Melanoma Antigen Gene Protein-A11 (MAGE-11) F-box Links the Androgen Receptor NH2-terminal Transactivation Domain to p160 Coactivators*

Androgen-dependent transcriptional activity by the androgen receptor (AR) and its coregulators is required for male reproductive development and function. In humans and other primates, melanoma antigen gene protein-A11 (MAGE-11) is an AR selective coregulator that increases AR transcriptional activity. Here we show that the interaction between AR and MAGE-11 is mediated by AR NH2-terminal FXXLF motif binding to a highly conserved MAGE-11 F-box in the MAGE homology domain, and is modulated by serum stimulation of mitogen-activated protein kinase phosphorylation of MAGE-11 Ser-174. The MAGE-11-dependent increase in AR transcriptional activity is mediated by a direct interaction between MAGE-11 and transcriptional intermediary factor 2 (TIF2) through the NH2-terminal region of TIF2, and by a MAGE-11 FXXIF motif interaction with an F-box-like region in activation domain 1 of TIF2. The results suggest that MAGE-11 functions as a bridging factor to recruit AR coactivators through a novel FXX(L/I)F motif-F-box interaction paradigm.

The androgen receptor (AR) 3 is a ligand-activated transcription factor that mediates the biological effects of testosterone and dihydrotestosterone (DHT). High affinity binding of androgen targets AR to the nucleus where it interacts with coregulators and androgen response element DNA associated with androgen-regulated genes. Naturally occurring functional knockouts of AR in the human population caused by AR gene mutations result in the androgen insensitivity syndrome, and demonstrate the functional importance of AR in male sex development and maturation (1). Gain-of-function AR somatic mutations identified in clinical specimens of prostate cancer support a critical role for AR during tumor progression to castration-recurrent growth (1)(2)(3)(4). The multidomain structure of AR includes the carboxyl-terminal ligand binding domain with activation function 2 (AF2), a hydrophobic interaction surface structurally dependent on bound androgen (5). AR AF2 binds LXXLL motifs of steroid receptor p160 coactivators (SRC), but interacts preferentially with the FXXLF motif in the AR NH 2 terminus (6,7). AR activation function 1 (AF1) in the unstructured NH 2 -terminal region interacts with multiple coregulatory proteins (8,9).
AR undergoes an androgen-dependent NH 2 -and COOHterminal (N/C) interaction mediated by the AR NH 2 -terminal FXXLF motif ( 23 FQNLF 27 ) binding to AF2 in the ligand binding domain (5, 10 -14). The ligand-dependent AR N/C interaction stabilizes AR and underlies the potency differences between testosterone and DHT (2,11,(13)(14)(15). The AR N/C interaction coordinates transcriptional activity between AF1 and AF2, in part by competitively inhibiting the SRC/p160 coactivator LXXLL motif binding to AF2 (15). The regulatory effect of the AR N/C interaction is modulated by melanoma antigen gene protein-A11 (MAGE-11), a MAGE gene family member and AR coregulator that binds the AR NH 2 -terminal FXXLF motif, competes with the AR N/C interaction, and enhances AR transcriptional activity by increasing accessibility of AF2 for SRC/ p160 coactivator recruitment (16).
MAGE-11 expression is limited to human and nonhuman primates (17,18), suggesting it arose during mammalian evolution as an additional control mechanism in the AR signaling network. Post-translational modification of MAGE-11 by phosphorylation of Thr-360, and subsequent monoubiquitinylation of Lys-240 and Lys-245 within the MAGE homology domain, a highly conserved region of the MAGE gene family, is induced by epidermal growth factor (EGF) and stabilizes the interaction with AR (19). The increase in MAGE-11 levels in castrationrecurrent prostate cancer during androgen deprivation therapy by DNA hypomethylation at the transcriptional start site supports the concept that MAGE-11 increases AR signaling in prostate cancer (20).
To understand the molecular mechanisms whereby MAGE-11 enhances AR transactivation, we pursued observa-Additional MAGE-11 F-box deletion mutants were created by double PCR mutagenesis to create an EcoRI/XhoI MAGE⌬329 -369 fragment that was inserted into the same sites of pSG5-HA to make pSG5-HA-MAGE⌬329 -369, and into EcoRI/SalI sites of FLAG-b to make FLAG-MAGE⌬329 -369.
