Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2277492&req=5

fig1: 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).

Mentions: Serines 104 and/or 106 have been shown to be phosphorylated by Cdk2/cyclin A and Cdk2/cyclin E (Trowbridge et al. 1997, Rogatsky et al. 1999), and GSK3 (Medunjanin et al. 2005). To further characterize ERα phosphorylation at these residues, we generated rabbit antisera specific for phosphorylation at S104 (α-PS104) or at S106 (α-PS106). S104 phosphorylation was stimulated following treatment of ERα-transfected COS-1 cells with E2 and PMA (Fig. 1A). ERα mutants in which S104 was substituted by alanine were not detected by α-PS104, whilst mutation of S106 (106A) reduced but did not abolish S104 phosphorylation. S106 phosphorylation was also stimulated by E2 and PMA, and blocked by mutants of S106 but not S104. Competition with phosphorylated peptides further confirmed that α-PS104 and α-PS106 are specific for ERα phosphorylated at S104 and S106 respectively. Detection of phopho-S104 ERα and phospho-S106 ERα were blocked by pre-incubation of the respective antiserum with a 100-fold excess of peptides containing a phosphorylated S104 (PS104 and PS104/6), and phosphorylated S106 (PS106 and PS104/6) respectively.


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)

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

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

fig1: 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).
Mentions: Serines 104 and/or 106 have been shown to be phosphorylated by Cdk2/cyclin A and Cdk2/cyclin E (Trowbridge et al. 1997, Rogatsky et al. 1999), and GSK3 (Medunjanin et al. 2005). To further characterize ERα phosphorylation at these residues, we generated rabbit antisera specific for phosphorylation at S104 (α-PS104) or at S106 (α-PS106). S104 phosphorylation was stimulated following treatment of ERα-transfected COS-1 cells with E2 and PMA (Fig. 1A). ERα mutants in which S104 was substituted by alanine were not detected by α-PS104, whilst mutation of S106 (106A) reduced but did not abolish S104 phosphorylation. S106 phosphorylation was also stimulated by E2 and PMA, and blocked by mutants of S106 but not S104. Competition with phosphorylated peptides further confirmed that α-PS104 and α-PS106 are specific for ERα phosphorylated at S104 and S106 respectively. Detection of phopho-S104 ERα and phospho-S106 ERα were blocked by pre-incubation of the respective antiserum with a 100-fold excess of peptides containing a phosphorylated S104 (PS104 and PS104/6), and phosphorylated S106 (PS106 and PS104/6) respectively.

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