GR stimulated the PSA (Fig.3 A) and probasin reporters (data not shown), and the MMTV (Fig. 3 B) and pGCΔ9 luciferase reporters (data not shown), with the weakest response from pGCΔ9. GR(LXXLL)3 caused greater ligand-induced transactivation of the PSA61-Luc reporter than did GR, or GR(LXXAA)3 in which the 3 LXXLL motifs were mutated to LXXAA (Fig. 3 A). Similar increases in gene activation by GR(LXXLL)3 were observed using the probasin-Luc and pGCΔ9-Luc reporters (data not shown). In contrast, response of the MMTV-luciferase reporter was similar for GR and the GR chimeras (Fig. 3 B). The results support observations above that transactivation of the androgen responsive enhancer/promoters from PSA and probasin depend on an agonist-induced N/C interaction for optimal transactivation compared with the MMTV enhancer/promoter. The increased response of pGCΔ9-Luc to GR(LXXLL)3 suggests that it shares some properties of PSA and probasin response elements that are sensitive to the effects of an N/C interaction. We next investigated whether the increase in AR transactivation of PSA61-Luc (Fig.4 A) and probasin-Luc by TIF2 required TIF2 binding to the AF2 region of the ligand binding domain. In contrast to TIF2, TIF2m123, a mutant in which the 3 LXXLL motifs were changed to LXXAA, was unable to coactivate AR or the AR N/C interaction mutants using the PSA61-Luc reporter (Fig.4 A). Transactivation in the presence of TIF2m123 was less than the control without TIF2 coexpression. Similar results were observed using the probasin and MMTV-Luc reporters (data not shown). The data suggest that coactivation by TIF2 of each of the enhance/promoters requires interaction of the TIF2 LXXLL motifs with the AR AF2 region and that TIF2m123 acts to a limited extent as a dominant negative inhibitor of endogenous TIF2. The lack of coactivation by TIF2m123 suggests, in addition, that an interaction between other regions of TIF2 and the AR NH2-terminal region (10He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 30Alen P. Claessens F. Verhoeven G. Rombauts W. Peeters B. Mol. Cell. Biol. 1999; 19: 6085-6097Crossref PubMed Scopus (217) Google Scholar, 31Bevan C.L. Hoare S. Claessens F. Heery D.M. Parker M.G. Mol. Cell. Biol. 1999; 19: 8383-8392Crossref PubMed Scopus (334) Google Scholar) were not sufficient to mediate increased transactivation. A specific role for AF2 in AR transactivation of the PSA61-Luc reporter by TIF2 was investigated using two AR AF2 mutants. Lysine 720 in helix 3 of the ligand binding domain is critical for TIF2 coactivator LXXLL motif binding, but not for the binding of the AR NH2-terminal FXXLF motif (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Glutamic acid 897 in helix 12 of the ligand binding domain is required for both TIF2 LXXLL motif binding and the N/C interactions (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). AR-FXXLF/WXXLF-K720A did not decrease AR-mediated transactivation of the PSA61-Luc reporter in the absence of TIF2 expression but blocked coactivation by TIF2 (Fig. 4 B). AR-FXXLF/WXXLF-E897K reduced inherent AR activity and blocked coactivation by TIF2. Furthermore, AR transactivation was not stimulated by TIF2 when AR had either of these AF2 mutations and where the N/C interaction was interrupted by mutations in the FXXLF and WXXLF motifs. The results indicate that TIF2 coactivation of AR requires binding to AF2. Previously we used a GST affinity matrix assay to determine the effect of androgen on the interaction of the FXXLF and WXXLF motifs with the ligand binding domain. We demonstrated an androgen-dependent interaction of the FXXLF motif with AF2, but results with the WXXLF motif were inconclusive (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Extensive binding in the absence of androgen of a 233-amino acid AR·GST fusion peptide containing the WXXLF motif raised the possibility that some of the binding was nonspecific (14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). We therefore prepared a fusion peptide with a shorter sequence containing the WXXLF motif. GST·AR-(412–460) bound 3-fold higher levels of 35S-labeled AR ligand binding domain residues 624–919 in the presence than in the absence of DHT (Fig.5 A, lanes 4 and5). In addition, mutation of residues LF to AA in the WXXLF motif in GST·AR-(412–460)WXXAA eliminated the androgen-dependent interaction (Fig.5 A, lanes 6 and 7). The results indicate that the WXXLF motif binds the AR ligand binding domain in an androgen-dependent manner. The predicted amphipathic α-helical structure of the WXXLF sequence and the hormone dependence of its interaction with the ligand binding domain suggested that the WXXLF motif interacts with the AF2 binding surface. We tested a series of AF2 mutants that were shown previously to decrease or eliminate TIF2 LXXLL motif binding (4Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1700) Google Scholar, 5Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar, 10He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) and the binding of the AR FXXLF motif in the N/C interaction (10He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 14He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). AR AF2 mutations E897K, V716R and K720A introduced into the 35S-labeled ligand binding domain each reduced or eliminated androgen-dependent binding of GST·AR-(412–460) (Fig. 5 A, lanes 9–22). Signal intensity of the wild-type control in the presence of androgen was 2-, 3-, and 4.5-fold greater than that of the E897K, V716R, and K720A mutants, respectively, in the presence of androgen, based on optical scanning of the film. The E897K ligand binding domain fragment mediated a 2-fold increase in binding of the WXXLF peptide in the presence of androgen relative to the no hormone control, but no androgen dependent increase in binding was detectable with the V716R and K720A ligand binding domain. Residues Glu-897, Val-716, and Lys-720 are part of the AF2 binding surface. The Val-716 side chain is in the AF2 hydrophobic cleft. Mutation to arginine (V716R) disrupts the hydrophobic binding surface and, as also shown previously in GST affinity matrix assays for FXXLF motif binding (10He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), is more disruptive than the Glu-897 mutation to WXXLF motif binding. The data provide evidence that the WXXLF motif binds the AF2 region of the ligand binding domain in an androgen-dependent manner. To further substantiate the androgen-dependent binding of the WXXLF motif to AF2, we used a peptide two-hybrid interaction assay previously described in HeLa cells (15He B. Minges J.T. Lee L.W. Wilson E.M. J. Biol. Chem. 2002; 277: 10
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