Figure 2.

PKA signaling inactivates CaMKK2 by phosphorylating Ser⁴⁹⁵ in the CaM-binding sequence. A, representative whole-protein TOF spectra of CaMKK2 purified from transfected COS7 cells treated with DMSO vehicle control for 20 min. B, representative whole-protein TOF spectra of CaMKK2 purified from transfected COS7 cells treated with 50 µm forskolin/500 µm IBMX for 20 min. C, phosphorylation of CaMKK2 on Ser¹⁰⁰, Ser⁴⁹⁵, and Ser⁵¹¹ purified from transfected COS7 cells pretreated with 20 µm H89 for 60 min, then treated with 50 µm forskolin/500 µm IBMX for 20 min. Phosphorylation and total CaMKK2 were detected using rabbit phospho-specific and mouse anti-FLAG antibodies, respectively. A representative immunoblot is shown. D, Ca²⁺-CaM activation of WT CaMKK2 and phosphorylation site mutants from transfected COS7 cells treated with DMSO vehicle control or 50 µm forskolin/500 µm IBMX for 20 min. CaMKK2 was immunoprecipitated and activity was measured in the presence or absence of 100 µm Ca²⁺ and 1 µm CaM. Data are mean ± S.D. (error bars), n = 3 independent experiments, and were statistically appraised by two-way ANOVA (treatment: F = 68.56, p < 0.0001; mutation: F = 6.624, p = 0.0041; interaction: F = 6.627, p = 0.0041) using Tukey's post-hoc multiple comparisons test. ***p = 0.0004, **p = 0.0018, *p = 0.0029, ##p = 0.0035. E, CaM overlay assay measuring binding of biotinylated CaM to WT CaMKK2 and phosphorylation site mutants purified from transfected COS7 cells treated with vehicle control (DMSO) or 50 µm forskolin/500 µm IBMX for 20 min. CaM binding and total CaMKK2 were visualized using fluorescent-labeled streptavidin and rabbit anti-FLAG antibody, respectively. Data are mean ± S.D. (error bars), n = 3 independent experiments, and were statistically appraised by two-way ANOVA (treatment: F = 200.3, p < 0.0001; mutation: F = 14.96, p < 0.0001; interaction: F = 27.86, p < 0.0001) using Tukey's post-hoc multiple comparisons test. ****p < 0.0001, ####p < 0.0001.