Regulation of human estrogen receptor alpha by phosphorylation

Estrogens are mitogens that stimulate the growth of both normal and transformed epithelial cells of the female reproductive system. The effect of estrogens is mediated through the estrogen receptors (ERs), ERα and ERβ that are ligand-regulated transcription factors. ERs are also phosphoproteins, which can be activated by both ligands and kinase activators or phosphatase inhibitors. Tamoxifen, a selective estrogen receptor modulator, functions as an ER antagonist in breast but an agonist in uterus. Acquired resistance to tamoxifen is a major problem in the clinical management of breast cancers. However, the exact mechanism driving resistance in selective tissues are unclear. In the current study, we report that co-expression of a constitutively active MEKK1, but not RAF or MEKK2, significantly increases the transcriptional activity of the receptor in endometrial and ovarian cancer cells. The expression of wild type MEKK1 and an active Rac1 which functions upstream of MEKK1 also increased the activity of the receptor while co-expression of dominant negative MEKK1 blocked the Rac1 induction, indicating that endogenous MEKK1 is capable of activating the receptor. Additional experiments demonstrated that the MEKK1-induced activation was mediated through both c-jun N-terminal kinases (JNK) and p38/Hog1 and was independent of the known phosphorylation sites on the receptor. p38, but not JNK, efficiently phosphorylated the receptor in immunocomplex kinase assays, suggesting that JNK may regulate other proteins that interact with ER. Consistent with this, JNK modulated the interaction of ER with TIF2 through the receptor's ligand-binding domain in response to estrogens. On the other hand, p38 was activated by estrogen in endometrial adenocarcinoma cells that are dependent on ERα expression. The activated p38 was found to phosphorylate the ER in vitro and in vivo at a novel threonine residue. Suppression of p38 activity or mutation of the threonine site inhibited the Crm1-dependent nuclear export of the receptor as well as its transcriptional activation by estrogens and MEKK1. The inhibition of p38 signal pathway by a specific chemical inhibitor blocked the biological activities of estrogens in regulating endogenous gene expression as well as endometrial cancer cell growth. More importantly, the expression of the constitutively active MEKK1 increased the agonistic activity of 4-hydroxytamoxifen and fully blocked its antagonistic activity in tamoxifen-resistant breast cancer cell line that is characterized by elevated level of p38. Therefore, these findings suggest that the uterine-specific agonistic activity of the tamoxifen compound may be determined by the status of kinases acting downstream of MEKK1. Taken together, our studies demonstrate the regulation of estrogen receptor phosphorylation by two MAP kinases that play a distinct role in ER activation and significantly contribute to the agonistic, activity of estrogens as well as tamoxifen in both endometrial and tamoxifen-resistant breast cancer cells.