Updated on 2025/07/16

写真a

 
TOKUMITSU Hiroshi
 
Organization
Faculty of Interdisciplinary Science and Engineering in Health Systems Professor
Position
Professor
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Degree

  • Ph.D. ( 1991.3   Nagoya University )

Research Interests

  • シグナル伝達

  • CaM-キナーゼカスケード

  • CREB

  • 細胞内カルシウム

  • タンパク質リン酸化酵素阻害剤

  • Numbl

  • タンパク質リン酸化

  • タンパク質リン酸化反応

  • タンパク質リン酸化酵素

  • 細胞内情報伝達

  • MLCK-A

  • STO-609

  • 線虫

  • Numb

  • カルモデュリン

  • SAD-kinase

  • CaM-kinase I

  • insulin

  • 包括脳ネットワーク

  • プロテインキナ-ゼ

  • インスリン

  • 糖尿病

  • glucose

  • グルコース

  • PREB

  • CaM-kinase kinase

  • CaMKK

  • CaM-kinase IV

  • TIM-062

  • KN-62

  • S100 protein

  • TIM-063

  • CaM-キナーゼ

  • リン酸化酵素カスケード

Research Areas

  • Life Science / Neuroscience-general

  • Life Science / Metabolism and endocrinology

  • Life Science / Pharmacology

  • Life Science / Functional biochemistry

Research History

  • Okayama University   学術研究院ヘルスシステム統合科学学域   Professor

    2021

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  • Okayama University   Graduate School of Interdisciplinary Science and Engineering in Health Systems   Professor

    2018

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  • Okayama University   Graduate School of Natural Science and Technology   Professor

    2012 - 2018

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  • Kagawa University   Faculty of Medicine   Associate Professor

    2007 - 2012

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  • Kagawa University   Faculty of Medicine   Associate Professor (as old post name)

    2003 - 2007

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Papers

  • Characterization of molecular mechanisms of CaMKKα/1 oligomerization. Reviewed International journal

    Shun Uenoyama, Hayato Nitta, Satomi Ohtsuka, Masaki Magari, Futoshi Suizu, Hiroshi Tokumitsu

    FEBS letters   599 ( 13 )   1914 - 1924   2025.5

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is an activating kinase for calcium/calmodulin-dependent protein kinase type 1 (CaMKI), calcium/calmodulin-dependent protein kinase type IV (CaMKIV), RAC-alpha serine/threonine-protein kinase (PKB), and AMP-activated protein kinase (AMPK) that has been reported to form an active oligomer in cells. Glutathione S-transferase (GST) pulldown assay from the extracts of COS-7 cells expressing GST- and His6-CaMKKα/1 mutants showed that the C-terminal region containing the autoinhibitory and calmodulin (CaM)-binding sequence (residues 438-463) is required for CaMKKα/1 homo-oligomerization. This was confirmed by the fact that the GST-CaMKKα/1 C-terminal domain (residues 435-505) directly interacted with EGFP-CaMKKα/1 residues 435-505 as well as with wild-type CaMKKα/1. Notably, once oligomerized in cells, CaMKKα/1 is neither exchangeable between the oligomeric complexes nor dissociated by Ca2+/CaM binding. These results support stable oligomerization of CaMKK in the cells by intermolecular self-association of its C-terminal region containing a regulatory domain.

    DOI: 10.1002/1873-3468.70078

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  • Development of a novel AAK1 inhibitor via Kinobeads-based screening. Reviewed International journal

    Akari Yoshida, Satomi Ohtsuka, Fumiya Matsumoto, Tomoyuki Miyagawa, Rei Okino, Yumeya Ikeda, Natsume Tada, Akira Gotoh, Masaki Magari, Naoya Hatano, Ryo Morishita, Ayano Satoh, Yukinari Sunatsuki, Ulf J Nilsson, Teruhiko Ishikawa, Hiroshi Tokumitsu

    Scientific reports   14 ( 1 )   6723 - 6723   2024.3

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and β/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor.

