180 R S THOMAS and others . Estrogen receptor phosphorylation by MAPK Figure 5 Effect of individual site mutations on ERa activity. COS-1 cells were co-transfected with ERE-3- TATA-luc, pRL-TK, and the wild-type ERa expression vector (ERa) or versions with alanine or glutamic acid substitutions, as indicated. Cells were treated and luciferase assays carried out as for Fig. 4. A parallel transfection series was assayed for ERa expression by immunoblot (a-ER). phosphorylation status at the respective adjacent site. Thus, in general, phosphorylation at these sites appears to be partly interdependent. We cannot, of course, rule out the possibility that these substitutions simply affect an ERa conformation required for efﬁcient phosphorylation. Previous studies have indicated that the agonist activity of tamoxifen (OHT) is due to activation of AF-1, whilst AF-2 function is inhibited (Tora et al. 1989b, Berry et al. 1990). In reporter gene assays using OHT, alanine substitution of S104, S106, or S118 inhibited ERa activity, with the greatest inhibition being observed with substitution of all three residues. Similarly, substitution of the individual sites by glutamic acid augmented ERa activity, with the greatest activity being seen for the triple mutant. Additionally, these sites appear to be important for estrogen-induced and ligand- independent ERa activities. These data suggest that phosphorylation at some or all three sites is critical for AF-1 activity and for the agonist action of OHT. Substitution of S118 to alanine resulted in a greater loss of function compared with that of S104 and S106, but glutamic acid substitutions suggested that achieving a high level of phosphorylation at S104 and S106 might have a greater impact upon ERa function than that at S118. These apparently contradictory results may be explained by functionally stronger S104 and S106 sites being phosphorylated at lower levels in COS-1 cells, relative to that at S118. It is additionally difﬁcult to assign Journal of Molecular Endocrinology (2008) 40, 173–184 levels of importance to these sites, since immunoblot analysis of ERa substitution mutants suggested that S104, S106, and S118 phosphorylation is partly inter- dependent. Our data instead suggest that these three sites, together, comprise a complex phosphorylated domain, involved in augmenting ERa activity. Reporter gene assays indicated that the activity of the ERa-4/6/18E mutant (and 4/6/18D mutant) in the absence of ligand was at a level similar to that seen for wild- type ERa in the presence of OHT, suggesting that OHT- induced phosphorylation at these residues may be sufﬁcient for AF-1 activity. However, the further stimu- lation of ERa-4/6/18E activity by tamoxifen in COS-1 cells, suggests that the agonist activity of OHT is not solely due to stimulation of phosphorylation at these sites. Indeed, acidic substitutions did not lead to a restoration of OHT-induced ERa activity in HeLa cells, in which ERa AF-1 activity is known to be low and associated with little or no stimulation by OHT (Tora et al. 1989b, Berry et al. 1990). These data further support the view that the agonist activity of OHT is not due simply to stimulation of AF-1 phosphorylation. Thus, although OHT agonist activity is dependent upon (and induces) phosphoryl- ation, other factors – possibly cell context-dependent expression of coactivators and corepressors (Shang & Brown 2002) – are also likely to be involved. Previous reports have shown that Cdk2 (Trowbridge et al. 1997, Rogatsky et al. 1999) and GSK3 (Medunjanin www.endocrinology-journals.org