PMC

Fig. 4. Rsk2 physically associates with ERα. (A) BHK cells were co-transfected with control vector or a vector encoding ERα, plus expression vectors encoding HA-tagged wild-type Rsk2, HA-Rsk2(Y707A) or HA-Rsk2(K100A/Y707A), then serum-starved, lysed, and ERα and associating proteins immunoprecipitated with anti-ERα antibody. Aliquots of the immunoprecipitates and lysates were immunoblotted. (B) BHK cells were co-transfected with a vector encoding HA-tagged HA-Rsk2(Y707A) plus control vector or a vector encoding wild-type ERα, or ERα(S167A). Immunoprecipitates and immunoblots were performed as in (A). (C) BHK cells were co-transfected as for (B) except that a vector encoding ERα(1–281) was used instead of ERα(S167A). Immunoprecipitates and immunoblots as in (A).

Fig. 1. Schematic of Rsk2 and ER constructs. (A) Wild-type Rsk2 contains two kinase domains separated by a linker region. Phos phorylation by MAPK or mutation of Y707A leads to activation of the CTKD (1). Autophosphorylation by the CTKD (2) leads to activation of the NTKD by PDK1 (3). The locations of residues for mouse Rsk2 are shown. (B) The domains and locations of the residues for human wild-type ERα, rat wild-type ERβ and the constructs used for this work are shown. A black bar indicates an inactivated region.

Fig. 7. Model of Rsk2 activation. (A) Schematic of Rsk2 activation of ERα-mediated transcription. See text for details. (B) Ribbon representation of the ERα HBD complexed to either estradiol or 4-OHT. These models were generated by RasMol and are based on the coordinates under Protein Data Bank entry codes 1ERE and 3ERT (; ). Ligands are represented in green. Residues have been colored pink (301–341), orange (H3), blue (363–371), purple (H5 and H6) and red (H12).

Fig. 3. Ser167 is important in Rsk2 activation of ERα-mediated transcription. (A) BHK cells were transfected with a vector encoding ERα, treated with E₂, EGF or vehicle, then lysed and aliquots immunoblotted. (B) BHK cells were transfected and treated with vehicle or E₂ as in Figure A except that an expression vector encoding the mutant ERα(S167A) was also used. Data were normalized as in Figure A. Means ± SE are shown. **P <0.005 and *P <0.05, obtained by comparing the response obtained with ERα and HA-Rsk2(Y707A) with that obtained with ERα(S167A) and HA-Rsk2(Y707A). The inset shows immunoblots of lysates of BHK cells transfected with ERα and HA-Rsk2(Y707A) or ERα(S167A) and HA-Rsk2(Y707A). (C) BHK cells were transfected and treated with vehicle or EGF as in Figure A except that an expression vector encoding ERα(1–281) was also used. Data were normalized initially as in Figure A, then expressed as the percentage increase relative to the vehicle control with ERα (1–281). Values are means ± SE. **P <0.005, obtained by comparing the response obtained with ERα and HA-Rsk2(Y707A) with that obtained with ERα(1–281) and HA-Rsk2(Y707A). The inset shows immunoblots of lysates of BHK cells transfected with ERα and HA-Rsk2(Y707A) or ERα(1–281) and HA-Rsk2(Y707A).

Fig. 5. Rsk2 specifically binds to the region from 326 to 394 of the HBD of ERα. (A) BHK cells were co-transfected with a vector encoding a deletion mutant of ERα fused to GFP and either HA-tagged RSK2(Y707A) or control vector, then treated with E₂ or vehicle. Immunoprecipitates were obtained as in Figure except that HA-Rsk2(Y707A) and associated proteins were immunoprecipitated using anti-HA antibody. Aliquots of the immunoprecipitates and lysates were immunoblotted. (B) BHK cells were transfected with a vector encoding HA-Rsk2(Y707A) and either control vector or a vector encoding wild-type ERα or the chimera ERαβ. Immunoprecipitates and immunoblots as in Figure . (C) BHK cells were transfected with a vector encoding a myc-tagged fragment of ERα and either HA-Rsk2(Y707A) or control vector. Immunoprecipitates and immunoblots as in (A). (D) BHK cells were transfected with a vector encoding a myc-tagged deletion fragment of ERα or ERβ. Additionally, the cells were co-transfected with either a vector encoding HA-Rsk2(Y707A) or control vector. Immunoprecipitates and immunoblots as in (A). (E) Schematic showing homology between the region in ERα that binds to Rsk2 and the analogous region of ERβ. The secondary structure of these regions is also shown, in which the rectangles represent helices ().

