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National Institute of Environmental Health Sciences

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Publication Detail

Title: In vivo and in vitro phosphorylation of the human estrogen receptor.

Authors: Arnold, S F; Obourn, J D; Yudt, M R; Carter, T H; Notides, A C

Published In J Steroid Biochem Mol Biol, (1995 Feb)

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.

PubMed ID: 7873451 Exiting the NIEHS site

MeSH Terms: Animals; Cell Line; Chromatography, High Pressure Liquid/methods; Cyclic AMP-Dependent Protein Kinases/metabolism; DNA-Activated Protein Kinase; DNA-Binding Proteins*; Estradiol/pharmacology; Humans; Molecular Weight; Nuclear Proteins; Phosphopeptides/analysis; Phosphorylation/drug effects; Protein Kinase C/metabolism; Protein-Serine-Threonine Kinases/metabolism; Receptors, Estrogen/chemistry; Receptors, Estrogen/isolation & purification; Receptors, Estrogen/metabolism*; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism*; Serine/metabolism; Spodoptera; Tumor Cells, Cultured

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