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Figure 6 | BMC Biochemistry

Figure 6

From: Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

Figure 6

Phosphorylation of ERα impacts receptor transcriptional activity. (A) 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 each novel phosphorylation site (S47A, S282A, S294A, and S559A). 24 hours post transfection, cells were incubated with vehicle (veh) or estradiol (10-8 M) overnight. Luciferase assays were performed and transcriptional activity was normalized to protein concentration and/or ERα expression by Western blot analysis. S47A exhibited similar transcriptional activity to wt ERα, whereas S294A resulted in suppressed transcriptional activity vehicle and estradiol. S282A and S559A displayed enhanced ligand independent transcriptional activity as compared to wt ERα. (B) HeLa cervical cancer cells were transfected with 500 ng of wt ERα or ERα phospho-mutant (S47A, S282A, S294A, S559A) expression plasmids. 24 hours post-transfection, cells were incubated with vehicle (veh), 10-8 estradiol (E2), for 3 hours. pS2 expression was measured by real-time RT-PCR, relative to GAPDH. S47A resulted in suppression of estradiol-induced pS2 expression, whereas S559A exhibited ligand-independent activation of ER. S282A and S294A displayed no statistical differences in pS2 mRNA. (C) HeLa cells were transfected with 500 ng of wt ERα or ERα phospho-mutant (S47A, S282A, S294A, and S559A) expression plasmids and incubated for 3 hours with estradiol (1-8M) at 18-24 hours post transfection. Transfection and incubation with estradiol were performed in parallel with those for RT-PCR (panel B). A and B represent composite results for 6 identical experiments. Statistical significance was determined using ANOVA and Fisher's LSD post-hoc analysis, p ≤ 0.05.

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