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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 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).
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fig5: 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).

Mentions: The above data demonstrate that serines 104, 106, and 118 are critical for AF-1 activity, and likely for AF-1 cooperativity with AF-2. In order to define the relative importance of these sites, single and double mutants were examined for their effect on ERα activity (Fig. 5). Substitution of S118 by alanine reduced OHT-stimulated ERα activity. Alanine substitution of 104A or 106A had little effect on OHT stimulation of ERα, although ERα-104A/106A was significantly less active than wild-type ERα. Interestingly, S104 or S106 mutations together with mutation of S118 reduced ERα activity more potently than the S118 substitution alone. On the basis of these findings, the order of importance of the three serines appears to be S118>S104>S106. Although mutation of S106 to alanine had very little effect on ERα activity, its substitution by glutamic acid resulted in the greatest stimulation in ERα activity, with the order of activity for the three mutants being S106>S104>S118, opposite to the relative activities observed with the alanine substitutions. Nevertheless, in agreement with the findings from the alanine mutants, multiple substitutions increased ERα activity correspondingly, again suggesting that all three phosphorylation sites are important for AF-1 activity.


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

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

fig5: 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).
Mentions: The above data demonstrate that serines 104, 106, and 118 are critical for AF-1 activity, and likely for AF-1 cooperativity with AF-2. In order to define the relative importance of these sites, single and double mutants were examined for their effect on ERα activity (Fig. 5). Substitution of S118 by alanine reduced OHT-stimulated ERα activity. Alanine substitution of 104A or 106A had little effect on OHT stimulation of ERα, although ERα-104A/106A was significantly less active than wild-type ERα. Interestingly, S104 or S106 mutations together with mutation of S118 reduced ERα activity more potently than the S118 substitution alone. On the basis of these findings, the order of importance of the three serines appears to be S118>S104>S106. Although mutation of S106 to alanine had very little effect on ERα activity, its substitution by glutamic acid resulted in the greatest stimulation in ERα activity, with the order of activity for the three mutants being S106>S104>S118, opposite to the relative activities observed with the alanine substitutions. Nevertheless, in agreement with the findings from the alanine mutants, multiple substitutions increased ERα activity correspondingly, again suggesting that all three phosphorylation sites are important for AF-1 activity.

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