Epidermal-growth-factor-dependent phosphorylation and ubiquitinylation of MAGE-11 regulates its interaction with the androgen receptor

Abstract

The androgen receptor (AR) is a ligand-activated transcription factor that interacts with coregulatory proteins during androgen-dependent gene regulation. Melanoma antigen gene protein 11 (MAGE-11) is an AR coregulator that specifically binds the AR NH(2)-terminal FXXLF motif and modulates the AR NH(2)- and carboxyl-terminal N/C interaction to increase AR transcriptional activity. Here we demonstrate that epidermal growth factor (EGF) signaling increases androgen-dependent AR transcriptional activity through the posttranslational modification of MAGE-11. EGF in the presence of dihydrotestosterone stabilizes the AR-MAGE complex through the site-specific phosphorylation of MAGE-11 at Thr-360 and ubiquitinylation at Lys-240 and Lys-245. The time-dependent EGF-induced increase in AR transcriptional activity by MAGE-11 is mediated through AR activation functions 1 and 2 in association with the increased turnover of AR and MAGE-11. The results reveal a dynamic mechanism whereby growth factor signaling increases AR transcriptional activity through the covalent modification of an AR-specific coregulatory protein. Sequence conservation of the MAGE-11 phosphorylation and ubiquitinylation sites throughout the MAGE gene family suggests common regulatory mechanisms for this group of cancer-testis antigens.

FIG. 1.

EGF stabilizes the MAGE-AR-DHT complex. (A) COS cells were transfected with 2 μg pCMV-AR and 4 μg Flagb empty vector (lanes 1 to 4) or 2 μg pCMV-AR and 4 μg Flag-MAGE (lanes 5 to 8). Cells were treated with and without 10 nM DHT and 100 ng/ml EGF for 24 h as indicated. Fresh medium was added the next day and after 2.5 h. Cells were harvested, and protein extracts were prepared in IP lysis buffer for immunoprecipitation using anti-Flag affinity resin. Transfer blots of the immunoprecipitates (upper two panels) and cell extracts (8 μg protein/lane; lower two panels) were developed using AR32 and anti-Flag antibodies. (B) COS cells were transfected with 2 μg pCMV-AR and 5 μg Flagb (lanes 1 to 4) or 2 μg pCMV-AR and 5 μg Flag-MAGE (lanes 5 to 18) and 24 h later placed in serum-free, phenol red-free medium without hormone. The next day the medium was replaced with and without 10 nM DHT and 10 ng/ml EGF and incubated at 37°C from 1 to 10 h as indicated. Anti-Flag immunoprecipitates (upper two panels) and cell extracts (10 μg protein/lane; lower two panels) were prepared and assayed as for panel A.

FIG. 2.

EGF regulates the turnover of AR and MAGE-11. (A) COS cells were transfected with 4 μg pCMV-AR (lanes 1 to 8), 4 μg pSG5-MAGE (lanes 9 and 10), and 4 μg pCMV-AR with 4 μg pSG5-MAGE (lanes 11 to 18). Cells were treated 24 h after transfection with and without 2, 5, and 10 nM DHT in the absence and presence of 100 ng/ml EGF as indicated. Cells were harvested 48 h later in IB lysis buffer and analyzed (10 μg protein/lane) on a 10% acrylamide gel containing SDS. The transfer blot was probed with AR32, MagAb13-26, and β-actin antibodies as indicated. (B) COS cells were transfected with 2 μg pSG5-MAGE without (lanes 1 to 8) and with 2 μg pCMV-AR (lanes 9 to 16). Twenty-four h after transfection, cells were placed in serum-free, phenol red-free medium in the absence (lanes 1 to 4 and 9 to 12) and presence (lanes 5 to 8 and 13 to 16) of 10 ng/ml EGF. The next day cells were placed in methionine-free medium with or without EGF and incubated for 30 min with [³⁵S]methionine. The medium was replaced with serum-free, methionine-containing medium with and without EGF, and cells were harvested at 0, 2.5, 5, and 8 h. Cell extracts containing 0.35 mg protein were immunoprecipitated using MagAb13-26, and the transfer blot was exposed to X-ray film. (C) Quantitation of ³⁵S labeling. The autoradiograph in panel B was scanned using an Image Pro Plus spectrophotometer, and the data are plotted on a semilog scale.

