HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 45, 37853-37867, November 11, 2005

This Article Abstract Full Text (PDF) All Versions of this Article: 280/45/37853    most recent M503850200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Song, L.-N. Articles by Gelmann, E. P. Search for Related Content PubMed PubMed Citation Articles by Song, L.-N. Articles by Gelmann, E. P. Social Bookmarking                      What's this?

Interaction of -Catenin and TIF2/GRIP1 in Transcriptional Activation by the Androgen Receptor^*

Liang-Nian Song and Edward P. Gelmann1

From the Departments of Oncology and Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20007-2197

Received for publication, April 8, 2005 , and in revised form, July 13, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The multifunctional oncoprotein -catenin interacts with the activation function-2 domain of androgen receptor (AR) to stimulate androgen receptor transcriptional activity, increase sensitivity, and broaden specificity of ligand interactions. -Catenin interacts with androgen receptor in close proximity to the binding groove for P160 coactivators such as transcriptional intermediary factor-2 (TIF2)/glucocorticoid receptor interacting protein-1 (GRIP1). -Catenin can also bind directly to TIF2/GRIP1. Both N- and C-terminal regions of -catenin are needed for optimal interaction with TIF2/GRIP1. We show that distinct residues of -catenin are responsible for both binding and functional interactions with androgen receptor and with TCF4, thus allowing the introduction of missense mutations that selectively affect these interactions. -Catenin and TIF2/GRIP1 are each able to mediate binding between the other and androgen receptor in functional interactions that enhance ligand-dependent transcription. The data strongly imply that AR, -catenin, and TIF2/GRIP1 bind in a three-way interaction that mediates transcription. Lastly, we observed that a -catenin C-terminal peptide containing 229 amino acids can bind TIF2/GRIP1 and AR but has a profound dominant inhibitory effect on ligand-dependent transcription. We propose that -catenin may play an integral role in formation of the androgen-receptor transcriptional complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
AR² activity is essential to the growth and progression of prostate cancer at all phases of the disease. A member of the steroid hormone receptor family, AR binds androgen and drives the transcription of androgen-responsive genes in a variety of tissues and cell types. Like the prostatic epithelium from which it is derived, prostate cancer cells are dependent on the presence of androgen when it first presents. Androgen ablation therapy is the first line of treatment for metastatic prostate cancer (1). The vast majority of cases show a clinical response to androgen ablation therapy (2). However, this clinical response is transient, and when the cancer recurs, it does so in the presence of castrate levels of androgen. Androgen-independent prostate cancer paradoxically still depends on AR. For example 30–40% of patients with advanced prostate cancer show amplification of the AR gene following androgen ablation therapy (3). Other specimens have shown point mutations within the AR ligand-binding domain that broaden ligand specificity to include anti-androgens and other steroid hormones (4). Point mutations in the N terminus may affect AR interaction with other proteins of the transcription complex (5, 6).

Another mechanism that affects AR signaling in advanced prostate cancer was suggested when our laboratory found that 5% of primary specimens had mutations in the -catenin gene (7). Further investigation into the interaction between AR and -catenin showed -catenin coimmunoprecipitates with AR, activates AR signaling in the presence of androgens, increases its affinity for other steroid hormones, and diminishes the effects of anti-androgens (8). AR affects -catenin translocation to the nucleus (9, 10) and specific regions -catenin and AR that interact to mediate AR activation and suppression of TCF4-mediated signaling (11–14).

-Catenin is a multifunctional protein that mediates WNT signaling and is an important oncogene in a variety of cancers (15). Oncogenic activation of -catenin involves disruption of phosphorylation sites at the N terminus of the protein that interrupt signals for recognition by ubiquitin ligases and protein degradation. Recently, it was shown that activation of the protease calpain is common in advanced prostate cancer and results in cleavage of -catenin near the N terminus to remove the phosphorylation sites and produce a 75-kDa protein. This finding further supports the notion that -catenin plays an important role in activation of AR signaling in advanced, androgen-independent prostate cancer.

Several studies have suggested interaction of components of the TCF4 transcription factors and androgen receptor transcription complex. A number of reports have described functional and physical interaction between androgen receptor and -catenin (9, 10, 12), cross-talk between AR-mediated and TCF4-mediated transcription (11, 16), and direct interaction of -catenin, TIF2/GRIP1, and AR in the region of the AR C-terminal AF-2 domain (11). Moreover, -catenin can also complex with TIF2/GRIP1 itself, suggesting a three-way interaction with AR and possible regulatory role for -catenin in androgen action (13).

Here we explore the structural elements of -catenin that determine interactions with TCF4 and AR and find that missense mutations of critical amino acids affect these two interactions differently. We proceed to show that either TIF2/GRIP1 or -catenin can each mediate binding of the other to AR and that together they enhance AR-dependent transcriptional activation. The data have implications for regulation of AR action and suggest strategies for intervention of AR signaling in androgen-independent prostate cancer.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmids and Plasmid Construction—pCMVhAR, pCMVhAR507–919, and GAL4/AR LBD were provided by Elizabeth Wilson, University of North Carolina, Chapel Hill, NC. The construction of pCMVhAR507–919-E893A, pCMVhAR507–919-M894A, pCMVhAR507–919-E897A, pCMVhAR507–919-Q902R, GAL4/AR LBD, GAL4/AR LBD-E893A, GAL4/AR LBD-M894A, GAL4/AR LBD-E897A, and GAL4/AR LBD-Q902R has been described before (11). GAL4/-catenin, GAL4/-catenin-ARM, and VP-16/-catenin have been reported earlier (11). GAL4/-cateninN and GAL4/-cateninC were provided by Salimuddin Shah and Stephen Byers, Georgetown University. VP-16/-catenin (S33A), VP-16/-catenin(15–552) (S33A), and VP-16/-catenin( 576–781) (S33A), were kindly provided by Andreas Hecht (Max Planck Institute, Freiberg, Germany) and have been described previously. pGEX-KT--catenin was kindly provided by David L. Rimm (Yale University, New Haven, CT). K312A, K312A/R386A, R386A, K435A, R469A, and H470A mutations in pcDNA--catenin, pGEX-KT--catenin, and VP-16/-catenin were made with the QuikChange site-directed mutagenesis kit as described before. All mutations were confirmed by nucleotide sequencing. TIF2 and TIF2 m123 have been described. TIF2, TIF2 5–1121, and VP-16/TIF2 were provided by Michael Stallcup (University of Southern California, Los Angeles). SubTIF2 was provided by Myles Brown (Dana-Farber Cancer Institute, Boston, MA). pFR-LUC, pM, pVP-16, pSG5, pBSK+, pcDNA3.1(+), the Renilla null luciferase reporter, and MMTV-luc have been described before (11). TCF4 plasmid and OT/OF reporter vectors were provided by Bert Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD).

