TY - JOUR
T1 - Antagonist-induced, activation function-2-independent estrogen receptor α phosphorylation
AU - Lipfert, Lorraine
AU - Fisher, John E.
AU - Wei, Nan
AU - Scafonas, Angela
AU - Su, Qin
AU - Yudkovitz, Joel
AU - Chen, Fang
AU - Warrier, Sudha
AU - Birzin, Elizabeth T.
AU - Kim, Seongkon
AU - Chen Qiang Tan, Helen Y.
AU - Schmidt, Azriel
AU - Dininno, Frank
AU - Rohrer, Susan P.
AU - Hammond, Milton L.
AU - Rodan, Gideon A.
AU - Freedman, Leonard P.
AU - Reszka, Alfred A.
PY - 2006/3
Y1 - 2006/3
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=33344475383&partnerID=8YFLogxK
U2 - 10.1210/me.2005-0190
DO - 10.1210/me.2005-0190
M3 - Article
C2 - 16223974
AN - SCOPUS:33344475383
SN - 0888-8809
VL - 20
SP - 516
EP - 533
JO - Molecular Endocrinology
JF - Molecular Endocrinology
IS - 3
ER -
