title: Estrogen receptor-alpha phosphorylated at Ser118 is present at the promoters of estrogen-regulated genes and is not altered due to HER-2 overexpression.
abstract: Detection of estrogen receptor (ER)-alpha phosphorylated at Ser(118) (P-Ser(118)-ER-alpha) may be an indicator of an intact ligand-dependent ER-alpha in breast tumors in vivo and may predict responsiveness to endocrine therapy. The current study addresses whether P-Ser(118)-ER-alpha is functionally involved in ER target gene transcription and if this is modulated by HER-2 overexpression. Using chromatin immunoprecipitation analysis, P-Ser(118)-ER-alpha was found associated with the promoters of several estrogen-regulated genes in MCF-7 breast cancer cells 30 minutes following estrogen treatment. Coactivators AIB1 and p300 were coimmunoprecipitated with P-Ser(118)-ER-alpha following estrogen treatment. The overexpression of HER-2 protein in MCF-7 cells did not affect estrogen induction of phosphorylation of Ser(118) or its presence at the promoters of several estrogen-regulated genes. U0126, an inhibitor of mitogen-activated protein kinase (MAPK) pathway, had no effect on P-Ser(118)-ER-alpha. The lack of effect of HER-2 overexpression on P-Ser(118)-ER-alpha expression in cell models is supported by similar levels of expression of P-Ser(118)-ER-alpha in ER(+)/HER-2-overexpressing and ER(+)/HER-2(-) breast tumors in vivo. Using inhibitors of cyclin-dependent kinase 7 (Cdk7), [(5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole and 2-(R)-1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine], and IkappaB kinase-alpha (IKK-alpha; BAY-11-7082), we show that IKK-alpha, but not Cdk7, is at least in part involved in estrogen-mediated phosphorylation at Ser(118) in MCF-7 cells. Our data provide direct evidence for a functional role of P-Ser(118)-ER-alpha in estrogen-regulated signaling and do not support the hypothesis that resistance of breast tumors to tamoxifen therapy involves ligand independent activation of ER-alpha due to constitutive phosphorylation of Ser(118) by constitutive activation of MAPK pathway.
authors.affiliation: Department of Biochemistry and Medical Genetics and Department of Pathology, Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Gregory E
authors.initials: GE
authors.lastname: Weitsman
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.forename: George P
authors.initials: GP
authors.lastname: Skliris
authors.forename: James R
authors.initials: JR
authors.lastname: Davie
authors.forename: Kanyarat
authors.initials: K
authors.lastname: Ung
authors.forename: Yulian
authors.initials: Y
authors.lastname: Niu
authors.forename: Linda
authors.initials: L
authors.lastname: Curtis-Snell
authors.forename: Ladislav
authors.initials: L
authors.lastname: Tomes
authors.forename: Peter H
authors.initials: PH
authors.lastname: Watson
authors.forename: Leigh C
authors.initials: LC
authors.lastname: Murphy
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis*; D014158:Transcription, Genetic; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D018844:Cyclin-Dependent Kinases; D004958:Estradiol; D047628:Estrogen Receptor alpha; D051550:I-kappa B Kinase; D018719:Erb-b2 Receptor Tyrosine Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human