GAL-MAGE-(85-205) and -(112-205) were created by PCR amplifying pSG5-MAGE and inserting EcoRI/XhoI-digested fragments into GAL0 digested with EcoRI and SalI. GAL-MAGE-(112-170) was created by PCR amplifying the parent template and ligating the EcoRI/SalI-digested fragment into the same sites of GAL0. GAL-MAGE-(251-272) wild-type and F260A,F264A mutant were created by cloning a small DNA insert into EcoRI and SalI sites of GAL0. All PCR-amplified regions were verified by DNA sequencing.
DNA Transfection-CWR-R1 prostate cancer cells were maintained and transfected using Effectene (Qiagen) as described (2,32). CWR-R1 cells (1.6 ϫ 10 5 cells/well) were transfected in 12-well plates with 0.1 g/well of MMTV-Luc or MMTV⌬-(Ϫ421-Ϫ364)-Luc in the absence and presence of 0.1 g of pSG5-MAGE-11 and/or 0.1 g of pSG5-TIF2. Medium was replaced 24 h later with serum-free, phenol red-free medium in the absence and presence of DHT and/or EGF and incubated at 37°C overnight.
For small interfering RNA (siRNA) studies, CWR-R1 cells (4 ϫ 10 5 /well) in 6-well plates were plated in 1 ml of antibioticfree medium without EGF and transfected using Lipofectamine 2000 reagent (Invitrogen) in antibiotic-free medium with 0.1 g of MMTV⌬-(Ϫ421-Ϫ364)-Luc and 0.2 g of pSG5 or pSG5-MAGE with and without 4 nM TIF2 siRNA-3 (GAUCA-GAAGUGACUAUUAA) or siCONTROL nontargeting siRNA pool (Dharmacon RNA Technologies). After 48 h at 37°C, cells were transferred to serum-free, phenol red-free medium containing antibiotics in the absence and presence of 0.1 DHT and 0.01 g/ml of EGF. After 24 h cells were lysed in 0.25 ml of buffer containing 1% Triton X-100, 2 mM EDTA, and 25 mM Tris phosphate, pH 7.8 (19). Luciferase activity was measured using an automated Lumistar Galaxy multiwell plate luminometer (BMG Labtech). Luciferase data are representative of at least three independent experiments, with the graphs showing the mean Ϯ S.E.
COS cells (4 ϫ 10 5 /6-cm dish) were transfected in antibioticfree medium using Lipofectamine 2000 with pSG5-MAGE, pSG5-TIF2, pCMV-AR, or empty parent vector in the absence and presence of 1 nM siRNA targeting MAGE-11, TIF2, AR, or nonspecific siCONTROL. The next day cells were placed to serum-free medium, and 24 h later extracts were analyzed by immunoblot as described below.
LAPC-4 human prostate cancer cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 2 mM L-glutamine, penicillin, streptomycin, 10% fetal bovine serum, and 1 nM methyltrienolone (R1881) (34). LAPC-4 cells (5 ϫ 10 5 cells/ well) in 6-well plates containing 1 ml of antibiotic-free medium without R1881 were transfected the following day using Lipofectamine 2000 with 0.1 g/well of MMTV⌬-(Ϫ421-Ϫ364)-Luc in the absence and presence of 3 nM siRNA. After 48 h at 37°C, cells were transferred to serum-free, phenol red-free medium containing antibiotics in the absence and presence of 0.1 nM DHT and 0.1 g/ml of EGF, and luciferase activity was measured 24 h later.
Human endometrial Ishikawa cells (10 5 cells/well) were transfected in 12-well plates using FuGENE 6 (Roche Applied Science) (19,33) with 0.1 g/well of PSA-Enh-Luc and 0.1 g of pSG5 empty vector, wild-type, and mutant pSG5-MAGE. The next day medium was replaced with serum-free, phenol redfree medium in the absence and presence of 1 nM DHT with and without 0.1 g/ml EGF. Twenty-four h later, luciferase activity was measured.
Monkey kidney CV1 cells (4.2 ϫ 10 5 /6-cm dish) were transfected using calcium phosphate (15) with 0.05 or 0.1 g of wildtype or mutant pCMV-AR and the indicated amounts of pSG5-TIF2, pSG5-MAGE, MMTV-Luc, or PSA-Enh-Luc. Immediately after transfection and 24 h later, cells were placed in serum-free, phenol red-free medium in the absence and presence of 1 nM DHT, incubated for 24 h at 37°C, and luciferase activity was measured.