    DOI: 10.1038/s41598-024-57051-9

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  • Transcriptional, biochemical, and immunohistochemical analyses of CaMKKβ/2 splice variants that co-localize with CaMKIV in spermatids. Reviewed International journal

    Satomi Ohtsuka, Yumi Miyai, Hiroyuki Mima, Masaki Magari, Yoichi Chiba, Futoshi Suizu, Hiroyuki Sakagami, Masaki Ueno, Hiroshi Tokumitsu

    Cell calcium   117   102820 - 102820   2024.1

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca2+-dependent physiological and pathophysiological pathways. Further, CaMKKβ/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKβ/2 splice variants (CaMKKβ-3 and β-3x). RT-PCR analyses revealed that mouse CaMKKβ-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKKβ-3 and β-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKβ-3 or β-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKβ-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKβ-1. We also observed the co-localization of CaMKKβ-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKβ-3 exhibited Ca2+/CaM-induced kinase activity similar to CaMKKβ-1. Conversely, we noted that CaMKKβ-3x impaired Ca2+/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKβ-1 or β-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKβ-3x showed the ability to phosphorylate downstream kinases, including CaMKIα, CaMKIV, and AMPKα in transfected cells comparable to CaMKKβ-1 and β-3. Collectively, CaMKKβ-3/-3x were identified as functionally active and could be bona fide CaMKIV-kinases in testes involved in the activation of the CaMKIV cascade in spermatids, resulting in the regulation of spermiogenesis.

    DOI: 10.1016/j.ceca.2023.102820

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  • PHI-1, an Endogenous Inhibitor Protein for Protein Phosphatase-1 and a Pan-Cancer Marker, Regulates Raf-1 Proteostasis Reviewed

    Jason A. Kirkbride, Garbo Young Nilsson, Jee In Kim, Kosuke Takeya, Yoshinori Tanaka, Hiroshi Tokumitsu, Futoshi Suizu, Masumi Eto

    Biomolecules   13 ( 12 )   1741 - 1741   2023.12

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:MDPI AG  

    Raf-1, a multifunctional kinase, regulates various cellular processes, including cell proliferation, apoptosis, and migration, by phosphorylating MAPK/ERK kinase and interacting with specific kinases. Cellular Raf-1 activity is intricately regulated through pathways involving the binding of regulatory proteins, direct phosphorylation, and the ubiquitin–proteasome axis. In this study, we demonstrate that PHI-1, an endogenous inhibitor of protein phosphatase-1 (PP1), plays a pivotal role in modulating Raf-1 proteostasis within cells. Knocking down endogenous PHI-1 in HEK293 cells using siRNA resulted in increased cell proliferation and reduced apoptosis. This heightened cell proliferation was accompanied by a 15-fold increase in ERK1/2 phosphorylation. Importantly, the observed ERK1/2 hyperphosphorylation was attributable to an upregulation of Raf-1 expression, rather than an increase in Ras levels, Raf-1 Ser338 phosphorylation, or B-Raf levels. The elevated Raf-1 expression, stemming from PHI-1 knockdown, enhanced EGF-induced ERK1/2 phosphorylation through MEK. Moreover, PHI-1 knockdown significantly contributed to Raf-1 protein stability without affecting Raf-1 mRNA levels. Conversely, ectopic PHI-1 expression suppressed Raf-1 protein levels in a manner that correlated with PHI-1’s inhibitory potency. Inhibiting PP1 to mimic PHI-1’s function using tautomycin led to a reduction in Raf-1 expression. In summary, our findings highlight that the PHI-1-PP1 signaling axis selectively governs Raf-1 proteostasis and cell survival signals.

    DOI: 10.3390/biom13121741

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  • Rapid detection of calmodulin/target interaction via the proximity biotinylation method. Reviewed International journal

    Kento Nlandu Nakamura, Haruki Yamauchi, Hiroyuki Mima, Yerun Chen, Satomi Ohtsuka, Masaki Magari, Ryo Morishita, Hiroshi Tokumitsu

    Biochemical and biophysical research communications   659   29 - 33   2023.4

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca2+-dependent biotinylation of a target protein kinase (Ca2+/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro, which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca2+-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity.

