Melanoma antigen gene protein-A11 (MAGE-11) F-box links the androgen receptor NH2-terminal transactivation domain to p160 coactivators

Abstract

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 NH(2)-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 NH(2)-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.

FIGURE 1.

Dependence of AR transactivation on MAGE-11 and TIF2. A, CWR-R1 cells were transfected with 0.1 μg of MMTV-Luc with or without 0.1 μg of pSG5-MAGE-11 or pSG5-TIF2. Cells were incubated in the absence and presence of DHT (shown as -log DHT concentration) and 0.1 μg/ml of EGF. B, COS cells were transfected using Lipofectamine 2000 with 1 μg of pSG5 or pSG5-MAGE (top panel), 1 μg of pSG5 or pSG5-TIF2 (middle panel), and 0.5 μg of pCMV5 or pCMV-AR (bottom panel) in the absence and presence of 1 nm nonspecific (NS) siRNA, MAGE-11 siRNA-2 and -3, TIF2 siRNA-3, and AR siRNA-3. Cell extracts were analyzed by immunoblot for MAGE-11 (10 μg of protein/lane), TIF2 (40 μg/lane), AR (30 μg/lane), and β-actin. C, CWR-R1 cells were transfected with 0.1 μg of MMTVΔ-(−421–−364)-Luc, 0.2 μg of pSG5 or pSG5-MAGE in the absence and presence of 4 nm nonspecific or TIF2 siRNA-3, and incubated in the absence and presence of 0.1 nm DHT and 0.01 μg/ml of EGF. D, LAPC-4 cells were transfected with 0.1 μg of MMTVΔ-(−421–−364)-Luc and 3 nm siRNA and incubated in the absence and presence of 0.1 nm DHT and 0.1 μg/ml EGF. E, top panel, CWR-R1 cells incubated for 48 h in serum-free medium were treated for the indicated times with 0.1 nm DHT and 10 ng/ml of EGF. Cell extracts in IB lysis buffer (200 μg of protein/lane) (lanes 2–8) were analyzed by immunoblot using 10 μg/ml of MAGE-Ab-(13–26), -(59–79), and -(94–108) antibodies overnight at 4 °C, and AR32 and β-actin antibodies (33). Extracts of COS cells expressing pCMV-AR (30 μg of protein) and pSG5-MAGE (0.25 μg of protein) served as controls (lane 1). Bottom panel, CWR-R1 cells were transfected with 4 nm siRNA and incubated for 6 h with or without 10 nm DHT and 10 ng/ml of EGF. RNA was extracted and analyzed by quantitative reverse transcription-PCR as described under “Experimental Procedures” for endogenous PSA mRNA relative to peptidylprolyl isomerase A.

FIGURE 2.

Requirement for a MAGE-11 F-box in AR transactivation. A, schematic diagram of full-length MAGE-11 showing the MAGE homology domain (MHD) with F-box residues 329–369, phosphorylation site Thr-360 within the F-box, Lys-240 and Lys-245 monoubiquitinylation (Ub) sites outside the F-box, and the NH₂-terminal nuclear localization signal (NLS). B, sequence homology between MAGE-11 F-box residues 329–369, cyclin F F-box residues 35–73, and MAGE family members, with conserved acidic residues in blue and hydrophobic residues in red. C, alanine mutagenesis of the MAGE F-box at conserved hydrophobic residues. D, Ishikawa cells were transfected with pCMV-AR (10 ng/well), 0.1 μg/well of PSA-Enh-Luc, and 0.1 μg of pSG5 empty vector (—), pSG5-MAGE wild-type (WT), or the indicated F-box mutant. Cells were incubated in the absence and presence of 1 nm DHT with or without 0.1 μg/ml of EGF. Bottom panel, Ishikawa cells were transfected with 2 μg of pSG5 (—), pSG5-MAGE WT, and the indicated F-box mutant. Cells were extracted in IB lysis buffer and 30 μg of protein/lane analyzed on immunoblots probed with antibody raised against FLAG-tagged human MAGE-11 (2 μg/ml). E, FLAG-b empty vector (—) or FLAG-AR (4 μg) were expressed in COS cells with pSG5-HA-MAGE-(112–429) WT or Δ329–369 F-box deletion (0.5 μg of DNA/10-cm dish). Cells were treated in the absence and presence of 10 nm DHT and 0.1 μg/ml EGF, lysed in IP lysis buffer, and incubated with FLAG resin overnight at 4 °C. Immunoprecipitates and cell extracts (25 μg of protein/lane) were analyzed by immunoblot using FLAG and HA antibodies.

