Phosphorylation at serines 104 and 106 by Erk1/2 MAPK is important for estrogen receptor-a activity Ross S Thomas, Naveed Sarwar, Fladia Phoenix, R Charles Coombes and Simak Ali Cancer Research UK Laboratories, Department of Oncology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK (Correspondence should be addressed to S Ali; Email: simak.ali@imperial.ac.uk) Abstract Phosphorylation of estrogen receptor-a (ERa) at speciﬁc residues in transcription activation function 1 (AF-1) can stimulate ERa activity in a ligand-independent manner. This has led to the proposal that AF-1 phosphorylation and the consequent increase in ERa activity could contribute to resistance to endocrine therapies in breast cancer patients. Previous studies have shown that serine 118 (S118) in AF-1 is phosphorylated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) in a ligand-independent manner. Here, we show that serines 104 (S104) and 106 (S106) are also phosphorylated by MAPK in vitro and upon stimulation of MAPK activity in vivo. Phosphorylation of S104 and S106 can be inhibited by the MAP-erk kinase (MEK)1/2 inhibitor U0126 and by expression of kinase-dead Raf1. Further, we show that, although S118 is important for the stimulation of ERa activity by the selective ER modulator 4-hydroxytamoxifen (OHT), S104 and S106 are also required for the agonist activity of OHT. Acidic amino acid substitution of S104 or S106 stimulates ERa activity to a greater extent than the equivalent substitution at S118, suggesting that phosphorylation at S104 and S106 is important for ERa activity. Collectively, these data indicate that the MAPK stimulation of ERa activity involves the phosphorylation not only of S118 but also of S104 and S106, and that MAPK-mediated hyperphosphorylation of ERa at these sites may contribute to resistance to tamoxifen in breast cancer. Journal of Molecular Endocrinology (2008) 40, 173–184 Introduction Estrogen receptor-a (ERa) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors, and is required for development and mainten- ance of the female and male reproductive systems, bone, and cardiovascular system, as well as being signiﬁcant for certain brain functions (Couse & Korach 1999). Additionally, estrogens stimulate breast cancer pro- gression and w70–80% of breast cancers express ERa. The presence of ERa correlates with likelihood of response to anti-estrogens, such as tamoxifen (Osborne 1998), and to aromatase inhibitors that inhibit estrogen biosynthesis (Henderson & Piccart-Gebhart 2005), demonstrating the importance of ERa in breast cancer progression. However, a proportion of patients with ERa- positive disease do not respond to endocrine therapy. Further, of those patients who respond, many who initially present with localized disease, and all with advanced breast cancer, relapse. Most resistant tumors remain ERa-positive and frequently respond to alternative anti- estrogen treatment, indicative of a continued role for ERa in breast cancer cell proliferation (Ali & Coombes 2002). Augmentation of ERa activity in the presence of estrogen or anti-estrogens, and/or ligand-independent activation of ERa, could provide an important mechanism for resistance to anti-estrogen therapies. In agreement with this possibility, a recent study shows that levels of S118 phosphorylation are increased following breast cancer recurrence following tamoxifen treatment (Sarwar et al. 2006). ERa regulates gene expression through direct binding to estrogen-response elements within the promoters of estrogen-regulated genes, or by recruit- ment to DNA through interaction with other transcrip- tion factors (Gronemeyer 1991, Bjornstrom & Sjoberg 2005). Transcriptional regulation by ERa is partly mediated by transcription activation function, AF-2, which is intrinsic to the ligand-binding domain (LBD). Estrogen binding to the LBD results in a confor- mational change that allows recruitment of coactivators to the LBD (Brzozowski et al. 1997, Shiau et al. 1998, Glass & Rosenfeld 2000). A second transcription activation function, AF-1, is encoded within the N-terminal 180 amino acids. AF-1 activity is promoter and cellular context dependent (Tzukerman et al. 1994), and can be inﬂuenced by the expression proﬁle of speciﬁc coactivators (Smith et al. 1997). Although there is evidence to suggest that phosphorylation of AF-1 alone can enhance activity (Bunone et al. 1996, Ignar-Trowbridge et al. 1996), ligand binding to AF-2 can also induce AF-1 activity (Metzger et al. 1992). This suggests that, in a basal state, AF-1 activity is blocked by AF-2. Finally, AF-1 and AF-2 can act synergistically in a Journal of Molecular Endocrinology (2008) 40, 173–184 DOI: 10.1677/JME-07-0165 0952–5041/08/040–173 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org This is an Open Access article distributed under the terms of the Society for Endocrinology’s Re-use Licence which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 173