Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase

Fukumoto, Yukio; Harada, Yuhei; Ohtsuka, Satomi; Kanayama, Naoki; Hatano, Naoya; Sakagami, Hiroyuki; Tokumitsu, Hiroshi

doi:10.1016/j.bbrc.2021.11.105

Cited by 3 publications

(2 citation statements)

References 37 publications

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“…coli BL21 (DE3) using pET–CaM (kindly provided by Dr. Nobuhiro Hayashi, Tokyo Institute of Technology, Yokohama, Japan) . His 6 -tagged CaMKKα (pME–His–CaMKKα) and CaMKKβ (pME–CaMKKβ) expression plasmids were constructed as described. , The anti-phospho-CaMKIα (at Thr177, clone 9H8) monoclonal antibody, anti-CaMKKα antibody, and anti-CaMKKβ antibody were generated. Furthermore, the anti-GST antibody (27457701V) was purchased from GE Healthcare (Buckinghamshire, UK).…”

Section: Methodsmentioning

confidence: 99%

Conformation-Dependent Reversible Interaction of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

et al. 2022

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Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), a Ca2+/CaM-dependent enzyme that phosphorylates and activates multifunctional kinases, including CaMKI, CaMKIV, protein kinase B/Akt, and 5′AMP-activated protein kinase, is involved in various Ca2+-signaling pathways in cells. Recently, we developed an ATP-competitive CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, Ohtsuka et al. Biochemistry 2020, 59, 1701–1710). To gain mechanistic insights into the interaction of CaMKK with TIM-063, we prepared TIM-063-coupled sepharose (TIM-127-sepharose) for association/dissociation analysis of the enzyme/inhibitor complex. CaMKKα/β in transfected COS-7 cells and in mouse brain extracts specifically bound to TIM-127-sepharose and dissociated following the addition of TIM-063 in a manner similar to that of recombinant GST–CaMKKα/β, which could bind to TIM-127-sepharose in a Ca2+/CaM-dependent fashion and dissociate from the sepharose following the addition of TIM-063 in a dose-dependent manner. In contrast to GST–CaMKKα, GST–CaMKKβ was able to weakly bind to TIM-127-sepharose in the presence of EGTA, probably due to the partially active conformation of recombinant GST–CaMKKβ without Ca2+/CaM-binding. These results suggested that the regulatory domain of CaMKKα prevented the inhibitor from interacting with the catalytic domain as the GST–CaMKKα mutant (residues 126–434) lacking the regulatory domain (residues 438–463) interacted with TIM-127-sepharose regardless of the presence or absence of Ca2+/CaM. Furthermore, CaMKKα bound to TIM-127-sepharose in the presence of Ca2+/CaM completely dissociated from TIM-127-sepharose following the addition of excess EGTA. These results indicated that TIM-063 interacted with and inhibited CaMKK in its active state but not in its autoinhibited state and that this interaction is likely reversible, depending on the concentration of intracellular Ca2+.

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Section: Methodsmentioning

confidence: 99%

Conformation-Dependent Reversible Interaction of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

et al. 2022

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“…Although CaMKK α/1 and β/2 had been considered monomeric kinases similar to other CaMKs including CaMKI and CaMKIV, Ling et al recently reported that FLAG-tagged CaMKKβ/2 and HA-tagged CaMKK2 β/2 mutant (Arg311Cys) might form a dimer or larger oligomer [ 74 ]. Consistent with this possibility, we demonstrated the oligomerization of both rat CaMKK isoforms in transfected cells by chemical crosslinking [ 75 ]. The CaMKKα/1 oligomer was catalytically active, although the mechanism and functional consequences of CaMKK oligomerization remain unknown.…”

Section: Domain Structure and Activation Of Camkkmentioning

confidence: 59%

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction

Tokumitsu

Sakagami

2022

IJMS

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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.

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Research advances on CaMKs-mediated neurodevelopmental injury

Kong,

Yang,

Yang

et al. 2024

Arch Toxicol

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Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase

Cited by 3 publications

References 37 publications

Conformation-Dependent Reversible Interaction of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

Conformation-Dependent Reversible Interaction of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction

Research advances on CaMKs-mediated neurodevelopmental injury

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Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase

Cited by 3 publications

References 37 publications

Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction

Research advances on CaMKs-mediated neurodevelopmental injury

Contact Info

Product

Resources

About

Conformation-Dependent Reversible Interaction of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063

Conformation-Dependent Reversible Interaction of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063