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

Lorraine Lipfert, John E. Fisher, Nan Wei, Angela Scafonas, Qin Su, Joel Yudkovitz, Fang Chen, Sudha Warrier, Elizabeth T. Birzin, Seongkon Kim, Helen Y. Chen Qiang Tan, Azriel Schmidt, Frank Dininno, Susan P. Rohrer, Milton L. Hammond, Gideon A. Rodan, Leonard P. Freedman, Alfred A. Reszka

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Estrogen receptor α (ERα) 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 wildtype ERα 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 ERα in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERα 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 ERα in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERα 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 ERα 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 ERα can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERα out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.

Original languageEnglish
Pages (from-to)516-533
Number of pages18
JournalMolecular Endocrinology
Volume20
Issue number3
DOIs
Publication statusPublished - 03-2006
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

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