Transcriptional Synergy between Melanoma Antigen Gene Protein-A11 (MAGE-11) and p300 in Androgen Receptor Signaling*

Androgen receptor (AR)-mediated gene regulation involves interactions with coregulatory proteins that include the melanoma antigen gene protein-A11 (MAGE-11). To understand the functional significance of sequence similarity between MAGE-11 and the adenovirus early protein E1A, we determined whether MAGE-11 contributes to AR transcriptional activity through an interaction with p300, a potent and ubiquitous transcriptional regulator. Here, we report that MAGE-11 interacts with the NH2-terminal region of p300 through the MAGE-11 MXXIF motif 185MXXIF189, with transcriptional activity depending on the MAGE-11 F-box and MAPK phosphorylation. The MAGE-11- and p300-dependent increase in AR transactivation required the NH2-terminal regions of AR and p300, p300 acetyltransferase activity, and the AR FXXLF motif 23FQNLF27 interaction with MAGE-11. MAGE-11 linked AR to p300 and the p160 coactivator, transcriptional intermediary protein 2 (TIF2). The p300 NH2-terminal FXXLF motif 33FGSLF37 was required for transcriptional activation by TIF2. Increased expression of p300 decreased the ubiquitinylation of MAGE-11 and transiently increased endogenous MAGE-11 levels. Autoacetylation of p300 and decreased acetylation of TIF2 were evident in the MAGE-11, p300, and TIF2 complex. The studies suggest that MAGE-11 links NH2-terminal domains of AR and p300 to promote transcriptional synergy through a cadre of FXXLF-related interacting motifs.

The androgen receptor (AR) 3 regulates gene transcription required for male sex development by binding androgens with high affinity and interacting with coregulatory proteins. AR transcriptional activity derives from activation function 1 (AF1) in the NH 2 -terminal region and the activation function 2 (AF2) hydrophobic surface in the ligand binding domain (Fig. 1). AR AF2 interacts with p160 coactivator LXXLL motifs and the AR NH 2 -terminal FXXLF motif 23 FQNLF 27 that mediates the androgen-dependent NH 2 -and carboxyl-terminal (N/C) interaction (1). The competitive relationship between AR FXXLF and p160 coactivator LXXLL motif binding to AF2 exerts a regulatory effect on p160 coactivator-induced AF2 activity that favors transactivation from the AR NH 2 -terminal AF1 region (2,3). These findings, together with the greater AR AF2 binding affinity for the AR FXXLF than coactivator LXXLL motifs (4), suggest that AF1 in the AR NH 2 -terminal region is the principal AR activation domain.
Although regulation of steroid receptors through the AF2 site is relatively well described, the molecular mechanisms by which coregulatory proteins mediate AR AF1 activity are not understood. One recently described AR coregulator that interacts with the AR NH 2 -terminal region, modulates the AR N/C interaction, and increases AR transcriptional activity is the melanoma antigen gene protein-A11 (MAGE-11). MAGE-11 binds the AR NH 2 -terminal FXXLF motif and interacts directly with p160 coactivators to increase AR transcriptional activity ( Fig. 1) (5,6). The interaction between AR and MAGE-11 is mediated by a MAGE-11 F-box and modulated by epidermal growth factor (EGF)-induced phosphorylation of Thr-360 within the F-box and monoubiquitinylation at lysine residues outside the F-box (6,7).
The functional importance of MAGE-11 as an AR coregulator is supported by its regulated expression in normal human reproductive physiology and deregulated expression in cancer. MAGE-11 levels increase by ϳ50-fold in the epithelium of the normal cycling human endometrium during the window of implantation (8) and to a similar extent in the CWR22 human prostate cancer xenograft during castration-recurrent growth. MAGE-11 levels were increased up to ϳ1000fold in a subset of patients with castration-recurrent prostate cancer (9). Increased expression of MAGE-11 provides a mechanism to enhance AR signaling and is consistent with the functional importance of AR during prostate cancer progression.
