178 R S THOMAS and others . Estrogen receptor phosphorylation by MAPK Phosphorylation at S104, S106, and S118 contributes to ERa activity The above data demonstrate that serines 104, 106, and 118 are critical for AF-1 activity, and likely for AF-1 cooperativity with AF-2. In order to deﬁne the relative importance of these sites, single and double mutants were examined for their effect on ERa activity (Fig. 5). Substitution of S118 by alanine reduced OHT-stimu- lated ERa activity. Alanine substitution of 104A or 106A had little effect on OHT stimulation of ERa, although ERa-104A/106A was signiﬁcantly less active than wild-type ERa. Interestingly, S104 or S106 mutations together with mutation of S118 reduced ERa activity more potently than the S118 substitution alone. On the basis of these ﬁndings, the order of importance of the three serines appears to be S118O S104OS106. Although mutation of S106 to alanine had very little effect on ERa activity, its substitution by glutamic acid resulted in the greatest stimulation in ERa activity, with the order of activity for the three mutants being S106OS104OS118, opposite to the relative activities observed with the alanine substitutions. Never- theless, in agreement with the ﬁndings from the alanine mutants, multiple substitutions increased ERa activity correspondingly, again suggesting that all three phos- phorylation sites are important for AF-1 activity. Activated Ras/Raf enhances ERa activity In order to conﬁrm the role of MAPK in the phosphorylation of S104 and S106, NIH-3T3 cells were co-transfected with reporter constructs and ERa, together with expression vectors encoding Ras or Raf (Fig. 6A). ERa activity was signiﬁcantly enhanced by constitutively active Ha-Ras (Ras-V12) and Raf1 (Raf- CAAX), and was inhibited by a kinase-dead mutant of Raf1 (Raf-S621A). By contrast, ERa-104A/106A/118A activity was not stimulated by Ras-V12 or Raf-CAAX. U0126 did not signiﬁcantly inhibit the activities of Figure 3 S104 and S106 are phosphorylated by Erk2 in vitro. (A) Puriﬁed GST-ERa was incubated with a panel of puriﬁed kinases, as indicated, according to manufacturer’s instructions, followed by immunoblotting with phospho-speciﬁc antibodies (a-PS104, a-PS106, and a-PS118) or a-ER. The ﬁlled arrows indicate the position of GST-ERa, and the open arrow indicates a non-speciﬁc product seen with a-PS106 antisera. (B) Puriﬁed GST-ERa was incubated with increasing amounts of puriﬁed Erk2 MAPK (0, 5, 10, 20, 50, and 100 ng) in the absence of ligand (NL) or in the presence of E2 (10 nM), followed by immunoblotting as before. (C) Puriﬁed GST, or wild-type GSaT-ERa-DLBD (ERa-DLBD) or GST-ERa-DLBD in which S104, S106, and/or S118 had been substituted by alanine (A), as indicated, were incubated with Erk2 in the presence of 32P-gATP, followed by SDS-PAGE and autoradiography of the dried gel. Immunoblotting a duplicate gel with a-ER was used to determine the relative levels of each mutant. The bar chart shows quantiﬁcation of each 32P signal relative to the respective total GST-ERa-DLBD level. Journal of Molecular Endocrinology (2008) 40, 173–184 www.endocrinology-journals.org