Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera.
Estrogen receptor alpha (ERalpha) 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 ERalpha 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 ERalpha 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 ERalpha is phosphorylated at S118 in vivo using immunoblotting of extracts prepared from a series of ERalpha-positive breast tumours.
Department of Cancer Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.
Dongsheng
D
Chen
Elinor
E
Washbrook
Naveed
N
Sarwar
Gaynor J
GJ
Bates
Paul E
PE
Pace
Vatsala
V
Thirunuvakkarasu
Jacqueline
J
Taylor
Richard J
RJ
Epstein
Frances V
FV
Fuller-Pace
Jean-Marc
JM
Egly
R Charles
RC
Coombes
Simak
S
Ali
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001943:Breast Neoplasms / Q000378:metabolism; D019556:COS Cells; D018844:Cyclin-Dependent Kinases; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000378:metabolism; D000077267:Fulvestrant; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D000906:Antibodies; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D004967:Estrogens; D011960:Receptors, Estrogen; D013629:Tamoxifen; C016601:afimoxifene; D000077267:Fulvestrant; D012694:Serine; D004958:Estradiol; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human