Faculty Profiles - TOKUMITSU Hiroshi

写真a

TOKUMITSU Hiroshi

Organization

Faculty of Interdisciplinary Science and Engineering in Health Systems Professor

Homepage

http://www.okayama-u.ac.jp/user/saibou/

External link

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

1999 - 2003

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（株）ヘリックス研究所主任研究員

1996 - 1999

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オレゴン健康科学大学 Vollum 研究所博士研究員

1993 - 1996

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DNAX 分子細胞生物学研究所博士研究員

1992 - 1993

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Nagoya University School of Medicine Research Assistant

1991 - 1994

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Nagoya University School of Medicine Special researcher of the Japan Society for the Promotion of Science

1991

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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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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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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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The immunoreceptor SLAMF8 promotes the differentiation of follicular dendritic cell‐dependent monocytic cells with B cell‐activating ability Reviewed

Masaki Magari, Miku Nishioka, Tomomi Hari, Sayaka Ogawa, Kaho Takahashi, Naoya Hatano, Naoki Kanayama, Junichiro Futami, Hiroshi Tokumitsu

FEBS Letters 596 ( 20 ) 2659 - 2667 2022.10

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Authorship：Last author Publishing type：Research paper (scientific journal) Publisher：Wiley

DOI： 10.1002/1873-3468.14468

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/1873-3468.14468
Substrate recognition by Arg/Pro-rich insert domain in calcium/calmodulin-dependent protein kinase kinase for target protein kinases. Reviewed International journal

Riku Kaneshige, Satomi Ohtsuka, Yuhei Harada, Issei Kawamata, Masaki Magari, Naoki Kanayama, Naoya Hatano, Hiroyuki Sakagami, Hiroshi Tokumitsu

The FEBS journal 289 ( 19 ) 5971 - 5984 2022.10

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Calcium/calmodulin-dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP-activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca2+ -signaling pathways. Mammalian cells expressing CaMKKα and CaMKKβ lacking Arg/Pro-rich insert domain (RP-domain) sequences showed impaired phosphorylation of AMPKα, CaMKIα, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild-type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKIα and CaMKIV; however, mutant LKB1 with the RP-domain sequences of CaMKKα and CaMKKβ inserted between kinase subdomains II and III acquired CaMKIα and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin-induced phosphorylation of hemagglutinin (HA)-CaMKIα at Thr177, HA-CaMKIV at Thr196, and HA-AMPKα at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase-dead mutants of CaMKK isoforms, but these dominant-negative effects were abrogated with RP-deletion mutants, suggesting that sequestration of substrate kinases by loss-of-function CaMKK mutants requires the RP-domain. This was confirmed by pulldown experiments that showed that dominant-negative mutants of CaMKKα and CaMKKβ interact with target kinases but not RP-deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP-domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP-domain may be a promising target for specific CaMKK inhibitors.

DOI： 10.1111/febs.16467

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

Hiroshi Tokumitsu, Hiroyuki Sakagami

International Journal of Molecular Sciences 23 ( 19 ) 11025 - 11025 2022.9

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Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI AG

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.

DOI： 10.3390/ijms231911025

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

Satomi Ohtsuka, Taisei Okumura, Yuna Τabuchi, Tomoyuki Miyagawa, Naoki Kanayama, Masaki Magari, Naoya Hatano, Hiroyuki Sakagami, Futoshi Suizu, Teruhiko Ishikawa, Hiroshi Tokumitsu

Biochemistry 61 ( 7 ) 545 - 553 2022.4

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Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society (ACS)

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+.

DOI： 10.1021/acs.biochem.1c00796

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Oligomerization of Ca²⁺/calmodulin-dependent protein kinase kinase Reviewed International journal

Yusei Fukumoto, Yuhei Harada, Satomi Ohtsuka, Naoki Kanayama, Masaki Magari, Naoya Hatano, Hiroyuki Sakagami, Hiroshi Tokumitsu

Biochemical and Biophysical Research Communications 587 160 - 165 2022.1

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

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/Ni-sepharose 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.

DOI： 10.1016/j.bbrc.2021.11.105

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A temporal Ca²⁺-desensitization of myosin light chain kinase in phasic smooth muscles induced by CaMKKβ/PP2A pathways. Reviewed

Toshio Kitazawa, Toshiyasu Matsui, Shuichi Katsuki, Akira Goto, Kai Akagi, Naoya Hatano, Hiroshi Tokumitsu, Kosuke Takeya, Masumi Eto

American Journal of Physiology-Cell Physiology 321 ( 3 ) C549 - C558 2021.9

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Language：English Publishing type：Research paper (scientific journal) Publisher：American Physiological Society

Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as digestive tract and urinary bladder, undergo a relaxation due to a decline of cellular [Ca²⁺] and a decreased Ca²⁺ sensitivity of LC20 phosphorylation, named Ca²⁺ desensitization. Here, we determined mechanisms underlying the temporal Ca²⁺ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of mouse ileum and urinary bladder. Upon the stimulation with pCa6.0 at 20°C, the contraction and the LC20 phosphorylation peaked within 30 sec and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, the LC20 kinase (MLCK) was inactivated, but no fluctuation in the LC20 phosphatase activity occurred, suggesting that the MLCK inactivation is a cause of the Ca²⁺-induced Ca²⁺-desensitization of LC20 phosphorylation. The MLCK inactivation was associated with phosphorylation at the calmodulin binding domain of the kinase. Treatment with antagonists for CaMKKß (STO-609 and TIM-063) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaMKII, AMPK nor PAK induced the MLCK inactivation in phasic smooth muscles. Conversely, PP2A inhibition amplified the phasic response. Signaling pathways through CaMKKß and PP2A may contribute to regulating the Ca²⁺ sensitivity of MLCK and the contractile response of phasic smooth muscles.

DOI： 10.1152/ajpcell.00136.2021

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Regulation of the tubulin polymerization-promoting protein by Ca²⁺/S100 proteins Reviewed

Seita Doi, Naoki Fujioka, Satomi Ohtsuka, Rina Kondo, Maho Yamamoto, Miwako Denda, Masaki Magari, Naoki Kanayama, Naoya Hatano, Ryo Morishita, Takafumi Hasegawa, Hiroshi Tokumitsu

Cell Calcium 96 102404 2021.7

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Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca²⁺/S100A6 Target Reviewed International journal

Maho Yamamoto, Rina Kondo, Haruka Hozumi, Seita Doi, Miwako Denda, Masaki Magari, Naoki Kanayama, Naoya Hatano, Ryo Morishita, Hiroshi Tokumitsu

Biomolecules 11 ( 4 ) 510 2021.3

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During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311-342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311-347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

DOI： 10.3390/biom11040510

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Development and Characterization of Novel Molecular Probes for Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609. Reviewed International journal

Satomi Ohtsuka, Yui Ozeki, Moeno Fujiwara, Tomoyuki Miyagawa, Naoki Kanayama, Masaki Magari, Naoya Hatano, Futoshi Suizu, Teruhiko Ishikawa, Hiroshi Tokumitsu

Biochemistry 59 ( 17 ) 1701 - 1710 2020.5

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

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKβ) with a similar potency (Ki = 0.35 μM for CaMKKα, and Ki = 0.2 μM for CaMKKβ) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50 of ∼0.3 μM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.