Fig. 6. Rsk2 activation requires AF2 activity. (A) BHK cells were co-transfected with an expression vector encoding ERα, ERβ, ERαβ or ERβα plus vectors encoding HA-Rsk2(Y707A) or the N-terminal-kinase-dead mutant, Rsk2(K100A/Y707A), as well as reporter and β-galactosidase expression vectors. The transfected cells were treated with vehicle or E₂ and the data analyzed as in Figure A. Data were normalized initially as in Figure A, then expressed as the percentage increase relative to the vehicle control with either ERβ, ERαβ or ERβα. Values are means ± SEM. ***P <0.0005, **P <0.01 and *P <0.05, obtained by comparing the response obtained with vector encoding Rsk2(K100A/Y707A) with that obtained with the vector encoding HA-Rsk2(Y707A). The inset shows immunoblots of lysates of BHK cells transfected with ERα and HA-Rsk2(Y707A) or ERαβ and HA-Rsk2(Y707A). (B) BHK cells were transfected, treated and the data analyzed as in (A) except that the cells were transfected with a vector encoding ERα or ERα-AF1. Values are means ± SEM. **P <0.01and *P <0.05, obtained by comparing the response obtained with ERα with that obtained with ERα-AF1. The inset shows immunoblots of lysates of BHK cells transfected with ERα and HA-Rsk2(Y707A) or ERα-AF1 and HA-Rsk2(Y707A). (C) BHK cells were transfected and serum-starved as in (A), then treated with either 1 µM ICI 182,780 or 0.5 µM 4-OHT. Control transfections were treated with vehicle or E₂. Data were normalized so that in the absence of activated Rsk2 the response to vehicle addition by ERα was 100. Data shown are from one experiment that is representative of two experiments each performed in quadruplicate. (D) BHK cells were co-transfected with a vector encoding HA-Rsk2(Y707A) plus a control vector or a vector encoding ERα, then serum-starved and treated with vehicle or 0.5 µM 4-OHT. Immunoprecipitates and immunoblots were performed as in Figure A. (E) BHK cells were co-transfected with a vector encoding ERα and either a control vector or a vector encoding HA-Rsk2(Y707A), then treated with E₂, 4-OHT or vehicle, lysed and aliquots immunoblotted.

Fig. 2. Constitutively active Rsk2, in the absence of active MAPK, specifically enhances ERα-mediated transcription. (A) BHK cells were co-transfected with ERα, an ERE-regulated luciferase reporter and β-galactosidase expression vectors. Additionally, the cells were transfected with a control vector or vectors encoding HA-Rsk2, HA-Rsk2(Y707A) or HA-Rsk2(K100A/Y707A), serum-starved and treated with vehicle (–), 10 nM estradiol (E₂), 100 ng/ml EGF or both E₂ and EGF. Luciferase and β-galactosidase activity were determined 19 h after hormone addition. The luciferase data were divided by the β-galactosidase activity to control for differences in transfection efficiency. To facilitate comparison between the different experiments, the data were normalized so that, in the absence of activated Rsk2, the response to vehicle addition by ERα was zero and the response of ERα to E₂ was 100. The inset shows immunoblots of lysates of BHK cells that were transfected with ERα, ERα and HA-Rsk2, or ERα and HA-Rsk2(Y707A). (B) BHK cells were transfected and treated as for (A) except that during hormone treatment either 2 µM of PD98059 or vehicle was also added. Data were normalized as in (A). (C) BHK cells were transfected and treated with vehicle or E₂ as for (A) except that an expression vector encoding ERβ was used. Data were normalized to those obtained for ERα as in (A), then expressed as the percentage increase relative to the vehicle control with ERβ. (D) MCF-7 cells were co-transfected with ERE-regulated luciferase reporter and β-galactosidase expression vectors. Additionally, the cells were transfected with a control vector or the vector encoding HA-Rsk2(Y707A). Transfected cells were serum-starved and treated with the indicated agent. After 5.5 h, the cells were lysed, and luciferase and β-galactosidase activity determined. Data were normalized as in (A). In each panel, values are means ± 1 SEM. ***P <0.0005, **P <0.01 and *P <0.05 (Student’s t-test) obtained by comparing the response obtained with vector control with that obtained with the vector encoding HA-Rsk2(Y707A).