Cell Culture and Transfection—Unless otherwise stated in the figure legends, all transfections were performed in CV-1 cells. Briefly, 24 h before transfection, cells were plated in 24-well microtiter plates (Falcon) at a cell density of 2–4 x 10⁴ cells per well. Transfection was performed with Lipofectamine Plus reagent (Invitrogen) with 10 ng of receptor-containing plasmid, 100 ng of reporter, 10 ng of Renilla null luciferase, and the indicated amounts of coregulators. Unless otherwise stated, transfection was routinely done with the total transfected DNA brought up to 300 ng/well with pBSK+ DNA. In experiments with different coregulator cDNA plasmids in different vectors, equimolar amounts of the corresponding plasmid vectors were cotransfected to control for artifacts of the vector DNA. After incubation for 16 h, the cells were washed and phenol red-free medium supplemented with 5% dextran charcoal-stripped fetal calf serum containing hormones or vehicle was added. The final concentration of vehicle ethanol was 0.1%. After a further 24 h, cells were lysed in 100 µl/well 1x passive lysis buffer (Promega, Madison, WI), and 30 µl of the cell lysates was used to assay for luciferase activity with the dual-luciferase assay system from Promega. The data were then normalized for the cotransfected Renilla activity. All samples were in triplicate, and each experiment described was repeated at least two times.

GST Pull-down Assays—293T cells were transfected with pCMVhAR, wild-type -catenin, and TCF4, or TIF2 alone with Lipofectamine. Cells were treated with or without 1 nM R1881 24 h after transfection. 48 h after transfection, cells were collected, washed with ice-cold phosphate-buffered saline, and lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 120 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM Na₃VO₄, supplemented with protease inhibitor mixture, Roche Molecular Biochemicals). The lysates were cleared by centrifugation (14,000 x g, 10 min at 4 °C). The total protein concentration of the extracts was measured with the DC protein assay (Bio-Rad). GST fusion proteins harboring wild-type -catenin, -catenin with point mutations (K312A, R386A, K435A, R469A, and H470A), or double mutation K312A/R386A were expressed in Escherichia coli BL21(DE3) (Stratagene) following induction with 0.5 mM isopropyl-1-thio--D-galactopyranoside for 1.5 h at 30 °C. Cells were sonicated, and proteins were clarified following the manufacturer's instruction (Amersham Biosciences). GST fusion proteins were purified on glutathione-Sepharose beads. For binding assays, GST/-catenin protein, or GST alone bound to glutathione-Sepharose beads was incubated with 500 µg of the 293T cell lysates for 2 h at 4 °C. After extensive washing, the retained proteins were eluted with sample buffer and resolved on a precast 4 to 20% Tris-glycine gel (Invitrogen). Western blotting was performed using antibodies against AR (N-20, Santa Cruz Biotechnology), -catenin (BD Biosciences Transduction Laboratories), TCF4 (clone 6H5–3, Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY), TIF2 (BD Biosciences Transduction Laboratories), and GST (Santa Cruz Biotechnology).

Western Blotting—For detection of the expression of pcDNA3-catenin and VP-16/-catenin mutants, SKBR3 cells were plated overnight in six-well plates. Cells were transiently cotransfected with 1 µg each of wild-type pcDNA3--catenin, VP-16/-catenin, or different mutants, together with 1 µg of GFP vector with a Lipofectamine method according to the manufacturer's protocol. 48 h after transfection, cell lysates were prepared as described in the previous paragraph. Aliquots of 30 µg of total proteins were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane (Bio-Rad), and probed with -catenin antibody (BD Biosciences Transduction Laboratories, 1:2000) and anti-GFP antibody (Clontech, 1:5000), followed by a secondary anti-mouse (-catenin) or anti-rabbit (GFP) immunoglobulinhorseradish peroxidase antibody (1:5000). Signal detection was performed with Supersignal West Pico chemiluminescent substrate (Pierce).

Coimmunoprecipitation—LNCaP cells were cultured in modified Iscove's minimal essential medium supplemented with 5% charcoalstripped serum for 48 h. Cells were then treated with 10 nM R1881 or ethanol. Cells were harvested at 24 h, washed with phosphate-buffered saline, and lysed by resuspension in 10 mM Hepes, pH 7.9, 1.5 mM MgCl₂,10mM KCl, 0.5 mM dithiothreitol, 10 mM -glycerophosphate, 1 mM sodium vanadate, 0.1% Triton X-100, and protease inhibitor mixture (Roche Molecular Biochemicals, Nutley, NJ). Nuclei were isolated by centrifugation (800 x g, 10 min at 4 °C) and resuspended in 2 volumes of 20 mM Hepes, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol, 10 mM -glycerophosphate, and protease inhibitors, incubated for 30 min at 4 °C on a rocker and collected for 30 min at 4 °C by microcentrifuge. Coimmunoprecipitation was done using Catch and Release version 2.0 reversible immunoprecipitation system (Upstate, Charlottesville, VA). Briefly, 500 µg of cell lysate was incubated with 2 µg of antibodies and 10 µl of affinity ligand at room temperature for 30 min in spin columns. Antibodies used for coimmunoprecipitation were rabbit immunoaffinity-purified anti-AR (PG-21, Upstate), monoclonal anti-TIF2 (BD Transduction Laboratories), and monoclonal anti--catenin (BD Transduction Laboratories). Columns were washed three times with wash buffer, and proteins bound to the columns were eluted with 70 µl of denaturing elution buffer. Samples were separated on 4–20% Tris-glycine gels, and Western blot analysis was performed as described with either monoclonal antibodies against AR (441, Santa Cruz Biotechnology), TIF2 (BD Transduction Laboratories), and -catenin (BD Transduction Laboratories) or AR antiserum (PG-21), GRIP1 (M-343, Santa Cruz Biotechnology), and -catenin (H-102, Santa Cruz Biotechnology). The AR PG-21 antiserum or monoclonal antibody 441 were used at 1:5000 dilution and all other the antibodies at 1:1000 dilution.

To perform coimmunoprecipitation with wild-type AR, mutant -catenin, and GRIP1, 293 cells were plated onto 100-mm² dishes and transfected with 3 µg each of wild-type AR, VP16/-catenin, or VP16/-catenin(K312A), together with or without wild-type GRIP1 or truncated GRIP1-(5–1121). Cells were treated with R1881 for 24 h prior to coimmunoprecipitation. Polyclonal anti-AR (PG-21) was used for coimmunoprecipitation and monoclonal antibodies against AR, TIF2, and VP16 (14–5, Santa Cruz Biotechnology) were used for Western blot analysis.

Statistical Analysis—Unless otherwise noted, values shown represent mean ± S.D. The differences between groups were analyzed for statistical significance by the two-tailed Student's t test using the program GraphPad Prism software version 4.02 (GraphPad Software, San Diego, CA). p < 0.05 was considered significant. In figures the following symbols are used to represent p values: ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001; and ^****, p < 0.0001.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
-Catenin Interactions with AR and TCF4—We showed previously that ligand-bound AR inhibited the transcriptional interaction between -catenin and a TCF-responsive promoter (11). Because the interaction of -catenin with both AR and TCF4 was blocked by inhibitor of -catenin and TCF (17, 18), we predicted that both AR and TCF4 would interact with the same region of -catenin (11). To test this hypothesis we generated missense mutations in -catenin to determine their effect on interactions with AR and TCF4. The location of these mutations is shown in the map in Fig. 1A. K312A and K435A disrupt each of the two lysine residue "charge buttons" that anchor the interaction with TCF proteins (19) and E-cadherin (20). The R386A mutation was chosen because Arg-386 is involved in binding to APC (21) and is also involved in the association with the Phe-21 residue of TCF4 (20). Arginine 469 and histidine 470 resides are required for the binding of TCF4/LEF1 by the formation of charge buttons with residues Lys-435, Asn-426, Lys-508, and Lys-312 (19, 20).