Immunoprecipitation was performed using 2-4 10-cm dishes/group of transfected COS-1 or HEK293 cells harvested in phosphate-buffered saline and pooled (15). Cells were solubilized in IP lysis buffer containing 0.05 M sodium fluoride. Lysates were precleared using 0.1 ml of agarose (Sigma) by rotating for 1 h at 4°C. Samples were transferred to 15 l of anti-FLAG M2 affinity-agarose (Sigma) and incubated at 4°C for 1 h or overnight. Samples were washed 3 times with IP lysis buffer, and protein was 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. Immunoprecipitates, protein extracts, and EZ-Run Prestained Rec protein ladder (Fisher Bioreagents) were separated on 8, 10, or 12% acrylamide gels containing SDS and transferred overnight to nitrocellulose membranes at 4°C.

MAGE-11 and TIF2 Interaction in AR Transactivation
In Vitro Binding and Kinase Assays-Glutathione S-transferase (GST) affinity matrix binding assays were performed with GST-TIF2 fusion proteins and 35 S-labeled MAGE-11 expressed from pSG5-MAGE-11 using the TNT T7 Quick Coupled transcription-translation system (Promega) (6,10). GST empty vector pGEX-4T-1 and GST-TIF2 fusion proteins were expressed in XL1-Blue Escherichia coli by incubating in the presence of 1 mM isopropyl 1-thio-␤-D-galactopyranoside for 3 h at 37°C. Cell pellets were resuspended in GST binding buffer containing 0.5% Nonidet P-40, 1 mM EDTA, 0.1 M NaCl, 20 mM Tris-HCl, pH 8.0, with 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma). Protein expression was normalized by Coomassie Blue staining of a mini-gel. Fusion proteins were incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for 1.5 h at 4°C, washed in GST binding buffer, and combined with 35 S-labeled MAGE-11 prepared using 25 Ci/sample of [ 35 S]methionine (PerkinElmer Life Sciences) and incubated for 2 h at 4°C. Beads were washed, eluted using 2ϫ SDS sample buffer, and analyzed on a 10% gel containing SDS.

Dependence of AR Transactivation on MAGE-11 and TIF2-
The ability of MAGE-11 to function as an AR coregulator in association with TIF2 was evaluated in CWR-R1 prostate cancer cells where the response to DHT was maximal at 0.1 nM DHT. Expression of MAGE-11 enhanced the dose-dependent increase in AR transactivation in CWR-R1 cells in response to DHT, and DHT and EGF, to a greater extent than TIF2 (Fig. 1A). Coexpression of MAGE-11 and TIF2 increased both ligand-dependent and ligand-independent AR activity to a greater extent than MAGE-11 or TIF2 alone. TIF2 siRNA-3 inhibited TIF2 expression (Fig. 1B), and the MAGE-11-dependent increase in AR transcriptional activity in CWR-R1 cells (Fig. 1C), supporting the synergistic effects of MAGE-11 and TIF2. In LAPC-4 prostate cancer cells, the DHT and EGF-dependent increase in AR transactivation was inhibited by MAGE-11 siRNA-2, but not siRNA-3 (Fig. 1D), consistent with their relative inhibitory effects on MAGE-11 expression ( Fig. 1B).
In CWR-R1 cells, endogenous AR was readily detected on immunoblots (Fig. 1E), whereas endogenous MAGE-11 was evident ϳ14 h after inducing quiescent cells to enter the cell cycle by treating with DHT and EGF (Fig. 1E, upper panel). The results suggest that endogenous MAGE-11 levels are low and cell cycle regulated, which may have hindered our attempts to coimmunoprecipitate endogenous MAGE-11 with AR or TIF2. However, we were able to demonstrate that siRNA knockdown of AR, MAGE-11, or TIF2 in CWR-R1 cells decreased the DHT and EGF-stimulated increase in PSA, an endogenous AR-regulated gene (Fig. 1E, lower panel). The results suggest that MAGE-11 functions coordinately with TIF2 to increase AR transactivation.
