174 R S THOMAS and others . Estrogen receptor phosphorylation by MAPK promoter- and cell-speciﬁc manner (Gronemeyer 1991, Tsai & O’Malley 1994, Beato et al. 1995). Tamoxifen is an example of a mixed estrogen agonist/antagonist, also termed selective ER modulators, which has been proposed to act by inhibiting the LBD/AF-2, whilst allowing activation of AF-1 (Berry et al. 1990, McDonnell et al. 1995, Brzozowski et al. 1997, Shiau et al. 1998). Structural studies have indicated that the inhibition of the LBD/AF-2 in the tamoxifen-bound ERa is due to the positioning of helix 12 in the hydrophobic cleft to which transcriptional coactivators, such as the p160 family members, are recruited, thereby preventing coactivator recruitment. The same cleft can also be involved in corepressor binding, and tamoxifen has been suggested to allow corepressor recruitment to the LBD (Nettles & Greene 2005). The nature of AF-1 activation by ligands such as tamoxifen, and in the absence of ligand, is unknown, but may involve phosphorylation of ERa. Previous studies have shown that in ovariectomized mice, uterine proliferation (which is normally estrogen- dependent) can be mediated by growth factors such as epidermal growth factor (EGF), and that the EGF-mediated uterine stimulation is ERa dependent (Ignar-Trowbridge et al. 1992, Curtis et al. 1996). ERa may become phosphory- lated in response to EGF and other growth factors, resulting in ligand-independent ERa activation (Ignar-- Trowbridge et al. 1993). Phosphorylation site mapping has demonstrated that serine 118 (S118) in AF-1 is phosphorylated by Erk1/2 MAPK, resulting in stimulation of ERa activity (Kato et al. 1995, Bunone et al. 1996), whilst phosphorylation of serine 167 (S167) in AF-1 by AKT and p90RSK also stimulates ERa activity ( Joel et al. 1998a, Campbell et al. 2001). The ERa AF-1 is additionally phosphorylated at serines 104 (S104) and/or 106 (S106) (Le Goff et al. 1994). Phosphorylation at these sites by Cdk2 (Trow- bridge et al. 1997, Rogatsky et al. 1999) and GSK3 (Medunjanin et al. 2005) has been reported, and our own data have previously indicated that these sites might also be targets for MAPK (Chen et al. 2002). Increased ERa activity due to AF-1 phosphorylation mediated by MAPK and/or other kinases could therefore be at least partly responsible for resistance to endocrine therapy in breast cancer. This hypothesis is supported by ﬁndings that stimulation of growth factor-regulated pathways can augment the agonist properties of tamoxifen (Smith 1998) and inhibit tamoxifen-induced growth suppression and apoptosis in breast cancer cells (Benz et al. 1993, Pietras et al. 1995, Campbell et al. 2001). Here, we investigate phosphorylation at serines 104 and 106 by Erk2 MAPK in vitro and in response to MAPK activity in vivo, and determine their contribution to ERa activity. Transcription reporter assays using ERa phosphorylation site mutants suggest that phosphorylation at all three sites Journal of Molecular Endocrinology (2008) 40, 173–184 contribute signiﬁcantly to AF-1 activity, and in particular are required for tamoxifen to act as an ERa agonist. Materials and methods Plasmids The expression vectors pSG5-HEG0 and pSG5-HE15, encoding full length and AF-2-truncated human ERa (ERa-DLBD) respectively have previously been described (Tora et al. 1989a). pGEX1lT-ERa and pGEX1lT-ERa- DLBD were generated by cloning ERa encoding EcoRI fragments from pSG5 into pGEX1lT (GE Healthcare UK Ltd, Little Chalfont, UK). ERa mutants were generated by site-directed mutagenesis, according to manufacturer’s protocols (Stratagene, La Jolla, CA, USA). pEF-RasV12, expression vector for constitutively active Ha-Ras, was kindly provided by Dr R Treisman (LRI, London, UK). pCMV-Raf CAAX and pCMV-Raf S621A were purchased from Clontech (Saint-Germaine-en-Laye, France). Antibodies Peptides corresponding to amino acids 97-112 of human ERa, having the sequence [C]-FPPLNSVP- SPSPLMLLH (phospho-S104) or [C]-FPPLNSVSPPSP- LMLLH (phospho-S106), were used to generate rabbit polyclonal antisera, as described (Chen et al. 2002). Antibodies speciﬁc for ERa phosphorylated at S118 (16J4, New England Biolabs, Hitchin, UK), ERa (6F11, Novocastra Laboratories, UK), MAPK (sc-93, Santa Cruz Biotechnology, Heidelberg, Germany) and P-MAPK (New England Biolabs) were obtained commercially. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse immunoglobulins were purchased from DAKO Ltd, Ely, UK. Immunoblot analysis Cells were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM) lacking phenol red and supplemented with 10% dextran-coated charcoal-stripped fetal calf serum (DSS) for 3 days prior to plating in six-well plates at 300 000 cells/well. Cells were transfected using Fugene 6 (Millipore, Wafford, UK), with 100 ng pSG5 empty vector or ERa expression construct, 5 ng empty vector or Ras/Raf expression construct, and 500 ng pBSC carrier DNA, as appropriate. After 48 h the cells were treated with ethanol carrier, estradiol (E2; 10 nM), 4-hydroxytamoxifen (OHT; 100 nM) or ICI182,780 (ICI; 100 nM) 30 min prior to harvest, phorbol 12- myristate 13 acetate (PMA; 100 nM) was added 15 min, and U0126 (10 mM) was added 60 min, prior to harves- ting, as appropriate. Cells were washed and harvested www.endocrinology-journals.org