FIG. 3.

MAGE-11 ubiquitinylation and dimerization. (A) Effects of EGF and AR on MAGE-11 ubiquitinylation. COS cells were transfected with 2 μg pSG5 empty vector (p5, lanes 1 and 2), 2 μg pSG5-MAGE in the absence (lanes 3 and 4) or presence (lanes 5 and 6) of 2 μg pCMV-AR, and 2 μg pCMV-AR alone (lanes 7 and 8) and treated 24 h later with and without 10 ng/ml EGF as indicated. The next day the medium was replaced with fresh medium with or without EGF. Cell extracts were prepared 2 h later in IB lysis buffer and analyzed (50 μg/lane) on a 10% acrylamide gel. The transfer blot was probed using MagAb59-79 antibody. (B) EGF-induced ubiquitinylation of MAGE-11. COS cells were transfected with 6 μg Flagb empty vector and 3 μg pSG5-HA-MAGE-112-429 (lanes 1, 2, and 6) or 6 μg FlagUb and 3 μg pSG5-HA-MAGE-112-429 (lanes 3 to 5) and treated 24 h later in the absence (lanes 1, 3, and 6) or presence (lanes 2, 4, and 5) of 100 ng/ml EGF. The next day, protein extracts were prepared in IP lysis buffer, and anti-Flag resin was used to immunoprecipitate FlagUb. Shown are immunoprecipitated proteins (IP; lanes 1 to 4) and cell extracts (25 μg protein/lane; lanes 5 and 6). The transfer blot was probed using HA tag antibody. (C) EGF- and AR-independent dimerization of MAGE-11. COS cells were transfected with 4 μg Flag-b and 2 μg pCMV-AR (lanes 1 to 4), 4 μg Flagb and 2 μg pSG5-HA-MAGE (lanes 5 and 6), 4 μg Flag-MAGE and 2 μg pSG5 (lanes 7 and 8), 4 μg Flag-MAGE and 2 μg pSG5-HA-MAGE (lanes 9 and 10), and 4 μg Flag-MAGE, 2 μg pCMV-AR, and 2 μg pSG5-HA-MAGE-11 (lanes 11 to 14). Cells were treated 24 h later with and without 10 nM DHT and 100 ng/ml EGF as indicated. The next day the media were exchanged, and 2 h later cells were harvested in IP lysis buffer and protein extracts were immunoprecipitated using anti-Flag M2 affinity gel. Immunoprecipitates (upper three panels) and cell lysates (10 μg protein/lane; lower four panels) were analyzed on 10% acrylamide gels containing SDS, and the transfer blots were probed using AR32, HA, Flag, and β-actin antibodies as indicated.

FIG. 4.

