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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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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.
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fig3: 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.

Mentions: In agreement with the above findings, purified Erk2 readily phosphorylated GST-ERα at S104, S106, and S118 (Fig. 3A). Cdk2 also phosphorylated S104 and S118, but did not appear to phosphorylate S106. GSK3 also phosphorylated S104, with longer exposures showing phosphorylation at S106, but not S118. However, Cdk2 and GSK3-mediated ERα phosphorylation appeared to be less significant than that mediated by Erk2. Similar results were obtained using recombinant ERα purified from SF9 insect cells and transiently transfected ERα in crude COS-1 extracts (data not shown). The presence of estrogen provided a moderate enhancement of Erk2-dependent phosphorylation (Fig. 3B), but confirmed that ERα can be phosphorylated by MAPK in the absence of ligand. To investigate the relative levels of phosphorylation at these sites, GST-ERα-ΔLBD and alanine substitution mutants were incubated with Erk2 in the presence of 32P-γATP (Fig. 3C). Substitution of S104, S106, or S118 by alanine resulted in significant reductions in Erk2 phosphorylation, to ∼40, 60, and 80% respectively, relative to wild-type ERα, with negligible detectable phosphorylation when all three sites were mutated, suggesting that S104, S106, and S118 are the only significant Erk2 phosphorylation sites within amino acids 1–281 of ERα. These data further support the possibility that phosphorylation at each of these sites influences phosphorylation at the other sites.


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)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: In agreement with the above findings, purified Erk2 readily phosphorylated GST-ERα at S104, S106, and S118 (Fig. 3A). Cdk2 also phosphorylated S104 and S118, but did not appear to phosphorylate S106. GSK3 also phosphorylated S104, with longer exposures showing phosphorylation at S106, but not S118. However, Cdk2 and GSK3-mediated ERα phosphorylation appeared to be less significant than that mediated by Erk2. Similar results were obtained using recombinant ERα purified from SF9 insect cells and transiently transfected ERα in crude COS-1 extracts (data not shown). The presence of estrogen provided a moderate enhancement of Erk2-dependent phosphorylation (Fig. 3B), but confirmed that ERα can be phosphorylated by MAPK in the absence of ligand. To investigate the relative levels of phosphorylation at these sites, GST-ERα-ΔLBD and alanine substitution mutants were incubated with Erk2 in the presence of 32P-γATP (Fig. 3C). Substitution of S104, S106, or S118 by alanine resulted in significant reductions in Erk2 phosphorylation, to ∼40, 60, and 80% respectively, relative to wild-type ERα, with negligible detectable phosphorylation when all three sites were mutated, suggesting that S104, S106, and S118 are the only significant Erk2 phosphorylation sites within amino acids 1–281 of ERα. These data further support the possibility that phosphorylation at each of these sites influences phosphorylation at the other sites.

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