ID | 63444 |
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Ohtsuka, Satomi
Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
Okumura, Taisei
Department of Science Education, Graduate School of Education, Okayama University
Τabuchi, Yuna
Department of Science Education, Graduate School of Education, Okayama University
Miyagawa, Tomoyuki
Department of Science Education, Graduate School of Education, Okayama University
Kanayama, Naoki
Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
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Magari, Masaki
Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
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Hatano, Naoya
Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
Sakagami, Hiroyuki
Department of Anatomy, Kitasato University School of Medicine
Suizu, Futoshi
Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University
Ishikawa, Teruhiko
Department of Science Education, Graduate School of Education, Okayama University
Tokumitsu, Hiroshi
Applied Cell Biology, Graduate School of Interdisciplinary Science & Engineering in Health Systems, Okayama University
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Abstract | 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. CaMKK alpha/beta 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-CaMKK alpha/beta, which could bind to TIM-127sepharose 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-CaMKK alpha, GST-CaMKK beta was able to weakly bind to TIM-127-sepharose in the presence of EGTA, probably due to the partially active conformation of recombinant GST-CaMKK beta without Ca2+/CaM-binding. These results suggested that the regulatory domain of CaMKK alpha prevented the inhibitor from interacting with the catalytic domain as the GST-CaMKK alpha 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, CaMKK alpha 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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Note | This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.biochem.1c00796
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Published Date | 2022-03-11
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Publication Title |
Biochemistry
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Volume | volume61
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Issue | issue7
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Publisher | American Chemical Society (ACS)
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Start Page | 545
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End Page | 553
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ISSN | 0006-2960
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NCID | AA00564599
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Content Type |
Journal Article
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language |
English
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OAI-PMH Set |
岡山大学
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Copyright Holders | Copyright © 2022 American Chemical Society
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File Version | author
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DOI | |
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Related Url | isVersionOf https://doi.org/10.1021/acs.biochem.1c00796
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