Primate-specific Melanoma Antigen-A11 Regulates Isoform-specific Human Progesterone Receptor-B Transactivation

Abstract

Background: Progesterone regulates the cyclic function of the human endometrium through its receptors and coregulatory proteins. Results: Primate-specific melanoma antigen-A11 (MAGE-11) interacts with the human progesterone receptor-B (PR-B) unique NH 2 -terminal region to coregulate progesterone-dependent gene activation. Conclusion: MAGE-11 is an isoform-specific coregulator of human PR-B. Significance: PR-B and progesterone regulation of human endometrium requires a primate-specific steroid receptor coregulator.

Melanoma Antigen-A11 (MAGE-A11) Enhances Transcriptional Activity by Linking Androgen Receptor Dimers

“…Coimmunoprecipitation was performed using two dishes each of COS1 cells (1.8 ϫ 10 6 /10-cm dish) expressing FLAG empty vector or FLAG-AR with pSG5-MAGE, pSG5-HA-p300, and/or AR mutants. One day after transfection using DEAE-dextran, cells were incubated in the absence or presence of 10 nM DHT with 50 or 100 ng/ml EGF for 24 h. Cell extracts and immunoprecipitates were prepared as described (5,6). Gels were calibrated using dual color Precision Plus Protein Standards (Bio-Rad) and probed using the following antibodies: peptide purified rabbit polyclonal MAGE-(94 -108) antibody against human MAGE-(94 -108) peptide (10 g/ml) (30), peptide purified rabbit polyclonal FLAG-MAGE antibody-1 against baculovirus-expressed FLAG-MAGE (0.5 g/ml for expressed MAGE-A11 and 10 g/ml for endogenous MAGE-A11) (7), rabbit polyclonal AR32 (33) and AR52 (15) anti-peptide antibodies (1 g/ml), rabbit polyclonal human p300 (C-20) sc-585 antibody (Santa Cruz Biotechnology; 1:200 dilution), mouse anti-FLAG M2 F3165 monoclonal antibody (Sigma; 1:2000 dilution), rabbit polyclonal HA antibody ab9110 (Abcam; 1:2000 dilution), and mouse monoclonal anti-␤-actin ab6276-100 (Abcam; 1:5000 dilution).…”

“…Lentivirus short hairpin RNA (shRNA) knockdown of MAGE-A11 was performed in LAPC-4 cells using virus prepared from the Open Biosystems TRC1 shRNA library (5). LAPC-4 cells (3 ϫ 10 6 /well) in 6-well plates were transduced with 0.15 ml of HEK293 cell medium containing ϳ10 6 lentivirus particles/ml.…”

“…COS1 cell extracts were prepared, analyzed on acrylamide gels containing SDS, and probed as described (5). Coimmunoprecipitation was performed using two dishes each of COS1 cells (1.8 ϫ 10 6 /10-cm dish) expressing FLAG empty vector or FLAG-AR with pSG5-MAGE, pSG5-HA-p300, and/or AR mutants.…”

“…The AR FXXLF motif region also serves as the interaction site for melanoma antigen-A11 (MAGE-A11), a primate-specific coregulator of human AR (4). MAGE-A11 also functions as an isoform-specific coregulator of full-length human progesterone receptor-B through an interaction with the progesterone receptor-B NH 2 -terminal LLXXVLXXLL motif, which is absent in the shorter A form (5). MAGE-A11 increases steroid receptor transcriptional activity in primates through interactions with the p300 histone acetyltransferase (6) and p160 steroid receptor coactivators (7).…”

Post-translational Down-regulation of Melanoma Antigen-A11 (MAGE-A11) by Human p14-ARF Tumor Suppressor

“…MAGE-A11 is a cancer-testis antigen that resides predominantly in the nucleus at low levels in normal human reproductive tract tissues and at higher levels in castration-resistant prostate cancer (1)(2)(3)(4)(5)(6). Although named initially for its identification in melanoma (7,8), MAGE-A11 is a coregulator of human AR and progesterone receptor-B (1,6,9). MAGE-A11 increases AR transcriptional activity by binding an extended AR NH 2 -terminal FXXLF motif region independent of AR binding the active androgens, testosterone or dihydrotestosterone (DHT) (1).…”