Estrogen receptor phosphorylation by MAPK . R S THOMAS and others 179 Figure 4 Effect of phosphorylation site mutations on ERa activity. (A and B) COS-1, (C) HeLa, and (D) MCF7 cells were co-transfected with the ERE-3-TATA-ﬁreﬂy luciferase reporter gene, a renilla luciferase control reporter gene, and expression vectors encoding wild-type ERa (ERa) or ERa in which S104, S106 and S118 were substituted by alanine (4/6/18A), glutamic acid (4/6/18E) or aspartic acid (4/6/18D), as indicated. Cells were treated with ethanol solvent (no ligand; NL), E2 (10 nM), OHT (100 nM) or ICI (100 nM), as indicated, except (B) where E2 was added at 0.01, 0.1, 1, and 10 nM and OHT at 0.1, 1, 10, or 100 nM. The cells were harvested for luciferase assays 20 h later. Results are presented as relative ratios of ﬁreﬂy to renilla control luciferase activities, as described in Materials and methods. (A) A parallel transfection series was assayed for ERa expression by immunoblot (a-ER). (D) MCF7 cells were additionally transfected with an empty vector control (K) in order to determine the contribution of endogenous ERa. ERa-104A/106A/118A or 104E/106E/118E, but did inhibit the activities of wt ERa and those encoding individual alanine mutants (Fig. 6B). Similar results were obtained by treatment with another MEK1/2 inhibitor, PD98059 (data not shown). These data suggest that the activation of the Ras/Raf/MEK/ MAPK signal transduction pathway results in ERa activation through phosphorylation at S104, S106, and S118. Discussion Phosphorylation at serine 118 in the AF-1 domain of ERa has been demonstrated by us and other researchers to be important for ERa activity (Ali et al. 1993, Le Goff et al. 1994, Kato et al. 1995, Bunone et al. 1996, Chen et al. 2000, 2002). Phosphorylation of serines 104 www.endocrinology-journals.org and 106 has also been shown to augment ERa activity (Le Goff et al. 1994), but their phosphorylation and function have not been investigated thoroughly. In order to investigate phosphorylation of these residues in greater detail, we generated phosphorylation site- speciﬁc antisera. The antisera demonstrated appropriate speciﬁcity for ERa phosphorylated at S104 or S106, determined using multiple point mutants of S104 and S106 and peptide competition of antibody binding, and conﬁrms that both S104 and S106 of ERa can be phosphorylated. Interestingly, phosphorylation at S104 appeared to be substantially reduced by substitutions at S106 and S118, and S106 phosphorylation reduced by S104E and S118A. S118 phosphorylation was also some- what reduced by S104 substitutions. The epitopes for the a-PS104 and a-PS106 antisera may overlap, but the peptide competition studies of antibody speciﬁcity go some way to suggest that these antisera are not inﬂuenced by the Journal of Molecular Endocrinology (2008) 40, 173–184