FIGURE 3.

Requirement for MAGE-11 F-box in AR transactivation by TIF2. A, CV1 cells were transfected with 0.1 μg of pCMV-AR and 5 μg of MMTV-Luc, 2 μg of pSG5-TIF2, 2 μg of pSG5-MAGE wild-type (WT) or L358A/L359A F-box mutant, and incubated in the absence and presence of 1 nm DHT. B, CV1 cells were transfected with 5 μg of MMTV-Luc and 0.1 μg of pCMV-ARΔ120–472 WT or the indicated mutant, 2 μg of pSG5-TIF2 (pSG5-T) and/or 2 μg of pSG5-MAGE (pSG5-M). Cells were incubated in the absence and presence of 1 nm DHT. Inset, pCMV-ARΔ120–472 WT and mutants (5 μg) were expressed in COS cells because expression was too low in CV1 cells. Cell extracts (30 μg of protein/lane) were analyzed on immunoblots probed with AR52 antibody. Bottom panel, schematic diagram of full-length human AR amino acid residues 1–919 with the NH₂-terminal FXXLF motif ²³FQNLF²⁷, activation function 1 (AF1), DNA binding domain (DBD), activation function 2 (AF2) in the ligand binding domain (LBD), and the ARΔ120–472 deletion mutant that lacks AF1. C, pCMV-ARΔ120–472 (0.1 μg) was expressed in CV1 cells with 5 μg of MMTV-Luc, 2 μg of pSG5-TIF2 and/or 2 μg of pSG5-MAGE WT or the indicated F-box mutant. Cells were incubated in the absence and presence of 1 nm DHT. D, pCMV-ARΔ120–472 (0.1 μg) was expressed in CV1 cells with 5 μg of MMTV-Luc in the absence and presence of 2 μg of pSG5-TIF2 and/or 2 μg of pSG5-MAGE WT or single residue F-box mutant. Cells were incubated in the absence and presence of 1 nm DHT. Bottom panel, CV1 cells were transfected with pSG5 (8 μg/10-cm dish) (—), pSG5-MAGE WT or the indicated single residue F-box mutant. Cell extracts in IB lysis buffer (95 μg of protein/lane) were analyzed on immunoblots probed with antibody raised against FLAG-tagged MAGE-11.

FIGURE 4.

Interaction between MAGE-11 F-box and AR FXXLF motif. A, GAL-MAGE wild-type (WT), F-box mutant, or GAL-MAGE-K236A/K240A/K245A (KA) (0.05 μg) were transfected in HeLa cells with 0.1 μg of 5×GAL4Luc3 and 0.1 μg of VP16 empty vector (—), VP-AR-(1–660) or VP-AR-(1–660)-L26A,F27A (LFAA) with a mutation in the AR FXXLF motif. Bottom panel, GAL-MAGE F-box or KA mutants, WT and GAL0 (—) (2 μg/10-cm dish) were expressed in HeLa cells. Cell extracts prepared in IB lysis buffer (150 μg of protein/lane) were analyzed on immunoblots probed using GAL antibody (1:100 dilution). B, HeLa cells were transfected with 0.1 μg of 5×GAL4Luc3, 0.05 μg of GAL-AR-(4–52), and 0.1 μg of VP16 (—), VP-MAGE WT, or the indicated mutant. Bottom panel, HeLa cells were transfected with VP-MAGE mutants (lanes 1–8), VP-MAGE WT (lane 9), and VP16 empty vector (—) (lane 10) (2 μg/10-cm dish). Cell extracts in IB lysis buffer (100 μg of protein/lane) were analyzed by immunoblot using VP16 antibody. C, HeLa cells were transfected with 0.05 μg of GAL-AR-(4–52) or GAL-AR-(16–36), 0.1 μg of 5×GAL4Luc3 and 0.1 μg of VP16 empty vector (—) or VP-MAGE WT or the indicated mutant. D, coimmunoprecipitation of endogenous Skp1 with FLAG-MAGE. FLAG empty vector (—) (3 μg), FLAG-MAGE (3 μg), and FLAG-MAGE-(112–429) (6 μg) were expressed in HEK293 cells. The next day cells were treated for 24 h with 100 ng/ml of EGF and 1 μm MG132. IP of an overnight incubation and cell extracts (35 μg of protein/lane) were analyzed using FLAG and Skp1 antibodies.

FIGURE 5.

