PMC

Both AF-1 and LBD/AF-2 domains of ERα are required for maximal induction of SRC-3 phosphorylation in response to E2. HEK293T cells were cotransfected with either Flag-SRC-3 (SRC-3) or Flag-SRC-3 phosphorylation mutant (A6) together with either the full-length wild-type ERα (ERα) (A and C), or ERα1-282 (N282) or ERα179-595 (179C) (A), or the full-length wild-type ERβ (ERβ) or ERβ143-530 (143C) (B), or ERα AF-1 phosphorylation mutant 3A (S104/106/118A) (C). Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. Immunoblots were probed with SRC-3 phosphorylation state-specific antibodies for T24, S505, S543, and S867. Total cellular levels of wild-type ER and its mutants and SRC-3 were also determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies.

The receptor-interacting domain of SRC-3 is required for estradiol-induced SRC-3 phosphorylation. HEK293T cells were transfected with either wild-type Flag-SRC-3 (SRC-3) or SRC-3 mutant (AAA) having all three LXXLL motifs in the receptor-interacting domain mutated to LXXAA, together with wild-type ERα. Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The level of SRC-3 phosphorylated at each of the six phosphorylation sites was assessed by immunoblotting with each SRC-3 phosphorylation state-specific antibody (rows 1 to 6). The level of SRC-3-bound ERα was determined by immunoblotting with an anti-ERα antibody (row 7). Total cellular levels of wild-type SRC-3 and its mutant and ERα were also determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies (rows 8 and 9).

Effects of different agonists and antagonists and their receptors on SRC-3 phosphorylation. HEK293T cells were transfected with Flag-tagged wild-type SRC-3 (SRC-3) or its phosphorylation-site mutant (A6) together with either ERα (A and B), ERβ (C), or progesterone receptor B (PR-B) (A). Forty-eight hours posttransfection, vehicle (−), 10 nM E2, 10 nM progesterone (P), 10 nM 4HT, 10 nM ICI 182,780 (ICI), or 10 nM Ral was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The levels of SRC-3 phosphorylated at S505 and S867 were assessed by Western blotting using SRC-3 phosphorylation state-specific antibodies. Total cellular levels of ERα, ERβ, SRC-3, and PR-B were also determined by immunoblotting total cell lysates separated by SDS-PAGE with the appropriate antibodies.

Estradiol induction of SRC-3 phosphorylation. (A) Schematic diagram of SRC-3. The known functional domains of SRC-3 are shown on top: bHLH/PAS, basic helix-loop-helix/Per-Arnt-Sim domain; RID, receptor-interacting domain; CID, CBP/p300-interacting domain; HAT, histone acetyltransferase domain. The identity and position of the six identified phosphorylated amino acids of SRC-3 are shown below. (B) Estradiol induced an ER-dependent change in electrophoretic mobility of SRC-3. HEK293T cells were transfected with plasmids for either Flag-tagged wild-type SRC-3 (SRC-3) or a Flag-SRC-3 mutant (A6) in which alanines were substituted for all six phosphorylation sites (S/T to A), and either ERα or ERβ, or empty vectors (V). Forty-eight hours posttransfection, 0.01% ethanol (−) or 10 nM estradiol (E2; +) was added for 1 h. Cells were then lysed and analyzed by Western blotting using an anti-SRC-3 antibody.

Estradiol-induced and ER-dependent SRC-3 phosphorylation does not require ER DNA binding. (A) HEK293T cells were cotransfected with pERE-E1b-Luc, and either the empty vector (Vector), or vectors for wild-type ERα (WT), or the EG (E203A/G204A) or CC (C202H/C205H) double ERα DNA-binding domain mutant. Twenty-four hours posttransfection, vehicle or E2 (10 nM) was added for an additional 20 h. The luciferase activity was assayed as described in Materials and Methods. (B) HEK293T cells were cotransfected with either Flag-SRC-3 wild type (SRC-3) or its phosphorylation mutant (A6), together with either the wild-type ERα (ERα), or the ERα double mutant EG or CC. Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The level of SRC-3 phosphorylated at S505, S543, and S867 was assessed by Western blotting using the SRC-3 phosphorylation state-specific antibody against each site (rows 1 to 3). The level of SRC-3-bound ERα was determined by immunoblotting with an anti-ERα antibody (row 4). Total cellular levels of wild-type ERα and its mutants and SRC-3 were also determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies (rows 5 and 6).

