Phosphorylation at serines 104 and 106 by Erk1/2 MAPK is important for estrogen receptor-alpha activity

Abstract

Phosphorylation of estrogen receptor-alpha (ERalpha) at specific residues in transcription activation function 1 (AF-1) can stimulate ERalpha activity in a ligand-independent manner. This has led to the proposal that AF-1 phosphorylation and the consequent increase in ERalpha activity could contribute to resistance to endocrine therapies in breast cancer patients. Previous studies have shown that serine 118 (S118) in AF-1 is phosphorylated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) in a ligand-independent manner. Here, we show that serines 104 (S104) and 106 (S106) are also phosphorylated by MAPK in vitro and upon stimulation of MAPK activity in vivo. Phosphorylation of S104 and S106 can be inhibited by the MAP-erk kinase (MEK)1/2 inhibitor U0126 and by expression of kinase-dead Raf1. Further, we show that, although S118 is important for the stimulation of ERalpha activity by the selective ER modulator 4-hydroxytamoxifen (OHT), S104 and S106 are also required for the agonist activity of OHT. Acidic amino acid substitution of S104 or S106 stimulates ERalpha activity to a greater extent than the equivalent substitution at S118, suggesting that phosphorylation at S104 and S106 is important for ERalpha activity. Collectively, these data indicate that the MAPK stimulation of ERalpha activity involves the phosphorylation not only of S118 but also of S104 and S106, and that MAPK-mediated hyperphosphorylation of ERalpha at these sites may contribute to resistance to tamoxifen in breast cancer.

Figure 1

Characterization of phospho-specific antisera by peptide competition and phosphorylation site substitutions. Lysates prepared from COS-1 cells transiently transfected with an empty expression vector (−), or expression vectors for wild-type ERα or ERα in which S104, S106, and/or S118 had been substituted by alanine (A) or glutamic acid (E), as indicated, were immunoblotted using antibodies for total ERα (α-ER), or for ERα phosphorylated at S104 (α-PS104) or S106 (α-PS106). Cells were treated with ethanol solvent (−), or 17β-estradiol (E₂; 10 nM) and 12-tetradecanoylphorbol-13-acetate (PMA; 100 nM), for 30 min prior to harvesting. (A) Replicate blots were incubated with primary antibody (no peptide), or antibody that had been pre-incubated with a 100-fold excess (10 μg/ml) of a peptide encompassing the ERα phosphorylation site; either unphosphorylated (unphos) or phosphorylated (PS104, PS106, or dual PS104/6) versions, as indicated. (B) Lysates were additionally immunoblotted using antibody for ERα phosphorylated at S118 (α-PS118). Levels of phospho-ERα were quantitated in relation to the respective total ERα level (boxed, below each immunoblot).

Figure 2

Phosphorylation of S104 and S106 is stimulated by ERα ligands and by activators of MAPK. Immunoblots of lysates prepared from COS-1 cells transfected with empty expression vector (−) or expression vector for ERα, were performed as for Fig. 1. Lysates were additionally immunoblotted using antibodies for total (α-MAPK) and phosphorylated (α-PMAPK) Erk1/2 MAPK. Levels of phospho-ERα were quantitated in relation to the respective total ERα level (boxed, below each immunoblot). (A) Cells were pre-incubated with U0126 (10 μM) for 1 h, followed by the addition of E₂ (10 nM), 4-hydroxytamoxifen (OHT, 100 nM), ICI 182 780 (ICI; 100 nM) or PMA (100 nM), as indicated, and cells harvested 30 min later. (B) Cells were transfected with expression vector for ERα, together with expression vectors for Ras-V12, Raf-CAAX or Raf-S621A, as indicated.

Figure 3

S104 and S106 are phosphorylated by Erk2 in vitro. (A) Purified GST-ERα was incubated with a panel of purified kinases, as indicated, according to manufacturer's instructions, followed by immunoblotting with phospho-specific antibodies (α-PS104, α-PS106, and α-PS118) or α-ER. The filled arrows indicate the position of GST-ERα, and the open arrow indicates a non-specific product seen with α-PS106 antisera. (B) Purified GST-ERα was incubated with increasing amounts of purified Erk2 MAPK (0, 5, 10, 20, 50, and 100 ng) in the absence of ligand (NL) or in the presence of E₂ (10 nM), followed by immunoblotting as before. (C) Purified GST, or wild-type GSαT-ERα-ΔLBD (ERα-ΔLBD) or GST-ERα-ΔLBD in which S104, S106, and/or S118 had been substituted by alanine (A), as indicated, were incubated with Erk2 in the presence of ³²P-γATP, followed by SDS-PAGE and autoradiography of the dried gel. Immunoblotting a duplicate gel with α-ER was used to determine the relative levels of each mutant. The bar chart shows quantification of each ³²P signal relative to the respective total GST-ERα-ΔLBD level.

Figure 4

Effect of phosphorylation site mutations on ERα activity. (A and B) COS-1, (C) HeLa, and (D) MCF7 cells were co-transfected with the ERE-3-TATA-firefly luciferase reporter gene, a renilla luciferase control reporter gene, and expression vectors encoding wild-type ERα (ERα) or ERα in which S104, S106 and S118 were substituted by alanine (4/6/18A), glutamic acid (4/6/18E) or aspartic acid (4/6/18D), as indicated. Cells were treated with ethanol solvent (no ligand; NL), E₂ (10 nM), OHT (100 nM) or ICI (100 nM), as indicated, except (B) where E₂ was added at 0·01, 0·1, 1, and 10 nM and OHT at 0·1, 1, 10, or 100 nM. The cells were harvested for luciferase assays 20 h later. Results are presented as relative ratios of firefly to renilla control luciferase activities, as described in Materials and methods. (A) A parallel transfection series was assayed for ERα expression by immunoblot (α-ER). (D) MCF7 cells were additionally transfected with an empty vector control (−) in order to determine the contribution of endogenous ERα.

Figure 5

Effect of individual site mutations on ERα activity. COS-1 cells were co-transfected with ERE-3-TATA-luc, pRL-TK, and the wild-type ERα expression vector (ERα) or versions with alanine or glutamic acid substitutions, as indicated. Cells were treated and luciferase assays carried out as for Fig. 4. A parallel transfection series was assayed for ERα expression by immunoblot (α-ER).

Figure 6

Effect of MAPK signaling activators and inhibitors on ERα activity. (A) NIH3T3 cells were co-transfected with ERE-3-TATA-luc, pRL-TK, and the wild-type ERα expression vector (ERα), or versions in which S104, S106, and S118 were substituted by alanine (4/6/18A) or glutamic acid (4/6/18E), as indicated. Cells were additionally co-transfected with empty expression vector (−) or expression vectors for Ras-V12, Raf-CAAX, or Raf-S621A, as shown. Cells were treated and luciferase assays carried out as for Fig. 4. (B) COS-1 cells were transfected with ERE-3-TATA-luc, pRL-TK, and the wild-type ERα expression vector (ERα) or versions with alanine or glutamic acid substitutions, as indicated. Cells were pre-incubated with U0126 (10 μM), 16 h following transfection, as indicated, for 1 h prior to addition of OHT, and harvested for luciferase assays 7 h later.