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Antagonist-Induced, Activation Function-2-Independent Estrogen Receptor Phosphorylation

Department of Molecular Endocrinology and Bone Biology, Merck Research Laboratories (L.L., J.E.F., N.W., A.S., Q.S., J.Y., F.C., S.W., E.T.B., A.S., S.P.R., L.P.F., A.A.R.), West Point, Pennsylvania 19486; Department of Medicinal Chemistry, Merck Research Laboratories (S.K., H.Y.C., Q.T., F.D., M.L.H.), Rahway, New Jersey 07065; and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine (G.A.R.), Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Dr. Alfred Reszka, Molecular Endocrinology and Bone Biology, Merck Research Laboratories, WP26A-1000, West Point, Pennsylvania 19486.

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

NURSA Molecule Pages Link:

Nuclear Receptors:   ERα

Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen  |  Raloxifene

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