Nuclear localization of ERα is not required for estradiol-induced SRC-3 phosphorylation. (A) HEK293T cells were cotransfected with GFP-tagged wild-type ERα (GFP-WT) or ERα NLS deletion mutant lacking aa 250 to 303 (GFP-ΔNLS), together with Flag-SRC-3. Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cell fixing and preparation were performed, and GFP fluorescence signals were determined microscopically (column 1). DNA was counterstained with DAPI (column 2), and the signals for GFP and DAPI were merged (column 3). (B) HEK293T cells were transfected with either Flag-SRC-3 wild type (SRC-3) or its phosphorylation mutant (A6), together with either wild-type ERα (WT), or ERα mutant (ΔNLS). Forty-eight hours posttransfection, E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The level of SRC-3 phosphorylated at each of the six phosphorylation sites was assessed by immunoblotting with each SRC-3 phosphorylation state-specific antibody (rows 1 to 6). The level of SRC-3-bound ERα was determined by immunoblotting with an anti-ERα antibody (row 7). Total cellular levels of ERα and SRC-3 were determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies (rows 8 and 9).

The coactivator-binding groove of ERα is required for estradiol-induced and ER-dependent SRC-3 phosphorylation. (A) HEK293T cells were cotransfected with pERE-E1b-Luc and either the empty vector (Vector), the ERα wild type (WT), the ERα single mutants (K362D as K, V376D as V, or L539A as L), the ERα double mutant (K362D/V376D as KV), or the ERα triple mutant (K362D/V376D/L539A as KVL). Twenty-four hours posttransfection, vehicle or E2 (10 nM) was added for an additional 20 h before cells were harvested for luciferase assays. The error bars represent the standard errors of the means of three individual experiments. (B) HEK293T cells were transfected with either Flag-SRC-3 or Flag-SRC-3 phosphorylation mutant (A6) together with full-length wild-type, single mutant, or triple mutant forms of ERα. Forty-eight hours posttransfection, vehicle or E2 (10 nM) was added for 1 h. Cells were then lysed, and Flag-tagged SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. The level of SRC-3 phosphorylated at S505 was assessed by Western blotting using an SRC-3 phosphorylation state-specific antibody. The level of SRC-3-bound ERα was determined by immunoblotting with an anti-ERα antibody. Total cellular levels of wild-type ERα and its mutants and SRC-3 were also determined by immunoblotting total cell lysates separated by SDS-PAGE with appropriate antibodies.

Estradiol-induced SRC-3 phosphorylation requires the presence of ER. (A) HEK293T cells were transfected with Flag-tagged SRC-3 (SRC-3) or its phosphorylation mutant (A6), in the absence or presence of ERα. Forty-eight hours later, 10 nM E2 (+) or 0.01% ethanol vehicle (−) was added for 1 h. Cells were then lysed, and Flag-SRC-3 was immunoprecipitated by anti-Flag antibody and separated by SDS-PAGE. Immunoblots were probed with antibodies against each of the six SRC-3 phosphorylation sites (rows 1 to 6). The amount of SRC-3-bound ERα was detected by probing with an anti-ERα antibody (row 7). The total amounts of ERα and SRC-3 in the lysates were determined by immunoblotting with an anti-ERα and an anti-SRC-3 antibody (rows 8 and 9, respectively). (B) Cells were transfected with plasmids for wild-type SRC-3 and ERβ, and the level of SRC-3 phosphorylated at each of the six sites was assessed as described above. (C) siRNA against ERα (siERα) or against luciferase (siLuc) was transfected into MCF-7 cells. Cells were treated as described above. Cells were then lysed, and endogenous SRC-3 was immunoprecipitated using an anti-SRC-3 antibody and separated by SDS-PAGE. The levels of SRC-3 phosphorylated at T24, S505, and S543 were assessed. (D) Cells were transfected as in panel A and treated with 10 nM E2 (+) or 0.01% ethanol (−) for various periods of time (min). The level of SRC-3 phosphorylated at T24, S505, and S543 and the amount of SRC-3-bound ERα were assessed. Shown in the bottom two rows of all the panels are total cell lysates separated by SDS-PAGE and probed with the anti-ERα, anti-ERβ, or anti-SRC-3 antibodies to assess the total cellular levels of ERα, ERβ, and SRC-3, respectively.