Region of ubiquitinylation in MAGE-11. (A) Schematic diagram of full-length MAGE-11 (amino acid residues 1 to 429) and fragments. The amino acid sequence is shown for the MAGE-11 nuclear localization signal (NLS; residues 18 to 22) and the amino acid 221 to 249 region that contains Lys-225, -236, -240, and -245. (B) Ubiquitinylation in the central region of MAGE-11. COS cells were transfected with 5 μg pSG5-HA-MAGE-2-429 (lanes 1 and 2), 1 μg pSG5-HA-MAGE-112-429 (lanes 3 and 4), 5 μg pSG5-HA-MAGE-2-252 (lanes 5 and 6), 1 μg pSG5-HA-MAGE-112-362 (lanes 7 and 8), or 5 μg pSG5-HA-MAGE-2-362 (lanes 9 and 10) and 24 h later treated in the absence or presence of 10 ng/ml EGF as indicated. The next day the media with and without EGF were replaced and again incubated for 2.5 h. After the second 2.5-h incubation, protein extracts were prepared in IB lysis buffer containing 50 mM NaF and phosphatase inhibitor cocktail 1 and 2 (Sigma) and analyzed (15 to 25 μg/lane) by immunoblotting using HA tag antibody. (C) Loss of the MAGE-11-dependent increase in AR transcriptional activity by MAGEΔ221-249. CV1 cells were transfected with 5 μg PSA-Enh-Luc and 0.1 μg pCMV-AR with and without 2 μg pSG5-MAGE and 2 μg pSG5-MAGEΔ221-249 as indicated. Cells were incubated in the absence and presence of 1 nM DHT for 48 h, and luciferase activity was determined. (D) Reduced interaction between AR and MAGEΔ221-249. COS cells were transfected with 2 μg pCMV-AR in the presence of 4 μg Flagb empty vector (lanes 1 to 4), 4 μg Flag-MAGE (lanes 5 to 8), and 4 μg Flag-MAGEΔ221-249 (lanes 9 to 12) and 24 h later cultured in the absence and presence of 10 nM DHT and 100 ng/ml EGF as indicated. The next day the media were replaced and cells were incubated an additional 2.5 h. Cells were extracted in IP lysis buffer and immunoprecipitated using anti-Flag affinity resin. Shown are immunoblots of immunoprecipitates (upper panel) and cell extracts (8 μg/lane; lower panel) probed with AR32 and anti-Flag antibodies. (E) Loss of AR interaction with MAGEΔ221-249. Luciferase activity was determined in two-hybrid assays using HeLa cells transfected with 0.1 μg 5XGAL4Luc3 and 0.05 μg GAL-MAGE or GAL-MAGEΔ221-249 with 0.05 μg VP16 or VP-AR1-660 as indicated and with 0.05 μg VP-MAGE or VP-MAGEΔ221-249 with 0.05 μg GAL-0 or GAL-AR16-36 as indicated. In the lower panel, immunoblots of extracts from COS cells transfected with 10 μg GAL-MAGE, GAL-MAGEΔ221-249, VP-MAGE, and VP-MAGEΔ221-249 are shown. After 24 h cells were placed in medium containing 1 μM MG132, a proteosome inhibitor which was readded in fresh medium the next day, and cells were harvested 1 h later. Protein extracts in IB lysis buffer (60 μg protein/lane) were separated on a 12% acrylamide gel containing SDS. Transfer blots were probed with GAL-4 (left two lanes) and VP16 antibodies (right two lanes).

FIG. 5.