Identification and Function of a MAGE-11 F-box-The carboxyl-terminal region of MAGE-11 contains sites of EGF-dependent phosphorylation and ubiquitinylation required for MAGE-11 to interact with AR ( Fig. 2A) (16,19). Examination of the MAGE-11 sequence revealed a predicted ␣-helical F-boxlike sequence IPEE⌿ 3 X⌿ 2 X 2 ⌿X⌿X 7 ⌿ 2 X 6 ⌿X 8 ⌿ 2 similar to cyclin F, where ⌿ is a hydrophobic residue and X is any amino acid. Sequence similarity with the F-box of cyclin F was noted throughout the MAGE gene family (Fig. 2B). The functional significance of MAGE-11 F-box residues 329 -369 was supported by the presence of the EGF-dependent Thr-360 Chk1 phosphorylation site within the F-box (underlined in Fig. 2B), because phosphorylation at Thr-360 is required for MAGE-11 to interact with AR (16,19).
The functional properties of the MAGE-11 F-box were investigated by creating alanine point mutations at conserved hydrophobic residues (Fig. 2C) and testing their effect on AR transactivation in Ishikawa cells, a human endometrial cell line that responds to EGF (Fig. 2D). The MAGE-11 F-box mutations reduced or eliminated the MAGE-11-dependent increase in AR transcriptional activity in response to DHT, and DHT and EGF. Coimmunoprecipitation of AR with FLAG-MAGE in the absence and presence of DHT and EGF was not eliminated by the single or double mutations within the MAGE-11 F-box (data not shown), although deletion of the F-box inhibited the coimmunoprecipitation of MAGE-11 with FLAG-AR (Fig. 2E). The results suggest that a MAGE-11 F-box dependent interaction increases AR transcriptional activity.

Synergistic Effects of TIF2 on AR AF2 Activity Depend on the MAGE-11 F-box-
The MAGE-11 and TIF2-dependent increase AR transcriptional activity that derives from AF2 in the ligand binding domain was investigated using full-length AR and AR⌬120 -472, a deletion mutant in which the AR NH 2terminal AF1 region was deleted (Fig. 3B, lower panel). AR⌬120 -472 retains the AR NH 2 -terminal FXXLF motif ( 23 FQNLF 27 ) that interacts with MAGE-11 and AR AF2. Androgen-dependent transcriptional activity of AR⌬120 -472 depends entirely on AF2.
In agreement with results in Fig. 2D, MAGE-11 F-box mutants, as shown for MAGE-L358A,L359A, eliminated the MAGE-11 and TIF2-dependent increase in AR transactivation (Fig. 3A). The ability of MAGE-11 and TIF2 to increase androgen-dependent AR⌬120 -472 activity (Fig. 3, B and C) demonstrates the synergistic effects of MAGE-11 and TIF2 on AF2 activity.
The dependence of AR⌬120 -472 transactivation on TIF2 binding to AF2 was demonstrated by the loss of activity caused by introducing the AR AF2 K720A or E897K charge clamp mutation that prevents TIF2 binding to AF2 (Fig. 3B), and by a TIF2 LXXLL motif mutant unable to bind AF2 (data not shown) (10). Transcriptional activity of AR⌬120 -472 was also eliminated by V33E, an AR mutation that inhibits AR FXXLF motif binding to MAGE-11 but not to AF2 (16) (Fig. 3B). This demonstrates that MAGE-11 binding to AR⌬120 -472 is required to relieve repression of AF2 activity caused by the AR N/C interaction.
The inhibitory effect of the AR N/C interaction on AF2 transcriptional activity was also indicated by the TIF2dependent increase in AR⌬120 -472-FXXAA activity (Fig. 3B, L26A,F27A). In this case, mutations in the AR FXXLF motif inhibit binding to both AF2 and MAGE-11 (6,16), allowing TIF2 activation of AF2. AR⌬120 -472 activity in the presence of TIF2 was also inhibited by the double and most of the single mutations in the MAGE-11 F-box (Fig. 3, C and D). The results indicate that the MAGE-11 F-box is required for the synergistic effects of MAGE-11 and TIF2 on AR AF2 transcriptional activity.
MAGE-11 F-box Binds the AR FXXLF Motif and Skp1-Mammalian two-hybrid assays provide evidence that the MAGE-11 F-box interacts with the AR FXXLF motif (Fig. 4). A series of single F-box mutations in GAL-MAGE eliminated the interaction with VP-AR-(1-660), an AR NH 2 -terminal fragment that contains the AR FXXLF motif and DNA binding domain (Fig. 4A). The AR FXXLF motif-dependent interaction with MAGE-11 was shown by loss of the interaction with VP-AR-(1-660)-FXXAA (LFAA) as reported previously (16). Interaction of the AR FXXLF motif with MAGE-11 was also eliminated by the MAGE-T360A mutation that disrupts a Chk1 phosphorylation site within the MAGE-11 F-box, and by mutations at monoubiquitinylation sites outside the F-box region (19).
