Regulation of androgen receptor-dependent transcription by coactivator MED1 is mediated through a newly discovered noncanonical binding motif

Abstract

Nuclear receptor (NR) activation by cognate ligand generally involves allosteric realignment of C-terminal α-helices thus generating a binding surface for coactivators containing canonical LXXLL α-helical motifs. The androgen receptor (AR) is uncommon among NRs in that ligand triggers an intramolecular interaction between its N- and C-terminal domains (termed the N/C interaction) and that coactivators can alternatively bind to surfaces in the AR N-terminal or hinge regions. The evolutionary conserved Mediator complex plays a key coregulatory role in steroid hormone-dependent transcription and is chiefly targeted to NRs via the LXXLL-containing MED1 subunit. Whereas MED1 has been demonstrated to serve as a key transcriptional coactivator for AR, the mechanisms by which AR recruits MED1 have remained unclear. Here we show that MED1 binds to a distinct AR N-terminal region termed transactivation unit-1 (Tau-1) via two newly discovered noncanonical α-helical motifs located between MED1 residues 505 and 537. Neither of the two MED1 LXXLL motifs is required for AR binding, whereas loss of the intramolecular AR N/C interaction decreases MED1 binding. We further demonstrate that mitogen-activated protein kinase phosphorylation of MED1 enhances the AR-MED1 interaction in prostate cancer cells. In sum, our findings reveal a novel AR-coactivator binding mechanism that may have clinical implications for AR activity in prostate cancer.

FIGURE 1.

AR recruits Mediator via interaction with MED1. A, LNCaP cells were transfected with MED1 siRNA or a nonspecific scrambled control siRNA and then probed by immunoblot with antibodies against MED1 and α-tubulin. B, androgen-starved LNCaP cells were transfected with siRNAs for 48 h and then treated with or without 10 nm DHT for 24 h. Whole cell extract was prepared and incubated with antibodies against AR conjugated with protein-A beads. Immunoprecipitated protein was then analyzed by immunoblot with antibodies indicated on the right of each panel. C and D, chromatin prepared from LNCaP cells (treated as described in B) was used for ChIP analyses using the indicated antibodies. Semi-quantitative PCR was performed using primer sets spanning AREs located at the Cdc6 promoter (C) or PSA enhancer (D).

FIGURE 2.

MED1 interacts with the AR Tau-1 domain. A, schematic representation of AR deletion and point mutants. Internal deletions are indicated by horizontal lines. B, D, and F, COS cells transfected with FLAG-tagged AR or AR mutant expression vectors were probed with an anti-FLAG antibody. Molecular mass markers (in kilodaltons) are indicated on the left. C, E, and G, androgen-starved COS cells were transiently transfected with FLAG-tagged AR or AR mutant expression vectors (indicated at the top of panels) along with a full-length HA-MED1 expression vector and then cultured with or without 10 nm R1881 for 24 h. Whole cell lysate was then prepared and incubated with anti-HA agarose beads. Immunoprecipitated protein was then analyzed by anti-FLAG and anti-MED1 immunoblotting.

FIGURE 3.

AR interacts with the MED1 N terminus independently of LXXLL motifs. A, schematic representation of MED1 deletion and point mutants. LXXLL motifs are indicated by black bars; LXXLL to LXXAA are indicated by open bars. ERK1/2 phosphorylation sites (threonines 1032 and 1457) are indicated by filled circles; ERK1/2 phosphorylation site mutations (Thr to Ala) are indicated by open circles. B–D, androgen-starved COS cells were transiently transfected with HA-tagged full-length MED1 (FL) or MED1 deletion mutant expression vectors (indicated above the panels) together with a full-length FLAG-AR expression vector and subsequently cultured with or without 10 nm R1881 for another 24 h. Whole cell lysate was then incubated with anti-HA agarose beads, and the precipitated proteins were probed with anti-AR, anti-HA, and anti-MED1 immunoblotting. Arrows indicate specific ectopically expressed MED1 full-length or truncated polypeptides.

FIGURE 4.

DNA-bound AR binds to the MED1 N terminus in vitro. A, schematic representation of biotinylated-ARE pull-down assay. B, purified baculovirus-expressed full-length MED1 and MED1 deletion mutants fractionated by SDS-PAGE and stained with Coomassie blue. C, MED1 and AR form a complex on DNA. Purified baculovirus-expressed full-length MED1 (bv-MED1-FL) and AR (bv-AR) were incubated with a biotinylated ARE corresponding to the androgen-responsive unit in the first intron of the C3(1) gene (40). DNA-bound protein complexes were precipitated with streptavidin beads, washed, fractionated by SDS-PAGE, and then immunoblotted with anti-MED1 and anti-AR antibodies. D, purified baculovirus-expressed full-length MED1 or MED1 deletion mutants were incubated together with bv-AR and a biotinylated ARE and processed as described in C.

FIGURE 5.

