title: In vivo and in vitro phosphorylation of the human estrogen receptor.
abstract: We report here that the human estrogen receptor (hER) overexpressed in Sf9 insect cells is phosphorylated similarly to hER from the human MCF-7 mammary carcinoma cell line. The recombinant and native hER labeled to steady-state with [32P]phosphate were purified to homogeneity using specific DNA-affinity chromatography followed by SDS-gel electrophoresis. Resolution of the hER tryptic digests by reverse phase-high performance liquid chromatography revealed that five [32P]phosphopeptides from the hER expressed in the Sf9 cells had retention times identical to five of the seven [32P]phosphopeptides from the hER in MCF-7 cells. Uniquely, a dephosphorylation of a single 32P-labeled peptide occurred in response to estradiol treatment of MCF-7 cells. In vitro protein kinase assays with the purified recombinant hER revealed that the DNA-dependent protein kinase (DNA-PK) phosphorylated the receptor and induced a decrease in the receptor's mobility as demonstrated by SDS-gel electrophoresis. In contrast, protein kinases A and C did not phosphorylate the purified recombinant hER. These results suggest that in the process of becoming transcriptionally active the estrogen receptor undergoes a dephosphorylation after estrogen-binding and subsequent phosphorylations, in part by the DNA-PK.
authors.affiliation: Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, NY 14642.
authors.forename: S F
authors.initials: SF
authors.lastname: Arnold
authors.forename: J D
authors.initials: JD
authors.lastname: Obourn
authors.forename: M R
authors.initials: MR
authors.lastname: Yudt
authors.forename: T H
authors.initials: TH
authors.lastname: Carter
authors.forename: A C
authors.initials: AC
authors.lastname: Notides
mesh_terms: D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004958:Estradiol / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D009687:Nuclear Proteins; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D018411:Spodoptera; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010748:Phosphopeptides; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D012694:Serine; D004958:Estradiol; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C