To further characterize MAGE-11 F-box binding to the AR FXXLF motif, VP-MAGE single residue F-box mutants were coexpressed with GAL-AR-(4 -52) or GAL-AR- (16 -36), two short AR NH 2 -terminal FXXLF motif peptides. VP-MAGE F-box mutations inhibited the interaction with the AR NH 2terminal peptides (Fig. 4, B and C). The interaction was also influenced by a mutation at a potential MAP kinase phosphorylation site at MAGE-11 Ser-174, which lies outside the F-box. VP-MAGE binding to GAL-AR-(4 -52) or GAL-AR- (16 -36) decreased with the MAGE-11 S174A mutation, but was unchanged by the S174D phosphomimetic.
MAGE-11 also interacted with Skp1 based on coimmunoprecipitation of endogenous Skp1 with FLAG-MAGE in HEK293 cells (Fig. 4D). This suggests an additional and more classical function of the MAGE-11 F-box.
The results indicate a MAGE-11 F-box interaction with the AR FXXLF motif that is influenced by phosphorylation at Thr-360 within the F-box, and potential phosphorylation at Ser-174 NH 2 -terminal to the F-box. MAGE-11 also interacts with Skp1, which suggests the MAGE-11 F-box serves as a binding site for Skp1, and MAGE-11 may associate with a Skp1-cullin-based E3 ubiquitin ligase complex.  5A). This, together with evidence that the S174A mutation inhibited MAGE-11 interaction with the AR FXXLF motif peptides, led us to investigate whether MAGE-11 is phosphorylated at Ser-174 by MAP kinase.
Interaction between MAGE-11 and TIF2-The studies suggested that MAGE-11 and TIF2 act synergistically to increase AR transactivation through AF2 (Figs. 1 and 3), but it remained unclear whether MAGE-11 could increase TIF2-dependent AR transcriptional activity independent of AF2. To address this, we made use of AR-(1-660), a constitutively active AR NH 2 -terminal fragment that contains AF1, but lacks the AR ligand binding domain and AF2.
The relatively low activity of AR-(1-660) increased ϳ2-fold with the coexpression of TIF2 (Fig. 6A). This agrees with previous evidence that AR activation by TIF2 in the absence of MAGE-11 depends largely on TIF2 LXXLL motif binding to AR AF2 (6). AR-(1-660) activity increased to a greater extent with the expression of MAGE-11, and increased further with the coexpression of MAGE-11 and TIF2. The MAGE-11 and TIF2dependent increase in AR-(1-660) activity was inhibited by MAGE-11 F-box mutations that interfere with MAGE-11 binding to the AR-(1-660) FXXLF motif (Fig. 6B).
The results raised the possibility of a direct interaction between MAGE-11 and TIF2 that could increase AR transcriptional activity in the absence and presence of androgen. An interaction between endogenous MAGE-11 and TIF2 in CWR-R1 or LAPC-4 cells could not be observed, possibly due to the low level cell cycle-dependent expression of MAGE-11 (Fig. 1E). Furthermore, the association between MAGE-11 and TIF2 may be transient like AR and MAGE-11 in the presence of DHT (16). However, an interaction between TIF2 and MAGE-11 was demonstrated by coimmunoprecipitation of HA-MAGE with FLAG-TIF2 (Fig. 7A), and by the coimmunoprecipitation of TIF2 with FLAG-MAGE (Fig. 7B). A direct interaction between MAGE-11 and TIF2 was supported by in vitro affinity matrix binding studies. MAGE-11 interacted to the greatest extent with the TIF2 NH 2 -terminal region present in GST-TIF2.0-(1-627), and to a lesser extent with TIF2 carboxyl-terminal fragments, some of which contain AD1-(1011-1179) (Fig. 7C). The results suggest that MAGE-11 interacts directly with TIF2 to increase AR transcriptional activity.
Interaction between MAGE-11 and the AD1 region of TIF2 was also demonstrated by the ability of MAGE-11 to increase the transcriptional activity of GAL-TIF2 fusion proteins (Fig.  9A). Both full-length MAGE-11 and MAGE-(112-429) increased the transcriptional activity of GAL-TIF2.1-(624 -1287) and GAL-TIF2.3-(624 -1179)-m123, where the latter contains mutations at each of the three TIF2 LXXLL motifs (Fig. 9B). These results support the in vitro binding and coimmunoprecipitation evidence that MAGE-11 interacts directly with TIF2 independent of the TIF2 LXXLL motifs.