Inhibition of MAGE-11 coregulator function by lysine mutations. (A) Decreased AR transcriptional activity by MAGE-11 lysine mutants. CV1 cells were transfected with 5 μg PSA-Enh-Luc and 0.1 μg pCMV-AR in the absence and presence of 2 μg pSG5-TIF2 and 2 μg pSG5-MAGE wild-type (WT) and the indicated lysine-to-alanine mutants. Cells were incubated for 48 h in the absence and presence of 1 nM DHT, and luciferase activity was measured. The lower panel shows immunoblot results with cell extracts from COS cells transfected with 4 μg pSG5 empty vector (−), wild-type (WT) pSG5-MAGE, or the indicated lysine-to-alanine mutants. Protein extracts (20 μg/lane) were separated on a 10% gel, and the transfer blot was probed with MagAb13-26 antibody. (B) Inability of MAGE-K240A and MAGE-K245A to increase AR AF2 activity. CV1 cells were transfected with 5 μg MMTV-Luc, 0.1 μg pCMV-ARΔ120-472 (with a deletion of the AR NH₂-terminal AF1 region) with 2 μg WT pSG5-MAGE alone, 2 μg pSG5-TIF2 alone, and 2 μg pSG5-TIF2 with 2 μg WT pSG5-MAGE and K225A, K236A, K240A and K245A mutants as indicated. Cells were incubated for 48 h in the absence and presence of 1 nM DHT, and luciferase activity was measured. (C) Effects of MAGE-11 lysine mutants on AR AF1 constitutive activity. HeLa cells were transfected with 0.1 μg PSA-Enh-Luc and 0.01 μg pCMV-AR1-660 in the absence and presence of 0.1 μg WT pSG5-MAGE and K236A, K240A, K245A, and K236A,K240A,K245A mutants as indicated. Cells were incubated for 48 h before measuring luciferase activity. (D) Two-hybrid interaction between AR and MAGE-11 lysine mutants. HeLa cells were transfected with 0.25 μg 5XGAL4Luc3 and 0.05 μg WT or the indicated lysine-to-alanine mutant in GAL-MAGE or VP-MAGE with 0.05 μg VP-AR1-660 or GAL-AR16-36. The next day cells were incubated for 48 h before measuring luciferase activity. (Inset) immunoblots of extracts from COS cells transfected with 8 μg GAL-0 (−) and WT and mutant GAL-MAGE (top panel) and pVP16 (−) and WT and mutant VP-MAGE (bottom panel), as indicated. After 24 h, cells were transferred to medium containing 1 μM MG132, which was readded in fresh medium the next day and cells were harvested 1 h later. Protein extracts in IB lysis buffer (50 μg/lane) were separated on 10% acrylamide gels containing SDS. Transfer blots were probed using GAL-4 (upper panel) and VP16 (lower panel) antibodies.

FIG. 6.

Requirement of MAGE-11 ubiquitinylation to interact with AR. (A) COS cells were transfected with 1 μg pCMV-AR in the presence of 4 μg Flagb empty vector (lanes 1 to 4), wild-type (WT) Flag-MAGE (lanes 5 to 8), Flag-MAGE-K225A,K236A,K240A,K245A (lanes 9 to 12), or Flag-MAGE-K240A (lanes 13 to 16). Cells were treated 24 h later in the absence and presence of 10 nM DHT and 100 ng/ml EGF as indicated. The next day media were replaced and cells were incubated for 2 h at 37°C. Flag-MAGE was immunoprecipitated using anti-Flag M2 affinity resin, and the immunoprecipitates (upper two panels) and cell extracts (10 μg/lane; lower two panels) were separated on 10% acrylamide gels. Transfer blots were probed using AR32 and anti-Flag M2 monoclonal antibodies. (B) COS cells were transfected with 6 μg Flagb (lanes 1, 4, 7, 10, and 13) (−) and 6 μg FlagUb as indicated with 2 μg WT pSG5-HA-MAGE-112-429 and the indicated lysine-to-alanine mutants. After 24 h cells were treated in the absence and presence of 100 ng/ml EGF as indicated. Cells were harvested the next day, and protein extracts were immunoprecipitated using anti-Flag M2 affinity resin. Immunoprecipitates were separated on 10% acrylamide gels containing SDS. Transfer blots were probed for MAGE-11 using HA tag antibody. X-ray film was exposed for 5 s (upper panel) and 20 min (lower panel). (C) In the left panel, COS cells were transfected with 3 μg pSG5-HA-MAGE-112-429 together with 6 μg Flagb (lanes 1 and 2), 6 μg Flag-Ub (lanes 3 and 4), and 6 μg Flag-Ub-KO, in which all ubiquitin lysine residues were changed to arginine (lanes 5 and 6). In the right panels, COS cells were transfected with 2 μg pCMV-AR together with 6 μg Flagb (lanes 7 to 10), 6 μg Flag-Ub and 3 μg pSG5-HA-MAGE-112-429 (lanes 11 to 14), and 6 μg Flag-Ub-KO and 3 μg pSG5-HA-MAGE-112-429 (lanes 15 to 18). Cells were incubated for 24 h in the absence and presence of 10 nM DHT and 10 ng/ml EGF as indicated, and extracts were prepared in IB (lanes 1 to 6) and IP (lanes 7 to 18) lysis buffer and analyzed by immunoblotting with AR32 and HA tag antibodies. (D) COS cells were transfected with 6 μg Flagb and 3 μg pSG5-HA-MAGE-112-429 (lane 1), 6 μg Flagb and 3 μg pSG5-HA-MAGE-112-429-K236A,K240A,K245A (HA-MAGE-112-3KA, lane 2), 6 μg Flag-Ub and 2 μg pCMV-AR (lanes 3 to 6), 6 μg Flag-Ub and 3 μg pSG5-HA-MAGE-112-429 in the absence (lanes 7 and 8) and presence (lanes 9 to 12) of 2 μg pCMV-AR, and 6 μg Flag-Ub with 3 μg pSG5-HA-MAGE-112-429-K236A,K240A,K245A (HA-MAGE-112-3KA) in the presence (lanes 13 to 16) and absence (lanes 17 and 18) of 2 μg pCMV-AR. Cells were incubated for 24 h in the absence and presence of 10 nM DHT and 100 ng/ml EGF as indicated and extracted in IP lysis buffer. The blots were probed with AR32, HA, and β-actin antibodies as indicated.

