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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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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.
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fig2: 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.

Mentions: S118 phosphorylation is stimulated by E2, as well as anti-estrogens (Joel et al. 1995, 1998b) and can be mediated by Cdk7 (Chen et al. 2000, 2002). Further, S118 can be phosphorylated by MAPK in response to EGF and PMA (Kato et al. 1995, Bunone et al. 1996, Chen et al. 2002). Phosphorylation of S104 and S106 was also stimulated by E2, OHT, ICI, and PMA (Fig. 2A). In the case of PMA, this phosphorylation was decreased by the MEK1/2 inhibitor U0126, and corresponds to a decrease in activated Erk1/2 MAPK. The phosphorylation induced by ligands was mostly unaffected by the addition of U0126, except that of S104 in response to OHT and ICI, which were reduced. Co-expression of ERα with constitutively active Ha-Ras (Ras-V12) or Raf1 (Raf-CAAX) resulted in enhanced phosphorylation of S104, S106, and S118, whereas the kinase-dead mutant of Raf1 (Raf-S621A) did not (Fig. 2B). Together, these data indicate that S104, S106, and S118 can be phosphorylated by Erk1/2 MAPK in response to activation of the upstream pathways, and also though MAPK-independent pathways upon ligand binding.


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)

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

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

fig2: 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.
Mentions: S118 phosphorylation is stimulated by E2, as well as anti-estrogens (Joel et al. 1995, 1998b) and can be mediated by Cdk7 (Chen et al. 2000, 2002). Further, S118 can be phosphorylated by MAPK in response to EGF and PMA (Kato et al. 1995, Bunone et al. 1996, Chen et al. 2002). Phosphorylation of S104 and S106 was also stimulated by E2, OHT, ICI, and PMA (Fig. 2A). In the case of PMA, this phosphorylation was decreased by the MEK1/2 inhibitor U0126, and corresponds to a decrease in activated Erk1/2 MAPK. The phosphorylation induced by ligands was mostly unaffected by the addition of U0126, except that of S104 in response to OHT and ICI, which were reduced. Co-expression of ERα with constitutively active Ha-Ras (Ras-V12) or Raf1 (Raf-CAAX) resulted in enhanced phosphorylation of S104, S106, and S118, whereas the kinase-dead mutant of Raf1 (Raf-S621A) did not (Fig. 2B). Together, these data indicate that S104, S106, and S118 can be phosphorylated by Erk1/2 MAPK in response to activation of the upstream pathways, and also though MAPK-independent pathways upon ligand binding.

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