176 R S THOMAS and others . Estrogen receptor phosphorylation by MAPK Figure 1 Characterization of phospho-speciﬁc antisera by peptide competition and phosphorylation site substitutions. Lysates prepared from COS-1 cells transiently transfected with an empty expression vector (K), or expression vectors for wild-type ERa or ERa in which S104, S106, and/or S118 had been substituted by alanine (A) or glutamic acid (E), as indicated, were immunoblotted using antibodies for total ERa (a-ER), or for ERa phosphorylated at S104 (a-PS104) or S106 (a-PS106). Cells were treated with ethanol solvent (K), or 17b-estradiol (E2; 10 nM) and 12-tetradecanoylphorbol-13-acetate (PMA; 100 nM), for 30 min prior to harvesting. (A) Replicate blots were incubated with primary antibody (no peptide), or antibody that had been pre-incubated with a 100-fold excess (10 mg/ml) of a peptide encompassing the ERa phosphorylation site; either unphosphorylated (unphos) or phosphorylated (PS104, PS106, or dual PS104/6) versions, as indicated. (B) Lysates were additionally immunoblotted using antibody for ERa phosphorylated at S118 (a-PS118). Levels of phospho-ERa were quantitated in relation to the respective total ERa level (boxed, below each immunoblot). by Cdk7 (Chen et al. 2000, 2002). Further, S118 can be phosphorylated by MAPK in response to EGF and PMA (Kato et al. 1995, Bunone et al. 1996, Chen et al. 2002). Phosphorylation of S104 and S106 was also stimulated by E2, OHT, ICI, and PMA (Fig. 2A). In the case of PMA, this phosphorylation was decreased by the MEK1/2 inhibitor U0126, and corresponds to a decrease in activated Erk1/2 MAPK. The phosphoryl- ation induced by ligands was mostly unaffected by the addition of U0126, except that of S104 in response to OHT and ICI, which were reduced. Co-expression of ERa with constitutively active Ha-Ras (Ras-V12) or Raf1 Journal of Molecular Endocrinology (2008) 40, 173–184 (Raf-CAAX) resulted in enhanced phosphorylation of S104, S106, and S118, whereas the kinase-dead mutant of Raf1 (Raf-S621A) did not (Fig. 2B). Together, these data indicate that S104, S106, and S118 can be phosphorylated by Erk1/2 MAPK in response to activation of the upstream pathways, and also though MAPK-independent pathways upon ligand binding. In agreement with the above ﬁndings, puriﬁed Erk2 readily phosphorylated GST-ERa at S104, S106, and S118 (Fig. 3A). Cdk2 also phosphorylated S104 and S118, but did not appear to phosphorylate S106. GSK3 also phosphorylated S104, with longer www.endocrinology-journals.org