DOI： 10.1021/acs.biochem.0c00149

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Phosphorylation and dephosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells. Reviewed International journal

Shota Takabatake, Yusei Fukumoto, Satomi Ohtsuka, Naoki Kanayama, Masaki Magari, Hiroyuki Sakagami, Naoya Hatano, Hiroshi Tokumitsu

Biochemical and Biophysical Research Communications 525 ( 1 ) 251 - 257 2020.2

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

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5'AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

DOI： 10.1016/j.bbrc.2020.02.056

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IL-34 cell surface localization regulated by the 78 kDa glucose-regulated protein facilitates the differentiation of monocytic cells. Reviewed

Sayaka Ogawa, Yukiko Matsuoka, Miho Takada, Kazue Matsui, Fumihiro Yamane, Eri Kubota, Shiori Yasuhara, Kentaro Hieda, Naoki Kanayama, Naoya Hatano, Hiroshi Tokumitsu, Masaki Magari

The Journal of Biological Chemistry 294 ( 7 ) 2386 - 2396 2019.2

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DOI： 10.1074/jbc.RA118.006226

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Regulation of Ca²⁺/calmodulin-dependent protein kinase kinase β by cAMP signaling. Reviewed International journal

Shota Takabatake, Satomi Ohtsuka, Takeyuki Sugawara T, Naoya Hatano, Naoki Kanayama, Masaki Magari, Hiroyuki Sakagami, Hiroshi Tokumitsu

iochimica et Biophysica Acta. General Subjects 1863 ( 4 ) 672 - 680 2019.1

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BACKGROUND: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation.

DOI： 10.1016/j.bbagen.2018.12.012

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Interaction of S100A6 with Target Proteins In Vitro and in Living Cells. Reviewed

Kyohei Sakane, Fuminori Yamaguchi, Mitsumasa Tsuchiya, Rina Kondo, Naoki Kanayama, Masaki Magari, Naoya Hatano, Ryoji Kobayashi, Hiroshi Tokumitsu

Methods in Molecular Biology (Clifton, N.J.) 1929 367 - 377 2019

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DOI： 10.1007/978-1-4939-9030-6_23

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ヘルスシステムの理解とその応用濾胞樹状細胞による抗体の親和性成熟の制御機構の解明(Interdisciplinary Science and Engineering in Health Systems Immunological functions of follicular dendritic cells on affinity maturation of antibody)

Magari Masaki, Ogawa Sayaka, Matsuoka Yukiko, Takada Miho, Kanayama Naoki, Tokumitsu Hiroshi

生物物理 58 ( Suppl.1-2 ) S197 - S197 2018.8

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Expression of Collagenase is Regulated by the VarS/VarA Two-Component Regulatory System in Vibrio alginolyticus. Reviewed International journal

Takehiko Mima, Kazuyoshi Gotoh, Yumiko Yamamoto, Keiko Maeda, Taku Shirakawa, Shunsuke Matsui, Yumi Murata, Takaki Koide, Hiroshi Tokumitsu, Osamu Matsushita

The Journal of Membrane Biology 251 ( 1 ) 51 - 63 2018.2

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Vibrio alginolyticus is an opportunistic pathogen in both humans and marine animals. Collagenase encoded by colA is considered to be one of the virulence factors. Expression of colA is regulated by multiple environmental factors, e.g., temperature, growth phase, and substrate. To elucidate the mechanism of regulation of colA expression, transposon mutagenesis was performed. VarS, a sensor histidine kinase of the two-component regulatory system, was demonstrated to regulate the expression of colA. VarA, a cognate response regulator of VarS, was also identified and shown to be involved in the regulation of colA expression. In vitro phosphorylation assays showed that phosphorylated VarS acted as a phosphoryl group donor to VarA. A site-directed mutagenesis study showed that the His300, Asp718 and His874 residues in VarS were essential for the phosphorylation of VarS, and the Asp54 residue in VarA was likely to receive the phosphoryl group from VarS. The results demonstrate that the VarS/VarA two-component regulatory system regulates the expression of collagenase in V. alginolyticus.

DOI： 10.1007/s00232-017-9991-9

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AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase Reviewed

Akihiro Nakanishi, Naoya Hatano, Yuya Fujiwara, Arian Sha'ri, Shota Takabatake, Hiroki Akano, Naoki Kanayama, Masaki Magari, Naohito Nozaki, Hiroshi Tokumitsu

JOURNAL OF BIOLOGICAL CHEMISTRY 292 ( 48 ) 19804 - 19813 2017.12

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DOI： 10.1074/jbc.M117.805085

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Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target Reviewed

Kyohei Sakane, Miyu Nishiguchi, Miwako Denda, Fuminori Yamagchi, Masaki Magari, Naoki Kanayama, Ryo Morishita, Hiroshi Tokumitsu

Biochemical and Biophysical Research Communications 491 ( 4 ) 980 - 985 2017.9

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DOI： 10.1016/j.bbrc.2017.07.161

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SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus Reviewed

Yuka Kawaguchi, Hiroaki Nariki, Naoko Kawamoto, Yuichi Kanehiro, Satoshi Miyazaki, Mari Suzuki, Masaki Magari, Hiroshi Tokumitsu, Naoki Kanayama

Biochemical and Biophysical Research Communications 485 ( 2 ) 261 - 266 2017.4

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DOI： 10.1016/j.bbrc.2017.02.097

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Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity Reviewed

Fuminori Yamaguchi, Mitsumasa Tsuchiya, Seiko Shimamoto, Tomohito Fujimoto, Hiroshi Tokumitsu, Masaaki Tokuda, Ryoji Kobayashi

Tohoku Journal of Experimental Medicine 240 ( 1 ) 67 - 78 2016.9

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DOI： 10.1620/tjem.240.67

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Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen Reviewed

Yusui Furuya, Miwako Denda, Kyohei Sakane, Tomoko Ogusu, Sumio Takahashi, Masaki Magari, Naoki Kanayama, Ryo Morishita, Hiroshi Tokumitsu

Cell Calcium 60 ( 1 ) 32 - 40 2016.7

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DOI： 10.1016/j.ceca.2016.04.004

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Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca²⁺/Calmodulin-dependent Protein Kinase Kinase Isoforms Reviewed

Yuya Fujiwara, Yoshinori Kawaguchi, Tomohito Fujimoto, Naoki Kanayama, Masaki Magari, Hiroshi Tokumitsu

The Journal of Biological Chemistry 291 ( 26 ) 13802 - 13808 2016.6

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DOI： 10.1074/jbc.M116.727867

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NUMB phosphorylation destabilizes p53 and promotes self-renewal of tumor-initiating cells by a NANOG-dependent mechanism in liver cancer Reviewed

Hifzur R. Siddique, Douglas E. Feldman, Chia-Lin Chen, Vasu Punj, Hiroshi Tokumitsu, Keigo Machida

Hepatology 62 ( 5 ) 1466 - 1479 2015.11

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DOI： 10.1002/hep.27987

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Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells Reviewed

Yuya Fujiwara, Yuri Hiraoka, Tomohito Fujimoto, Naoki Kanayama, Masaki Magari, Hiroshi Tokumitsu

Biochemistry 54 ( 25 ) 3969 - 3977 2015.6

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DOI： 10.1021/acs.biochem.5b00149

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Compartmentalized AMPK Signaling Illuminated by Genetically Encoded Molecular Sensors and Actuators Reviewed

Takafumi Miyamoto, Elmer Rho, Vedangi Sample, Hiroki Akano, Masaki Magari, Tasuku Ueno, Kirill Gorshkov, Melinda Chen, Hiroshi Tokumitsu, Jin Zhang, Takanari Inoue

Cell Reports 11 ( 4 ) 657 - 670 2015.4

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DOI： 10.1016/j.celrep.2015.03.057

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Oxidized S100A4 inhibits the activation of protein phosphatase 5 through S100A1 in MKN-45 gastric carcinoma cells Reviewed

Mitsumasa Tsuchiya, Fuminori Yamaguchi, Seiko Shimamoto, Tomohito Fujimoto, Hiroshi Tokumitsu, Masaaki Tokuda, Ryoji Kobayashi

International Journal of Molecular Medicine 34 ( 6 ) 1713 - 1719 2014.12

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DOI： 10.3892/ijmm.2014.1947

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Ca2+/S100 proteins inhibit the interaction of FKBP38 with Bcl-2 and Hsp90 Reviewed