FIG. 7.

Phosphorylation sites in MAGE-11. (A) Effects of λ-phosphatase on the electrophoretic mobility of MAGE-11 serine mutants. COS cells were transfected with 2 μg wild-type (WT) pSG5-HA-MAGE-112-362 and with the indicated serine-to-alanine mutants. The next day cells were incubated for 24 h in the absence and presence of 100 ng/ml EGF as indicated. Media with and without EGF were replaced and cells were incubated an additional 2 h at 37°C and harvested in 0.2 ml IP lysis buffer without sodium fluoride. Protein extracts (2 μg) from EGF-treated cells were incubated in 40 μl for 1 h at 4°C with and without 400 IU λ-phosphatase (New England Biolabs). Samples were analyzed on 12% acrylamide gels containing SDS, and transfer blots were probed with HA tag antibody. Shown are the major (arrow) and slower-migrating ubiquitinylated forms of WT and mutant HA-MAGE-112-362. (B) MAGE-T360A lacks coactivator activity. CV1 cells were transfected with 5 μg PSA-Enh-Luc and 0.1 μg pCMV-hAR with and without 1 μg WT pSG5-MAGE and the S168A,S170A,S174A,S181A,S199A,S208A (6-SA) and T360A mutants as indicated. Cells were incubated in the absence and presence of 1 nM DHT for 48 h, and luciferase activity was determined. In the lower panel, expression levels of WT and mutant pSG5-MAGE were determined by transfecting COS cells with 4 μg pSG5 empty vector (lane 1), WT pSG5-MAGE (lane 2), and the 6-SA (lane 3) and T360A (lane 4) mutants. Protein extracts (2 μg/lane) were analyzed by immunoblotting using MagAb59-79 antibody. (C) Lack of AR AF2 transcriptional response to MAGE-T360A. CV1 cells were transfected with 5 μg MMTV-Luc and 0.1 μg pCMV-ARΔ120-472 in the absence and presence of 2 μg pSG5-TIF2 and 2 μg WT pSG5-MAGE, 6-SA, and T360A mutant as indicated. Cells were incubated in the absence and presence of 1 nM DHT for 48 h, and luciferase activity was measured. (D) Lack of AR AF1 transcriptional response to MAGE-T360A. HeLa cells were transfected with 0.1 μg PSA-Enh-Luc with 0.01 μg pCMV5 empty vector (p5) and 0.01 μg pCMV-AR1-660 in the absence and presence of 0.1 μg WT pSG5-MAGE, 6-SA, or T360A mutants as indicated. Luciferase activity was measured 48 h later. (E) Phosphorylation at MAGE-11 Thr-360. COS cells were transfected with 5 μg WT pSG5-HA-MAGE-112-429 and the T360A mutant and incubated for 24 h in the absence and presence of 100 ng/ml EGF and again in fresh medium for 2 h. Cells were extracted in IP lysis buffer, and equivalent amounts of protein were immunoprecipitated using an anti-HA affinity matrix (15 μl/470 μg protein). Immunoprecipitates were analyzed using HA tag antibody (1/10 of immunoprecipitate) and antiphosphothreonine antibody (9/10 of immunoprecipitate). Immunoblots were quantitated using an Image-Pro Plus spectrophotometer to obtain the ratio of phosphothreonine antibody to HA antibody reactivity. (F) Chk1 kinase phosphorylation of MAGE-11 Thr-360. An in vitro kinase assay was performed using GST-Chk1 kinase together with GST-²¹⁰GLYRSPSMPE²¹⁹, which is a GST-Cdc25C truncated fusion peptide substrate for Chk1 kinase (BioVision Inc.) (lane 1), GST-0 empty vector (lane 2), WT GST-MAGE-³⁵⁴EPKRLLTQN³⁶² (lane 3), and GST-MAGE-³⁵⁴EPKRLLAQN³⁶² T360A mutant (lane 4) (upper panel). Reactions were performed as described in Materials and Methods at 30°C for 30 min. The ∼30-kDa band is phosphorylated WT GST-MAGE-354-362, and the ∼82-kDa band appears to be autophosphorylated GST-Chk1 kinase. The rehydrated stained gel indicated equivalent protein loading (lower panel).

