Phosphorylation of human estrogen receptor α at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera

Dongsheng Chen, Elinor Washbrook, Naveed Sarwar, Gaynor J. Bates, Paul E. Pace, Vatsala Thirunuvakkarasu, Jacqueline Taylor, Richard J. Epstein, Frances V. Fuller-Pace, Jean Marc Egly, R. Charles Coombes, Simak Ali (Lead / Corresponding author)

Research output: Contribution to journalArticle

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Abstract

Estrogen receptor α (ERα) is a transcription factor that regulates expression of target genes in a ligand-dependent manner. Activation of gene expression is mediated by two transcription activation functions AF-1 and AF-2, which act in a promoter- and cell-specific manner. Whilst AF-2 activity is regulated by estrogen (E2) binding, the activity of AF-1 is additionally modulated by phosphorylation at several sites. One of these phosphorylation sites, serine 118 (S118) is of particular interest as its mutation significantly reduces ERα activity. Previous studies have shown that S118 can be phosphorylated by the ERK1/2 mitogen activated protein kinases (MAPK) and by the cyclin-dependent protein kinase Cdk7. In this study we use antisera that specifically recognize ERα phosphorylated at S118 to demonstrate that MAPK phosphorylates S118 in a ligand-independent manner, whereas Cdk7 mediates E2-induced phosphorylation of S118. E2 stimulation of S118 phosphorylation was observed within 10 min of its addition and was maximal at 10−7 M E2. S118 phosphorylation was maximal at 30 min but then declined, such that by 180 min following E2 addition little S118 phosphorylation was evident. S118 phosphorylation was also induced by the partial estrogen antagonist 4-hydroxytamoxifen, but not by the complete antagonist ICI 182, 780. S118 phosphorylation upon addition of the MAPK inducers EGF or PMA followed the expected time courses. Finally, we show that ERα is phosphorylated at S118 in vivo using immunoblotting of extracts prepared from a series of ERα-positive breast tumours.

Original languageEnglish
Pages (from-to)4921-4931
Number of pages11
JournalOncogene
Volume21
Issue number32
DOIs
Publication statusPublished - 1 Jan 2002

Fingerprint

Estrogen Receptors
Serine
Immune Sera
Signal Transduction
Phosphorylation
Furylfuramide
Mitogen-Activated Protein Kinases
Ligands
Estrogen Antagonists
Gene Expression
Cyclin-Dependent Kinases
Mitogen-Activated Protein Kinase 1
Immunoblotting
Epidermal Growth Factor
Transcriptional Activation
Estrogens
Transcription Factors
Breast Neoplasms
Mutation

Keywords

  • Breast cancer
  • Estrogen receptor α
  • MAP kinase
  • Phosphorylation
  • TFIIH

Cite this

Chen, Dongsheng ; Washbrook, Elinor ; Sarwar, Naveed ; Bates, Gaynor J. ; Pace, Paul E. ; Thirunuvakkarasu, Vatsala ; Taylor, Jacqueline ; Epstein, Richard J. ; Fuller-Pace, Frances V. ; Egly, Jean Marc ; Coombes, R. Charles ; Ali, Simak. / Phosphorylation of human estrogen receptor α at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. In: Oncogene. 2002 ; Vol. 21, No. 32. pp. 4921-4931.
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title = "Phosphorylation of human estrogen receptor α at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera",
abstract = "Estrogen receptor α (ERα) is a transcription factor that regulates expression of target genes in a ligand-dependent manner. Activation of gene expression is mediated by two transcription activation functions AF-1 and AF-2, which act in a promoter- and cell-specific manner. Whilst AF-2 activity is regulated by estrogen (E2) binding, the activity of AF-1 is additionally modulated by phosphorylation at several sites. One of these phosphorylation sites, serine 118 (S118) is of particular interest as its mutation significantly reduces ERα activity. Previous studies have shown that S118 can be phosphorylated by the ERK1/2 mitogen activated protein kinases (MAPK) and by the cyclin-dependent protein kinase Cdk7. In this study we use antisera that specifically recognize ERα phosphorylated at S118 to demonstrate that MAPK phosphorylates S118 in a ligand-independent manner, whereas Cdk7 mediates E2-induced phosphorylation of S118. E2 stimulation of S118 phosphorylation was observed within 10 min of its addition and was maximal at 10−7 M E2. S118 phosphorylation was maximal at 30 min but then declined, such that by 180 min following E2 addition little S118 phosphorylation was evident. S118 phosphorylation was also induced by the partial estrogen antagonist 4-hydroxytamoxifen, but not by the complete antagonist ICI 182, 780. S118 phosphorylation upon addition of the MAPK inducers EGF or PMA followed the expected time courses. Finally, we show that ERα is phosphorylated at S118 in vivo using immunoblotting of extracts prepared from a series of ERα-positive breast tumours.",
keywords = "Breast cancer, Estrogen receptor α, MAP kinase, Phosphorylation, TFIIH",
author = "Dongsheng Chen and Elinor Washbrook and Naveed Sarwar and Bates, {Gaynor J.} and Pace, {Paul E.} and Vatsala Thirunuvakkarasu and Jacqueline Taylor and Epstein, {Richard J.} and Fuller-Pace, {Frances V.} and Egly, {Jean Marc} and Coombes, {R. Charles} and Simak Ali",
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Chen, D, Washbrook, E, Sarwar, N, Bates, GJ, Pace, PE, Thirunuvakkarasu, V, Taylor, J, Epstein, RJ, Fuller-Pace, FV, Egly, JM, Coombes, RC & Ali, S 2002, 'Phosphorylation of human estrogen receptor α at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera', Oncogene, vol. 21, no. 32, pp. 4921-4931. https://doi.org/10.1038/sj.onc.1205420

