Figure 4. Effect of phosphomimetic MEF2D and nonphosphorylatable MEF2D mutations on MEF2 activity. A, Each of the four ATM-phosphorylatable sites in MEF2D contributes to full activation after DNA damage. HEK293T cells were cotransfected with the following expression plasmids: two reporter constructs, MEF2-luc and Renilla, plus wt MEF2D, nonphosphorylatable MEF2D mutant (T259A/S275A/S294A/S314A), MEF2D mutant containing three putative ATM-phosphorylatable sites and a single alanine substitution (T259A, S275A, S294A, or S314A), or pcDNA3 vector control. One day after transfection, cells were exposed to etoposide (Eto) or control (untreated) conditions before measuring luciferase activity (n = 3 independent experiments). *p < 0.01 (ANOVA). NS, Not statistically significant. Data are mean ± SEM. B, Effect of nonphosphorylatable MEF2D mutations on MEF2 activity after serum induction. NIH 3T3 cells were cotransfected with two reporter constructs, MEF2-luc and Renilla, plus wt-MEF2D, nonphosphorylatable MEF2D mutant (T259A/S275A/S294A/S314A), or pcDNA3 vector control. The transfected cells were serum starved for 30 h and then treated with serum for 2 h before preparation of cell lysates for luciferase assays (n = 3 independent experiments). *p < 0.01 (ANOVA). Data are mean ± SEM. C, Effect of phosphomimetic MEF2D mutations on MEF2 activity in HEK293T cells. Cells were cotransfected with two reporter constructs, MEF2-luc and Renilla, plus wt-MEF2D, nonphosphorylatable MEF2D mutant (T259A/S275A/S294A/S314A), phosphomimetic MEF2D mutant (T259D/S275D/S294D/S314D), or pcDNA3 vector control. One day after transfection, transfected cells were exposed to etoposide or control (untreated) conditions before measuring luciferase activity. Basal luciferase activity of the untreated vector control was arbitrarily set to 1, and all other values were normalized to this reference point (n = 3 independent experiments). *p < 0.01 (ANOVA). Data are mean ± SEM. D, Effect of phosphomimetic MEF2D mutations on MEF2 activity after ATM knockdown in cerebellar granule cells. Cells were cotransfected with shRNA-1 and two reporter constructs, MEF2-luc and Renilla, plus wt-MEF2D, nonphosphorylatable MEF2D mutant (T259A/S275A/S294A/S314A), phosphomimetic MEF2D mutant (T259D/S275D/S294D/S314D), or pcDNA3 vector control. Three days after transfection, cells were exposed to etoposide or control (untreated) conditions before measuring luciferase activity. Basal luciferase activity of the untreated vector control was arbitrarily set to 1, and other values were normalized to this reference point (n = 3 independent experiments). *p < 0.01 (ANOVA). Data are mean ± SEM. Protein expression levels of wt and MEF2D mutant constructs are shown to the right of each panel.