Seiko Shimamoto, Mitsumasa Tsuchiya, Fuminori Yamaguchi, Yasuo Kubota, Hiroshi Tokumitsu, Ryoji Kobayashi

BIOCHEMICAL JOURNAL 458 ( 1 ) 141 - 152 2014.2

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DOI： 10.1042/BJ20130924

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Suramin is a Novel Activator of PP5 and Biphasically Modulates S100-Activated PP5 Activity Reviewed

Fuminori Yamaguchi, Sho Yamamura, Seiko Shimamoto, Hiroshi Tokumitsu, Masaaki Tokuda, Ryoji Kobayashi

Applied Biochemistry and Biotechnology 172 ( 1 ) 237 - 247 2014.1

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DOI： 10.1007/s12010-013-0522-6

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Ca²⁺/S100 proteins regulate HCV virus NS5A-FKBP8/FKBP38 interaction and HCV virus RNA replication Reviewed

Joji Tani, Seiko Shimamoto, Kyoko Mori, Nobuyuki Kato, Kohji Moriishi, Yoshiharu Matsuura, Hiroshi Tokumitsu, Mitsumasa Tsuchiya, Tomohito Fujimoto, Kiyohito Kato, Hisaaki Miyoshi, Tsutomu Masaki, Ryoji Kobayashi

Liver International : Official Journal of the International Association for the Study of the Liver 33 ( 7 ) 1008 - 1018 2013.8

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DOI： 10.1111/liv.12151

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Ca²⁺/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein) Reviewed

Seiko Shimamoto, Yasuo Kubota, Fuminori Yamaguchi, Hiroshi Tokumitsu, Ryoji Kobayashi

The Journal of Biological Chemistry 288 ( 10 ) 7158 - 7168 2013.3

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DOI： 10.1074/jbc.M112.436758

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In vitro substrate phosphorylation by Ca²⁺/calmodulin-dependent protein kinase kinase using guanosine-5 '-triphosphate as a phosphate donor Reviewed

Saki Yurimoto, Tomohito Fujimoto, Masaki Magari, Naoki Kanayama, Ryoji Kobayashi, Hiroshi Tokumitsu

BMC Biochemistry 13 27 2012.12

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DOI： 10.1186/1471-2091-13-27

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S100 proteins modulate protein phosphatase 5 function: a link between Ca²⁺ signal transduction and protein dephosphorylation. Reviewed

Fuminori Yamaguchi, Yoshinori Umeda, Seiko Shimamoto, Mitsumasa Tsuchiya, Hiroshi Tokumitsu, Masaaki Tokuda, Ryoji Kobayashi

The Journal of Biological Chemistry 287 ( 17 ) 13787 - 13798 2012.4

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DOI： 10.1074/jbc.M111.329771

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Local Application of Neurotrophins Specifies Axons Through Inositol 1,4,5-Trisphosphate, Calcium, and Ca2+/Calmodulin-Dependent Protein Kinases Reviewed

Shinichi Nakamuta, Yasuhiro Funahashi, Takashi Namba, Nariko Arimura, Marina R. Picciotto, Hiroshi Tokumitsu, Thomas R. Soderling, Akira Sakakibara, Takaki Miyata, Hiroyuki Kamiguchi, Kozo Kaibuchi

Science Signaling 4 ( 199 ) ra76 2011.11

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DOI： 10.1126/scisignal.2002011

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Generation of Autonomous Activity of Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase beta by Autophosphorylation Reviewed

Hiroshi Tokumitsu, Naoya Hatano, Tomohito Fujimoto, Said Yurimoto, Ryoji Kobayashi

Biochemistry 50 ( 38 ) 8193 - 8201 2011.9

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DOI： 10.1021/bi201005g

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Analysis of CaM-kinase signaling in cells Reviewed

Gary A. Wayman, Hiroshi Tokumitsu, Monika A. Davare, Thomas R. Soderling

Cell Calcium 50 ( 1 ) 1 - 8 2011.7

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DOI： 10.1016/j.ceca.2011.02.007

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Identification of a novel CaMKK substrate Reviewed

Tomohito Fujimoto, Naoya Hatano, Naohito Nozaki, Saki Yurimoto, Ryoji Kobayashi, Hiroshi Tokumitsu

Biochemical and Biophysical Research Communications 410 ( 1 ) 45 - 51 2011.6

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DOI： 10.1016/j.bbrc.2011.05.102

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Exendin-4 regulates GLUT2 expression via the CaMKK/CaMKIV pathway in a pancreatic beta-cell line Reviewed

Ke Chen, Xiao Yu, Koji Murao, Hitomi Imachi, Junhua Li, Tomie Muraoka, Hisashi Masugata, Guo-Xing Zhang, Ryoji Kobayashi, Toshihiko Ishida, Hiroshi Tokumitsu

Metabolism: Clinical and Experimental 60 ( 4 ) 579 - 585 2011.4

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DOI： 10.1016/j.metabol.2010.06.002

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Regulation of nuclear localization signal-importin alpha interaction by Ca²⁺/S100A6 Reviewed

Maki Takata, Seiko Shimamoto, Fuminori Yamaguchi, Masaaki Tokuda, Hiroshi Tokumitsu, Ryoji Kobayashi

FEBS Letters 584 ( 22 ) 4517 - 4523 2010.11

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DOI： 10.1016/j.febslet.2010.09.052

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Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein Reviewed

Hiroshi Tokumitsu, Naoya Hatano, Mitsumasa Tsuchiya, Saki Yurimoto, Tomohito Fujimoto, Naoki Ohara, Ryoji Kobayashi, Hiroyuki Sakagami

Biochemical Journal 431 ( 1 ) 81 - 91 2010.10

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DOI： 10.1042/BJ20100637

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Inactivation of Ca²⁺/calmodulin-dependent protein kinase I by S-glutathionylation of the active-site cysteine residue. Reviewed International journal

Toshie Kambe, Tao Song, Tsuyoshi Takata, Naoya Hatano, Yoshiaki Miyamoto, Naohito Nozaki, Yasuhito Naito, Hiroshi Tokumitsu, Yasuo Watanabe

FEBS Letters 584 ( 11 ) 2478 - 84 2010.6

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We show that Ca(2+)/calmodulin(CaM)-dependent protein kinase I (CaMKI) is directly inhibited by its S-glutathionylation at the Cys(179). In vitro studies demonstrated that treatment of CaMKI with diamide and glutathione results in inactivation of the enzyme, with a concomitant S-glutathionylation of CaMKI at Cys(179) detected by mass spectrometry. Mutagenesis studies confirmed that S-glutathionylation of Cys(179) is both necessary and sufficient for the inhibition of CaMKI by diamide and glutathione. In transfected cells expressing CaMKI, treatment with diamide caused a reversible decrease in CaMKI activity. Cells expressing mutant CaMKI (179CV) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKI via its modification at Cys(179) is an important mechanism in processing calcium signal transduction in cells.

