FIGURE 5.

EF2K stability is regulated by PKA signaling. A, PC12 cells were treated with the selective PKA activator 6-BNZ cAMP (100 μm) for 0–6 h, and EF2K and GAPDH levels in lysates were assessed by SDS-PAGE and immunoblot. B, graph of summary data. A one-way ANOVA with Tukey's post-hoc tests demonstrated that EF2K levels were significantly reduced after 1 h of 6-BNZ cAMP treatment (68.84 ± 6.35, n = 3, *, p < 0.05), and these levels subsequently recovered to baseline. C, PC12 cells were transfected with either wild-type GFP-EF2K or 365D or 499E mutant forms and treated with Chx (30 μg/ml) for 0–6 h to assess their stability. EF2K and GAPDH levels in lysates were assessed by SDS-PAGE and immunoblot. D, graph of summary data. A two-way ANOVA revealed significant main effects of the form of GFP-EF2K transfected and chase time, as well as a significant interaction between transfection and time. Bonferroni post-hoc tests indicated a significantly increased degradation rate of the 499E mutant relative to that of wild-type GFP-EF2K at 2 and 4 h (2 h: 48.32 ± 11.35, n = 3, ***, p < 0.001; 4 h: 40.81 ± 5.25, n = 3, **, p < 0.01), and this rate later recovered to wild-type levels. The 365D mutant did not exhibit a different level of degradation than wild-type GFP-EF2K at any time point.