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. 2010 Apr 1;24(7):708-19.
doi: 10.1101/gad.568410.

CARM1 mediates the ligand-independent and tamoxifen-resistant activation of the estrogen receptor alpha by cAMP

Sophie Carascossa  1 , Peter Dudek, Bruno Cenni, Pierre-André Briand, Didier Picard
Affiliations

CARM1 mediates the ligand-independent and tamoxifen-resistant activation of the estrogen receptor alpha by cAMP

Sophie Carascossa et al. Genes Dev. .

Abstract

The estrogen receptor alpha (ERalpha) is activated as a transcription factor by both estrogen and a large variety of other extracellular signals. The mechanisms of this ligand-independent activation, notably by cAMP signaling, are still largely unknown. We now close the gap in the signaling pathway between cAMP and ERalpha. Whereas the direct phosphorylation of ERalpha by the cAMP-activated protein kinase A (PKA) is dispensable, the phosphorylation of the coactivator-associated arginine methyltransferase 1 (CARM1) by PKA at a single serine is necessary and sufficient for direct binding to the unliganded hormone-binding domain (HBD) of ERalpha, and the interaction is necessary for cAMP activation of ERalpha. Sustained PKA activity promoting a constitutive interaction may contribute to tamoxifen resistance of breast tumors. Binding and activation involve a novel regulatory groove of the ERalpha HBD. As a result, depending on the activating signal, ERalpha recruits different coactivator complexes to regulate alternate sets of target genes.