DOI： 10.1016/j.febslet.2010.04.059

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Exendin-4 regulates pancreatic ABCA1 transcription via CaMKK/CaMKIV pathway Reviewed

Junhua Li, Koji Murao, Hitomi Imachi, Hisashi Masugata, Hisakazu Iwama, Satoshi Tada, Guo-Xing Zhang, Ryoji Kobayashi, Toshihiko Ishida, Hiroshi Tokumitsu

Journal of Cellular and Molecular Medicine 14 ( 5 ) 1083 - 1087 2010.5

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DOI： 10.1111/j.1582-4934.2009.00955.x

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S100 proteins regulate the interaction of Hsp90 with Cyclophilin 40 and FKBP52 through their tetratricopeptide repeats Reviewed

Seiko Shimamoto, Yasuo Kubota, Hiroshi Tokumitsu, Ryoji Kobayashi

FEBS Letters 584 ( 6 ) 1119 - 1125 2010.3

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DOI： 10.1016/j.febslet.2010.02.055

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Breast cancer cells expressing stem cell markers CD44+ CD24 lo are eliminated by Numb-1 peptide-activated T cells Reviewed

Takashi Mine, Satoko Matsueda, Yufeng Li, Hiroshi Tokumitsu, Hui Gao, Cristopher Danes, Kwong-Kwok Wong, Xinhui Wang, Soldano Ferrone, Constantin G. Ioannides

Cancer Immunology, Immunotherapy 58 ( 8 ) 1185 - 1194 2009.8

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DOI： 10.1007/s00262-008-0623-1

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Identification and Characterization of Wolframin, the Product of the Wolfram Syndrome Gene (WFS1), as a Novel Calmodulin-Binding Protein Reviewed

Saki Yurimoto, Naoya Hatano, Mitsumasa Tsuchiya, Kiyohito Kato, Tomohito Fujimoto, Tsutomu Masaki, Ryoji Kobayashi, Hiroshi Tokumitsu

Biochemistry 48 ( 18 ) 3946 - 3955 2009.5

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DOI： 10.1021/bi900260y

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Interactions of S100A2 and S100A6 with the tetratricopeptide repeat proteins, Hsp90/Hsp70-organizing protein and kinesin light chain Reviewed

Seiko Shimamoto, Maki Takata, Masaaki Tokuda, Fumikazu Oohira, Hiroshi Tokumitsu, Ryoji Kobayashi

The Journal of Biological Chemistry 283 ( 42 ) 28246 - 28258 2008.10

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DOI： 10.1074/jbc.M801473200

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Calmodulin-kinases: Modulators of neuronal development and plasticity Reviewed

Gary A. Wayman, Yong-Seok Lee, Hiroshi Tokumitsu, Alcino Silva, Thomas R. Soderling

Neuron 59 ( 6 ) 914 - 931 2008.9

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DOI： 10.1016/j.neuron.2008.08.021

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The role of calcium/calmodulin-dependent protein kinase cascade on MIP-1 alpha gene expression of ATL cells Reviewed

Kensuke Matsumoto, Koji Murao, Hitomi Imachi, Takamasa Nishiuchi, Wenming Cao, Xiao Yu, Junhua Li, Rania A. M. Ahmed, Hisakazu Iwama, Ryoji Kobayashi, Hiroshi Tokumitsu, Toshihiko Ishida

Experimental Hematology 36 ( 4 ) 390 - 400 2008.4

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DOI： 10.1016/j.exphem.2007.11.013

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Activation of SAD kinase by Ca²⁺/calmodulin-dependent protein kinase kinase Reviewed

Tomohito Fujimoto, Saki Yurimoto, Naoya Hatano, Naohito Nozaki, Noriyuki Sueyoshi, Isamu Kameshita, Akihiro Mizutani, Katsuhiko Mikoshiba, Ryoji Kobayashi, Hiroshi Tokumitsu

Biochemistry 47 ( 13 ) 4151 - 4159 2008.4

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DOI： 10.1021/bi702528r

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14-3-3 Proteins directly regulate Ca²⁺/calmodulin-dependent protein kinase kinase alpha through phosphorylation-dependent multisite binding. Reviewed

Tohru Ichimura, Masato Taoka, Yasukazu Hozumi, Kaoru Goto, Hiroshi Tokumitsu

FEBS Letters 582 ( 5 ) 661 - 665 2008.3

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DOI： 10.1016/j.febslet.2008.01.037

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Distinct developmental expression of two isoforms of Ca²⁺/calmodulin-dependent protein kinase kinases and their involvement in hippocampal dendritic formation Reviewed

Akifumi Kamata, Hiroyuki Sakagami, Hiroshi Tokumitsu, Masashi Sanda, Yuji Owada, Kohji Fukunaga, Hisatake Kondo

Neuroscience Letters 423 ( 2 ) 143 - 148 2007.8

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DOI： 10.1016/j.neulet.2007.07.003

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Spatiotemporal expression of four isoforms of Ca²⁺/calmodulin-dependent protein kinase I in brain and its possible roles in hippocampal dendritic growth Reviewed

Akifumi Kamata, Hiroyuki Sakagami, Hiroshi Tokumitsu, Yuji Owada, Kohji Fukunaga, Hisatake Kondo

Neuroscience Research 57 ( 1 ) 86 - 97 2007.1

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DOI： 10.1016/j.neures.2006.09.013

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Knockdown of nuclear Ca²⁺/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish Reviewed

Takaki Nimura, Noriyuki Sueyoshi, Atsuhiko Ishida, Yukihiro Yoshimura, Makoto Ito, Hiroshi Tokumitsu, Yasushi Shigeri, Naohito Nozaki, Isamu Kameshita

Archives of Biochemistry and Biophysics 457 ( 2 ) 205 - 216 2007.1

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DOI： 10.1016/j.abb.2006.09.034

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Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2 Reviewed

Hiroshi Tokumitsu, Naoya Hatano, Shigeyuki Yokokura, Yuka Sueyoshi, Naohito Nozaki, Ryoji Kobayashi

FEBS Letters 580 ( 24 ) 5797 - 5801 2006.10

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DOI： 10.1016/j.febslet.2006.09.043

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Phosphorylation of Numb family proteins - Possible involvement of Ca²⁺/calmodulin-dependent protein kinases Reviewed

Hiroshi Tokumitsu, Naoya Hatano, Hiroyuki Inuzuka, Yuka Sueyoshi, Shigeyuki Yokokura, Toru Ichimura, Naohito Nozaki, Ryoji Kobayashi

The Journal of Biological Chemistry 280 ( 42 ) 35108 - 35118 2005.10

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DOI： 10.1074/jbc.M503912200

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Role of calcium-calmodulin-dependent protein kinase cascade in thyrotropin (TSH)-releasing hormone induction of TSH and prolactin gene expression Reviewed

Koji Murao, Hitomi Imachi, Wen M Cao, Xiao Yu, Hiroshi Tokumitsu, Hiroyuki Inuzuka, Norman C W Wong, Margaret A Shupnik, Ryoji Kobayashi, Toshihiko Ishida

Endocrinology 145 ( 11 ) 4846 - 4852 2004.11

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DOI： 10.1210/en.2004-0544

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Mechanism of the generation of autonomous activity of Ca²⁺/calmodulin-dependent protein kinase IV Reviewed

Hiroshi Tokumitsu, Naoya Hatano, Hiroyuki Inuzuka, Shigeyuki Yokokura, Naohito Nozaki, Ryoji Kobayashi

The Journal of Biological Chemistry 279 ( 39 ) 40296 - 40302 2004.9

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DOI： 10.1074/jbc.M406534200

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Calcium/calmodulin-dependent protein kinase I inhibits neuronal nitric-oxide synthase activity through serine 741 phosphorylation. Reviewed International journal

Tao Song, Naoya Hatano, Mariko Horii, Hiroshi Tokumitsu, Fuminori Yamaguchi, Masaaki Tokuda, Yasuo Watanabe

FEBS Letters 570 ( 1-3 ) 133 - 7 2004.7

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We demonstrate here that neuronal nitric-oxide synthase (nNOS) is phosphorylated and inhibited by a constitutively active form of Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM-K I1-293). Substitution of Ser741 to Ala in nNOS blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser741 by Ser to Asp mutation resulted in decreased binding of and activation by CaM, since the mutation was within the CaM-binding domain. CaM-K I1-293 gave phosphorylation of nNOS at Ser741 in transfected cells, resulting in 60-70% inhibition of nNOS activity. Wild-type CaM-K I also did phosphorylate nNOS at Ser741 in transfected cells, but either CaM-K II or CaM-K IV did not. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser741 on nNOS.

