In vivo and in vitro phosphorylation of the human estrogen receptor

J Steroid Biochem Mol Biol. 1995 Feb;52(2):159-71. doi: 10.1016/0960-0760(94)00166-j.

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

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

Substances

  • DNA-Binding Proteins
  • Nuclear Proteins
  • Phosphopeptides
  • Receptors, Estrogen
  • Recombinant Proteins
  • Serine
  • Estradiol
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C