Figure 7
Mutation of S46 and S46/47 impacts receptor transcriptional activity. (A) 105 ERα (-) HeLa cells were cotransfected with 100 ng ERE2-TK-luciferase reporter and 200 ng wt ERα (wt), or serine to alanine mutants of ERα for S46 (S46A), S47A (S47A), or both S46 and S47 (S46/47A). 24 hours post transfection, cells were incubated with vehicle (veh) or estradiol (10-8 M) for 18-24 hours. Luciferase assays were performed to determine the relative transcriptional activity of ERα and ERα phospho-mutants. Transcriptional activity was normalized to protein concentration and ERα expression by Western blot analysis. These studies demonstrate that S46A and S46/47A lead to substantial inhibition of ERα mediated gene expression, whereas the activity of S47A remains similar to that of wt ERα. (B) Western blot analysis demonstrating expression of wt ERα, S46A, S47A, and S46/47A. 105 HeLa cells were transfected with 500 ng of wt ERα or ERα phospho-mutant (S46A, S47A, or S46/47A) expression plasmids and incubated for 5 hours with estradiol (1-8M) at 18-24 hours post transfection. S47A shows an electrophoretic upshift not evident with wt ERα, S46A, or S46/47A. Panel A represents the composite of 3 experiments. Statistical significance was determined using ANOVA and Fisher's LSD post-hoc analysis, p ≤ 0.05.