In a mammalian two-hybrid assay, the AR ligand R1881 induced an interaction between wild-type VP-16/-catenin and GAL4/AR LBD as we previously showed (11). Missense mutant proteins VP-16/-catenin(K312A), VP-16/-catenin(R386A), and the double mutant VP-16/-catenin(K312A/K386A) did not interact with GAL4/AR LBD in the presence of R1881. However, three other missense mutations, K435A, R469A, and H470A did not affect the interaction with GAL4/AR LBD to as great a degree (Fig. 2A). These results suggest that Lys-312 and Arg-386 are required for the interaction between -catenin and ligand-bound bound AR. To make sure that the variation of the binding activity of different VP-16/-catenin mutants was not due to the changes in protein expression levels, we did a parallel Western blot assay and showed that the expression levels of different constructs were not substantially different (Fig. 2A).

The mammalian two-hybrid binding data correlated with transcriptional activation of an AR-responsive reporter construct. In a transient transactivation assay we examined the effect of different -catenin mutant constructs on the ligand-dependent activation of an N-terminal truncated AR-(507–919). We used the truncated AR-(507–919) instead of a full-length construct, because the truncated receptor is essentially inactive in the absence of coactivators such as TIF2/GRIP1 or -catenin, and therefore provides a qualitative read-out of the transcriptional interaction between AR and -catenin constructs. As shown in Fig. 2B, wild-type -catenin can effectively activate the truncated AR in the presence of R1881. In contrast, -catenin point mutants K312A, R386A, and the double mutant K312A/R386A could not activate the receptor. The other three -catenin point mutants, K435A, R469A, and H470A, could activate the receptor to the wild-type level. Western blot showed that the expression of different VP-16/-catenin mutants were at the comparable levels. These results are consistent with the notion binding of -catenin to AR is required for coactivation as we have previously suggested.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Schematic maps of two proteins used in this report as expression constructs or as fusion constructs with VP16 or GAL4. A, -catenin map showing the location of the missense mutations inserted individually or in pairs to affect the charge buttons in the armadillo repeat region. In addition, the deletion constructs that are studied as fusion proteins with VP-16 or GAL4 are shown. B, TIF2/GRIP1 constructs are shown in alignment with the full-length protein sequence with the functional domains indicated.

AR and TCF4 can compete for binding of -catenin, and each has an inhibitory effect on the transcriptional activity of the other in transfection experiments (11). -Catenin(K435A) had lost nearly all interaction with TCF4 and retained interaction with AR, whereas -catenin(K312A) had the opposite effect. We therefore compared the effects of K312A and K435A with wild-type -catenin on the ligand dose dependence of AR-mediated transcription. We observed that at 10 pM DHT, which did not activate AR alone, there was activation in the presence of either wild-type or K435A mutant -catenin. This result underscores the finding that -catenin can support AR transcriptional activity at castrate levels of DHT. -Catenin(K435A) had a more potent effect on AR activation than wild type -catenin, consistent with the notion that the absence of competitive binding to TCF family proteins decreased competition for -catenin (Fig. 2D). -Catenin(K312A) had very little activity in the same experiment and was not differentiable from the control plasmid at DHT concentrations <100 pM and not significantly different at all concentrations.

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Interaction of -catenin with AR and TCF4. A, activation of a GAL4-responsive promoter in a mammalian two-hybrid interaction of GAL4/AR LBD and VP16/-catenin missense mutant constructs. The panel at the right shows the expression levels of VP-16/-catenin constructs and GFP as a control. p values were calculated for comparisons versus -catenin. B, MMTV-Luc reporter assay of AR-(507–919)-dependent transcription with VP-16/-catenin missense mutant constructs. The panel at the right shows the expression levels of VP-16/-catenin constructs and GFP as a control. p values were calculated for comparisons versus -catenin. The absence of p values indicates no statistically significant difference from -catenin. C, OT-Luc activity in the presence of TCF4 and -catenin constructs. p values were calculated for comparisons versus -catenin. The absence of p values indicates no statistically significant difference from -catenin. D, MMTV-Luc reporter assay of exogenous wild-type full-length AR transfected into 293T cells with control plasmid, -catenin, or the -catenin mutant indicated. DHT concentrations are shown on the horizontal axis. For all data points at DHT concentrations 1 pM the activities for -catenin and -catenin(435A) were statistically different from the pcDNA3 vector control and -catenin(K312A) was not.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Competitive binding of AR and TCF4 for -catenin. 293T cells were transfected with expression vectors for AR, TCF4. Cell lysates were isolated after 24-h treatment with 10 nM R1881. GST--catenin constructs shown at the top were produced in bacteria as described under "Materials and Methods." Protein complexes formed in the presence or absence of 10 nM R1881 were isolated on glutathione-complexed beads and analyzed by Western blotting with the antibodies indicated on the left.

The presence of R1881 and exogenous AR expression markedly reduced availability of TCF4 to bind GST/-catenin fusion proteins. This observation is best explained by the ligand-dependent interaction of AR and TCF4, which decreased the availability of TCF4 for binding to the GST/-catenin fusion constructs (22). The amounts of GST fusion proteins used in the pull-down assays are shown at the bottom of Fig. 3 and were comparable among the different lysates. Thus differences in AR or TCF4 binding to GST/-catenin fusion proteins was not due to the differences in fusion proteins bound to the glutathione-agarose beads.

Interaction of -Catenin with TIF2/GRIP1—We previously showed that -catenin and TIF2/GRIP1 synergistically activated AR-mediated transcription. Li et al. (13) obtained complementary results and further showed that the AD2 domain of GRIP1 mediated a physical interaction with -catenin and was necessary for synergism with -catenin in activating AR. However, the region of -catenin required for the interaction with TIF2/GRIP1 has not been determined. We performed mammalian two-hybrid assays with GAL4 fusion proteins harboring various regions of -catenin and VP-16/GRIP1. A GAL4/-catenin fusion protein bound strongly to the VP-16/GRIP1 fusion protein. GAL4 fusion proteins with either N- or C-terminal -catenin deletions also bound to VP-16/GRIP1. However, a GAL4/-catenin fusion construct with the armadillo repeat alone (141–668) showed no detectable binding (Fig. 4A). To control for differences in the basal transcriptional activities of the GAL4/-catenin constructs we plotted the fold induction of reporter activity induced by the interactions with VP-16/GRIP1 relative to VP-16 alone. Transcription induced by interactions of VP-16/GRIP1 with GAL4/-catenin-(1–668) and GAL4/-catenin-(141–781) was equal or greater than transcription induction with GAL4/-catenin (Fig. 4A, inset). VP-16/GRIP1 did not induce any increase in reporter activity mediated by GAL4/-catenin-(141–668) that contained the armadillo repeat region alone. The data suggest that the primary sites of interaction between -catenin and TIF2/GRIP1 resided in the C and N termini of -catenin. We next confirmed the result of Li et al. (13) that the AD2 domain of GRIP1 is required for the direct interaction with and -catenin (Fig. 4B). They had shown that the region of GRIP1 from 1122–1462 mediated nearly complete binding to -catenin. We used a slightly shorter construct and found that the interaction with -catenin was attenuated 50% compared with wild-type GRIP1 (see constructs in Fig. 1B).