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Figures

Figure 1.
Figure 1.
The role of ERα domains and phosphorylation sites. These luciferase reporter assays were carried out in ERα-negative cells. Data shown are averages of at least triplicate samples, expressed relative to that of the very first sample in each panel. (A) Schematic representation of ERα domains and corresponding Gal4 DNA-binding domain fusion proteins. (B) Response of ERα deletion mutants to cAMP/PKA. (C) PKA response of ERα domains expressed as chimeras with the DNA-binding domain of Gal4. (D) cAMP responses of ERα phosphorylation mutants. SKBr3 (B,C) and 293T (D) cells were used for the experiments. (wt) Wild-type; (cAMP) 8-Br-cAMP.
Figure 2.
Figure 2.
A new surface of the ERα HBD is required for the cAMP response. (A) G400-centered views of a portion of the ERα HBD dimer structure. The model was generated with the data of PDB file 1A52. The left panel is a cartoon view showing the relative positions of the mutagenized amino acids (G400, R412, L429, S433, S463, and S464). The panel on the right shows a surface view, which is tilted slightly to the right relative to the view in the left panel. The two subunits were colored in orange and purple. (B,C) Ser 464 is a key determinant of the cAMP response of full-length ERα. Graphs of luciferase reporter assays in B and C show cAMP and E2 responses, respectively, of wild-type (WT) and mutant ERα in transfected 293T cells. Where indicated with a plus sign (+), cells were treated with 8-Br-cAMP (cAMP) or E2.
Figure 3.
Figure 3.
CARM1 interacts with and is required for the cAMP response of ERα. (A) The effect of overexpression of CARM1 on the activity of endogenous ERα was determined with a luciferase reporter by transient transfection of MCF7 cells. (B) CARM1 interacts with ERα in MCF7 cells in a cAMP-dependent manner. Co-IP experiment of endogenous proteins. Cells were stimulated with 8-Br-cAMP 2 h before lysis. Equal amounts of extracts (bottom panel: Ponceau S-stained filter) were immunoprecipitated with an antibody against ERα or control (Ctrl) antibodies, and immunoblots were probed with antibodies to the endogenous CARM1 (top panel) or ERα (middle panel). (IgH) Heavy chain of antibodies. (C) E2 and cAMP induce the recruitment of CARM1 to the pS2 promoter. DNA gel visualizing the PCR products of a ChIP experiment with CARM1 and control (Ctrl) antisera. (D,E) CARM1 is necessary for the cAMP response of endogenous ERα in MCF7-SH cells. (D) Luciferase assay of cells cotransfected with the indicated shRNA constructs. Graphs show averages of the means of three independent experiments, each with triplicate samples. (E) Quantitative RT–PCR analysis of the endogenous ERα target gene pS2 with EEF1A1 internal standard. In this case, stable RNAi was obtained by infection with corresponding lentiviral preparations. Each data point represents the average of the data of three independent experiments, standardized to the value of the control shRNA sample, arbitrarily set to 1. In contrast to the induction by E2 ([#] P > 0.37), the cAMP induction is significantly reduced by the CARM1 knockdown ([##] P < 0.057).
Figure 4.
Figure 4.
Potentiation of ERα mutants by CARM1 and interaction correlate. (A) cAMP-induced but not basal activities of Gal4.ERα are potentiated by CARM1 overexpression. Luciferase reporter gene assays in 293T cells. (B) cAMP responses of mutant Gal4.ERα chimeras without and with coexpressed CARM1. (C) Co-IP of mutant Gal4.ERα chimeras with CARM1. HA-tagged CARM1 and the indicated wild-type (WT) or mutant versions of Gal4.ERα were coexpressed in 293T cells. Cells were stimulated with 8-Br-cAMP 1 h before lysis. Equal amounts of extracts (bottom panel: Ponceau-stained filter of gel run in parallel; bottom middle panel: immunoblot probed with an anti-Gal4 antibody) were immunoprecipitated with an anti-HA antibody. Immunoblots were probed with antibodies to Gal4 (top panel) or to the HA tag (top middle panel).
Figure 5.
Figure 5.
CARM1 is phosphorylated by PKA in vivo and in vitro. (A) CARM1 immunoblot of an IP with an antibody against substrates phosphorylated by PKA (“IP PKA substrates”). MCF7 cells were treated with 8-Br-cAMP or vehicle 2 h before lysis. (B) CARM1 S448 is the main target of PKA in vivo. Wild-type (wt) and mutant HA-CARM1 were expressed in 293T cells and analyzed as in A, except that the membrane was probed with an anti-HA antibody. (C) In vitro phosphorylation of CARM1 by PKA. Recombinant purified His6-tagged CARM1 (wt or S448A as indicated) was phosphorylated with the catalytic β subunit of PKA and analyzed by immunoblotting with a monoclonal against phospho-Ser/Thr.
Figure 6.
Figure 6.
Direct interaction of CARM1 with ERα depends on direct phosphorylation of CARM1 by PKA. (A) In vitro phosphorylated CARM1 interacts directly with the ERα HBD. Following phosphorylation of His6-tagged CARM1 (wild type, S448A, or S448E) by PKA, its interaction with the ERα HBD was assessed by a GST pull-down experiment. Inputs represent 20% of each reaction. (B) The S448A CARM1 phosphorylation mutant is defective for stimulation of the cAMP response of ERα. Luciferase reporter gene assays showing stimulation of cAMP responses of ERα by wild-type and mutant CARM1 in cotransfected 293T cells. Note that expression levels of the three CARM1 versions were similar (data not shown).
Figure 7.
Figure 7.
Increased PKA activity promotes the constitutive and tamoxifen-resistant interaction of ERα and CARM1 in LCC2 cells. (A) Immunoblot showing constitutive interaction. Equal amounts of MCF7 and LCC2 cell extracts (bottom panel: Ponceau staining) were immunoprecipitated with an anti-CARM1 antibody, and immunoblots were probed with antibodies to the endogenous ERα (top panel) or CARM1 (middle panel). (B) CARM1 is constitutively phosphorylated in LCC2 cells. The experiment was done as described in the legend for Figure 5A. (C) The constitutive interaction between CARM1 and ERα in LCC2 cells depends on PKA activity and is not affected by OHT. Wild-type (wt) MCF7 and LCC2 cells were treated with 8-Br-cAMP, OHT, 8-Br-cAMP + OHT, or H89 4 h before lysis. Extracts were immunoprecipitated with an anti-ERα antibody, and immunoblots were probed with antibodies to endogenous CARM1 (top and middle panels) or ERα (bottom panel). (D) OHT activates ERα in LCC2 in a PKA- and CARM1-dependent fashion. Luciferase reporter gene assays of endogenous ERα in cells cotransfected with shRNA constructs and treated as indicated. Activities are expressed relative to that of the control sample for each transfection. (E) Quantitative RT–PCR analysis of the endogenous ERα target gene pS2 with EEF1A1 internal standard. In this case, stable RNAi was obtained by infection with corresponding lentiviral preparations. The color code of the bars is the same as in D. Each data point represents the average of the data of three independent experiments, standardized to the values of the control samples of the two cell lines that were arbitrarily set to 1. The differences of the highlighted comparisons are highly significant ([#] P < 0.01; [##] P < 0.03).
Figure 8.
Figure 8.
Model of CARM1-mediated activation of ERα by cAMP signaling. For comparison, the E2-induced indirect Grip1-mediated recruitment of CARM1 is illustrated. Irrespective of the presence of OHT, PKA-phosphorylated CARM1 interacts with ERα and may function as a pioneer factor, allowing the subsequent binding of others. (AF2 coact) Transcriptional coactivators that are recruited to AF2 (Grip1 being one of them). The scheme also highlights that the two signals induce the assembly of different complexes, resulting in distinct transcriptional programs.
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