DOI： 10.1016/j.febslet.2004.05.083

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The role of calcium/calmodulin-dependent protein kinase cascade in glucose upregulation of insulin gene expression Reviewed

Xiao Yu, Koji Murao, Yoshitaka Sayo, Hitomi Imachi, Wen M Cao, Shouji Ohtsuka, Michio Niimi, Hiroshi Tokumitsu, Hiroyuki Inuzuka, Norman C W Wong, Ryoji Kobayashi, Toshihiko Ishida

Diabetes 53 ( 6 ) 1475 - 1481 2004.6

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DOI： 10.2337/diabetes.53.6.1475

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Regulation of axonal extension and growth cone motility by calmodulin-dependent protein kinase I Reviewed

Gary A Wayman, Stefanie Kaech, Wilmon F Grant, Monika Davare, Soren Impey, Hiroshi Tokumitsu, Naohito Nozaki, Gary Banker, Thomas R Soderling

Journal of Neuroscience 24 ( 15 ) 3786 - 3794 2004.4

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DOI： 10.1523/JNEUROSCI.3294-03.2004

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S100A1 is a novel molecular chaperone and a member of the Hsp70/Hsp90 multichaperone complex Reviewed

Miki Okada, Takashi Hatakeyama, Hideaki Itoh, Naoki Tokuta, Hiroshi Tokumitsu, Ryoji Kobayashi

The Journal of Biological Chemistry 279 ( 6 ) 4221 - 4233 2004.2

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DOI： 10.1074/jbc.M309014200

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Regulatory mechanism of Dictyostelium myosin light chain kinase A Reviewed

Hiroshi Tokumitsu, Naoya Hatano, Hiroyuki Inuzuka, Yumi Ishikawa, Taro QP Uyeda, Janet L Smith, Ryoji Kobayashi

The Journal of Biological Chemistry 279 ( 1 ) 42 - 50 2004.1

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DOI： 10.1074/jbc.M309621200

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Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox Reviewed

Miki Okada, Hideaki Itoh, Takashi Hatakeyama, Hiroshi Tokumitsu, Ryoji Kobayashi

The Biochemical Journal 374 433 - 441 2003.9

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DOI： 10.1042/BJ20030351

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Identification and characterization of novel components of a Ca²⁺/calmodulin-dependent protein kinase cascade in HeLa cells Reviewed

Yumi Ishikawa, Hiroshi Tokumitsu, Hiroyuki Inuzuka, Maki Murata-Hori, Hiroshi Hosoya, Ryoji Kobayashi

FEBS Letters 550 ( 1-3 ) 57 - 63 2003.8

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DOI： 10.1016/S0014-5793(03)00817-2

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Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation of neuronal nitric oxide synthase. Reviewed International journal

Yasuo Watanabe, Tao Song, Katsuyoshi Sugimoto, Mariko Horii, Nobukazu Araki, Hiroshi Tokumitsu, Tohru Tezuka, Tadashi Yamamoto, Masaaki Tokuda

The Biochemical Journal 372 ( Pt 2 ) 465 - 71 2003.6

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Post-synaptic density-95 (PSD-95) is a neuronal scaffolding protein that associates with N -methyl-D-aspartate (NMDA) receptors and links them to intracellular signalling molecules. In neurons, neuronal nitric oxide synthase (nNOS) binds selectively to the second PDZ domain (PDZ2) of PSD-95, thereby exhibiting physiological activation triggered via NMDA receptors. We have demonstrated previously that Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM-K IIalpha) directly phosphorylates nNOS at residue Ser(847), and can attenuate the catalytic activity of the enzyme in neuronal cells [Komeima, Hayashi, Naito and Watanabe (2000) J. Biol. Chem. 275, 28139-28143]. In the present study, we examined how CaM-K II participates in the phosphorylation by analysing the functional interaction between nNOS and PSD-95 in cells. The results showed that PSD-95 directly promotes the nNOS phosphorylation at Ser(847) induced by endogenous CaM-K II. In transfected cells, this effect of PSD-95 required its dual palmitoylation and the PDZ2 domain, but did not rely on its guanylate kinase domain. CaM-K Ialpha and CaM-K IV failed to phosphorylate nNOS at Ser(847) in transfected cells. Thus PSD-95 mediates cellular trafficking of nNOS, and may be required for the efficient phosphorylation of nNOS at Ser(847) by CaM-K II in neuronal cells.

DOI： 10.1042/BJ20030380

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A single amino acid difference between alpha and beta Ca²⁺/calmodulin-dependent protein kinase kinase dictates sensitivity to the specific inhibitor, STO-609 Reviewed

Hiroshi Tokumitsu, Hiroyuki Inuzuka, Yumi Ishikawa, Ryoji Kobayashi

The Journal of Biological Chemistry 278 ( 13 ) 10908 - 10913 2003.3

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DOI： 10.1074/jbc.M213183200

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Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo Reviewed

F Suizu, Y Fukuta, K Ueda, T Iwasaki, H Tokumitsu, H Hosoya

BIOCHEMICAL JOURNAL 367 ( 2 ) 335 - 345 2002.10

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DOI： 10.1042/BJ20020536

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A CaMK cascade activates CRE-mediated transcription in neurons of Caenorhabditis elegans Reviewed

Yoshishige Kimura, Ethan E Corcoran, Koh Eto, Keiko Gengyo-Ando, Masa-Aki Muramatsu, Ryoji Kobayashi, Jonathan H Freedman, Shohei Mitani, Masatoshi Hagiwara, Anthony R Means, Hiroshi Tokumitsu

EMBO Reports 3 ( 10 ) 962 - 966 2002.10

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DOI： 10.1093/embo-reports/kvf191

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Transcriptional regulation of nuclear orphan receptor, NOR-1, by Ca²⁺/calmodulin-dependent protein kinase cascade Reviewed

Hiroyuki Inuzuka, Hiroshi Tokumitsu, Naganari Ohkura, Ryoji Kobayashi

FEBS Letters 522 ( 1-3 ) 88 - 92 2002.7

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DOI： 10.1016/S0014-5793(02)02890-9

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STO-609, a specific inhibitor of the Ca²⁺/calmodulin-dependent protein kinase kinase Reviewed

Hiroshi Tokumitsu, Hiroyuki Inuzuka, Yumi Ishikawa, Masahiko Ikeda, Ikutaro Saji, Ryoji Kobayashi

The Journal of Biological Chemistry 277 ( 18 ) 15813 - 15818 2002.5

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DOI： 10.1074/jbc.M201075200

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Interaction of S100 proteins with the antiallergic drugs, olopatadine, amlexanox, and cromolyn: Identification of putative drug binding sites on S100A1 protein Reviewed

Miki Okada, Hiroshi Tokumitsu, Yasuo Kubota, Ryoji Kobayashi

Biochemical and Biophysical Research Communications 292 ( 4 ) 1023 - 1030 2002.4

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DOI： 10.1006/bbrc.2002.6761

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Differential regulatory mechanism of Ca²⁺/calmodulin-dependent protein kinase kinase isoforms Reviewed

Hiroshi Tokumitsu, Masato Iwabu, Yumi Ishikawa, Ryoji Kobayashi

Biochemistry 40 ( 46 ) 13925 - 13932 2001.11

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Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca²⁺/calmodulin-dependent kinase kinase peptide Reviewed

Hirofumi Kurokawa, Masanori Osawa, Hiroyuki Kurihara, Naoko Katayama, Hiroshi Tokumitsu, Mark B Swindells, Masatsune Kainosho, Mitsuhiko Ikura

Journal of Molecular Biology 312 ( 1 ) 59 - 68 2001.9

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DOI： 10.1006/jmbi.2001.4822

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Regulatory mechanism of Ca²⁺/calmodulin-dependent protein kinase kinase Reviewed