The ability of MAGE-11 to increase the constitutive activity of AR-(1-660), an NH 2 -terminal and DNA binding fragment, when MAGE-11 itself lacks inherent transcriptional activity, provided support for the idea that MAGE-11 functions as an AR coregulator through direct interactions with coactivators. Based on sequence similarity between MAGE-11 and the adenovirus early protein E1A, an oncogenic protein that interacts strongly with p300, we investigated the possible interaction between MAGE-11 and p300, a ubiquitous transcriptional regulator and potent acetyltransferase (Fig. 1). p300 activates gene transcription by acetylating histones and transcription factors and as a coactivator of promoter-specific nuclear receptors (10). Studies described here address the mechanisms underlying the MAGE-11-dependent increase in AR transcriptional activity arising from AR NH 2 -terminal AF1.
Expression Studies-Monkey kidney CV1 cells (4 ϫ 10 5 /6-cm dish) were transfected using calcium phosphate DNA precipitation (3) with 0.1 g of pCMV-AR and the indicated amounts of wild-type and mutant pSG5-MAGE, pSG5-TIF2, and pSG5-HA-p300 and 3 or 5 g PSA-Enh-Luc. After transfection and 24 h later, cells were placed in serum-free, phenol red-free media in the absence and presence of 1 nM dihydrotestosterone (DHT). The next day, the medium was replaced, and 24 h later cells were extracted in 0.25 ml of luciferase lysis buffer containing 1% Triton X-100, 2 mM EDTA, and 25 mM Tris phosphate, pH 7.8. Luciferase activity was measured using an automated Lumistar Galaxy multiwell plate luminometer (BMG Labtech). Luciferase activity shown is representative of at least three independent experiments, and the graphs show the mean Ϯ S.E. Two-hybrid transfection assays were performed in HeLa human epithelial cervical carcinoma cells using FuGENE 6 (Roche Applied Science) by plating 5 ϫ 10 4 cells/well in 12-well plates (4). Cells were transfected with 0.1 g/well 5ϫGAL4 Luc3 and 0.05 g GAL-p300 fragments with 0.05 g of pVP16 empty vector or VP-MAGE. The day after transfection, cells were transferred to serum-free medium and incubated overnight at 37°C before luciferase activity was measured.
SiRNA was transfected into COS cells (4 ϫ 10 5 /well in 6well plates) using Lipofectamine 2000 (Invitrogen) in the absence of antibiotics with 1 g of pSG5-MAGE, 1 g of pSG5-TIF2, or 2 g of pSG5-HA-p300 in the absence and presence of 5 nM MAGE-11 siRNA-2 GCACUGAUCCUGCAUGCUAUU, FIGURE 1. Functional interaction between AR, MAGE-11, p300, and TIF2. AR transcriptional activity is increased through interactions with MAGE-11, p300, and TIF2. AR contains a centrally located DNA binding domain (DBD) and activation function 1 (AF1) region in the NH 2 -terminal region. AR NH 2terminal FXXLF motif 23 FQNLF 27 interacts with the AR activation domain 2 (AF2) region of the ligand binding domain (LBD) in the androgen-dependent AR N/C interaction and with the MAGE-11 F-box residues 329 -369 (6). MAGE-11 contains MXXIF motif 185 MDAIF 189 that interacts with the NH 2 -terminal region of p300 and FXXIF motif 260 FPEIF 264 that interacts with TIF2 (6). A p300 NH 2 -terminal FXXLF motif 33 FGSLF 37 interacts with TIF2, and TIF2 LXXLL motif 745 LRYLL 749 interacts with AF2 in the AR ligand binding domain. These motif-driven interactions occur in a temporal and/or competitive manner and are influenced by the cell cycle and post-translation modification that includes phosphorylation, ubiquitinylation, and acetylation. The acetylase activity of p300 is required for MAGE-11 and TIF2 to increase AR transcriptional activity.
