Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) is the activating kinase for multiple downstream kinases, including CaM-kinase I (CaMKI), CaM-kinase IV (CaMKIV), protein kinase B (PKB/Akt), and 5′AMP-kinase (AMPK), through the phosphorylation of their activation-loop Thr residues in response to increasing the intracellular Ca²⁺ concentration, as CaMKK itself is a Ca²⁺/CaM-dependent enzyme. The CaMKK-mediated kinase cascade plays important roles in a number of Ca²⁺-dependent pathways, such as neuronal morphogenesis and plasticity, transcriptional activation, autophagy, and metabolic regulation, as well as in pathophysiological pathways, including cancer progression, metabolic syndrome, and mental disorders. This review focuses on the molecular mechanism underlying CaMKK-mediated signal transduction in normal and pathophysiological conditions. We summarize the current knowledge of the structural, functional, and physiological properties of the regulatory kinase, CaMKK, and the development and application of its pharmacological inhibitors.

The Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5′-AMP–activated protein kinase (AMPK) phosphorylation cascade affects various Ca²⁺-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 Ca²⁺ concentrations. Moreover, the Ca²⁺/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 in vitro, leading to reduced autonomous, but not Ca²⁺/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr¹⁴⁴ phosphorylation by activated AMPK converts CaMKKβ into a Ca²⁺/CaM-dependent enzyme as shown by completely Ca²⁺/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 Thr¹⁴⁴ phosphorylation–dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr¹⁴⁴ antibody revealed phosphorylation of Thr¹⁴⁴ 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 Ca²⁺-dependent AMPK activation by CaMKKβ.

Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr¹⁷² in the AMPKα subunit more efficiently than CaMKKα, with a lower K_m (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu³⁵⁸ in CaMKKβ/Ile³²² in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.

FKBP38 (FK506-binding protein 38), a membrane-anchored TPR (tetratricopeptide repeat)-containing immunophilin, regulates signalling pathways such as cell survival, apoptosis, proliferation and metastasis. However, the mechanisms that regulate the activity of FKBP38 are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hop [Hsp70 (heat-shock protein of 70 kDa)/Hsp90-organizing protein], kinesin-light chain, Tom70 (translocase of outer mitochondrial membrane 70), FKBP52, CyP40 (cyclophilin 40), CHIP (C-terminus of Hsc70-interacting protein) and PP5 (protein phosphatase 5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore we have hypothesized that Ca2+/S100 proteins can interact with FKBP38 and regulate its function. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, S100B and S100P specifically interact with FKBP38 and inhibit the interaction of FKBP38 with Bcl-2 and Hsp90. Overexpression of permanently active S100P in Huh-7 cells inhibited the interaction of FKBP38 with Bcl-2, resulting in the suppression of Bcl-2 stability. The association of the S100 proteins with FKBP38 provides a Ca2+-dependent regulatory mechanism of the FKBP38-mediated signalling pathways.