Hiroshi Tokumitsu, Masa-aki Muramatsu, Mitsuhiko Ikura, Ryoji Kobayashi

The Journal of Biological Chemistry 275 ( 26 ) 20090 - 20095 2000.6

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Identification of tranilast-binding protein as 36-kDa microfibril-associated glycoprotein by drug affinity chromatography, and its localization in human skin Reviewed

Hiromi Furuichi, Kayoko Yamashita, Miki Okada, Tetsuhiko Toyoshima, Yuiro Hata, Shigehiko Suzuki, Toshifumi Itano, Tsuyoshi Shishibori, Hiroshi Tokumitsu, Ryoji Kobayashi

Biochemical and Biophysical Research Communications 270 ( 3 ) 1002 - 1008 2000.4

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DOI： 10.1006/bbrc.2000.2480

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A novel target recognition revealed by calmodulin in complex with Ca²⁺-calmodulin-dependent kinase kinase Reviewed

Masanori Osawa, Hiroshi Tokumitsu, Mark B Swindells, Hiroyuki Kurihara, Masaya Orita, Tadao Shibanuma, Toshio Furuya, Mitsuhiko Ikura

Nature Structural Biology 6 ( 9 ) 819 - 824 1999.9

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Ca²⁺/calmodulin-dependent protein kinase cascade in Caenorhabditis elegans - Implication in transcriptional activation Reviewed

Koh Eto, Naomi Takahashi, Yoshishige Kimura, Yasuhiko Masuho, Ken-ichi Arai, Masa-aki Muramatsu, Hiroshi Tokumitsu

The Journal of Biological Chemistry 274 ( 32 ) 22556 - 22562 1999.8

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Substrate recognition by Ca²⁺/calmodulin-dependent protein kinase kinase - Role of the Arg-Pro-rich insert domain Reviewed

Hiroshi Tokumitsu, Naomi Takahashi, Koh Eto, S Yano, Thomas R Soderling, Masa-aki Muramatsu

The Journal of Biological Chemistry 274 ( 22 ) 15803 - 15810 1999.5

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Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway Reviewed

Shigetoshi Yano, Hiroshi Tokumitsu, Thomas R Sodeling

Nature 396 ( 6711 ) 584 - 587 1998.12

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Identification of mouse ULK1, a novel protein kinase structurally related to C-elegans UNC-51 Reviewed

Jin Yan, Hidehito Kuroyanagi, Asato Kuroiwa, Yo-ichi Matsuda, Hiroshi Tokumitsu, Toshifumi Tomoda, Takuji Shirasawa, Masa-aki Muramatsu

Biochemical and Biophysical Research Communications 246 ( 1 ) 222 - 227 1998.5

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DOI： 10.1006/bbrc.1998.8546

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The Ca²⁺/calmodulin-dependent kinase type IV is involved in the CD5-mediated signaling pathway in human T lymphocytes Reviewed

Sonja I Gringhuis, Lou FMH de Leij, Gary A Wayman, Hiroshi Tokumitsu, Edo Vellenga

The Journal of Biological Chemistry 272 ( 50 ) 31809 - 31820 1997.12

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Calcium/calmodulin-dependent protein kinase kinase: Identification of regulatory domains Reviewed

Hiroshi Tokumitsu, Gary A Wayman, Masa-aki Muramatsu, Thomas R Soderling

Biochemistry 36 ( 42 ) 12823 - 12827 1997.10

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Inhibitory cross-talk by cAMP kinase on the calmodulin-dependent protein kinase cascade Reviewed

Gary A Wayman, Hiroshi Tokumitsu, Thomas R Soderling

The Journal of Biological Chemistry 272 ( 26 ) 16073 - 16076 1997.6

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Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade Reviewed

Herve Enslen, Hiroshi Tokumitsu, Philip JS Stork, Roger J Davis, Thomas R Soderling

Proceedings of the National Academy of Sciences of the United States of America 93 ( 20 ) 10803 - 10808 1996.10

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Requirements for calcium and calmodulin in the calmodulin kinase activation cascade Reviewed

Hiroshi Tokumitsu, Thomas R Soderling

The Journal of Biological Chemistry 271 ( 10 ) 5617 - 5622 1996.3

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Characterization of a Ca²⁺/calmodulin-dependent protein kinase cascade. Molecular cloning and expression of calcium/calmodulin-dependent protein kinase kinase Reviewed

Hiroshi Tokumitsu, Herve Enslen, Thomas R Soderling

The Journal of Biological Chemistry 270 ( 33 ) 19320 - 19324 1995.8

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The long amino-terminal tail domain of annexin XI is necessary for its nuclear localization Reviewed

Akihiro Mizutani, Naoko Watanabe, Toshinori Kitao, Hiroshi Tokumitsu, Hiroyoshi Hidaka

Archives of Biochemistry and Biophysics 318 ( 1 ) 157 - 165 1995.4

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DOI： 10.1006/abbi.1995.1216

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Phosphorylation of CREB by CaM-kinase IV activated by CaM-kinase IV kinase Reviewed

Herve Enslen, Hiroshi Tokumitsu, Thomas R Soderling

Biochemical and Biophysical Research Communications 207 ( 3 ) 1038 - 1043 1995.2

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NFATx, a novel member of the nuclear factor of activated T cells family that is expressed predominantly in the thymus Reviewed

Esteban S. Masuda, Yoshiyuki Naito, Hiroshi Tokumitsu, Dave Campbell, Fumiko Saito, Charles Hannum, Ken-Ichi Arai, Naoko Arai

Molecular and Cellular Biology 15 ( 5 ) 2697 - 2706 1995

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DOI： 10.1128/MCB.15.5.2697

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Activation mechanisms for Ca²⁺/calmodulin-dependent protein kinase IV. Identification of a brain CaM-kinase IV kinase Reviewed

Hiroshi Tokumitsu, Debra A Brickey, John Glod, Hiroyoshi Hidaka, James Sikela, Thomas R Ssoderling

The Journal of Biological Chemistry 269 ( 46 ) 28640 - 28647 1994.11

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Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells: involvement of the conserved lymphokine element 0 Reviewed

AkioTsuboi, EstebanS.Masuda, YoshiyukiNaito, Hiroshi Tokumitsu, Ken-ichi Arait, Naoko Arai

Molecular Biology of the Cell 5 ( 1 ) 119 - 128 1994.1

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The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT Reviewed

Esteban S Masuda, Hiroshi Tokumitsu, Akio Tsuboi, Joseph Shlomai, Peggy Hung, K en-ichi Arai, Naoko Arai

Molecular and Cellular Biology 13 ( 12 ) 7399 - 7407 1993.12

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Binding site of annexin XI on the calcyclin molecule. Reviewed

Masato Watanabe, Yuko Ando, Hiroshi Tokumitsu, Hiroyoshi Hidaka

Biochemical and Biophysical Research Communications 196 ( 3 ) 1376 - 1382 1993.11

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Purification of the 120 kDa component of the human nuclear factor of activated T cells (NF-AT): reconstitution of binding activity to the cis-acting element of the GM-CSF and IL-2 promoter with AP-1 Reviewed

Hiroshi Tokumitsu, Esteban S Masuda, Akio Tsuboi, Ken-ichi Arai, Naoko Arai

Biochemical and Biophysical Research Communications 196 ( 2 ) 737 - 744 1993.10

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Phosphorylation of annexin XI (CAP-50) in SR-3Y1 cells Reviewed

Akihiro Mizutani, Hiroshi Tokumitsu, Ryoji Kobayashi, Hiroyoshi Hidaka

The Journal of Biological Chemistry 268 ( 21 ) 15517 - 15522 1993.7

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Calcyclin-binding site located on the NH2-terminal domain of rabbit CAP-50 (annexin XI): functional expression of CAP-50 in Escherichia coli Reviewed

