In vivo and in vitro phosphorylation of the human estrogen receptor.
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.
Arnold
S F
SF
Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, NY 14642.
Obourn
J D
JD
Yudt
M R
MR
Carter
T H
TH
Notides
A C
AC
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
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