AR binds to a novel noncanonical α-helical array in the MED1 RBD. A, schematic representation of the GST-MED1-RBD (amino acids 501–738) and mutant derivative fusion proteins. LXXLL motifs are indicated by black bars; LXXLL to LXXAA are indicated by open bars. B, predicted secondary structure of MED1 amino acid residues 501–635. Five α-helical motifs (α1, α2, α3, α4, and α5) are indicated by horizontal cylinders. C, schematic representation of the GST-MED1-RBD1 (amino acids 501–635) and mutant derivative fusion proteins. The α-helices α1, α2, α3, α4, and α5 are indicated by black boxes. D and E, purified GST-MED1 fusion proteins were fractionated by SDS-PAGE and stained with Coomassie blue. F–H, GST pulldown assays were carried out by incubating [³⁵S]methionine-labeled AR (labeled in the presence of 10 nm DHT) together with GST-MED1-RBD and mutant derivative fusion proteins (see A and C). The bound proteins were detected by autoradiography. I, sequence alignment of the identified AR-binding noncanonical α-helical array in MED1 of different species.

FIGURE 6.

The tandem noncanonical α-helical array in the MED1 RBD is required for transcriptional coactivation of AR. A, COS cells were androgen-starved and transfected with expression vectors for HA-MED1 or HA-MED1Δα1,α2 (see Fig. 3A for schematic representation) along with full-length FLAG-AR and subsequently cultured with or without 10 nm R1881 for another 24 h. Whole cell lysate was then incubated with anti-HA agarose beads, and the precipitated proteins were probed with anti-AR and anti-MED1 immunoblotting. B, androgen-starved LNCaP cells were transfected with expression vectors for wild-type MED1, MED1Δα1,α2 or an empty vector control together with a MMTV-Luc reporter template. 3 h post-transfection, cells were treated with or without 10 nm R1881 for another 24 h. Whole cell lysate was then prepared and assayed for luciferase reporter activity and for MED1 expression by immunoblotting (shown at the bottom). C, androgen-starved LNCaP cells were transfected with expression vectors for wild-type MED1, MED1-CΔ454, MED1-CΔ918, or an empty vector control along with the MMTV-Luc reporter. Cells were treated with or without R1881 and assayed for luciferase reporter activity as outlined in A. Luciferase values were normalized by using a β-galactosidase expression vector as internal control and are presented as the mean ± S.E. of triplicate transfections.

FIGURE 7.

ERK1 phosphorylation of MED1 enhances its association with AR. A, COS cells were androgen-starved and then transiently transfected with expression vectors for HA-MED1 or MED1-ERK mutant together with full-length FLAG-AR and subsequently cultured with or without 10 nm R1881 for another 24 h. Whole cell lysate was then incubated with anti-HA agarose beads, and the precipitated proteins were probed with anti-AR and anti-MED1 immunoblotting. B, purified baculovirus-expressed full-length MED1 (bv-MED1-FL) or MED1-ERK mutant (bv-MED1-Erk mut) were incubated in kinase buffer containing (+) or lacking (−) purified ERK1 and then probed by immunoblot using anti-phospho-threonine (α-p-Thr) and anti-MED1 antibodies. C, purified bv-MED1-FL and bv-MED1-Erk mut were preincubated in kinase buffer containing (+) or lacking (−) purified ERK1. The entire kinase reactions were then incubated with bv-AR and a biotinylated ARE. DNA-bound protein complexes were precipitated with streptavidin beads, washed, fractionated by SDS-PAGE, and then immunoblotted with anti-MED1 and anti-AR antibodies. 30% of the bv-MED1-FL and bv-MED1-Erk mut protein input were loaded in lanes 1 and 2.

FIGURE 8.

ERK phosphorylation of MED1 stabilizes protein expression in prostate cancer cells. A–C, prostate cancer cell lines 1532T (A), DU145 (B), and LNCaP (C) were transiently transfected with expression vectors for ERK2-L73P/S151D, MKK1-NΔ4, MKK1–8E, or empty vector controls as indicated. Whole cell extract was prepared, and equivalent amounts of protein were probed by immunoblot using anti-MED1 and anti-tubulin antibodies.

FIGURE 9.

ERK phosphorylation of MED1 promotes its recruitment to the PSA enhancer. A–F, LNCaP cells were androgen-starved for 48 h and then treated with or without DHT and EGF (A, C, and E) or DHT and U0126 (B, D, and F) as indicated. A and B, chromatin was prepared and immunoprecipitated with anti-MED1 antibodies or nonspecific IgG. Semi-quantitative PCR was then performed using primer sets spanning the PSA distal enhancer. Image processing and quantification of the semi-quantitative PCR data were performed using Quantity One software (Bio-Rad). The results are presented as relative -fold induction of the enrichment over nonspecific IgG and normalized to input. C and D, PSA mRNA expression was determined by RT-PCR in real-time. Values were normalized to β-actin expression. E and F, equal amounts of whole cell extract were probed by immunoblot with antibodies against MED1 and α-tubulin. Error bars in A–D represent the S.D. calculated from three independent experiments.