Hiroshi Tokumitsu, Akihiro Mizutani, Hiroyoshi Hidaka

Archives of Biochemistry and Biophysics 303 ( 2 ) 302 - 306 1993.6

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Site-directed mutation makes rabbit calcyclin dimer Reviewed

uhko Ando, Masato Watanabe, Hajime Akatsuka, Hiroshi Tokumitsu, Hiroyoshi Hidaka

FEBS Letters 314 ( 2 ) 109 - 113 1992.12

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Molecular cloning of rabbit CAP-50, a calcyclin-associated annexin protein Reviewed

Hiroshi Tokumitsu, AkihiroMizutani, Masa-akiMuramatsu, TakashiYokota, Ken-ichiArai, HiroyoshiHidaka

Biochemical and Biophysical Research Communications 186 ( 3 ) 1227 - 1235 1992.8

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Specific binding of CAP-50 to calcyclin. Reviewed International journal

H Minami, H Tokumitsu, A Mizutani, Y Watanabe, M Watanabe, H Hidaka

FEBS letters 305 ( 3 ) 217 - 9 1992.7

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CAP-50, a calcyclin-associated protein with an apparent molecular mass of 50 kDa, was purified and proved to be a novel annexin [Tokumitsu, H. et al. (1992) J. Biol. Chem. 267, 8919-8924]. We examined the binding of CAP-50 to other Ca(2+)-binding proteins which have two of four EF-hand structures, by a co-precipitation assay with phospholipid (phosphatidylserine). Among nine Ca(2+)-binding proteins (calcyclin, S-100 proteins, p11, calgizzarin, calvasculin, calmodulin and troponin C) examined, only calcyclin interacted with CAP-50. These results clearly show that the interaction of CAP-50 to calcyclin is specific, i.e. other Ca(2+)-binding proteins with the EF-hand structure could not substitute for calcyclin, thereby suggesting the possible role in specific regulation of the function of CAP-50 by Ca2+/calcyclin.

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CAP-50, a newly identified annexin, localizes in nuclei of cultured fibroblast 3Y1 cells Reviewed

Akihiro Mizutani, Nobuteru Usuda, Hiroshi Tokumitsu, HiroyukMiinami, KiyoshiYasui, Ryoji Kobayashi, Hiroyoshi Hidak

The Journal of Biological Chemistry 267 ( 19 ) 13498 - 13504 1992.7

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A calcyclin-associated protein is a newly identified member of the Ca²⁺/phospholipid-binding proteins, annexin family Reviewed

Hiroshi Tokumitsu, Akihiro Mizutani, Hiroyuki Minami, Ryoji Kobayashi, Hiroyoshi Hidaka

The Journal of Biological Chemistry 267 ( 13 ) 8919 - 8924 1992.5

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Acidic calmodulin binding protein, ACAMP-81, is MARCKS protein interacting with synapsin I Reviewed

Akihiro Mizutani, Hiroshi Tokumitsu, HiroyoshiHidaka

Biochemical and Biophysical Research Communications 182 ( 3 ) 1395 - 1401 1992.2

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Identification of two subtypes of protein kinase C in human placenta Reviewed

Seiji Nomura, Hiroshi Tokumitsu, Shigehiko Mizutani, Osamu Narita, Yutaka Tomada, Hiroyoshi Hidaka

Placenta 12 ( 6 ) 605 - 613 1991.11

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Ca²⁺/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells Reviewed

Motomu Terasawa, Hiroshi Tokumitsu, Ryoji Kobayashi, Hiroyoshi Hidaka

The Journal of Biochemistry 110 ( 3 ) 417 - 422 1991.9

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A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins Reviewed

Hiroshi Tokumitsu, Ryoji Kobayashi, Hiroyoshi Hidaka

Archives of Biochemistry and Biophysics 288 ( 1 ) 202 - 207 1991.7

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Phosphorylation of bovine brain 81-kDa acidic calmodulin binding protein (ACAMP-81) in vitro Reviewed

Hiroshi Tokumitsu, Akihiro Mizutani, Masato Watanabe, Hiroyoshi Hidaka

Archives of Biochemistry and Biophysics 286 ( 1 ) 94 - 98 1991.4

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KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II Reviewed

Hiroshi Tokumitsu, Takashi Chijiwa, Masatoshi Hagiwara, Akihiro Mizutani, Motomu Terasawa, Hiroyoshi Hidaka

The Journal of Biological Chemistry 265 ( 8 ) 4315 - 4320 1990.3

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Novel and selective inhibitors of CaM-kinase II and other calmodulin-dependent enzymes Reviewed

Hiroyoshi Hidaka, Masato Hagiwara, Hiroshi Tokumitsu

Advances in Experimental Medicine and Biology 269 159 - 162 1990

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Anti-gizzard MLCK monoclonal antibody MM13 inhibits superprecipitation and phosphorylation of bovine aortic smooth muscle actomyosin Reviewed

Hiroshi Tokumitsu, Masatoshi Hagiwara, Koji Onoda, Hiroyoshi Hidaka

The Journal of Biochemistry 106 ( 3 ) 511 - 514 1989.9

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Purification and characterization of 81K, heat stable calmodulin-binding protein from bovine brain Reviewed

Hiroshi Tokumitsu, Akihiro Mizutani, Seiji Nomura, Masato Watanabe, Hiroyoshi Hidaka

Biochemical and Biophysical Research Communications 163 ( 1 ) 581 - 588 1989.8

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Monoclonal antibody assessment of tissue- and species-specific myosin light chain kinase isozymes Reviewed

Masatoshi Hagiwara, Hiroshi Tokumitsu, Koji Onoda, Toshio Tanaka, Masaaki Ito, Nobuo Kato, Hiroyoshi Hidaka

The Journal of Biochemistry 106 ( 1 ) 71 - 75 1989.7

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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

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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

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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

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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

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

Hiroshi Tokumitsu

Journal of Japanese Biochemical Society 90 ( 4 ) 452 - 461 2018.8

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DOI： 10.14952/SEIKAGAKU.2018.900452

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インターラクトーム解析法を用いたヒトS100A6標的分子の探索

坂根恭平, 西口みゆ, 古谷雄穂, 傳田美和子, 山口文徳, 曲正樹, 金山直樹, 森下了, 徳光浩

日本生化学会大会(Web) 88th 1P0204 (WEB ONLY) - [1P0204] 2015.12

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Ca²⁺/Calmodulin結合型転写因子の網羅的同定

太尾田泰成, 大西和貴, 古谷雄穂, 傳田美和子, 金山直樹, 曲正樹, 森下了, 徳光浩

日本生化学会大会(Web) 88th 3P0185 (WEB ONLY) - [3P0185] 2015

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ヒトCalmodulin標的分子の網羅的同定

古谷雄穂, 西口みゆ, 傳田美和子, 曲正樹, 金山直樹, 森下了, 徳光浩

日本生化学会大会(Web) 87th 4T14A-01(3P-324) (WEB ONLY) - 01] 2014.10

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S 100 PROTEINS MODULATE PROTEIN PHOSPHATASE 5 FUNCTION : A LINK BETWEEN Ca2+ SIGNAL TRANSDUCTION AND PROTEIN DEPHOSPHORYLATION

Fuminori Yamaguchi, Yoshinori Umeda, Seiko Shimamoto, Mitsumasa Tsuchiya, Hiroshi Tokumitsu, Masaaki Tokuda, Ryoji Kobayashi

JOURNAL OF PHYSIOLOGICAL SCIENCES 63 S139 - S139 2013

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[Small-molecule modulator of calcium signaling]. Reviewed