    DOI: 10.1016/j.bbrc.2023.03.072

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Books

  • Ca²⁺/calmodulin-dependent protein kinase kinase —カルシウムシグナル伝達から創薬へ—

    徳光 浩( Role: Sole author ,  全て)

    生化学(公益財団法人 日本生化学会)  2018.8 

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  • Ca²⁺/カルモデュリン依存性蛋白質リン酸化酵素カスケ-ドと生理機能

    徳光 浩( Role: Sole author)

    蛋白質核酸酵素(共立出版)  1998.4 

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MISC

  • CaMKKの基質認識機構の解明と特異的阻害分子開発への応用

    川俣一晟, 美馬光志, 山内陽生, 北前勝哉, 山内香奈, 大塚里美, 曲正樹, 金山直樹, 徳光浩

    日本生化学会大会(Web)   95th   2022

  • CaMKK阻害剤(TIM-063)を用いた阻害剤プロテオミクス解析

    大塚里美, 波多野直哉, 奥村太晟, 澤直樹, 田邊史子, 傳田美和子, 金山直樹, 曲正樹, 森下了, 石川彰彦, 徳光浩

    日本生化学会大会(Web)   94th   2021

  • 新規S100A6標的分子(HMG20A)の同定と相互作用解析

    山本真穂, 近藤里奈, 傳田美和子, 土居青太, 金山直樹, 曲正樹, 森下了, 徳光浩

    日本生化学会大会(Web)   92nd   2019

  • 筋肉特異的Calmodulin結合分子,Striated Muscle Activator of Rho Signaling(STARS)の分子間相互作用解析

    赤木魁, 田中啓之, 金山直樹, 曲正樹, 波多野直哉, 徳光浩

    日本分子生物学会年会プログラム・要旨集(Web)   42nd   2019

  • CaMKKβのリン酸化/脱リン酸化による動的制御機構の解明

    高畠翔太, 福本侑世, 金山直樹, 曲正樹, 波多野直哉, 徳光浩

    日本分子生物学会年会プログラム・要旨集(Web)   42nd   2019

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Research Projects

  • CaMKKシグナル伝達の制御機構解明とそれに基づく分子標的薬創製

    Grant number:21H02429  2021.04 - 2024.03

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    徳光 浩, 石川 彰彦, 渡辺 泰男, 曲 正樹

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    Grant amount:\16380000 ( Direct expense: \12600000 、 Indirect expense:\3780000 )

    本研究では、細胞内Ca2+を二次伝達因子とする細胞内シグナル伝達機構において、神経発生、遺伝子発現制御から代謝応答まで多岐に渡る生体機能調節を担う制御酵素として見出されたタンパク質リン酸化酵素であるCaMKKの分子制御機構の解明とその分子基盤に立脚したCaMKK阻害薬の創製を研究目的としている。本年度の研究実績として、消化管平滑筋におけるカルシウム脱感作反応においてCaMKKを介したリン酸化カスケード反応の関与が、新たに開発したCaMKK阻害剤TIM-063を用いることで明らかとなった(Kitazawa et al. Am J Physiol Cell Physiol 2021)。さらにCaMKKと阻害剤TIM-063の物理的相互作用について、TIM-063誘導体(TIM-127)を架橋したセファロース担体を用いることにより詳細に解析した。その結果、CaMKKと阻害剤TIM-063の相互作用は、酵素のカルシウム/calmodulin結合に依存しており、不活性型のコンフォメーションをとるCaMKKは阻害剤に結合しないこと、さらにはCaMKK/阻害剤結合はCaMKKの活性化状態に依存して可逆的であることを証明することに成功した(Ohtsuka et al. Biochemsitry 2022)。これまでCaMKKはその分子構造が単量体と考えられていたが、本研究において培養細胞に遺伝子導入したCaMKKアイソフォームは、多量体を形成することを細胞膜透過性架橋剤を用いることで明らかにした。さらに、この遺伝子導入細胞より単離した多量体CaMKKは、リン酸化酵素として酵素活性を有することも併せて証明することができた(Fukumoto et al. Biochem Biophys Res Commun 2022)。

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  • Regulatory mechanism of transcription by ciliary proteins.