To confirm the physical interaction between -catenin and TIF2/GRIP1, we performed a set of GST-pull-down assays with GST fusion proteins harboring full-length -catenin. Five single missense mutations and one double mutation were introduced into the GST fusion constructs to determine whether missense mutations that affected -catenin interaction with either AR or TCF4 had any effect on interaction with TIF2/GRIP1. As shown in Fig. 4C, TIF2 bound very strongly to most of the GST/-catenin fusion proteins. GST/-catenin(H470A) that bound TIF2 weakly. Exposure of the filter to an anti-GST antibody showed that the amounts of GST fusion proteins used in each interaction were comparable (Fig. 4C). Because the armadillo repeat domain is not required for the interaction between -catenin and TIF2, it is not surprising that most of the point mutants in the armadillo repeat domain did not interfere with binding of -catenin to TIF2 in a GST-pull-down assay. The effect of the missense H470A mutation on TIF2 binding is not as yet explained. Importantly, the regions of -catenin that interacted with AR were not the same that interacted with TIF2/GRIP1. The data were consistent with our proposed model that the three proteins interact by direct three-way binding during AR-mediated transcription.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. Interaction of -catenin and TIF2/GRIP1. A, mammalian two-hybrid assay of VP-16/GRIP1 and GAL4/-catenin constructs. The inset shows the relative luciferase activity of each construct normalized to the VP-16 control interaction. For each GAL4 construct the relative luciferase activity for the VP-16/GRIP1 was significantly different from VP-16. p values are shown for comparisons indicated. B, mammalian two-hybrid assay of VP-16/GRIP1 constructs with GAL4/-catenin. VP-16/GRIP1 and VP-16/GRIP1-(1141–1464) were both significantly different from VP-16. C, GST-pull-down assay similar to Fig. 3 with GST--catenin mutant constructs and TIF2 expressed in 293T cells. D, mammalian two-hybrid assay with VP-16/GRIP1 and GAL4/-catenin competed with-catenin mutant constructs as shown. p values for assays that were significantly different from VP-16/GRIP1 are shown in the figure.

TIF2/GRIP1 and -Catenin Cooperate in AR Binding and Transactivation—-Catenin, TIF2/GRIP1, and AR can form a complex in the presence of androgen and -catenin and TIF2/GRIP1 can synergize as coactivators of AR-mediated transcription (11, 13). Therefore changes in intracellular levels of both TIF2/GRIP1 and -catenin can affect the activity of AR that is modulated by the presence of the other molecule. To demonstrate that either TIF2 or -catenin could mediate the binding of the other to AR, we employed -catenin missense mutations that were shown in Figs. 1 and 2 to affect either association with AR or interaction with TCF4. We carried out mammalian two-hybrid assays with VP-16 fusion proteins containing full-length, wild-type VP-16/-catenin, VP-16/-catenin(K312A), an AR interaction mutant, or VP-16/-catenin(K435A), a TCF4 interaction mutant. We found that wild-type VP-16/-catenin, VP-16/-catenin(K435A), and TIF2 all induced reporter activity, indicating these proteins interacted with GAL4/AR LBD in a ligand-dependent manner, whereas VP-16/-catenin(K312A) did not interact with GAL4/AR LBD due to the disruption of interaction with AR LBD by the K312A mutation (Fig. 5A, top panel). Cotransfection of TIF2 with either wild-type VP-16/-catenin or VP-16/-catenin(K435A), enhanced reporter activity to a greater degree than with either coactivator alone. Even though VP-16/-catenin(K312A) is deficient in AR binding, TIF2 plus VP-16/-catenin(K312A) induced a greater degree of GAL4 reporter induction than TIF2 alone (Fig. 5A, top panel). TIF2m123 is a mutant of TIF2 deficient in AR interaction (24). TIF2m123 and VP-16/-catenin(K312A) together had no effect on GAL4/AR LBD, because both have inactivation of AR binding. TIF2m123 reduced the binding interactions of both VP-16/-catenin and VP-16/-catenin(K435A) by approximately one-third to one-half, suggesting that binding of TIF2 to the AF-2 groove is critical for the synergism with VP-16/-catenin for activation of GAL4/AR LBD. In each case TIF2 enhanced the interaction of the VP-16/-catenin fusion construct and TIF2m123 inhibited the interaction (Fig. 5A, top panel).

View larger version (31K): [in this window] [in a new window]   FIGURE 5. TIF2/GRIP1 and -catenin can each mediate binding of the other to AR. A and B, upper and lower panels showing mammalian two-hybrid (upper) and AR-mediated transcription from MMTV-Luc reporter construct (lower) assays with the indicated constructs. In upper panel A significant p values are shown for the comparisons to VP-16/-catenin and VP-16/-catenin(K435A) as indicated. In lower panel A p values are shown for comparisons to the value at the left of each bracket.In panel B statistically significant differences are shown by the brackets and p values as indicated. C, modified mammalian two-hybrid assay in CV-1 cells transfected with GAL4/-catenin, the FR-Luc reporter plasmid, and the TIF2/GRIP1 expression constructs indicated. Each of the constructs is statistically different from the activity with the pSG5 empty vector.

The interaction of TIF2 and -catenin was further examined with a coactivator mutant subGRIP1, in which the three NR boxes have been changed to CoRNR nuclear receptor corepressor binding motifs (24). The subGRIP1 construct alone showed no binding interaction with GAL4/AR LBD and induced 90% inhibition of binding between GAL4/AR LBD and VP-16/-catenin demonstrating that subGRIP1 was able to block binding by complexing with -catenin, because sub-GRIP1 cannot bind to AR LBD (24) (Fig. 5B, top panel). In contrast subGRIP1 reduced by half, but did not abrogate, the transcriptional interaction between AR-(507–919) and VP-16/-catenin. subGRIP1 had no effect on AR-dependent transcription but was not only unable to synergize the interaction of AR and VP-16/-catenin but inhibited transcription either by competing for -catenin or by inhibiting AR LBD directly (Fig. 5B, bottom panel). This result was also consistent with the notion that optimal AR-mediated transcriptional activity required binding of both -catenin and TIF2/GRIP1 to the AR.

Both TIF2m123 and subGRIP1 retained the ability to bind -catenin as shown by a modified mammalian two-hybrid assay in which we cotransfected cells with GAL4/-catenin, the FR-Luc reporter plasmid, and the TIF2/GRIP1 mutants. As shown in Fig. 5C, wild-type GRIP1, subGRIP1, and TIF2m123 induced significant increases in reporter activity as compared with control vector, indicating that both subGRIP1 and TIF2m123 bound -catenin.

