title: Antagonist-induced, activation function-2-independent estrogen receptor alpha phosphorylation.
abstract: Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.
authors.affiliation: Molecular Endocrinology and Bone Biology, Merck Research Laboratories, WP26A-1000, West Point, Pennsylvania 19486, USA.
authors.forename: Lorraine
authors.initials: L
authors.lastname: Lipfert
authors.forename: John E
authors.initials: JE
authors.lastname: Fisher
authors.forename: Nan
authors.initials: N
authors.lastname: Wei
authors.forename: Angela
authors.initials: A
authors.lastname: Scafonas
authors.forename: Qin
authors.initials: Q
authors.lastname: Su
authors.forename: Joel
authors.initials: J
authors.lastname: Yudkovitz
authors.forename: Fang
authors.initials: F
authors.lastname: Chen
authors.forename: Sudha
authors.initials: S
authors.lastname: Warrier
authors.forename: Elizabeth T
authors.initials: ET
authors.lastname: Birzin
authors.forename: Seongkon
authors.initials: S
authors.lastname: Kim
authors.forename: Helen Y
authors.initials: HY
authors.lastname: Chen
authors.forename: Qiang
authors.initials: Q
authors.lastname: Tan
authors.forename: Azriel
authors.initials: A
authors.lastname: Schmidt
authors.forename: Frank
authors.initials: F
authors.lastname: Dininno
authors.forename: Susan P
authors.initials: SP
authors.lastname: Rohrer
authors.forename: Milton L
authors.initials: ML
authors.lastname: Hammond
authors.forename: Gideon A
authors.initials: GA
authors.lastname: Rodan
authors.forename: Leonard P
authors.initials: LP
authors.lastname: Freedman
authors.forename: Alfred A
authors.initials: AA
authors.lastname: Reszka
mesh_terms: D000818:Animals; D016227:Benzoquinones; D019556:COS Cells; D002522:Chlorocebus aethiops; D018844:Cyclin-Dependent Kinases / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004717:Endometrium / Q000166:cytology / Q000187:drug effects; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D005260:Female; D000077267:Fulvestrant; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D047029:Lactams, Macrocyclic; D020781:Matrix Metalloproteinase 1 / Q000187:drug effects / Q000235:genetics; D009929:Organ Size / Q000187:drug effects; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D014599:Uterus / Q000187:drug effects; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D016227:Benzoquinones; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D020845:Selective Estrogen Receptor Modulators; D000077267:Fulvestrant; D012694:Serine; D004958:Estradiol; D018844:Cyclin-Dependent Kinases; D020781:Matrix Metalloproteinase 1; C001277:geldanamycin; D000095043:Cyclin-Dependent Kinase-Activating Kinase