FIG. 8.

Functional requirement of MAGE-11 Thr-360. (A) Reduced AR interaction with MAGE-T360A. COS cells were transfected with 2 μg pCMV-AR together with 4 μg Flagb empty vector (lanes 1 to 4), Flag-MAGE (lanes 5 to 9), or Flag-MAGE-T360A (lanes 9 to 12). The next day cells were treated for 24 h and again for 2.5 h in the absence and presence of 10 nM DHT and 100 ng/ml EGF as indicated. Transfer blots containing the immunoprecipitates (top panel) and cell extracts (15 μg protein/lane) (bottom panel) were probed with anti-Flag M2 monoclonal, AR32, and β-actin antibodies. (B) Reduced ubiquitinylation of MAGE-T360A. COS cells were transfected with 6 μg Flagb (−) and FlagUb as indicated with 3 μg wild-type (WT) pSG5-HA-MAGE-112-429 (lanes 1 to 3), and the T360A (lanes 4 to 6), K236A,K240A,K245A (lanes 7 to 9), and S168A,S170A,S174A,S181A,S199A,S208A (6-SA; lanes 10 to 12) mutants. Cells were incubated the next day in the absence and presence of 100 ng/ml EGF and 24 h later harvested for immunoprecipitation using anti-Flag affinity resin. The blot was probed using HA tag antibody. (C) Two-hybrid assay showing loss of MAGE-T360A interaction with AR. HeLa cells were transfected with 0.1 μg 5XGAL4Luc3 and 0.05 μg WT GAL-MAGE, the T360A and K240A,K245A mutants, and pVP16 empty vector or VP-AR1-660 (VP-AR) as indicated, and with 0.05 μg WT VP-MAGE and the T360A and K240A,K245A mutants with 0.05 μg GAL-0 empty vector or GAL-AR16-36 (GAL-AR) as indicated. Luciferase activity was determined 48 h later. The lower panel shows protein extracts that were analyzed by immunoblotting from COS cells transfected with 8 μg GAL-0 empty vector, WT GAL-MAGE, and the T360A and K240A,K245A mutants (50 μg protein/lane) and pVP16 empty vector, WT VP-MAGE, and the T360A and K240A,K245A mutants (60 μg protein/lane) as indicated. Transfer blots were probed using GAL-4 (left panel) and VP16 (right panel) antibodies.

