182 R S THOMAS and others . Estrogen receptor phosphorylation by MAPK et al. 2005) can phosphorylate S104 and/or S106. However, whilst investigating the phosphorylation of S118, we had previously observed that mutation of S104 and S106 reduced the levels of ERa phosphorylation by MAPK in vitro (Chen et al. 2002). In agreement with this, we show here that S104 and S106 phosphorylation was stimulated by PMA and inhibited by the MEK1/2 inhibitor U0126, in transfected COS-1 cells. Further, co-transfection with activated Ras-V12 or Raf-CAAX, but not dominant-negative Raf-S621A, stimulated S104 and S106 phosphorylation. Together, these results suggest that serines 104 and 106 are phosphorylated by Erk1/2 MAPK. In vitro kinase experiments conﬁrmed that, in addition to phosphorylating S118, Erk2 could also directly phosphorylate S104 and S106. Of the other kinases tested, Cdk2 was able to phosphorylate S104 and S118, and GSK3 able to phosphorylate S104, but to levels considerably lower than that achieved by Erk2. However, Cdk2 and/or GSK3 may phosphorylate S104 and/or S106 in vivo. Indeed, ligand-stimulated phos- phorylation was in most cases insensitive to U0126, suggesting the involvement of other kinases. However, OHT- and ICI-induced phosphorylation of S104 did appear to partly involve MAPK, raising the possibility that multiple kinases are differentially involved, depending on the speciﬁc ligand. In reporter gene assays, constitutively active Ras and Raf stimulated ERa activity in the absence of ligand or in the presence of OHT. Substitution of S104/S106/S118 by alanine prevented ERa activation by Ras and Raf, indicating that these sites are required for MAPK stimulation of ERa activity. Furthermore, U0126 inhib- ited ERa activity, as did dominant-negative Raf, but had little effect on triple S104, S106, and S118 mutants. These results suggest that the inhibitory effect of Raf-S621A and U0126 on ERa activity was due to the inhibition of phosphorylation at some or all of the three sites, and further implicates MAPK in the phosphorylation of S104, S106, and S118. Phosphorylation of S104, S106, and S118 could also be induced by ERa ligands; E2, OHT, and ICI in COS-1 cells. The in vitro kinase assays indicated that ligand-binding results in marginally more efﬁcient phosphorylation of ERa by MAPK, perhaps due to the altered conformation of ERa and/or unmasking of potential MAPK docking site(s) (Obenauer et al. 2003). However, ligand-stimulated phosphorylation of ERa in vivo was largely insensitive to U0126, and may be mediated by Cdk2 and/or GSK3 (Trowbridge et al. 1997, Rogatsky et al. 1999, Medunjanin et al. 2005). In conclusion, phosphorylation of S104, S106, and S118 is important for ERa AF-1 activity, as displayed by enhanced ligand-independent, and E2- and OHT-depen- dent, activities. This enhanced activity is not due to ligand hypersensitivity. No one site is critical, but lack of phosphorylation at all of the sites together results in near complete loss of AF-1 activity and prevents the Journal of Molecular Endocrinology (2008) 40, 173–184 agonist action of OHT. Additionally, phosphorylation of these sites occurs in a partially interdependent manner and phosphorylation at each site appears to act via a similar mechanism to enhance ERa activity, suggesting that this region constitutes a phospho-regulated domain of cooperative MAPK phosphorylation sites. Activation of the EGF receptor and ErbB2 pathways, which signal through MAPK, has been associated with more aggressive breast cancer phenotypes and poor patient prognosis (Ross & Fletcher 1998, Arteaga 2001). These pathways have additionally been linked to the tamoxifen resistance phenotype (Benz et al. 1993, Kurokawa et al. 2000, Gee et al. 2001, Kurokawa & Arteaga 2003, Shou et al. 2004). The evidence presented here suggests that modulation of ERa phosphorylation can determine whether or not tamoxifen acts as an ERa agonist or antagonist, and that hyperphosphorylation may result in tamoxifen-induced activities at levels high enough to support the growth of cells that depend upon ERa activity, such as those found in the majority of breast cancers. Acknowledgements We thank the members of the laboratory for helpful discussions, and Dr L Buluwela for discussion of the work and critical reading of this manuscript. This work was made possible by grants from Cancer Research UK and the Breast Cancer Research Trust. The authors declare that there is no conﬂict of interest that would prejudice the impartiality of this scientiﬁc work. References Ali S & Coombes RC 2002 Endocrine-responsive breast cancer and strategies for combating resistance. Nature Review Cancer 2 101–112. Ali S, Metzger D, Bornert JM & Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO Journal 12 1153–1160. Arteaga CL 2001 The epidermal growth factor receptor: from mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. Journal of Clinical Oncology 19 32S–40S. Beato M, Herrlich P & Schutz G 1995 Steroid hormone receptors: many actors in search of a plot. Cell 83 851–857. Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, Shepard HM & Osborne CK 1993 Estrogen-dependent, tamoxifen- resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Research and Treatment 24 85–95. Berry M, Metzger D & Chambon P 1990 Role of the two activating domains of the oestrogen receptor in the cell-type and promoter- context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO Journal 9 2811–2818. Bjornstrom L & Sjoberg M 2005 Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Molecular Endocinology 19 833–842. Bocquel MT, Kumar V, Stricker C, Chambon P & Gronemeyer H 1989 The contribution of the N- and C-terminal regions of steroid receptors to activation of transcription is both receptor and cell- speciﬁc. Nucleic Acid Research 17 2581–2595. www.endocrinology-journals.org