Serum stimulation of MAP kinase phosphorylation of MAGE-11 Ser-174. A, MAGE-11 Ser-Pro sites Ser-168, Ser-170, Ser-174, and Ser-181 (underlined) include consensus MAP kinase site ¹⁷²PQSP¹⁷⁵. B, GAL-MAGE-(112–205) wild-type (WT) and mutants and GAL-MAGE-(112–170) (5 μg) were expressed in COS cells. Cell extracts in IP lysis buffer without sodium fluoride (35 μg) were incubated in the absence and presence of 800 IU of λ-phosphatase for 1 h at 4 °C, and the immunoblot probed with GAL antibody. C, GAL0 (—), GAL-MAGE-(112–205) WT, or S174A mutant (5 μg) were expressed in COS cells. Immediately after transfection and the next day, serum-free medium containing 1 μm MG132 and U0126 was added. Cells were extracted in IB lysis buffer containing phosphatase mixture inhibitors 1/2. Protein extracts (35 μg of protein/lane) were analyzed on immunoblots probed using GAL and β-actin antibodies. D, GAL-MAGE-(112–205) (5 μg) was expressed in COS cells incubated in the absence (lanes 1, 3, 5, and 7) or presence of 10% fetal bovine serum (lanes 2, 4, 6, and 8). Cells were harvested at 0, 30, 60, and 120 min in IB lysis buffer. Cell extracts (35 μg of protein/lane) were analyzed by immunoblots probed with GAL and β-actin antibodies. E, ERK1 in vitro phosphorylation of MAGE-11 Ser-174. In vitro kinase assay (top panel) was performed as described (19) at 30 °C for 30 min with 0.16 μCi/μl of [γ-³²P]ATP, 0.1 μg of active ERK1 kinase and purified GST0 (lane 1), GST-MAGE-(171–179) WT (lane 2), and S174A mutant (lane 3), and ERK1 substrate modeled after myelin basic protein (37) (lane 4). The dried gel was exposed to x-ray film for 16 h, and rehydrated for Coomassie Blue staining to demonstrate equivalent protein loading (bottom panel).

FIGURE 6.

MAGE-11 and TIF2-dependent increase in AR AF1 activity. A, CV1 cells were transfected with 5 μg of PSA-Enh-Luc and 0.05 μg of pCMV5 empty vector (p5) or pCMV-AR-(1–660) in the absence and presence of 0.1 μg of pSG5-MAGE and/or 2 μg of pSG5-TIF2. B, CV1 cells were transfected with 5 μg of PSA-Enh-Luc and 0.05 μg of p5 or 0.05 μg of pCMV-AR-(1–660) in the absence and presence of 2 μg of pSG5-TIF2 and/or 1 μg of pSG5-MAGE WT or the indicated F-box mutant.

FIGURE 7.

Interaction between MAGE-11 and TIF2. A, FLAG-b empty vector (—) (8 μg) and 5 μg of pSG5-HA-MAGE (lane 1), or 8 μg of FLAG-TIF2 and 5 μg of pSG5-HA-MAGE (lane 2) were expressed in COS cells. Cells were incubated overnight in the presence of 0.1 μg/ml EGF and 1 μm MG132, harvested in IP lysis buffer, and incubated with FLAG resin overnight at 4 °C. Immunoprecipitates and cell extracts (40 μg of protein/lane) were analyzed on immunoblots probed with HA and TIF2 antibodies. B, pSG5-TIF2 (8 μg) was expressed with 4 μg of FLAG-b empty vector (—) or 4 μg of FLAG-MAGE in COS cells. Cells were incubated overnight in the presence of 0.1 μg/ml EGF and 1 μm MG132, harvested in IP lysis buffer, and incubated with FLAG resin overnight at 4 °C. Immunoprecipitates and cell extracts (50 μg of protein/lane) were analyzed on immunoblots probed using FLAG and TIF2 antibodies. C, in vitro GST affinity matrix assays using the indicated GST-TIF2 fusion fragments expressed in E. coli and ³⁵S-labeled MAGE-11 expressed from pSG5-MAGE-11 using the TnT T7 Quick Coupled transcription-translation system. Input lane contains 2% of the total ³⁵S-labeled MAGE-11 used in the reactions.

FIGURE 8.

