Regulation of estrogen receptor nuclear export by ligand-induced and p38-mediated receptor phosphorylation

Estrogens are female sex steroid hormones that control development, maintenance, and regulation of the female reproductive phenotype and behavior. They also stimulate the growth of normal and transformed epithelial cells of the female reproductive systems. The effect of estrogens is mediated through both estrogen receptors α and β (ERα and ERβ), which belong to the nuclear hormone receptor superfamily, a group of ligand-regulated, zinc finger-containing transcription factors (11, 40). The superfamily includes not only receptors for classical steroids such as estrogens, androgens, progesterones, and glucocorticoids, but also receptors for steroid analogues and nonsteroid ligands such as vitamin D, thyroid, and retinoic acids, as well as orphan receptors for which the ligand is unknown. Unlike the thyroid and vitamin D receptors, which reside in the nucleus in the absence of ligands, receptors for classical steroids such as ERα are targeted to the nucleus after binding with estrogens or selective estrogen receptor modulators such as tamoxifen. In contrast, the pure ERα antagonist ICI 182,780 directs the ERα to the cytoplasm (6). Of importance in this respect are three clusters of basic amino acids, similar to the nuclear localization signals found in simian virus 40 large T antigen, which were identified in the DNA binding domain and the hinge region of ERα (44). The nuclear localization signals are constitutively active and do not seem to explain the estrogen effect on the ERα nuclear localization (44). It is therefore possible that estrogen-induced targeting of ERα to the nucleus is mediated through other mechanisms. Studies in recent years have provided increasing evidence that nuclear localization is also controlled through nuclear export signals (17, 38, 42). A number of studies on exported proteins have shown that typical nuclear export signals are hydrophobic, leucine-rich sequences that signal the nuclear export complex containing exportin/Crm1 and RanGTP to transfer nuclear export signal-carrying proteins to the cytoplasm (8, 12, 29, 37). Besides ligands, nonsteroid stimuli such as kinase activators, phosphatase inhibitors, neurotransmitters, and growth factors were also shown to activate the ERα (35). They either activate the receptor ligand independently or enhance the ligand-induced activity. Consistent with the cross talk with kinase/phosphatase pathways, ERα has been found to be phosphorylated at different sites by various kinases, including the external signal-regulated kinase (4, 19), cyclin A-CDK2 (32), c-SRC (25), protein kinase A (5), and pp90RSK1 (16). Except for tyrosine-537, all known ERα phosphorylation sites are on serine residues (1). Single-site mutation or simultaneous mutation at multiple sites reduced the transcriptional activity of the receptor (13). With the exception of the serine-236 phosphorylation by protein kinase A (5), most studies indicated the general role of ERα phosphorylation to be the regulation of the transcriptional activity of the receptor by modulating the interaction between the ERα activation domains and transcriptional coactivators (9, 28, 39). In the present studies, we report that, in ERα-expressing endometrial cancer cells, 17β-estradiol activates the p38 mitogen-activated protein kinase (MAPK) pathway, which in turn mediates the phosphorylation of the ERα on threonine-311 (Thr311), promoting the receptor's nuclear localization and interaction with steroid receptor coactivators. Additional studies show that Thr311 phosphorylation in ERα is a critical determinant for its transcriptional and biological activities in endometrial cancer cells.