Mutations in the MAGE-11 F-box inhibited the ability of MAGE-11 to increase the AD1 activity of GAL-TIF2.8 (Fig.  9D). This appears to reflect a decreased transcriptional response of GAL-TIF2.8 AD1 to p300, which is known to interact with TIF2. Alone, p300 resulted in a small increase in GAL-TIF2.8 activity. In the presence of MAGE-11, p300 increased TIF2 AD1 activity in a MAGE-11 F-box-dependent manner (Fig. 9E). The results indicate that the MAGE-(165-298) region interacts with the NH 2 -terminal and AD1 regions of TIF2, and that the MAGE-11 F-box influences TIF2 AD1 activation by p300. MAGE-11 FXXIF Motif-Within the MAGE-(165-298) region that interacts with TIF2 is a predicted ␣-helical FXXIF motif with the sequence 260 FPEIF 264 flanked by charged residues similar to the AR FXXLF motif sequence 23 FQNLF 27 (Fig. 10A). The AD1 region of TIF2 also contains a predicted ␣-helical sequence with spacing of hydrophobic residues similar to the F-box in  cyclin F, Skp2, and MAGE-11 (Fig. 10B). We therefore investigated whether the MAGE-11 FXXIF motif contributes to the interaction with TIF2 AD1.

DISCUSSION
Interaction between MAGE-11 and TIF2-MAGE-11 increases AR transcriptional activity in part by increasing the recruitment of SRC/p160 coactivators (10,21,39). In the absence of MAGE-11, the AR N/C interaction competitively inhibits SRC/p160 coactivator binding to AF2, which shifts the dominant activation function from AF2 to AF1 (5) (Fig. 12A). MAGE-11 relieves the AR N/C interaction-induced repression of AR AF2 activity by binding the AR FXXLF motif and increasing AF2 accessibility for SRC/p160 coactivator binding. Here we show that MAGE-11 also interacts directly with TIF2 to . Cells were incubated with 0.1 g/ml of EGF and 1 M MG132, extracted in IP lysis buffer, and incubated with FLAG resin for 1 h at 4°C. Immunoprecipitated proteins (upper panel) and cell extracts (lower panels) (25 g of protein/lane) were analyzed on immunoblots probed using HA and FLAG antibodies. E, HeLa cells were transfected with 0.05 g of GAL-MAGE-(85-205) wild-type (WT), S174A or S174D mutant, 0.1 g of 5ϫGAL4Luc3, and 0.05 g of VP16 empty vector (-) or VP-TIF2.0-(1-627). Bottom panel, GAL0 empty vector (-) (2 g) and 2 g of GAL-MAGE-(85-205) WT, S174A, or S174D mutant were expressed in HeLa cells. Cell extracts (200 g of protein/lane) were analyzed on immunoblots probed using a GAL antibody. DECEMBER 11, 2009 • VOLUME 284 • NUMBER 50 increase both ligand-dependent and ligand-independent AR activity.

MAGE-11 and TIF2 Interaction in AR Transactivation
The results suggest that MAGE-11 functions as a bridging factor to stabilize and recruit SRC/p160 coactivators in a manner less dependent on AR binding of androgen. MAGE-11 interacts with the NH 2 -terminal region of TIF2, and with TIF2 AD1 through a MAGE-11 FXXIF motif (Fig. 12B). TIF2, SRC1, and TRAM1 each increases the activity of the AR-(1-660) NH 2 -terminal and the DNA binding domain fragment to a greater extent in the presence of MAGE-11. SRC/p160 coacti-vator interaction with the AR NH 2terminal region (10,21) may be facilitated by their interaction with MAGE-11.
MAGE-11 Is an F-box Protein-MAGE-11 is a member of a multigene MAGE family whose functions are largely uncharacterized. MAGE-11 is expressed in the human male and female reproductive tracts, and undergoes phosphorylation and monoubiquitinylation to interact with the AR FXXLF motif (16,19,33). Well characterized steroid receptor coactivator interaction motifs include the LXXLL motifs of SRC/p160 coactivators (40), FXXLF motifs of putative AR coactivators (7), bromodomains, and others (41). MAGE-11 residues 329 -369 function as an F-box interaction site for the AR FXXLF motif (Fig. 12), and a MAGE-11 FXXIF motif interacts with an F-box-like sequence in the AD1 region of TIF2.