These data thus far suggested that -catenin and TIF2/GRIP1 could each mediate the binding of the other to a complex with AR, but that interaction of both with AR was required for a synergistic effect on AR-dependent transcription. To explore this interaction further we examined the activity of a GRIP1 construct that did not interact with -catenin (13). We performed a modified mammalian two-hybrid assay with GAL4/AR LBD, VP-16/-catenin, and full-length GRIP1 or GRIP1-(5–1121) that deleted the second activating domain that is required for interaction with -catenin (see Fig. 1B) (13). When cells were cotransfected with VP-16/-catenin and wild-type full-length GRIP1, the reporter activity was 8.5- and 11.4-fold higher than the activities induced individually by VP-16/-catenin and TIF2, respectively, indicating a strong synergistic binding of these two factors to agonist bound AR (Fig. 6A). When cells were cotransfected with VP-16/-catenin and GRIP1-(5–1121) the reporter activity was 4.3- and 2.5-fold of that seen with transfection of the individual VP-16/-catenin and GRIP1-(5–1121) constructs, respectively (Fig. 6A).

Loss of the second GRIP1 activation domain diminished the coactivation of AR (Fig. 6B). In this assay VP16/-catenin enhanced the effects of both GRIP1 and GRIP1-(5–1121) to nearly identical degrees (1.17 to 1.18-fold) showing that the attenuation of AR binding by loss of the AD2 domain did not affect interaction with AR-mediated transactivation.

When the binding assay of Fig. 6A was repeated with the mutant VP-16/-catenin(K312A) that abrogated the direct -catenin-AR interaction, in place of the wild type -catenin construct, we observed a result qualitatively very similar (Fig. 6C). This implied that binding of VP-16/-catenin(K312A) to the transcription complex was mediated entirely by either GRIP1 or GRIP1-(5–1121). The data thus far suggest that TIF2/GRIP1 interaction directly with AR can facilitate the effect of -catenin on AR-mediated transactivation, and that either TIF2/GRIP1 or -catenin mediated binding between AR and the other protein.

To further characterize the interactions between AR, TIF2/GRIP1, and -catenin in transcription we exploited four AR missense mutations, E8983A, M894A, E897V, and Q902R, known to affect the interaction of the AR AF2 domain with either TIF2/GRIP1 or -catenin (11). AR(E893A) was generated in the laboratory and found to selectively disrupt AR binding to -catenin and attenuate AR interaction with TIF2/GRIP1. AR(M894A) and AR(E897V), also laboratory-derived, and AR(Q902R), a prostate cancer mutation (25), all have substantial defects in TIF2/GRIP1 binding and varying degrees of altered -catenin interactions. A Q902K mutation was found to be associated with partial androgen insensitivity in one family and was found to decrease sensitivity to R1881 and to decrease interaction with TIF2 (25). It is not clear how the Q902R mutation was selected in a patient with advanced prostate cancer, however, the patient with the Q902R mutation experienced disease progression just 1 month after orchiectomy for a recurrent Gleason score 8 stage C tumor (25). It is therefore possible that the Q902R mutation was not selected by prostate cancer treatment. The relative activities of the AR LBD mutants with -catenin and TIF2/GRIP1 in mammalian two-hybrid and AR transcriptional assays are shown graphically in Fig. 7A based on data we obtained previously (11).

In mammalian two-hybrid assays GRIP1 alone did not significantly affect reporter activation by GAL4/AR LBD(M894A), E897V, or Q902R. VP-16/-catenin, on the other hand, interacted with these three constructs to varying degrees (Fig. 7B). VP16/-catenin was able to engage GRIP1 in the transcriptional complex with these AR-interacting mutants as indicated by the augmentation of the VP-16/-catenin interaction with the addition of GRIP1 (Fig. 7B). The enhancing effect of GRIP1 on the interaction of -catenin with the GAL4/AR LBD mutant constructs was 16-fold for GAL4/AR LBD(M894A), 47-fold for GAL4/AR LBD(E897V), and 19-fold for GAL4/AR LBD(Q902R). VP-16/-catenin did not demonstrate an interaction with GAL4/AR LBD(E893A) and had no effect on the interaction of GAL4/AR LBD(E893A) with GRIP1 (Fig. 7B). The data show that -catenin can recruit TIF2 to AR and overcome reduced affinity of TIF2 for the AF-2 binding groove. In the case of the mutant AR LBD(E893A) TIF2 had no appreciable effect on the recruitment of -catenin to AR. Because of the overall attenuation of activity by the E893A mutation, a cooperative effect of -catenin and TIF2/GRIP1 may not be possible with this construct.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. TIF2/GRIP1 and -catenin can each mediate binding of the other to AR. Mammalian two-hybrid (A) and AR-mediated transcription (B) from MMTV-Luc reporter assays with the indicated constructs. C, mammalian two-hybrid assay as in panel A with VP-16/-catenin(K312A). p values for comparisons are shown as indicated.

To confirm further the notion of a three-way interaction between AR, -catenin, and TIF2/GRIP1, the GAL4/AR LBD(E897V) mutant that did not bind TIF2/GRIP1 but had the most robust interaction with a GRIP1-VP-16/-catenin complex was used in mammalian two-hybrid experiments that tested the effects of -catenin mutants with compromised AR and TIF2/GRIP1 binding. As expected GRIP1 and GRIP1-(5–1121) had minimal interaction with GAL4/AR LBD(E897V) (Fig. 7D). VP-16/-catenin bound to the mutant fusion protein receptor in the presence of hormone and recruited both GRIP1 and GRIP1-(5–1121) to enhance binding and AR-mediated transcription (Fig. 7, D and E). The mutant VP-16/-catenin(K312A) that has minimal interaction with wild-type AR did not bind to GAL4/AR LBD(E897V). VP-16/-catenin(K312A) had no interaction with GRIP1-(5–1121), and a low degree of interaction with GAL4/AR LBD(E897V) when GRIP1 was added. The weak effect of GRIP1 and VP-16/-catenin(K312A) was most likely due to the residual binding of GRIP1 to the mutant AR constructs. These data further show that the binding of VP-16/-catenin to AR was critical for the enhancement of the interaction with mutant GRIP1.

When AR was not able to interact efficiently with both -catenin and TIF2/GRIP1 then either of the two cofactors could compete with AR for binding of the other and, paradoxically, inhibit AR-mediated transcription. This is demonstrated in Fig. 7F where VP-16/-catenin was able to compete GRIP1 off AR and inhibit transcription from AR-(507–919) (E893) in a dose-dependent manner. In a similar manner, GRIP1, whose binding to AR is compromised by the M894A mutation, was able to compete for VP-16/-catenin and reduce AR-mediated transcription in a dose-dependent manner.

-Catenin Domains That Interact with TIF2/GRIP1 and AR—We wanted to determine the regions of -catenin required for the two-way interaction with AR and TIF2/GRIP1. Loss of the C-terminal domain of -catenin reduces the interaction with AR by 40%, and deletion of armadillo repeats 5 and 6 completely disrupts binding of -catenin to AR (12). We speculate that optimal interaction with AR requires both the charged surface of the armadillo repeat helical backbone and the C-terminal domain similar to the interaction of -catenin with TATA binding protein (27). We performed mammalian two-hybrid assays with GAL4/AR LBD, GRIP1, and VP-16 fusion proteins harboring the full length, N-terminal deleted -catenin(15–552), or C-terminal deleted -catenin (1–574) (constructs shown in Fig. 1A). The two -catenin truncation mutants do not interact with AR in the presence of androgen (11). Both GRIP1 and -catenin(S33A) interacted with GAL4/AR LBD to induce GAL4-dependent transcription (Fig. 8A). Together, VP-16/-catenin(S33A) and GRIP1 synergized to interact with GAL4/AR in a ligand-dependent manner. When cells were cotransfected with N- or C-terminal -catenin deletion mutants together with GRIP1 a much stronger reporter activity was seen, suggesting that GRIP1 was able to recruit -catenin to the transcriptional complex on the androgen-responsive promoter. The reporter activity in the presence of the N-terminal deletion mutant VP-16/-catenin(S33A)(15–552) and GRIP1 was indistinguishable from the effect of VP-16/-catenin(S33A) and GRIP1. On the other hand, the effect of VP-16/-catenin(S33A)-(1–574) and GRIP1 was much less, consistent with the notion that the C terminus of -catenin is important for the physical interaction with GRIP1.

