Phosphorylation of the human estrogen receptor. Identification of hormone- regulated sites and examination of their influence on transcriptional activity

We have used a transient transfection system with a cytomegalovirus-based vector expressing high levels of biologically active human estrogen receptor (ER) in COS-1 cells to study the phosphorylation of human ER and to identify major hormone-regulated phosphorylation sites. The features of phosphorylation of the wild-type ER were very similar to those previously observed for the endogenous ER in uterine cells: The ER exhibited a basal level of phosphorylation which was increased ~3-4-fold by estrogen (estradiol) and by antiestrogens (hydroxytamoxifen and ICI164,384), and phosphorylation was increased to an almost similar extent by activation of either protein kinase A or C signal transduction pathways with cholera toxin plus isobutyl methylxanthine (CT+IBMX) or phorbol ester 12-O- tetradecanoylphorbol-13-acetate (TPA), respectively. Phosphoamino acid analysis revealed that the phosphorylation occurred exclusively on serine residues in all cases. Tryptic phosphopeptide analysis of ER, using a two- dimensional peptide mapping procedure, revealed similar patterns for ER in cells treated with estradiol, antiestrogens or TPA; with CT+IBMX treatment, the same phosphopeptides were seen, but the relative phosphorylation of the different ER phosphotryptic peptides differed. In ER deleted of the NH₂- terminal A and B (A/B) domains, estrogen and antiestrogen-stimulated phosphorylations were abolished, while the phosphorylation induced by CT+IBMX was maintained. This suggests that sites of phosphorylation enhanced by estradiol and antiestrogen, but not those induced by CT+IBMX, are located in the A/B domain. These results were further confirmed by comparing the tryptic phosphopeptide patterns of wild-type and A/B-deleted receptor upon estradiol and CT+IBMX treatments, and then by site-directed mutagenesis, by substituting alanines for the serine residues in the A/B domain (Ser¹⁰⁴, Ser¹⁰⁶, Ser¹¹⁸, Ser¹⁵⁴, and Ser¹⁶⁷) involved in known protein kinase consensus sequences. Comparison of the tryptic phosphopeptide patterns of wild-type ER and these mutant ERs allowed us to identify serine 104 and/or serine 106 and serine 118, all three being part of a serine-proline motif, the preferred substrate of proline-directed protein kinase, as major ER phosphorylation sites. When tested with two estrogen-responsive reporter gene constructs in several cell types, the mutant S104A, S106A, S118A showed a ~40% reduction in transactivation activity in response to E₂, while the mutants S118A and S104A, S106A alone showed a ~15% decrease in transactivation. Our studies identify several serines in the NH₂-terminal portion of the human ER as being major hormone-regulated phosphorylation sites. The location of these sites in one of the two transactivation domains of the ER is consistent with their modulatory effects on transcriptional activity of the human ER.