The ϳ40 amino acid F-box, named for a weakly conserved hydrophobic repeat first identified in cyclin F (42), is present in the S-phase kinase-associated protein Skp2, which interacts   (43). Acidic and basic residues (blue) flank the FXXLF-like motifs (red). B, F-box related sequences have predicted ␣-helical structure and relatively conserved spacing of two acidic residues (blue) and multiple hydrophobic residues (red). F-box-like sequences similar to the cyclin F F-box-(35-73) include MAGE-11 F-box-(329 -369) that interacts with the AR FXXLF motif, Skp2 F-box-(99 -139) that interacts with a region that contains the Skp1 FXXIL motif (43), and TIF2 AD1 F-box-(1073-1112) that interacts with the MAGE-11 FXXIF motif.
Based on our studies, MAGE-11 and its family members are F-box proteins that may function as components of the SCF complex. MAGE-11 would be in the FBX class of F-box proteins because it lacks a WD40 LxGH…D/N(X) 5 (W/F/ Y)(D/N) repeat sequence (55). MAGE-11 shares size and sequence similarity with Skp2, but lacks the repeating leucine-rich ␤-strand and ␣-helix arrangement of Skp2 (56). The carboxyl-terminal position of the MAGE-11 40-amino acid F-box (amino acids 329 -369) in the conserved MAGE homology domain differs from the NH 2 -terminal position of the F-box in components of the SCF complex. On the other hand, poxvirus ankyrin repeat proteins interact with Skp1 and have a 30-residue F-box-like sequence in the carboxyl-terminal region (57). The conserved nature of the MAGE-11 F-box across the MAGE family, the MAGE-11 F-box interaction with the AR FXXLF motif, the F-box requirement for the MAGE-11-dependent increase in GAL-TIF2 transcriptional activity, and evidence that MAGE-11 interacts with Skp1, suggest that the MAGE-11 F-box has multiple binding partners involved in domain sharing.
F-box proteins have functions other than as bait for the SCF complex (47). For example, the F-box protein MoKA is an KLF7 coregulator independent of the SCF complex and ubiquitination (58). MAGE-11 binding to the AR FXXLF motif is associated with increased AR transcriptional activity and increased degradation of AR and MAGE-11 (15,16,19,59). Agonist-induced down-regulation of other steroid receptors (49, 60 -63) occurs for AR through its association with MAGE-11. Like Skp2, MAGE-11 is expressed at low levels in a cell cycle-dependent manner, although MAGE-11 levels increase in human endometrium during the window of receptivity to implantation, and in castration-recurrent prostate cancer (20,33).
Whether the phosphorylation and monoubiquitinylationdependent MAGE-11 F-box interaction with the AR FXXLF motif involves the action of an SCF ubiquitin E3 ligase complex remains to be established. Substrates such as c-Myc and cyclin E, which bind the FBXW7 F-box protein that binds Skp1 in the SCF complex, are targeted for ubiquitinylation by phosphorylation within a phosphodegron (64). MAGE-11 is phosphorylated within the F-box by checkpoint kinase Chk1, which triggers monoubiquitinylation at lysines 240 and 245 (19), and by MAP kinase in response to serum. Monoubiquitinylation, which has been shown to mediate interactions between regulatory components of the proteosome required for transcription (65), may be required for an SCF function of MAGE-11. MAGE-11 F-box binding to the AR FXXLF motif, and MAGE-FXXIF motif binding to an F-box-like sequence in AD1 of TIF2, suggests a novel F-box-FXX(L/I)F protein interaction paradigm. Selectivity for the MAGE-11 F-box-AR FXXLF motif interaction is supported by the lack of MAGE-11 binding to FXXLF motifs present in other AR coactivators, even though these same FXXLF motifs interact with AR AF2 (7,16). Single amino acid mutations disrupt the MAGE-11 F-box-AR FXXLF motif and MAGE-11 FXXIF-TIF2 AD1 interactions, although these same mutations did not eliminate coimmunoprecipitation of the complex. This suggests that the protein structure outside the region to some extent compensates for single residue F-box mutations.