Immunoprecipitation was performed by transfecting COS cells as above using two 10-cm plates/group. For protein acetylation assays, COS cells were treated for 1 h prior to harvest with histone deacetylase inhibitor 5 mM nicotinamide with and without 5 mM sodium butyrate. Cell lysates were prepared in 0.25 volume of immunoprecipitation (IP) lysis buffer containing 1% Triton X-100, 0.5% deoxycholate, 0.15 M NaCl, 0.05 M NaF, 1 mM EDTA, and 50 mM Tris, pH 7.6, with 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, complete protease inhibitor mixture (Roche Applied Science) with or without histone deacetylase inhibitors 5 mM nicotinamide and 5 mM sodium butyrate. Lysates were diluted 4-fold with IP buffer lacking deoxycholate and precleared for 1 h at 4°C with 0.1 ml of Sepharose CL-4B (Sigma). To coimmunoprecipitate endogenous p300 with FLAG-MAGE, human embryonic kidney 293 (HEK293) cells maintained in minimal essential medium containing 2 mM L-glutamine, 10% fetal bovine serum, and penicillin and streptomycin (15 ϫ 10 6 cells/10-cm dish, seven dishes/ group) were transfected using DEAE-dextran. HEK293 cell extracts were prepared in 0.5 volume of IP lysis buffer and diluted with an equal volume of IP buffer without deoxycholate prior to preclearing for 10 min at 4°C with 50 l of Sepharose CL-4B. COS and HEK293 cell extracts were transferred to 15 l of anti-FLAG M2-agarose (Sigma) for 2 h or overnight incubation at 4°C. The next day, samples were pelleted and washed with IP lysis buffer without deoxycholate, resuspended in 0.05 ml of 2ϫ SDS sample buffer containing 3.3% SDS, 10% 2-mercaptoethanol, 10% glycerol, and 0.12 M Tris-HCl, pH 6.8, incubated for 5 min at 90°C, and analyzed on immunoblots as described above.

MAGE-11 Increases p300-dependent AR Transcriptional
Activity-To determine whether MAGE-11 increases AR transcriptional activity through mechanisms that involve p300, AR was expressed in the absence and presence of MAGE-11 and p300 with a PSA-Enh-Luc reporter vector that contains an androgen-responsive enhancer (15). Androgen-dependent AR activity increased to a greater extent with the coexpression of MAGE-11 than with p300. Transcriptional activity increased further when MAGE-11 and p300 were coexpressed ( Fig. 2A) and was greatest when TIF2 was also coexpressed (Fig. 2, A and B).
Dependence on MAGE-11 for the TIF2-induced increase in AR activity could be explained by previous findings that MAGE-11 interacts with TIF2 (6) and that binding of MAGE-11 to the AR FXXLF motif overcomes an inhibitory effect of the AR N/C interaction on TIF2 binding to AF2 ( Fig. 1) (3). The synergistic effects of MAGE-11 and p300 on transcriptional activity mediated by the AR NH 2 -terminal region suggested that MAGE-11 may interact directly with p300.
A requirement for the MAGE-11 F-box to increase GAL-p300-(2-300) transcriptional activity was supported by the loss in activity associated with a series of single amino acid MAGE-11 F-box mutants (Fig. 4C) that also decreased MAGE-11 binding to the AR FXXLF motif (6). Mutation of MAPK site Ser-174 blocked the MAGE-(112-429)-dependent increase in GAL-p300-(2-300) activity and was recovered with
A direct interaction between MAGE-11 and the p300 NH 2terminal region was supported by in vitro binding studies in which GST-p300-(2-357) interacted with 35 S-labeled MAGE-11 (Fig. 4E). In addition, endogenous p300 coimmunoprecipitated with FLAG-MAGE in HEK293 cells (Fig. 4F). However, the low levels and cell cycle-dependent expression of endogenous MAGE-11 (see Fig. 11A) hindered further analysis of these protein-protein interactions.
The results suggest that the carboxyl-terminal region of MAGE-11 interacts with the NH 2 -terminal region of p300 and increases p300 transcriptional activity through mechanisms that depend on the MAGE-11 F-box and MAPK phosphorylation. The apparent inhibitory effect of the MAGE-11 NH 2 -terminal region on GAL-p300 transcriptional activity (Fig. 4B) suggested the presence of an auto-regulatory mechanism.
MAGE-11 MXXIF Motif Interacts with p300-To further characterize the MAGE-11 interaction with p300, transcriptional activation of GAL-luciferase was measured after expressing full-length p300 with a series of GAL-MAGE fragments (Fig. 5A). GAL-MAGE and GAL-MAGE-(112-429) were activated to only a limited extent by p300 (Fig. 5B). In contrast, p300 strongly in which the 185 MXXIF 189 motif was intact, had no effect on p300-dependent activity. However, activity decreased with the GAL-MAGE-(85-152)-I97A,F98A 94 IXXIF 98 motif mutant that lacked the 185 MXXIF 189 motif (Fig. 5C). The MAGE-11 185 MXXIF 189 motif was also required for p300 to increase AR transcriptional activity. Mutations in either the MXXIF or IXXIF motif in full-length MAGE-11 did not significantly decrease AR transcriptional activity when assayed using the PSA-Enh-Luc reporter without coexpression of p300 or TIF2 (Fig. 5D). However, mutations in the 185 MXXIF 189 motif minimized or eliminated the MAGE-11-dependent increase in AR transcriptional activity with the expression of p300 and/or TIF2. AR activity was similar to wild-type with mutations in the MAGE-11 94 IXXIF 98 motif.
