Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor alpha

The estrogen receptor (ER) regulates the expression of target genes in a li gand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LED. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ER alpha (hER alpha) is enhance d through phosphorylation of the Ser(118) residue by mitogen-activated prot ein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protei n (p68) which interacted with the A/B domain but not with the LBD of hER al pha. Phosphorylation of hER alpha Ser(118) potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 an d the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ER alpha in a cell-type-specific manner. Howev er, it did not potentiate AF-1 or AF-2 of ERP, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the R NA helicase activity previously ascribed to p68 is dispensable for the ER a lpha AF-1 coactivator activity and that p68 binds to CBP in vitro. Furtherm ore, the interaction region for p68 in the ER alpha A/B domain was essentia l for the full activity of hER alpha AF-1. Taken together, these findings s how that p68 acts as a coactivator specific far the ER alpha AF-1 and stron gly suggest that the interaction between p68 and the hER alpha A/B domain i s regulated by MAPK-induced phosphorylation of Ser(118).