The FXX(L/I)F motifs in AR and MAGE-11, and the uncharacterized FXXIY motif in TIF2, are each flanked by charged residues that may facilitate protein-protein interactions by increasing the solubility of the hydrophobic region. AR FXXLF motif binding to AF2 is influenced by complementary charged clusters that surround the AF2 region of AR (24). The TIF2 FXXIY motif 333 FSQIY 337 and Skp1 FXXIL motif 101 FELIL 105 within the interaction region for Skp2 (Fig. 10A) are also flanked by charged residues (43). Although functions for the TIF2 and Skp1 FXXLF-like motifs have not yet been demonstrated, the presence of flanking charged residues support a role in mediating protein-protein interactions. The ubiquitinylation-dependent interaction between the AR FXXLF motif and the MAGE-11 F-box may be relevant to other MAGE gene family members with sequence homology within the MAGE-11 F-box.
MAGE-11 Thr-360 phosphorylation by checkpoint kinase Chk1 within the MAGE-11 F-box is required for its interaction with the AR FXXLF motif. AR FXXLF motif binding to MAGE-11 is modulated by serum stimulation of MAP kinase phosphorylation at MAGE-Ser-174, although mutation of Ser-174 did not eliminate the ability of MAGE-11 to increase AR transcriptional activity (19). MAGE-11 Ser-174 appears to be a post-translational regulatory site phosphorylated by ERK1, based on the inhibitory effect of the S174A mutation in the context of shorter AR NH 2 -terminal fragments (19), and the greater transcriptional activity of GAL-MAGE-11 fusion proteins containing the S174D phosphomimetic. Conformational changes in nuclear receptor coregulators are regulated by peptidyl-prolyl isomerase 1 (Pin1), which targets phosphorylated Ser/Thr-Pro residues (74,75). Pin1, which is highly expressed in metastatic prostate cancer, may exert regulatory effects on MAGE-11 mediated by MAP kinase phosphorylation at Ser-174.
AR Reactivation in Prostate Cancer-AR becomes reactivated during androgen deprivation therapy and prostate cancer progression to castration-recurrent growth (76,77). This involves mechanisms that include increases in mitogen signaling, SRC/p160 coactivator levels (32), sensitivity to low levels of androgen (19,32,73,78), and local tissue androgen synthesis (79). Of the coregulators that interact with AR (80), both the SRC/p160 coactivators and MAGE-11 levels increase in castration-recurrent prostate cancer (20,81,82). MAGE-11 mRNA FIGURE 12. Interactions between AR, MAGE-11, and TIF2. A, schematic diagram of dynamic interactions between AR (blue), MAGE-11 (orange), and TIF2 (light blue) in the context of the AR antiparallel dimer bound to DNA. Indicated is the AR NH 2 -terminal (N) activation function 1 (AF1), AR carboxyl-terminal (C) activation function 2 (AF2), and the AR FXXLF motif interaction site for MAGE-11 and AF2 in the N/C interaction. B, detailed schematic diagram of interactions between MAGE-11 (orange) F-box residues 329 -369 in the carboxyl-terminal MAGE homology domain (MHD) with the AR (blue) NH 2 -terminal FXXLF motif 23 FQNLF 27 , which is modulated by phosphorylation in the MAGE-11 F-box at Thr-360, monoubiquitinylation (Ub) at Lys-240 and -245 (19), and serum stimulation of MAP kinase phosphorylation of MAGE-11 Ser-174 outside the F-box. AR transcriptional activity is increased by MAGE-11 F-box binding of the AR FXXLF motif, which competitively inhibits AR FXXLF motif binding to AF2 in the AR N/C interaction. This exposes AF2 in the AR ligand binding domain (LBD) for TIF2 (light blue) LXXLL (LX) motif binding. MAGE-11 also increases AR transcriptional activity through direct interactions with the TIF2 NH 2 -terminal region and with AD1 mediated in part by MAGE-11 FXXIF motif 260 FPEIF 264 . levels can increase by ϳ1000-fold in clinical specimens of castration-recurrent prostate cancer compared with benign prostate, and by ϳ50-fold in the castration-recurrent CWR22 human prostate cancer xenograft (20). Increased expression of MAGE-11 in prostate cancer during androgen deprivation therapy is associated with DNA hypomethylation at CpG sites in the MAGE-11 promoter and may also result from increased cyclic AMP signaling. The ability of MAGE-11 to increase AR transcriptional activity by interacting with TIF2 suggests that increased levels of both coactivators contribute to AR reactivation in castration-recurrent prostate cancer.