FIG. 9.

Effects of MAGE-11 on AR transcriptional activity in Ishikawa cells. (A) Ishikawa cells (10⁵/well in 12-well plates) were transfected using FuGENE-6 with 0.1 μg PSA-Enh-Luc, 0.01 μg pCMV-AR, and 0.1 μg pSG5, wild-type pSG5-MAGE, or the S168A,S170A,S174A,S181A,S199A,S208A (6-SA), T360A, and K236A,K240A,K245A mutants as indicated. Cells were incubated in the absence and presence of 1 nM DHT and 100 ng/ml EGF for 24 h as indicated, and luciferase activity was measured. (B) Time dependence of the EGF-induced increase in AR1-660 activity. Ishikawa cells (7.5 × 10⁴/well) were plated (day 1) in separate 12-well plates according to the time of harvest and transfected (day 2) with 0.1 μg MMTV-Luc and 0.05 μg pCMV5 (p5) and 0.05 μg pCMV-AR1-660 as indicated using FuGENE-6. On day 3, cells were cultured in the absence and presence of 10 ng/ml EGF. The next day (day 4), the media were exchanged and cells were harvested for luciferase activity (day 3, 24 h) or further incubated for the indicated times in the absence and presence of 10 ng/ml EGF. Cells were solubilized in luciferase lysis buffer and lysates were stored at −20°C until analysis for luciferase activity, which was stable to freeze-thaw. (C) Inhibition of AR transcriptional activity by MAGE-11 siRNA. Ishikawa cells (10⁵/well in 12-well plates) were transfected using Lipofectamine 2000 with 0.1 μg MMTV-Luc and 0.025 μg pCMV-AR in the absence and presence of 2 nM MAGE-11 siRNA-2 and siRNA-3 as indicated. Twenty-four h after transfection, cells were placed in serum-free, phenol red-free medium without hormone treatment. After 24 h the medium was replaced with fresh medium with and without 0.1 nM DHT and 10 ng/ml EGF as indicated. Cells were cultured another 24 h, and luciferase activity was measured. (Inset) Inhibition of MAGE-11 expression by siRNA. COS cells (4 × 10⁵/well in six-well plates) were transfected using Lipofectamine 2000 in Dulbecco's modified Eagle's medium without antibiotics with 0.5 μg pSG5 (p5, lane 1) and 0.5 μg pSG5-MAGE in the absence (lane 2) and presence of 10 nM MAGE-11 siRNA-1 (lane 3), siRNA-2 (lane 4), and siRNA3 (lane 5). Cells were extracted in IB lysis buffer and analyzed (10 μg/lane) on immunoblots using MagAb13-26 and β-actin antibodies.

FIG. 10.

Sequence alignment of the MAGE superfamily. Amino acid sequence alignment was performed using PredictProtein (http://www.predictprotein.org) and is shown for MAGE-11 of the MAGE-A subfamily, the first four members of the MAGE-A subfamily, and the primary member of MAGE subfamilies B to H. Amino acid residues are color coded for lysine and arginine (red), aspartic and glutamic acid (blue), glycine, alanine, valine, leucine, isoleucine, proline, and methionine (green), cysteine (yellow), and Thr-360 (purple). Also indicated for MAGE-11 is the nuclear localization signal (NLS, residues 18 to 23), the first and second (Met-111) methionine residues, the MAGE homology domain (MHD) residues 222 to 421, Lys-240 and Lys-245 ubiquitinylation sites, and the Thr-360 phosphorylation site. Absence of sequence and minimal interior spacing for optimal sequence alignment are designated by a dash. In two instances, lowercase letters signify deletion of an intervening proline.