View larger version (39K): [in this window] [in a new window]   FIGURE 7. TIF2/GRIP1 and -catenin interactions with AR AF-2 domain mutants. A, graphic representation of relative mammalian two-hybrid interactions ("binding") and MMTV-luciferase activation for four AR AF-2 domain mutants with -catenin and TIF2/GRIP1 as previously published (11). B, mammalian two-hybrid assays of AR AF-2 domain mutants with VP-16/-catenin and GRIP1. p values are shown for significant differences of comparisons at the ends of the brackets. C, MMTV-Luc assays with constructs shown in panel B. p values are shown for significant differences of comparisons at the ends of the brackets. Mammalian two-hybrid (D) and MMTV-Luc reporter (E) assays of AR LBD(E897V) with GRIP1 and -catenin mutant constructs. In D and E significant differences are indicated between the value at the far left of each bracket and the values below the asterisks. F, MMTV-Luc reporter assay demonstrating competition for binding VP-16/-catenin between two AR AF-2 mutant constructs and GRIP1. The numbers under the histograms indicate the nanograms of DNA in the transient transfection. The brackets show significant differences compared with the values at the far left end of the respective brackets.

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Effect of -catenin truncations on AR and GRIP1 interaction. Mammalian two-hybrid (A) and MMTV-Luc reporter (B) assays of AR, GRIP1, and subgenomic -catenin constructs.

The formation of a trimeric complex between AR, TIF2/GRIP1, and -catenin was strongly suggested by the mammalian two-hybrid data, particularly by the experiments where binding between any two of the constituents was inactivated by missense mutation. Further confirmation of the formation of a trimer was obtained by coimmunoprecipitation of endogenous proteins from LNCaP cell nuclear extracts from cells exposed or not to R1881. First we demonstrated that three monoclonal antibodies against AR, TIF2, and -catenin could be used simultaneously to detect their respective antigens (Fig. 9A). We saw no nonspecific bands, even with long Western blot exposures. Because the level of AR expression in LNCaP cells is very high, we used 1:5000 dilutions of AR antibodies in all the Western blot analysis. In the Western blot of -catenin, we detected the 75-kDa -catenin cleavage product induced by calpain as previously described (28). The relative densities of the input proteins are shown in the top panel of Fig. 9B.

After immunoprecipitated with polyvalent AR antiserum both TIF2 and -catenin could be detected by Western blot independent of prior exposure to R1881 (Fig. 9B). This is in agreement with the fact that p160 coactivators can interact directly with the AR N terminus in a ligand-independent manner via a conserved glutamine-rich region (29). We have shown that -catenin binding to AR is ligand-dependent, but we cannot say whether the ligand-independent coimmunoprecipitation of AR with -catenin is mediated by an intervening TIF2/GRIP1 molecule. Densitometric analysis showed that more TIF2 and -catenin were pulled down in the presence of R1881 than in its absence. Similarly, AR and -catenin were pulled down by TIF2 antibody, and there was no substantial difference between the R1881 treated and untreated samples. When -catenin antibody was used to isolate the immunoprecipitate, AR and TIF2 could be pulled down and more AR was detected in the presence of R1881. However, the amount of TIF2/GRIP1 pulled down by -catenin antibody was comparable in R1881-treated and untreated groups, suggesting the association of TIF2 and -catenin is independent of ligand binding to AR.

The formation of a three-way physical complex between AR, -catenin, and TIF2/GRIP1 was also examined with exogenous mutant proteins expressed in 293T cells. Here we used AR, GRIP1, or GRIP1-(5–1121) that has attenuated -catenin interaction, and VP-16/-catenin or VP-16/-catenin(K312A) that has reduced AR interaction. Both full-length, wild-type GRIP1, and truncated GRIP1-(5–1121) could be pulled down by AR antibody in the presence of R1881 (Fig. 10A). When cells were cotransfected with both GRIP1 and VP16/-catenin, much more GRIP1 could be pulled down (Fig. 10A, lanes 1 and 3). This observation is in agreement with our mammalian two-hybrid and transactivation assay results showing that GRIP1 and VP-16/-catenin could synergistically coactivate AR-mediated reporter activity (Fig. 5A). More interestingly, when VP-16/-catenin(K312A), a mutant that cannot bind AR, was used, similar results were obtained (Fig. 10A, lanes 1 and 4), suggesting that -catenin could be recruited to the GRIP1-AR complex by association with GRIP1 independently of AR. When GRIP1-(5–1121) was used, VP-16/-catenin and VP-16/-catenin(K312A) could still enhance the association of GRIP1 with AR to a limited degree (Fig. 10A, lanes 2, 5, and 6). This result is also in agreement with our mammalian two-hybrid and transactivation assays (Fig. 6C) and suggests that even though AD2 of TIF2/GRIP1 is the major region required for the direct association with -catenin other regions in the N terminus of TIF2/GRIP1 may also be involved in the interaction (13).

We also asked whether GRIP1 could mediate the interaction of AR with -catenin. To this end, VP-16/-catenin(K312A) was transfected into 293T cells with or without GRIP1. After R1881 treatment, whole cell lysates were precipitated with polyclonal AR antibody (PG-21). As a positive control, wild-type VP-16/-catenin was pulled down by AR antibody (Fig. 10B, lane 1). In contrast, VP-6/-catenin(K312A) could not be pulled down by AR antibody under identical conditions (Fig. 10B, lane 2). However, when VP-16/-catenin(K312A) was cotransfected with GRIP1, VP-16/-catenin(K312A) could be pulled down efficiently (Fig. 10B, lane 3). These results demonstrated that -catenin could be recruited not only directly by AR but also indirectly by GRIP1.

View larger version (23K): [in this window] [in a new window]   FIGURE 9. Coimmunoprecipitation of AR, TIF2/GRIP1, and -catenin. A, simultaneous detection of endogenous AR, TIF2/GRIP1, and -catenin from LNCaP cells. B, immunoprecipitation and Western blotting of the indicated proteins. The histograms show relative densities in each experiment normalized to the input material in the top panel.