    Grant number:19H03447  2019.04 - 2022.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    Suizu Futoshi

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    Grant amount:\17420000 ( Direct expense: \13400000 、 Indirect expense:\4020000 )

    Inversin accumulates in a Cajal body-like structure in the nucleus, which is one of the centers of transcriptional activity, and has a specific transcriptional regulatory activity. This strongly suggests that the ciliary protein Inversin functions as a novel transcriptional regulator. In addition, it was clarified that during the induction of cyst formation in the unicellular ciliate Colpoda cucullus, rapid fragmentation and expression change of the ciliate constituent protein beta-tubulin occurred and it was taken up into the body. From the dynamic changes of ciliary proteins, it is considered that the localization, structure, and functional changes of ciliary proteins and reabsorption lead to the reuse of amino acids and nucleic acids for cyst formation. In addition, it is presumed that changes in the transcriptional control function of ciliary proteins are important because they acquire the properties of low temperature, low pH, and UV resistance with the rapid change to cysts.

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  • Regulation of CaMKKbeta/AMPK signaling and drug development

    Grant number:18K06113  2018.04 - 2021.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Tokumitsu Hiroshi

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    Grant amount:\4290000 ( Direct expense: \3300000 、 Indirect expense:\990000 )

    We have obtained the results regarding CaMKK-mediated intracellular signal transduction as follows; (1) CaMKKβ is phosphorylated at Thr144 in HeLa cells upon stimulation with isoproterenol, indicating the regulatory phosphorylation of CaMKKβ by cAMP-mediated signaling. (2) CaMKKβ is also rapidly dephosphorylated in the cells suggesting the dynamic regulation of CaMKKβ through phosphorylation/dephosphorylation. (3) We have succeeded to develop a novel CaMKK inhibitor (TIM-063) and an inactive analogue (TIM-062) which could be useful for evaluating the physiological roles of CaMKK-mediated signaling pathways.

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  • Improvement of an animal cell-displaying technology by using a splicing factor

    Grant number:15H04196  2015.04 - 2019.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)  Grant-in-Aid for Scientific Research (B)

    Kanayama Naoki

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    Grant amount:\16640000 ( Direct expense: \12800000 、 Indirect expense:\3840000 )

    We have been developing an animal cell-displaying technology using the hypermutating B cell line DT40. In this study, we elucidated a function of a splicing factor, SRSF1, which we have previously found that is essential for hypermutation of the immunoglobulin gene, and developed a method to increase hypermutation efficiency by manipulation of SRSF1 expression.

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  • Phosphorylation-dependent regulatory mechanism of calmoudlin-kinase cascade

    Grant number:26440056  2014.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)  Grant-in-Aid for Scientific Research (C)

    Tokumitsu Hiroshi, HATANO Naoya

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    Grant amount:\5070000 ( Direct expense: \3900000 、 Indirect expense:\1170000 )

    Intracellular Ca2+ plays an important role in the cellular signal transduction system as a second messenger. Calmodulin (caM) is one of Ca2+-binding proteins, which activates multifunctional CaM-kinases. In this research, we examined the molecular mechanism of substrate recognition for Ca2+/calmodulin-dependent protein kinase kinase beta based on the structure-function analysis.

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Class subject in charge

  • Signal Transduction and Drug Development (2024academic year) Prophase  - その他

  • General Biotechnology and Drug Discovery Technology (2024academic year) Prophase  - 木1~2

  • Advanced Internship for Interdisciplinary Medical Sciences and Engineering (2024academic year) Year-round  - その他

  • Technical English for Interdisciplinary Medical Sciences and Engineering (2024academic year) Late  - その他

  • Introduction to Interdisciplinary Science and Engineering in Health Systems (2024academic year) Prophase  - 火1~2

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Academic Activities

  • 日本学術振興会特別研究員等審査会専門委員

    Role(s):Review, evaluation

    日本学術振興会研究者養成課特別研究員等審査会  2020 - 2021

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    Type:Scientific advice/Review 

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  • 日本生化学会評議員・代議員

    Role(s):Planning, management, etc.

    日本生化学会  2018

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    Type:Academic society, research group, etc. 

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