Novel Interactions between FOXM1 and CDC25A Regulate the Cell Cycle
Figure 5
CDC25A regulated FOXM1 transcriptional activity through CDK1-mediated phosphorylation sites.
(A) CDC25A activated the transcriptional activity of FOXM1 in U2OS cells. U2OS cells were co-transfected with pACT-FOXM1 or/and pACT-CDC25A, along with the pGL3-6×FOXM1-Luc plasmids containing 6 FOXM1 DNA binding sequences, and pRL-SV40, a Renilla luciferase reporter vector was used as the control. Data were normalized to Renilla luciferase activities and are presented as the mean ± SD (N = 3). *** p<0.001. (B) siRNA-mediated inhibition of CDK1 blocked CDC25A-mediated FOXM1 transcriptional activity. U2OS cells were co-transfected with pACT-CDC25A, pACT-FOXM1 and pGL3-6×FOXM1-Luc plasmids, or pACT-FOXM1 and pACT-CDC25A together with siRNAs against CDK1, CDK2, CDK4 or CDK6 or control siRNA, and pRL-SV40 was used as the normalizing control. Forty-eight hours after transfection, the cells were lysed, and firefly and Renilla luciferase activities were measured. Data were normalized to Renilla luciferase activities and are presented as the mean ± SD (N = 3). Data were subjected to one-way ANOVA (significance level α = 0.05) and Dunnett's multi-comparison post-hoc tests (CDK siRNA vs. CTR siRNA). *** p<0.001. (C) CDC25A activated FOXM1 transcriptional activity via the CDK phosphorylation sites T600, T611, and T620 and a LXL docking motif at L656. U2OS cells were co-transfected with pACT-CDC25A and pACT-FOXM1 (wild-type or the mutations of phosphorylation sites/LXL motif), along with pGL3-6×FOXM1-Luc plasmids. pRL-SV40 was used as the normalizing control. Data were normalized to Renilla luciferase activities and are presented as the mean ± SD (N = 3). Data were subjected to one-way ANOVA (significance level α = 0.05) and Dunnett's multi-comparison post-hoc tests (mutations vs. wild-type). *** p<0.001.