The results suggest that MAGE-11 MXXIF motif sequence 185 MDAIF 189 is a primary interaction site for p300 (Fig. 1), and the IXXIF motif sequence 94 ITQIF 98 is a secondary interaction site. The inability of p300 to increase GAL-MAGE or GAL-MAGE-(112-429) activity while smaller fragments were strongly activated by p300 (Fig. 5B) suggested that the MAGE-11 carboxyl-terminal region has an inhibitory effect. Functional Requirement for AR and p300 NH 2 -terminal FXXLF Motifs-p300 has an NH 2 -terminal FXXLF motif sequence 33 FGSLF 37 in a similar relative position to the AR FXXLF motif (Fig. 6A). Evidence that the p300 FXXLF motif was required to enhance GAL-p300-(2-300) activity (Fig. 3C), in addition to the close proximity of flanking charged residues (Fig. 6A), suggested that the p300 NH 2 -terminal FXXLF motif was functionally important. Because previous studies demonstrated AR activation of androgen-responsive enhancer/promoters depends on the AR FXXLF motif (25), we investigated the requirement for NH 2 -terminal FXXLF motifs in the AR interaction with p300.
Multiple Functions of the AR FXXLF Motif-Previous studies have demonstrated the requirement for the androgen-dependent AR N/C interaction between the AR FXXLF motif and AF2 in AR activation of androgen-responsive enhancer/ promoters (1,25). These findings, together with the requirement for the AR FXXLF motif in functional interactions involving MAGE-11 and p300, provided evidence that the AR N/C interaction influences AR transactivation involving p300.
To investigate this further, we determined the effect of MAGE-11 and p300 using an experimental paradigm that depended on the androgen-dependent AR N/C interaction. AR-(507-919) is an AR DNA and ligand binding domain fragment that lacks the NH 2 -terminal region and retains high affinity androgen binding (26). AR-(507-919) alone did not activate a PSA-Enh-Luc reporter in the presence of DHT with or without coexpression of MAGE-11 or p300 alone or together (Fig. 8A). However, expression of AR-(507-919) with AR-(1-503), an AR NH 2 -terminal fragment that lacks the AR DNA and ligand binding domains, resulted in androgen-dependent activity that was increased by expressing MAGE-11 or p300 and was highest when all were expressed together. An interaction between AR-(507-919) and AR-(1-503) was required for transcriptional activation and depended on AR FXXLF motif binding to AF2 in the ligand binding domain (1). Dependence on the AR N/C interaction for the increase in activity by MAGE-11 and p300 was shown by loss of AR-(507-919) activity expressed with AR FXXLF motif mutant AR-(1-503)-L26A,F27A with or without MAGE-11 or p300 alone or together.
The requirement for endogenous p300 in AR N/C interaction-dependent AR transactivation was demonstrated further using p300 siRNA. p300 siRNA-1, -3, and -4 each decreased p300 levels (Fig. 8B) and decreased the androgen-dependent activity of AR-(507-919) and AR-(1-503) coexpressed with MAGE-11 in the presence of DHT (Fig. 8C). Specificity of siRNA inhibition was indicated by wild-type activity associated with nonspecific siRNA and p300 siRNA-2 that did not reduce p300 levels.
To demonstrate further that MAGE-11 functionally links AR and p300 through the AR FXXLF motif, GAL-AR- (16 -36), which contains only the AR NH 2 -terminal FXXLF motif region, was expressed with wild-type or mutant MAGE-11 and p300 alone or together in the presence of a GAL-luciferase reporter.
The results suggest that AR transcriptional activity is enhanced by the ability of MAGE-11 to link AR and p300 by binding the same AR FXXLF motif required to mediate the AR N/C interaction. The results support the notion that AR transcriptional activation by p300 is mediated through sequential AR FXXLF motif interactions involving the AR AF2 site and MAGE-11.
