ID | 63074 |
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Fukumoto, Yusei
Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University
Harada, Yuhei
Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University
Ohtsuka, Satomi
Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University
Kanayama, Naoki
Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University
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Magari, Masaki
Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University
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Hatano, Naoya
Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University
Sakagami, Hiroyuki
Department of Anatomy, Kitasato University School of Medicine
Tokumitsu, Hiroshi
Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University
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Abstract | Ca2+/calmodulin-dependent protein kinase kinases (CaMKKα and β) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His–CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG–CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST–CaMKKα/His–CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Nisepharose and was observed in a Ca2+/CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG–CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling.
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Keywords | CaMKK
oligomerization
Ca2+-signaling
phosphorylation
CaM kinase cascade
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Note | © 2021 Elsevier Inc. This manuscript version is made available under the CC-BY-NC-ND 4.0 License. http://creativecommons.org/licenses/by-nc-nd/4.0/. This is the accepted manuscript version. The formal published version is available at [https://doi.org/10.1016/j.bbrc.2021.11.105] .
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Published Date | 2022-1
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Publication Title |
Biochemical and Biophysical Research Communications
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Volume | volume587
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Publisher | Elsevier BV
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Start Page | 160
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End Page | 165
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ISSN | 0006-291X
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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 | © 2021 Elsevier Inc.
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File Version | author
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Related Url | isVersionOf https://doi.org/10.1016/j.bbrc.2021.11.105
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Citation | http://creativecommons.org/licenses/by-nc-nd/4.0/
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Funder Name |
Japan Society for the Promotion of Science
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助成番号 | JP21H02429
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