Estrogen receptor phosphorylation by MAPK . R S THOMAS and others 175 directly into Laemmli buffer and immunoblotting was performed, as described (Sarwar et al. 2006). For peptide competition experiments, the primary antibodies were pre-incubated with 10 mg/ml of the appropriate peptide (Chen et al. 2002). Quantitation of phospho-ERa signal was performed by densitometry of scanned ﬁlms using ImageQuant software, and expressed relative to the respective total ER level. Kinase assays The glutathione-S-transferase (GST) fusion proteins, GST-ERa and GST-ERa-DLBD were expressed in Escherichia coli (Rosetta strain, Merck Chemicals Ltd, Nottingham, UK) following induction with 0. 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 h at 25 8C. Cells (50 ml) were lysed with 10 ml XTractor buffer (BD Biosciences, Oxford, UK) containing 1 mM dithiothreitol (DTT) and protease inhibitors, and the lysates cleared by high-speed centrifugation. The lysates were incubated with glutathione-sepharose beads at 4 8C for 1 h, and the beads were washed thrice with TBS containing 1 mM DTT and protease inhibitors. GST-ERa beads were resuspended in 1! kinase buffer, 200 mM ATP, GE2 (10 nM) and kinase (Erk2 and GSK3, New England Biolabs, UK; Cdk2/cyclin A, Cdk2/cyclin E, Cdk4/cyclin D1, Cdk7/cyclin H/MAT1, AKT1, and AKT3; New England Biolabs), according to manufacturer’s instructions. For radioactive kinase assays, 50 mM cold ATP and 10 mM 32PgATP (Amersham) was used. Reactions were incubated at 30 8C for 30 min and processed by SDS-PAGE followed by autoradiography or immunoblot. Reporter assays Cells were grown in DMEM lacking phenol red and supplemented with 10% DSS for 3 days prior to plating in 24-well plates at 50 000 cells/well. Cells were transfected using Fugene 6 (Roche), with 100 ng pERE3-TATA-luc and pRL-TK reporters, 10 ng pSG5 empty vector or ERa expression construct, 50 ng empty vector or Ras/Raf expression vector, and 500 ng pBSC carrier DNA. After 4 h, the medium was replaced with fresh media containing ethanol carrier, E2, OHT or ICI 182 780, at concentrations indicated in ﬁgures. After a further 20 h, the cells were harvested and luciferase levels determined using Dual-Glo reagents (Promega). For experiments in which U0126 was used, 10 nM U0126 was added 1 h prior to the addition of ligands and the cells were harvested after a further 7 h. Fireﬂy luciferase levels were corrected for transfection efﬁciency using corresponding renilla luciferase levels. The activity for wild-type ERa in the absence of ligand was taken as one, with all other activities shown relative to this. All experiments were www.endocrinology-journals.org independently repeated at least four times, and the data presented as mean values with S.E.M. error bars. Results Antisera display speciﬁcity for ERa phosphorylated at S104 and S106 Serines 104 and/or 106 have been shown to be phosphorylated by Cdk2/cyclin A and Cdk2/cyclin E (Trowbridge et al. 1997, Rogatsky et al. 1999), and GSK3 (Medunjanin et al. 2005). To further characterize ERa phosphorylation at these residues, we generated rabbit antisera speciﬁc for phosphorylation at S104 (a-PS104) or at S106 (a-PS106). S104 phosphorylation was stimulated following treatment of ERa-transfected COS-1 cells with E2 and PMA (Fig. 1A). ERa mutants in which S104 was substituted by alanine were not detected by a-PS104, whilst mutation of S106 (106A) reduced but did not abolish S104 phosphorylation. S106 phosphorylation was also stimulated by E2 and PMA, and blocked by mutants of S106 but not S104. Competition with phosphorylated peptides further conﬁrmed that a-PS104 and a-PS106 are speciﬁc for ERa phosphorylated at S104 and S106 respectively. Detection of phopho-S104 ERa and phospho-S106 ERa were blocked by pre-incubation of the respective antiserum with a 100-fold excess of peptides contai- ning a phosphorylated S104 (PS104 and PS104/6), and phosphorylated S106 (PS106 and PS104/6) respectively. Investigation of phosphorylation using mutants in which S104, S106, and S118 were substituted by alanine or glutamic acid, either singly or together, showed that S104 phosphorylation was inﬂuenced by the status of S106 and S118 (Fig. 1B). This result could potentially be explained by a reduced efﬁcacy of a-PS104 binding when S106 or S118 are not phosphorylated, but this is not supported by the fact that a-PS104 binding was competed similarly by both the PS104 and PS104/6 peptides (Fig. 1A). Hence, phosphorylation at S106 and S118 may be important for subsequent phosphorylation of S104. S106 phosphorylation was lower in the case of S118A, but not S104A, suggesting a role for S118 in S106 phosphorylation. In the case of S118, the data suggest that S104, but not S106 inﬂuences S118 phosphorylation. Together, these ﬁndings are indica- tive of crosstalk between S104, S106, and S118, which regulates phosphorylation at these sites. Phosphorylation at S104 and S106 is induced by ERa ligands and PMA, and by activated Raf/Ras S118 phosphorylation is stimulated by E2, as well as anti- estrogens (Joel et al. 1995, 1998b) and can be mediated Journal of Molecular Endocrinology (2008) 40, 173–184