Phosphorylation of human estrogen receptor α at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. / Chen, Dongsheng; Washbrook, Elinor; Sarwar, Naveed; Bates, Gaynor J.; Pace, Paul E.; Thirunuvakkarasu, Vatsala; Taylor, Jacqueline; Epstein, Richard J.; Fuller-Pace, Frances V.; Egly, Jean Marc; Coombes, R. Charles; Ali, Simak (Lead / Corresponding author).

In: Oncogene, Vol. 21, No. 32, 01.01.2002, p. 4921-4931.

Research output: Contribution to journalArticle

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T1 - Phosphorylation of human estrogen receptor α at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera

AU - Chen, Dongsheng

AU - Washbrook, Elinor

AU - Sarwar, Naveed

AU - Bates, Gaynor J.

AU - Pace, Paul E.

AU - Thirunuvakkarasu, Vatsala

AU - Taylor, Jacqueline

AU - Epstein, Richard J.

AU - Fuller-Pace, Frances V.

AU - Egly, Jean Marc

AU - Coombes, R. Charles

AU - Ali, Simak

PY - 2002/1/1

Y1 - 2002/1/1

N2 - Estrogen receptor α (ERα) is a transcription factor that regulates expression of target genes in a ligand-dependent manner. Activation of gene expression is mediated by two transcription activation functions AF-1 and AF-2, which act in a promoter- and cell-specific manner. Whilst AF-2 activity is regulated by estrogen (E2) binding, the activity of AF-1 is additionally modulated by phosphorylation at several sites. One of these phosphorylation sites, serine 118 (S118) is of particular interest as its mutation significantly reduces ERα activity. Previous studies have shown that S118 can be phosphorylated by the ERK1/2 mitogen activated protein kinases (MAPK) and by the cyclin-dependent protein kinase Cdk7. In this study we use antisera that specifically recognize ERα phosphorylated at S118 to demonstrate that MAPK phosphorylates S118 in a ligand-independent manner, whereas Cdk7 mediates E2-induced phosphorylation of S118. E2 stimulation of S118 phosphorylation was observed within 10 min of its addition and was maximal at 10−7 M E2. S118 phosphorylation was maximal at 30 min but then declined, such that by 180 min following E2 addition little S118 phosphorylation was evident. S118 phosphorylation was also induced by the partial estrogen antagonist 4-hydroxytamoxifen, but not by the complete antagonist ICI 182, 780. S118 phosphorylation upon addition of the MAPK inducers EGF or PMA followed the expected time courses. Finally, we show that ERα is phosphorylated at S118 in vivo using immunoblotting of extracts prepared from a series of ERα-positive breast tumours.

AB - Estrogen receptor α (ERα) is a transcription factor that regulates expression of target genes in a ligand-dependent manner. Activation of gene expression is mediated by two transcription activation functions AF-1 and AF-2, which act in a promoter- and cell-specific manner. Whilst AF-2 activity is regulated by estrogen (E2) binding, the activity of AF-1 is additionally modulated by phosphorylation at several sites. One of these phosphorylation sites, serine 118 (S118) is of particular interest as its mutation significantly reduces ERα activity. Previous studies have shown that S118 can be phosphorylated by the ERK1/2 mitogen activated protein kinases (MAPK) and by the cyclin-dependent protein kinase Cdk7. In this study we use antisera that specifically recognize ERα phosphorylated at S118 to demonstrate that MAPK phosphorylates S118 in a ligand-independent manner, whereas Cdk7 mediates E2-induced phosphorylation of S118. E2 stimulation of S118 phosphorylation was observed within 10 min of its addition and was maximal at 10−7 M E2. S118 phosphorylation was maximal at 30 min but then declined, such that by 180 min following E2 addition little S118 phosphorylation was evident. S118 phosphorylation was also induced by the partial estrogen antagonist 4-hydroxytamoxifen, but not by the complete antagonist ICI 182, 780. S118 phosphorylation upon addition of the MAPK inducers EGF or PMA followed the expected time courses. Finally, we show that ERα is phosphorylated at S118 in vivo using immunoblotting of extracts prepared from a series of ERα-positive breast tumours.

KW - Breast cancer

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