Dependence on p300 Acetyltransferase Activity-The decrease in the MAGE-11-dependent GAL-AR-(16 -36) activity with the p300 acetyltransferase mutant led us to investigate the requirement for p300 acetyltransferase activity in the MAGE-11-dependent AR activity. AR transcriptional activity in the presence of MAGE-11 or TIF2 alone or together increased with p300 but not with p300-D1399Y when assayed using a PSA-Enh-Luc reporter (Fig. 9A). Similarly, the p300-dependent increase in GAL-MAGE-(85-205) and -(140 -205) activity was eliminated with the p300-D1399Y acetylation mutant (Fig. 9B).
Because protein stability can be influenced by acetylation or ubiquitinylation of lysine residues (28), we determined whether p300 acetyltransferase activity influenced MAGE-11 ubiquitinylation. Coexpression of HA-MAGE-(112-429) with FLAG-ubiquitin followed by immunoprecipitation using a FLAG-affinity resin demonstrated the ubiquitinylation of MAGE-11 (Fig. 9C) as reported previously (7). Coexpression of p300 or the p300-D1399Y acetyltransferase mutant inhibited the ubiquitinylation of MAGE-(112-429). However, there was no evidence that MAGE-11 was acetylated by p300 (data not shown), although acetylated forms of TIF2 and p300 were present in the complex with MAGE-11 that were not detected with p300-D1399Y and MAGE-11 (Fig. 10A). Acetylation of TIF2 was reduced with the coexpression of MAGE-11 and was not detected with p300-D1399Y (Fig. 10B).
The results indicate that p300 acetyltransferase activity is required for MAGE-11 and p300 to increase AR transcriptional activity. Although MAGE-11 was not acetylated by p300 even though there was evidence for the acetylation of both p300 and TIF2, p300 inhibited MAGE-11 ubiquitinylation independent of p300 acetyltransferase activity, and MAGE-11 had an inhibitory effect on TIF2 acetylation.
p300 Increases Endogenous MAGE-11 Levels and Is Associated with an Androgen-responsive Enhancer-Evidence that p300 inhibits the ubiquitinylation of MAGE-11 (Fig. 9C) raised the possibility that p300 might influence endogenous MAGE-11 levels. To pursue this further, p300 was expressed in COS cells, and protein levels were determined at the indicated times in serum-free medium. Endogenous MAGE-11 was not detected without the coexpression of p300 but was evident in a time-dependent manner when p300 was expressed (Fig. 11A). Highest levels of MAGE-11 occurred at 7 h and declined sub-sequently as p300 levels decreased. The time-and p300-dependent transient increase in endogenous MAGE-11 suggested that p300 influences the cell cycle expression of MAGE-11.
To obtain evidence that endogenous p300 is associated with AR and MAGE-11 at an androgen-responsive gene enhancer, ChIP assays were performed using LAPC-4 prostate cancer cells that express AR, MAGE-11, p300, and increased levels of PSA in response to androgen. The extent of amplified DNA using PSA upstream enhancerspecific primers indicated an increased association of AR, p300, and MAGE-11 in the presence of 10 nM DHT.

