Estrogen receptor phosphorylation by MAPK . R S THOMAS and others 177 Figure 2 Phosphorylation of S104 and S106 is stimulated by ERa ligands and by activators of MAPK. Immunoblots of lysates prepared from COS-1 cells transfected with empty expression vector (K) or expression vector for ERa, were performed as for Fig. 1. Lysates were additionally immunoblotted using antibodies for total (a-MAPK) and phosphorylated (a-PMAPK) Erk1/2 MAPK. Levels of phospho-ERa were quantitated in relation to the respective total ERa level (boxed, below each immunoblot). (A) Cells were pre-incubated with U0126 (10 mM) for 1 h, followed by the addition of E2 (10 nM), 4-hydroxytamoxifen (OHT, 100 nM), ICI 182 780 (ICI; 100 nM) or PMA (100 nM), as indicated, and cells harvested 30 min later. (B) Cells were transfected with expression vector for ERa, together with expression vectors for Ras-V12, Raf-CAAX or Raf-S621A, as indicated. exposures showing phosphorylation at S106, but not S118. However, Cdk2 and GSK3-mediated ERa phosphorylation appeared to be less signiﬁcant than that mediated by Erk2. Similar results were obtained using recombinant ERa puriﬁed from SF9 insect cells and transiently transfected ERa in crude COS-1 extracts (data not shown). The presence of estrogen provided a moderate enhancement of Erk2-dependent phosphorylation (Fig. 3B), but conﬁrmed that ERa can be phosphorylated by MAPK in the absence of ligand. To investigate the relative levels of phosphorylation at these sites, GST-ERa-DLBD and alanine substitution mutants were incubated with Erk2 in the presence of 32P-gATP (Fig. 3C). Substitution of S104, S106, or S118 by alanine resulted in signiﬁcant reductions in Erk2 phosphorylation, to w40, 60, and 80% respectively, relative to wild-type ERa, with negligible detectable phosphorylation when all three sites were mutated, suggesting that S104, S106, and S118 are the only signiﬁcant Erk2 phosphorylation sites within amino acids 1–281 of ERa. These data further support the possibility that phosphorylation at each of these sites inﬂuences phosphorylation at the other sites. www.endocrinology-journals.org S104, S106, and S118 are important for ERa activity In reporter gene assays, substitution of S104, S106, and S118 by alanine (4/6/18A) resulted in a reduction in ERa activity in the presence of E2 by nearly 50%, whilst substitution with glutamic or aspartic acid, to ‘mimic’ phosphorylation at these sites, stimulated ERa activity two- to threefold (Fig. 4A). ERa activity for the alanine mutant in the presence of OHT was reduced by O80%, and was two- to threefold higher for the 4/6/18E and 4/6/18D mutants. Signiﬁcant ERa activation (seven- fold relative to wild-type ERa) was also observed in the absence of ligand for 4/6/18E and 4/6/18D. These data suggest that phosphorylation at one or more of these residues is critical for AF-1 activity and for the agonist activity of OHT. Western analysis of total ERa levels indicated relatively equal levels of expression across the mutants and across different ligand treat- ments. ICI is generally thought to decrease ERa levels, but in many cases a change in solubility is mistaken for degradation (Lipfert et al. 2005); the assays here have measured total ERa levels. To examine whether acidic substitutions in ERa resulted in ligand hypersensitivity, we examined ERa activity by reporter assay in the presence of increasing concentrations of estrogen and OHT (Fig. 4B). The acidic ERa mutant displayed an enhanced activity, of similar magnitude compared with wild-type ERa, at all ligand concentrations. Hence, although the acidic mutant could yield equivalent levels of activity at lower ligand concentrations compared with that of wild-type ERa at higher ligand concentrations . (compare 4/6/18E at 0 1 nM E2 with wt at 1 nM E2, and 4/6/18E at 1 mM OHT with wt at 100 mM OHT), there was no evidence for enhanced ligand sensitivity. ERa AF-1 is only weakly active in HeLa cells, with the majority of ERa activity in HeLa cells being due to AF-2 (Bocquel et al. 1989). Further, the lack of AF-1 activity correlates with lack of agonist activity for OHT in HeLa cells (Bocquel et al. 1989, Berry et al. 1990). Triple acidic substitution stimulated ERa activity in the absence of ligand, as well as in the presence of E2 (Fig. 4C). Activity in the presence of OHT was also increased, but was not signiﬁcantly greater than that obtained in the absence of ligand. Hence, acidic substitutions in AF-1 enhanced ERa activity in HeLa cells in a similar manner to COS-1 cells, but did not activate an OHT response. Finally, in the estrogen responsive and ERa-positive MCF7 breast cancer cell line, transfection of ERa stimulated reporter gene activity over and above the activity observed for endogenous ERa (Fig. 4D). Reporter gene activity for the glutamic and aspartic acid mutants was signiﬁcantly greater in the absence of ligand or in the presence of E2 or OHT, compared with the activities obtained for wild-type ERa, generally following what was seen using COS-1 cells. Journal of Molecular Endocrinology (2008) 40, 173–184