MAGE-11 and TIF2 interaction domains. A, schematic diagram of full-length MAGE-11 and deletion fragments, with nuclear localization signal (NLS, residues 18–22), monoubiquitinylation sites Lys-240 and -245, Ser-174 and Thr-360 phosphorylation sites (19), MAGE homology domain (MHD), and the F-box region. B, FLAG-b empty vector (—) (8 μg) (lanes 1–5) or 8 μg of FLAG-TIF2 (lanes 6–10) were expressed in COS cells with 0.5 μg of pSG5-HA-MAGE-(112–429), 1 μg of pSG5-HA-MAGE-(112–307), or 2 μg of pSG5-HA-MAGE-(165–307), -(112–298), and –(112–276). Cells were incubated with 0.1 μg/ml of EGF and 1 μm MG132, solubilized in IP lysis buffer and immunoprecipitated using FLAG affinity resin overnight at 4 °C. IP (upper panel) and cell extracts (lower panels) (15 μg of protein/lane for HA-MAGE, 55 μg of protein/lane for FLAG-TIF2) were analyzed on immunoblots probed using HA and FLAG antibodies. C, FLAG-b empty vector (lanes 1 and 2) (—), FLAG-TIF2 (lanes 3 and 4), FLAG-TIF2.0-(1–627), or FLAG-TIF2.8-(1011–1179) (8 μg) were expressed in COS cells with 5 μg of pSG5-HA-MAGE or 0.5 μg of pSG5-HA-MAGE-(112–429). Cells were incubated with 0.1 μg/ml of EGF and 1 μm MG132, extracted in IP lysis buffer, and incubated with FLAG affinity resin overnight at 4 °C. Immunoprecipitates (upper panel) and cell extracts (lower panels) (25 μg of protein/lane) were analyzed on immunoblots probed using FLAG and HA antibodies. D, FLAG-b empty vector (—) (8 μg) (lanes 1–3) and FLAG-TIF2.0-(1–627) (lanes 4–6) were expressed in COS cells with 1 μg of pSG5-HA-MAGE-(112–429), 2 μg of pSG5-HA-MAGE-(165–429), or 2 μg of pSG5-HA-MAGE-(112–307). Cells were incubated with 0.1 μg/ml of EGF and 1 μm 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 μg of protein/lane) were analyzed on immunoblots probed using HA and FLAG antibodies. E, HeLa cells were transfected with 0.05 μg of GAL-MAGE-(85–205) wild-type (WT), S174A or S174D mutant, 0.1 μg of 5×GAL4Luc3, and 0.05 μg of VP16 empty vector (—) or VP-TIF2.0-(1–627). Bottom panel, GAL0 empty vector (—) (2 μg) and 2 μg of GAL-MAGE-(85–205) WT, S174A, or S174D mutant were expressed in HeLa cells. Cell extracts (200 μg of protein/lane) were analyzed on immunoblots probed using a GAL antibody.

FIGURE 9.

MAGE-11 increases TIF2 AD1 activity. A, schematic diagram of GAL-TIF2 fusion proteins showing three LXXLL motifs (LX) beginning at residues 641, 690, and 745, activation domain 1 (AD1) residues 1011–1131, glutamine (Q)-rich region 1131–1179, and activation domain 2 (AD2) residues 1288–1464. B, HeLa cells were transfected with 0.1 μg of 5×GAL4Luc3 and 0.05 μg of GAL-TIF2.0-(1–627), GAL-TIF2.1-(624–1287), and GAL-TIF2.3-(624–1179)-m123, which contains L644E,L645A,L693A,L694A,L748A,L749A mutations in the LXXLL motifs, or GAL-TIF2.2-(1288–1464), and 0.1 μg of pSG5 empty vector (—), pSG5-HA-MAGE or pSG5-HA-MAGE-(112–429). C, HeLa cells were transfected with 0.1 μg/well of 5×GAL4Luc3 and 2 ng of GAL-TIF2.8-(1011–1179) or GAL-TIF2.12-(940–1131), which contain AD1 and 0.1 μg of pSG5 (—), pSG5-HA-MAGE, or pSG5-HA-MAGE-(112–429). D, GAL-TIF2.8-(1011–1179) (2 ng), which contains AD1 was expressed in HeLa cells with 0.1 μg/well of 5×GAL4Luc3, 0.1 μg of pSG5 (—), or 0.1 μg of pSG5-HA-MAGE-(112–429) wild-type (WT) or the indicated F-box mutant. Bottom panel, HeLa cells were transfected with 2 μg of pSG5, pSG5-HA-MAGE-(112–429) WT, and the indicated F-box mutant. Cell extracts (150 μg of protein/lane) were analyzed on immunoblots probed with HA antibody. E, HeLa cells were transfected with 0.1 μg/well of 5×GAL4Luc3, 2 ng of GAL-TIF2.8-(1011–1179), and 0.05 μg of pSG5 (—), pSG5-HA-p300, or pSG5-HA-MAGE-(112–429) WT or V337A/L338A F-box mutant.