DISCUSSION
AR Transcriptional Regulation by MAGE-11 and p300-We have demonstrated transcriptional synergy between AR, the AR coregulator MAGE-11, and p300, a ubiquitous and potent transcriptional regulator and acetyltransferase (22,29,30). MAGE-11 links the NH 2 -terminal regions of AR and p300 and increases AR transcriptional activity dependent on p300 acetyltransferase activity. A transcription complex involving AR, MAGE-11, p300, and TIF2 was mediated through FXXLF-like motif interactions. The AR FXXLF motif functions in the androgen-dependent AR N/C interaction (1, 2) and binds MAGE-11 ( Fig. 1) (1, 5). The MAGE-11-dependent link between AR and p300 was evident from the transcriptional activity of NH 2 -and car-  A, coimmunoprecipitation of acetylated TIF2 and p300 with FLAG-MAGE was performed in COS cells transfected with 4 g of FLAG empty vector (Ϫ) or FLAG-MAGE in the absence and presence of 5 g of pSG5 empty vector (Ϫ) or pSG5-TIF2, and 5 g of WT pSG5-HA-p300 or D1399Y mutant. Cells were incubated with 0.1 g/ml EGF for 24 h prior to harvest and with 5 mM nicotinamide 1 h prior to harvest. Cell extracts prepared in IP lysis buffer containing 5 mM nicotinamide were immunoprecipitated overnight at 4°C. Immunoprecipitates and cell extracts (50 g of protein/lane) were probed using FLAG, p300, TIF2, and acetylated lysine antibodies. B, inhibition of p300-mediated acetylation of TIF2 by MAGE-11 was determined by expressing 5 g of FLAG empty vector (Ϫ) or FLAG-TIF2 with 5 g of pSG5 empty vector (Ϫ), pSG5-HA-p300, or D1399Y mutant with and without 1 g of pSG5-MAGE. Cells were incubated in serum-free media with 0.1 g/ml EGF for 24 h prior to harvest and with 5 mM nicotinamide and 5 mM sodium butyrate for 1 h prior to harvest in IP lysis buffer containing 5 mM nicotinamide and 5 mM sodium butyrate. Immunoprecipitates and cell extracts (30 g of protein/lane for p300, 50 g/lane for acetylated lysine, 70 g/lane for FLAG-TIF2 and MAGE-11) were probed on transblots using antibodies for FLAG-MAGE-11, FLAG, and TIF2, p300, and acetylated lysine.
boxyl-terminal AR fragments that depend on the AR N/C interaction and by the increase in activity of GAL-AR- (16 -36), an AR FXXLF motif fusion peptide.
The findings suggest that the AR FXXLF motif mediates an increase in AR transcriptional activity not only through its interaction with AF2 in the AR N/C interaction but also by recruiting MAGE-11, which in turn interacts with the NH 2terminal region of p300. The critical function of the AR N/C interaction is supported by AR AF2 site mutations that disrupt AR FXXLF motif binding and AR function (19). The studies support the supposition that a single interaction motif undergoes multiple transient interactions during the process of ARmediated transcription. Dependence of androgen-responsive enhancer/promoters on the AR NH 2 -terminal FXXLF motif (25) may be explained by multiple FXXLF motif-mediated interactions that lead to recruitment of p300. It is noteworthy that the reported ability of p300 to increase AR transcriptional activity made use of prostate cancer cell lines (31) that have relatively higher levels of AR and MAGE-11.
Interaction sites for FXXLF-related motifs can also serve multiple functions that differ in specificity. For example, the AF2 site in the AR ligand binding domain binds the AR NH 2terminal FXXLF motif, p160 coactivator LXXLL motifs (Fig. 1), and FXXLF-related motifs in putative AR coactivators (25,32), but it does not bind the p300 NH 2 -terminal FXXLF motif (18). In contrast, the MAGE-11 F-box binds the AR FXXLF motif but does not bind several FXXLF motifs present in putative AR coactivators (6) or in p300. The MAGE-11 F-box was required to increase the activity of GAL-p300-(2-300) through a mechanism that remains to be established. The MAGE-11 F-box at amino acid residues 329 -369 begins with the sequence 329 IPEE 332 followed by a conserved arrangement of hydrophobic amino acid residues similar to the F-box of cyclin F (6). p300 contains a similar 43 LPDE 46 sequence positioned close to the 33 FGSLF 37 FXXLF motif but with a different arrangement of hydrophobic residues than the MAGE-11 F-box. Deletion of this putative p300 F-box region in GAL-p300- (48 -200) increased the interaction with full-length AR and was influenced by the AR FXXLF motif. This suggested that an F-boxlike sequence in the NH 2 -terminal region of p300 may interact with another protein that competes with AR binding to p300. The existence of multiple overlapping motifs and binding surfaces is consistent with MAGE-11 and p300 coordinating temporal interactions during AR transactivation.
Both MAGE-11 and p300 are downstream targets of MAPK (6,33) and p300 is phosphorylated in a time-dependent manner in response to EGF (34). In response to serum stimulation, MAGE-11 is phosphorylated by MAPK at Ser-174, a conserved residue in the MAGE-A subfamily (6). The MAGE-S174A mutant eliminated the functional interaction between MAGE-11 and p300 that was restored with the S174D phosphomimetic. The MAGE-11 S174A mutation also diminished the MAGE-11 interaction with the AR FXXLF motif (6). Inhibition of GAL-p300 activity by MAGE-S174A suggests that phosphorylation influences the ability of p300 to function with MAGE-11 to modulate AR transcriptional activity. The results suggest that MAPK phosphorylation at Ser-174 regulates several important aspects of MAGE-11 function.