View larger version (28K): [in this window] [in a new window]   FIGURE 10. Coimmunoprecipitation of AR, TIF2/GRIP1, and -catenin. A and B, expression vectors with the indicated plasmids were transfected into 293T cells and coimmunoprecipitation and Western blotting was done as indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Our findings demonstrate that -catenin interacts with AR via different amino acid residues than it interacts with TCF4, but we confirm that the two interactions compete for -catenin binding. Based on our results and others', there appears to be a complex interaction between TCF4 and AR transcription factors. Indeed, the following binding interactions have been demonstrated: AR--catenin (8–12); AR-TCF4 (22); -catenin-TIF2 (11, 13) in addition to the well established interactions of AR with p160 coactivators like TIF2 and the obligate interaction of -catenin and TCF4 in the initiation of TCF-4-dependent transcription (15). Our data also show, through targeted mutational analysis, that the three-way complex of AR, -catenin, and TIF2 supports a higher level of transcriptional activation than the complex of AR with either of the two alone. Furthermore, either the two coactivators can tether the other to AR resulting, in most instances, in enhanced transcriptional activation.

The physical interaction of AR and peptide binding motifs of nuclear receptor coactivators is mediated by a shallow cleft in the ligand-bound AR (31). This implies that the binding between AR and TIF2/GRIP1 may need to be secured by additional molecular interactions. The interaction of AR and TIF2/GRIP1 may be mediated in part by interactions of TIF2/GRIP1 domains outside the NR box with regions of AR outside the AF-2 domain. We propose that -catenin has the potential to mediate this interaction by binding simultaneously to AR and TIF2/GRIP1. Moreover, -catenin by itself can activate AR-mediated transcription.

These findings have potential significance for AR-mediated signaling in prostate cancer progression. In the rare cancers with -catenin mutations there is a direct effect on AR action early in the disease (7, 32). In other instances APC may undergo gene methylation or mutation, resulting in -catenin activation (33). Other components of the -catenin regulatory pathway have also been disrupted in prostate cancer models (33). Inherited APC mutations may predispose to prostate cancer (34). And -catenin is cleaved by calpain activation in advanced prostate cancer and may be activated as a result (28). Interestingly, reduced -catenin expression in localized prostate cancer is associated with a shorter interval to biochemical relapse and may indicate that alternative pathways for AR activation result in more aggressive disease (35).

Because androgen receptor signaling is essential through all phases of prostate cancer development and progression, one might expect that the molecular pathology of prostate cancer might include genetic alterations of AR coactivators. Thus far this has not been demonstrated, although some have found that expression of p160 steroid receptor coactivator family members is enhanced in advanced prostate cancer. Overexpression of coactivators is expected to enhance AR action, broaden ligand specificity, and mediate AR agonism by the anti-androgen hydroxyflutamide (36). However, in other cohorts different coactivators have been implicated (37). Moreover, to our knowledge genetic change of p160 coactivator genes has not been found. -Catenin is therefore one of the few AR coactivators that is implicated in prostate cancer pathogenesis both by mutation and by post-translational modification (7, 28, 32).

Lastly, we encountered a surprising inhibition of AR-mediated transcription by the C-terminal domain of -catenin. The precise mechanism of this inhibitory effect was not elucidated, but the data imply that the C-terminal domain fragment of -catenin has a profound inhibitory interaction with AR. The inhibitory effect was not reversed to a significant degree by supplementation with either CBP or p300, two components of the transcriptional complex that bind to the -catenin C terminus. If the mechanism of inhibition is due to the binding of the -catenin C terminus to AR, as implied by the data in Fig. 8, the binding must have quite high affinity, as it was incompletely competed by excess full-length -catenin. The competition by full-length -catenin did reflect a dose response. On the other hand, the effect of the -catenin C terminus on AR-mediated transcription was only partially reversed by TIF2/GRIP1 in a manner that suggested a noncompetitive interaction (Fig. 9, A and C). Further experiments will identify the minimal inhibitory region of -catenin and will elucidate the mechanism of this profound AR inhibition. The mechanism of this inhibition may have relevance in instructing the design of a new class of anti-androgens.

View larger version (36K): [in this window] [in a new window]   FIGURE 11. Inhibition of AR-mediated transcription by VP-16/-catenin(15–552). A, MMTV-Luc reporter assay in the presence of AR-(507–919) and GRIP1 with titration of VP-16/-catenin(15–552). B, MMTV-Luc reporter assay driven by VP-16/AR-(507–919) and inhibited by VP-16/-catenin(15–552). C, MMTV-Luc reporter assay with AR-(507–919) showing inhibition by VP-16/-catenin(15–552) without reversal by increasing GRIP1 input plasmid. D, MMTV-Luc reporter assay with AR-(507–919) showing inhibition by VP-16/-catenin(15–552) with low level reversal in a dose-dependent manner by increasing -catenin input plasmid. E, MMTV-Luc reporter assay with AR-(507–919) showing lack of effect of p300 transfection on transcriptional inhibition by VP-16/-catenin(15–552). F, MMTV-Luc reporter assay with VP-16/AR-(507–919) showing lack of effect of p300 or CBP transfection on transcriptional inhibition by VP-16/-catenin(15–552).

	FOOTNOTES

¹ To whom correspondence should be addressed: Depts. of Oncology and Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, 3800 Reservoir Rd., NW, Washington, DC 20007-2197. Tel.: 202-444-2207; Fax: 202-444-1229; E-mail: Gelmanne{at}georgetown.edu.