FIGURE 10.

FXXLF and F-box related sequences in AR, MAGE-11, TIF2, Skp1, and Skp2. A, FXXLF-like motifs include AR-(15–36) FXXLF motif ²³FQNLF²⁷ that mediates the androgen-dependent AR N/C interaction with AF2 (6) and interaction with MAGE-11 (7). MAGE-11-(252–273) FXXIF motif ²⁶⁰FPEIF²⁶⁴ interacts with the AD1 region of TIF2. TIF2-(325–346) FXXIY motif ³³³FSQIY³³⁷ function is unknown. Skp1-(93–114) FXXIL motif ¹⁰¹FELIL¹⁰⁵ is in a region that interacts with the Skp2 F-box (43). Acidic and basic residues (blue) flank the FXXLF-like motifs (red). B, F-box related sequences have predicted α-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.

FIGURE 11.

MAGE-11 FXXIF motif-dependent interaction with TIF2. A, top panels, FLAG-b empty vector (lane 1) (—) or FLAG-TIF2.8-(1011–1179) (lanes 2–4) (8 μg) were expressed in COS cells with 0.5 μg of pSG5-HA-MAGE-(112–429) wild-type (WT) (lanes 1 and 2) or the indicated mutant (lanes 3 and 4). Cells were incubated with 0.1 μg/ml of EGF and 1 μm MG132 and immunoprecipitated overnight at 4 °C using FLAG resin. IP (upper panel) and cell extracts (25 μg of protein for HA-MAGE, 50 μg of protein for FLAG-TIF2.8, lower panels) were analyzed on immunoblots using HA and FLAG antibodies. Bottom panels, HeLa cells were transfected with 2 μg of pSG5 (—), pSG5-HA-MAGE-(2–429) WT, F260A, or F264A mutant (left panel), and pSG5-HA-MAGE-(112–429) WT, F260A, or F264A mutant, and pSG5 (—) (right panel). Cells extracts in IB lysis buffer (150 μg of protein/lane) were analyzed on immunoblots using HA antibody. B, GAL-TIF2.1-(624–1287) or GAL-TIF2.3m123 (TIF2-(624–1179)-L644E,L645A,L693A,L694A,L748A,L749A) (0.05 μg) were transfected in HeLa cells with 0.1 μg of pSG5 (—), pSG5-HA-MAGE-(112–429) WT or mutant, and 0.1 μg of 5×GAL4Luc3. C, CV1 cells were transfected with 0.1 μg of pCMV-AR and 5 μg of PSA-Enh-Luc with and without 2 μg of pSG5-TIF2 and 1 μg of WT or mutant pSG5-HA-MAGE. Cells were incubated in the absence and presence of 1 nm DHT. Bottom panel, CV1 cells were transfected with 8 μg of pSG5 (–), pSG5-HA-MAGE-(112–429) WT, F260A, or F264A mutant. Cells extracts in IB lysis buffer (200 μg of protein/lane) were analyzed on immunoblots probed with HA antibody. D, CV1 cells were transfected with 0.1 μg of pCMV-ARΔ120–472 with and without 2 μg of pSG5-TIF2, 2 μg of WT or mutant pSG5-HA-MAGE, and 5 μg of MMTV-Luc. Cells were incubated in the absence and presence of 1 nm DHT. Bottom panel, CV1 cells were transfected with 8 μg of pSG5 (—), pSG5-HA-MAGE WT, F151A, F260A, or F264A mutant. Cell extracts in IB lysis buffer (150 μg/lane) were analyzed on immunoblots using HA antibody. E, HeLa cells were transfected with 0.1 μg of 5×GAL4Luc3, 50 ng of GAL0 empty vector (—), WT or mutant GAL-MAGE-(251–272), and 0.1 μg of VP16 empty vector (—), VP-TIF2.0-(1–627), VP-TIF2.1-(624–1287), or VP-TIF2.2-(1288–1464). Bottom panel, HeLa cells were transfected with 2 μg of GAL0 (lane 1), GAL-MAGE-(251–272) WT (lane 2), or F260A,F264A mutant (lane 3). Cell extracts (100 μg of protein/lane) were analyzed on immunoblots using GAL antibody.

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 NH₂-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) NH₂-terminal FXXLF motif ²³FQNLF²⁷, 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 NH₂-terminal region and with AD1 mediated in part by MAGE-11 FXXIF motif ²⁶⁰FPEIF²⁶⁴.