The NH 2 -terminal region of p300 was implicated previously in ligand-dependent nuclear receptor signaling, although the interaction sites were not defined (10,37). The closely related cyclic AMP-response element-binding protein-binding protein (CBP) (10, 29) mediated ligand-independent effects of the AR NH 2 -terminal region through the CBP 271-452-amino acid region (31), and the CBP steroid receptor coactivator 1 (SRC1) interaction domain carboxyl-terminal residues 2058 -2130 interact with SRC1 activation domain 1 (AD1) residues 926 -960 conserved among p160 coactivators (36,38). We found that the ability of MAGE-11 and p300 to increase AR transcriptional activity depended on the p300 NH 2 -terminal FXXLF motif sequence 33 FGSLF 37 . However, the p300 FXXLF motif did not interact directly with MAGE-11 but was required for a functional interaction with the NH 2 -terminal region of TIF2, and for the MAGE-11-dependent increase in GAL-p300 transcriptional activity.
p300 acetyltransferase activity has multiple targets that include acetylation of histone tail lysine residues that open the chromatin structure for active transcription. p300 also acetylates non-histone proteins such as p160 coactivators, p53 and ␤-catenin (29, 39 -42). TIF2 was reported to be acetylated by CBP, and the p160 activator for thyroid hormone and retinoid receptors ACTR was acetylated by p300, which modulated the interaction of activator for thyroid hormone and retinoid receptors with nuclear receptors (43). p300 acetyltransferase activity was also linked to AR signaling. AR was acetylated at FIGURE 11. Transient increase and chromatin association of endogenous MAGE-11. A, COS cells were transfected with 6 g of pSG5-HA-p300 or pSG5 empty vector and the next day transferred to serum-free phenol red-free medium and 24 h later to fresh serum-free medium. Cells were harvested at the indicated times, and cell extracts were prepared in immunoblot lysis buffer, and 40 g of protein/lane were analyzed on a transblot probed using p300 antibody and 10 g/ml each of MAGE-11 antipeptide antibodies 13-26, 59 -79, and 94 -108. B, ChIP analysis of AR, p300, and MAGE-11 at the PSA androgen-responsive enhancer was performed in LAPC-4 cells incubated for 24 h with and without 10 nM DHT and cross-linked using formaldehyde. Sonicated DNA was immunoprecipitated using 10 g of normal rabbit IgG (lanes 2 and 3), AR H-280 (lanes 4 and 5), p300 (lanes 6 and 7), and MAGE-11 antibodies (lanes 8 and 9), with a no template control (lane 1).
Lys-632 and -633 by p300 and p/CAF, a p300-associated protein (44). However, these basic residues are part of the AR bipartite nuclear targeting signal (45) so that inhibitory effects on AR nuclear transport may complicate interpretation of the consequences of acetylation on AR transcriptional activity. The dependence of MAGE-11 on p300 acetyltransferase activity to increase AR transcriptional activity was associated with the acetylation of p300 and TIF2.
Protein stability and function are influenced by the competing and complementary effects of acetylation and ubiquitinylation at lysine residues (28). For example, p300 regulates the levels of p53 by promoting p53 degradation through an association with the MDM2 E3-like ubiquitin ligase (46). Although MAGE-11 was not acetylated by p300, p300 inhibited MAGE-11 ubiquitinylation through an acetylation-independent mechanism. The transient increase in endogenous MAGE-11 when p300 was expressed appeared to be cell cycleregulated, in agreement with evidence that MAGE-11 is cell cycle-regulated in CWR-R1 prostate cancer cells (6).
MAGE-11 is monoubiquitinylated at Lys-240 and -245 and undergoes polyubiquitinylation in association with its degradation (7). Although the E3 ligase that ubiquitinates MAGE-11 has not been identified, some evidence suggests that MAGE-11 itself functions as part of a ubiquitin ligase complex. MAGE-11 contains an F-box similar to the F-box in S-phase kinase-associated protein 2 (Skp2) and MAGE-11 interacted with Skp1 (6). p300 was reported to have E4 ubiquitin ligase activity (47) that could influence the ubiquitinylation of MAGE-11.