² The abbreviations used are: AR, androgen receptor; TIF2, transcriptional intermediary factor 2; GRIP1, glucocorticoid receptor interacting protein-1; CMV, cytomegalovirus; LBD, ligand binding domain; GST, glutathione S-transferase; GFP, green fluorescent protein; DHT, dihydrotestosterone; MMTV, murine mammary tumor virus.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Tyrrell, C. J., Kaisary, A. V., Iversen, P., Anderson, J. B., Baert, L., Tammela, T., Chamberlain, M., Webster, A., and Blackledge, G. (1998) Eur. Urol. 33, 447-456[CrossRef][Medline] [Order article via Infotrieve]
2. Sadi, M. V., Walsh, P. C., and Barrack, E. R. (1991) Cancer 67, 3057-3064[CrossRef][Medline] [Order article via Infotrieve]
3. Visakorpi, T., Hyytinen, E., Koivisto, P., Tanner, M., Keinanen, R., Palmberg, C., Palotie, A., Tammela, T., Isola, J., and Kallioniemi, O. P. (1995) Nat. Genet. 9, 401-406[CrossRef][Medline] [Order article via Infotrieve]
4. Taplin, M. E., Bubley, G. J., Ko, Y. J., Small, E. J., Upton, M., Rajeshkumar, B., and Balk, S. P. (1999) Cancer Res. 59, 2511-2515[Abstract/Free Full Text]
5. Miyamoto, H., Yeh, S., Wilding, G., and Chang, C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7379-7384[Abstract/Free Full Text]
6. Tilley, W. D., Buchanan, G., Hickey, T. E., and Bentel, J. M. (1996) Clin. Cancer Res. 2, 277-285[Abstract/Free Full Text]
7. Voeller, H. J., Truica, C. I., and Gelmann, E. P. (1998) Cancer Res. 58, 2520-2523[Abstract/Free Full Text]
8. Truica, C. I., Byers, S., and Gelmann, E. P. (2000) Cancer Res. 60, 4709-4713[Abstract/Free Full Text]
9. Mulholland, D. J., Cheng, H., Reid, K., Rennie, P. S., and Nelson, C. C. (2002) J. Biol. Chem. 277, 17933-17943[Abstract/Free Full Text]
10. Pawlowski, J. E., Ertel, J. R., Allen, M. P., Xu, M., Butler, C., Wilson, E. M., and Wierman, M. E. (2002) J. Biol. Chem. 277, 20702-20710[Abstract/Free Full Text]
11. Song, L. N., Herrell, R., Byers, S., Shah, S., Wilson, E. M., and Gelmann, E. P. (2003) Mol. Cell Biol. 23, 1674-1687[Abstract/Free Full Text]
12. Yang, F., Li, X., Sharma, M., Sasaki, C. Y., Longo, D. L., Lim, B., and Sun, Z. (2002) J. Biol. Chem. 277, 11336-11344[Abstract/Free Full Text]
13. Li, H., Kim, J. H., Koh, S. S., and Stallcup, M. R. (2004) J. Biol. Chem. 279, 4212-4220[Abstract/Free Full Text]
14. Shah, S., Hecht, A., Pestell, R., and Byers, S. W. (2003) J. Biol. Chem. 278, 48137-48145[Abstract/Free Full Text]
15. Morin, P. J. (1999) BioEssays 21, 1021-1030[CrossRef][Medline] [Order article via Infotrieve]
16. Mulholland, D. J., Read, J. T., Rennie, P. S., Cox, M. E., and Nelson, C. C. (2003) Oncogene 22, 5602-5613[CrossRef][Medline] [Order article via Infotrieve]
17. Tago, K., Nakamura, T., Nishita, M., Hyodo, J., Nagai, S., Murata, Y., Adachi, S., Ohwada, S., Morishita, Y., Shibuya, H., and Akiyama, T. (2000) Genes Dev. 14, 1741-1749[Abstract/Free Full Text]
18. Graham, T. A., Clements, W. K., Kimelman, D., and Xu, W. (2002) Mol. Cell 10, 563-571[CrossRef][Medline] [Order article via Infotrieve]
19. Graham, T. A., Weaver, C., Mao, F., Kimelman, D., and Xu, W. (2000) Cell 103, 885-896[CrossRef][Medline] [Order article via Infotrieve]
20. Huber, A. H. and Weis, W. I. (2001) Cell 105, 391-402[CrossRef][Medline] [Order article via Infotrieve]
21. von Kries, J. P., Winbeck, G., Asbrand, C., Schwarz-Romond, T., Sochnikova, N., Dell'Oro, A., Behrens, J., and Birchmeier, W. (2000) Nat. Struct. Biol. 7, 800-807[CrossRef][Medline] [Order article via Infotrieve]
22. Amir, A. L., Barua, M., McKnight, N. C., Cheng, S., Yuan, X., and Balk, S. P. (2003) J. Biol. Chem. 278, 30828-30834[Abstract/Free Full Text]
23. Voegel, J. J., Heine, M. J., Tini, M., Vivat, V., Chambon, P., and Gronemeyer, H. (1998) EMBO J. 17, 507-519[CrossRef][Medline] [Order article via Infotrieve]
24. Shang, Y., Myers, M., and Brown, M. (2002) Mol. Cell 9, 601-610[CrossRef][Medline] [Order article via Infotrieve]
25. Taplin, M. E., Bubley, G. J., Shuster, T. D., Frantz, M. E., Spooner, A. E., Ogata, G. K., Keer, H. N., and Balk, S. P. (1995) N. Engl. J. Med. 332, 1393-1398[Abstract/Free Full Text]
26. Umar, A., Berrevoets, C. A., Van, N. M., van, L. M., Verbiest, M., Kleijer, W. J., Dooijes, D., Grootegoed, J. A., Drop, S. L., and Brinkmann, A. O. (2005) J. Clin. Endocrinol. Metab. 90, 507-515[Abstract/Free Full Text]
27. Piedra, J., Martinez, D., Castano, J., Miravet, S., Dunach, M., and de Herreros, A. G. (2001) J. Biol. Chem. 276, 20436-20443[Abstract/Free Full Text]
28. Rios-Doria, J., Kuefer, R., Ethier, S. P., and Day, M. L. (2004) Cancer Res. 64, 7237-7240[Abstract/Free Full Text]
29. Bevan, C. L., Hoare, S., Claessens, F., Heery, D. M., and Parker, M. G. (1999) Mol. Cell Biol. 19, 8383-8392[Abstract/Free Full Text]
30. Hecht, A., Vleminckx, K., Stemmler, M. P., van Roy, F., and Kemler, R. (2000) EMBO J. 19, 1839-1850[CrossRef][Medline] [Order article via Infotrieve]
31. Hur, E., Pfaff, S. J., Payne, E. S., Gron, H., Buehrer, B. M., and Fletterick, R. J. (2004) PLoS. Biol. 2, E274[CrossRef][Medline] [Order article via Infotrieve]
32. Chesire, D. R., Ewing, C. M., Sauvageot, J., Bova, G. S., and Isaacs, W. B. (2000) Prostate 45, 323-334[CrossRef][Medline] [Order article via Infotrieve]
33. Gerstein, A. V., Almeida, T. A., Zhao, G., Chess, E., Shih, I., Buhler, K., Pienta, K., Rubin, M. A., Vessella, R., and Papadopoulos, N. (2002) Genes Chromosomes Cancer 34, 9-16[CrossRef][Medline] [Order article via Infotrieve]
34. Lehrer, S., McCurdy, L. D., Stock, R. G., Kornreich, R., Stone, N. N., and Eng, C. (2000) Cancer Genet. Cytogenet. 122, 131-133[CrossRef][Medline] [Order article via Infotrieve]
35. Horvath, L. G., Henshall, S. M., Lee, C. S., Kench, J. G., Golovsky, D., Brenner, P. C., O'neill, G. F., Kooner, R., Stricker, P. D., Grygiel, J. J., and Sutherland, R. L. (2005) Int. J. Cancer 113, 415-422[CrossRef][Medline] [Order article via Infotrieve]
36. Culig, Z., Comuzzi, B., Steiner, H., Bartsch, G., and Hobisch, A. (2004) J. Steroid Biochem. Mol. Biol. 92, 265-271[CrossRef][Medline] [Order article via Infotrieve]
37. Linja, M. J., Porkka, K. P., Kang, Z., Savinainen, K. J., Janne, O. A., Tammela, T. L., Vessella, R. L., Palvimo, J. J., and Visakorpi, T. (2004) Clin. Cancer Res. 10, 1032-10401[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				L.-N. Song and E. P. Gelmann Silencing Mediator for Retinoid and Thyroid Hormone Receptor and Nuclear Receptor Corepressor Attenuate Transcriptional Activation by the {beta}-Catenin-TCF4 Complex J. Biol. Chem., September 19, 2008; 283(38): 25988 - 25999. [Abstract] [Full Text] [PDF]

				C. K. Yang, J. H. Kim, and M. R. Stallcup Role of the N-Terminal Activation Domain of the Coiled-Coil Coactivator in Mediating Transcriptional Activation by {beta}-Catenin Mol. Endocrinol., December 1, 2006; 20(12): 3251 - 3262. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 280/45/37853    most recent M503850200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Song, L.-N. Articles by Gelmann, E. P. Search for Related Content PubMed PubMed Citation Articles by Song, L.-N. Articles by Gelmann, E. P. Social Bookmarking                      What's this?