Antagonist-induced, activation function-2-independent estrogen receptor alpha phosphorylation

Mol Endocrinol. 2006 Mar;20(3):516-33. doi: 10.1210/me.2005-0190. Epub 2005 Oct 13.

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.

MeSH terms

  • Animals
  • Benzoquinones
  • COS Cells
  • Chlorocebus aethiops
  • Cyclin-Dependent Kinase-Activating Kinase
  • Cyclin-Dependent Kinases / drug effects
  • Cyclin-Dependent Kinases / metabolism
  • Cytoplasm / drug effects
  • Cytoplasm / metabolism
  • Endometrium / cytology
  • Endometrium / drug effects
  • Estradiol / analogs & derivatives
  • Estradiol / pharmacology
  • Estrogen Antagonists / pharmacology*
  • Estrogen Receptor alpha / agonists
  • Estrogen Receptor alpha / antagonists & inhibitors*
  • Estrogen Receptor alpha / metabolism*
  • Female
  • Fulvestrant
  • HSP90 Heat-Shock Proteins / antagonists & inhibitors
  • HSP90 Heat-Shock Proteins / drug effects
  • HSP90 Heat-Shock Proteins / metabolism
  • Humans
  • Lactams, Macrocyclic
  • Matrix Metalloproteinase 1 / drug effects
  • Matrix Metalloproteinase 1 / genetics
  • Organ Size / drug effects
  • Phosphorylation
  • Promoter Regions, Genetic
  • Quinones / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Selective Estrogen Receptor Modulators / pharmacology
  • Serine / metabolism
  • Uterus / drug effects

Substances

  • Benzoquinones
  • Estrogen Antagonists
  • Estrogen Receptor alpha
  • HSP90 Heat-Shock Proteins
  • Lactams, Macrocyclic
  • Quinones
  • Selective Estrogen Receptor Modulators
  • Fulvestrant
  • Serine
  • Estradiol
  • Cyclin-Dependent Kinases
  • Matrix Metalloproteinase 1
  • geldanamycin
  • Cyclin-Dependent Kinase-Activating Kinase