A Primate-specific AR Transcriptional Scaffold-The evolution of MAGE-11 has resulted in its exclusive expression within the primate lineage. This suggests a novel mechanism has arisen in primates to increase AR signaling that is facilitated by interactions between AR, MAGE-11, p300, and p160 coactivators. MAGE-11 may function as a transcription factor scaffold assembly protein for AR. A similar function as a transcription factor nucleation site has been attributed to p300 that may increase gene expression at select enhancers by bridging between DNA-binding nuclear receptors and transcription factors to the basal transcriptional machinery (48,49). Many transcription factors, nuclear receptors, and signaling proteins interact with p300, and the levels of p300 are thought to be rate-limiting for transcription (50). Competition may exist in recruiting p300 to nuclear receptor-regulated enhancers. Interaction between MAGE-11 and p300 could enhance AR-mediated gene regulation through the more efficient recruitment of a limiting set of proteins that include p300, p160 coactivators, and components of the general transcriptional machinery. p300 and CBP were shown to increase androgen-dependent and ligand-independent AR transactivation in prostate cancer (44,51,52). However, p300 protein levels decrease in prostate cancer cells in response to androgen (53), and p300 mRNA levels were unchanged in the CWR22 human prostate cancer xenograft during tumor progression to castration-recurrent growth when AR mRNA levels increase ϳ5-fold (9). In contrast, MAGE-11 mRNA levels increased ϳ50-fold in the castration-recurrent CWR22 tumor, and up to 1000-fold in a subset of castration-recurrent prostate cancer tissue specimens. The increase in MAGE-11 in castration-recurrent prostate cancer provides a mechanism for AR to more effectively recruit p300 and TIF2 and increase transactivation of AR target genes when circulating androgen levels are low.
MAGE-11 and the Adenovirus Early Protein, E1A-p300 was first identified based on its interaction with the adenovirus early oncoprotein E1A that targets p300 during viral infection (31,35,54,55). Sequence similarity between the NH 2 -terminal regions of MAGE-11 and E1A suggested that MAGE-11 may share properties of E1A and interact with p300. Binding to E1A decreased p300 transcriptional activity through the modification of histone acetyltransferase activity (35, 56 -58). MAGE-11 does not interact with the histone acetyltransferase domain of p300 but inhibits p300 acetylation of TIF2. Inhibition of p300 histone acetyltransferase activity was also associated with p300 phosphorylation at Ser-89 (59), suggesting that allosteric effects extend from the p300 NH 2 -terminal region that could be influenced by MAGE-11.
E1A has a modular structure of interaction motifs for transcriptional regulators associated with the induction of the S-phase of the cell cycle, cell immortalization, cell transformation, and transcriptional regulation (60 -62). Based on evidence that E1A binds p300 and promotes cell growth, it was suggested that p300 functions as a tumor suppressor (29). The E1A FX(D/E)XXXL motif sequence 66 FPDSVML 72 was reported to interact with one of several domains in p300/CBP (63). MAGE-11 contains a similar FX(D/E)XXXL motif sequence 66 FREQANL 72 at the same relative position to the NH 2 terminus as E1A. However, single amino acid mutations in the MAGE-11 FX(D/E)XXXL motif did not interfere with p300 binding (data not shown), suggesting that other MAGE-11 regions possibly homologous to E1A bind p300 (60). Instead, interaction between MAGE-11 and p300 depended on the MAGE-11 MXXIF motif 185 MDAIF 189 (Fig. 1) and secondarily on IXXIF motif 94 ITQIF 98 , as well as the MAGE-11 F-box and MAPK phosphorylation. The MAGE-11 185 MXXIF 189 motif and Ser-174 MAPK site are in close proximity and homologous to a region in E1A. The reported E1A 66 FX(D/E)XXXL 72 interaction site for p300 is preceded by 62 VSQIF 66 which, based on sequence homology and position, could be a previously unrecognized E1A interaction site for p300.
Although there are no known human genomic counterparts for E1A, sequence similarity between MAGE-11 and E1A suggests that MAGE-11 shares some functional properties with E1A. The late evolutionary species-specific divergence of the MAGE gene family among primates suggests that like E1A, MAGE-11 may have hijacked transcription factor binding sites required for its function as an AR-selective coregulator.