The Ca/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca concentrations. Moreover, the Ca/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation , leading to reduced autonomous, but not Ca/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr phosphorylation by activated AMPK converts CaMKKβ into a Ca/CaM-dependent enzyme as shown by completely Ca/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr antibody revealed phosphorylation of Thr in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca-dependent AMPK activation by CaMKKβ.

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.

Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca 2+-dependent cellular processes including neuronal, metabolic, and pathophysiological pathways. Herein we developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analog (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and β) with a similar potency (Ki = 0.35 µM for CaMKKα and Ki = 0.2 µM for CaMKKβ) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV and endogenous AMPKα in HeLa cells with an IC 50 of ~0.3 µM, and it suppressed the CaMKK isoforms-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.