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

Thomas RS, Sarwar N, Phoenix F, Coombes RC, Ali S - J. Mol. Endocrinol. (2008)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Laboratories, Department of Oncology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK.

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.

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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.
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fig6: 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.

Mentions: In order to confirm the role of MAPK in the phosphorylation of S104 and S106, NIH-3T3 cells were co-transfected with reporter constructs and ERα, together with expression vectors encoding Ras or Raf (Fig. 6A). ERα activity was significantly enhanced by constitutively active Ha-Ras (Ras-V12) and Raf1 (Raf-CAAX), and was inhibited by a kinase-dead mutant of Raf1 (Raf-S621A). By contrast, ERα-104A/106A/118A activity was not stimulated by Ras-V12 or Raf-CAAX. U0126 did not significantly inhibit the activities of ERα-104A/106A/118A or 104E/106E/118E, but did inhibit the activities of wt ERα and those encoding individual alanine mutants (Fig. 6B). Similar results were obtained by treatment with another MEK1/2 inhibitor, PD98059 (data not shown). These data suggest that the activation of the Ras/Raf/MEK/MAPK signal transduction pathway results in ERα activation through phosphorylation at S104, S106, and S118.


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

Thomas RS, Sarwar N, Phoenix F, Coombes RC, Ali S - J. Mol. Endocrinol. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2277492&req=5

fig6: 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.
Mentions: In order to confirm the role of MAPK in the phosphorylation of S104 and S106, NIH-3T3 cells were co-transfected with reporter constructs and ERα, together with expression vectors encoding Ras or Raf (Fig. 6A). ERα activity was significantly enhanced by constitutively active Ha-Ras (Ras-V12) and Raf1 (Raf-CAAX), and was inhibited by a kinase-dead mutant of Raf1 (Raf-S621A). By contrast, ERα-104A/106A/118A activity was not stimulated by Ras-V12 or Raf-CAAX. U0126 did not significantly inhibit the activities of ERα-104A/106A/118A or 104E/106E/118E, but did inhibit the activities of wt ERα and those encoding individual alanine mutants (Fig. 6B). Similar results were obtained by treatment with another MEK1/2 inhibitor, PD98059 (data not shown). These data suggest that the activation of the Ras/Raf/MEK/MAPK signal transduction pathway results in ERα activation through phosphorylation at S104, S106, and S118.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Laboratories, Department of Oncology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK.

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.

Show MeSH
Related in: MedlinePlus