Regulation of Ca ²⁺ /calmodulin-dependent protein kinase kinase β by cAMP signaling

Background: Ca ²⁺ /calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5’-AMP-activated protein kinase (AMPK), controlling Ca ²⁺ -dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca ²⁺ /CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. Methods: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. Results: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca ²⁺ /CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca ²⁺ /CaM-dependent enzyme. Conclusion: cAMP/PKA signaling may confer Ca ²⁺ -dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. General significance: Our results suggest a novel cross-talk between cAMP/PKA and Ca ²⁺ /CaM/CaMKKβ signaling through regulatory phosphorylation.