Kobayashi R, Tokumitsu H, Fujimoto T

Nihon rinsho. Japanese journal of clinical medicine 70 Suppl 8 277 - 280 2012.11

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CaMKKの自己リン酸化による活性調節メカニズム

徳光浩, 横倉沙紀, 本, 波多野直哉, 藤本智仁, 小林良二

日本生化学会大会プログラム・講演要旨集 84回 3T10p - 6 2011.9

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ATPアナログを用いたCaMKKの新規標的基質分子の同定

藤本智仁, 波多野直哉, 野崎直仁, 横倉沙紀, 本, 小林良二, 徳光浩

日本生化学会大会プログラム・講演要旨集 84回 3P - 0376 2011.9

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新しいニューロン性カルモジュリン結合蛋白PRG-1(可塑性関連遺伝子1)(PRG-1 (plasticity related gene 1), a novel neuronal calmodulin-binding protein)

徳光浩, 波多野直哉, 土屋光正, 揺本沙紀, 藤本智仁, 小原直樹, 小林良二, 阪上洋行

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 3P - 0346 2010.12

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Calmodulin-Kinases: Modulators of Neuronal Development and Plasticity (vol 59, pg 914, 2008)

Gary A. Wayman, Yong-Seok Lee, Hiroshi Tokumitsu, Alcino J. Silva, Thomas R. Soderling

NEURON 64 ( 4 ) 590 - 590 2009.11

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DOI： 10.1016/j.neuron.2009.11.012

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Exendin-4 regulates glucokinase expression by CaMKK/CaMKIV pathway in pancreatic beta-cell line Reviewed

K. Murao, J. Li, H. Imachi, T. Muraoka, H. Masugata, G. X. Zhang, R. Kobayashi, T. Ishida, H. Tokumitsu

DIABETES OBESITY & METABOLISM 11 ( 10 ) 939 - 946 2009.10

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DOI： 10.1111/j.1463-1326.2009.01067.x

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新規カルモデュリン結合タンパク質、PRG-1の同定と生化学的解析

土屋光正, 波多野直哉, 揺本沙紀, 倉, 小林良二, 阪上洋行, 徳光浩

日本生化学会大会プログラム・講演要旨集 82回 4T4a - 2 2009.9

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機能的プロテオミクス法を用いたカルモデュリン結合タンパク質の網羅的探索

揺本沙紀, 横倉, 藤本智仁, 波多野直哉, 土屋光正, 中野重治, 掛川寿夫, 小林良二, 徳光浩

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 81回・31回 1T15 - 12 2008.11

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Signal transduction of CaM-kinase cascade and its specific inhibitor

H Tokumitsu

JOURNAL OF PHARMACOLOGICAL SCIENCES 94 38P - 38P 2004

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Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide

M Okada, D Takezawa, S Tachibanaki, S Kawamura, H Tokumitsu, R Kobayashi

BIOCHEMICAL JOURNAL 375 ( 1 ) 87 - 97 2003.10

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DOI： 10.1042/BJ20030376

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STO-609, a specific inhibitor of the Ca2+/calmodulin-dependent protein kinase kinase. (vol 277, pg 15813, 2002)

H Tokumitsu, H Inuzuka, Y Ishikawa, M Ikeda, Saji, I, R Kobayashi

JOURNAL OF BIOLOGICAL CHEMISTRY 278 ( 6 ) 4368 - 4368 2003.2

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The Ca2+/Calmodulin-dependent Kinase Type IV is involved in the glucose-induced glucokinase transcription in INS-1 cells

Y Sayo, K Murao, S Ohtuka, H Tokumitsu, H Imachi, H Hosokawa, WM Cao, M Sato, T Ishida

DIABETES 51 A393 - A393 2002.6

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新規ミオシン調節軽鎖キナーゼとして同定されたCaM‐KIの解析

水津太, 福田康朗, 上田こずえ, 菊池麻子, 村田(堀)麻希, 徳光浩, 細谷浩史

生化学 73 ( 8 ) 812 2001.8

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The Ca2+/calmodulin-dependent kinase type IV is involved in the glucose-induced insulin transcription in INS-1 cells

Y Sayo, K Murao, H Tokumitsu, H Imachi, H Hosokawa, M Sato, T Ishida, J Takahara

DIABETES 49 A255 - A255 2000.5

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CaM-kinase cascade in C. elegans

ETO Ko, KIMURA Yoshishige, TAKAHASHI Naomi, MURAMATSU Masa-aki, ARAI Ken-ichi, TOKUMITSU Hiroshi

21 561 - 561 1998.12

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Structure and Function of CaM-kinase kinase

TOKUMITSU Hiroshi, TAKAHASHI Naomi, OSAWA Masanori, IKURA Mitsuhiko, MURAMATSU Masa-aki

21 535 - 535 1998.12

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CiNii Article

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MUTAGENESIS STUDY OF CAM-KINASE-IV

H TOKUMITSU, D BRICKEY, TR SODERLING

FASEB JOURNAL 8 ( 7 ) A1224 - A1224 1994.4

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CLE0, THE GM-CSF PROMOTER CIS-ACTING ELEMENT, MEDIATES INDUCTION SIGNALS IN T-CELLS AND IS RECOGNIZED BY FACTORS RELATED TO AP1 AND NF-AT

N ARAI, ES MASUDA, H TOKUMITSU, K ARAI, N ARAI

JOURNAL OF CELLULAR BIOCHEMISTRY 17 - 17 1994.1

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CLEO, A COMPLEX CIS-ACTING ELEMENT REGULATING GM-CSF EXPRESSION IN T-CELLS

E MASUDA, H TOKUMITSU, A TSUBOI, Y NAITO, K ARAI, N ARAI

JOURNAL OF IMMUNOLOGY 150 ( 8 ) A51 - A51 1993.4

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DIFFERENTIAL REGULATION OF LYMPHOKINE GENE INDUCTION IN TH1 AND TH2

Y NAITO, MATSUDA, I, H TOKUMITSU, ES MASUDA, A TSUBOI, RL COFFMAN, K ARAI, N ARAI

JOURNAL OF IMMUNOLOGY 150 ( 8 ) A273 - A273 1993.4

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INVOLVEMENT OF REL/NF-KAPPA-B PROTEINS IN ACTIVATION OF GM-CSF GENE IN T-CELLS

A TSUBOI, ES MASUDA, H TOKUMITSU, K ARAI, N ARAI

JOURNAL OF CELLULAR BIOCHEMISTRY 149 - 149 1993.1

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SEARCH FOR THE FUNCTIONAL SUBSTRATE PROTEINS OF PROTEIN-KINASES AND THEIR SELECTIVE INHIBITORS

H HIDAKA, M WATANABE, H TOKUMITSU

BIOLOGY AND MEDICINE OF SIGNAL TRANSDUCTION 24 485 - 490 1990

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SEARCH FOR THE FUNCTIONAL SUBSTRATE PROTEINS OF PROTEIN-KINASES AND THEIR SELECTIVE INHIBITORS

H HIDAKA, M WATANABE, H TOKUMITSU

ADVANCES IN SECOND MESSENGER AND PHOSPHOPROTEIN RESEARCH 24 485 - 490 1990

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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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Therapeutic strategy for lipotoxicity of pancreatic beta-cells

Grant number：24591352 2012.04 - 2015.03

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)

MURAO Koji, IMACHI Hitomi, TOKUMITSU Hiroshi, OHMORI Koji

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Grant amount：\5200000 （ Direct expense: \4000000 、 Indirect expense：\1200000 ）

The reasons for beta-cell dysfunction in type 2 diabetes are incompletely understood. Recently, abnormalities in cholesterol metabolism have emerged as a potential contributor to beta-cell dysfunction. ATP-binding cassette transporter A1 (ABCA1), a cytoplasmic membrane protein, is a pivotal regulator of lipid efflux from cells to apolipoproteins and plays an important role in reverse cholesterol transport. A recent study reported that mice with specific inactivation of the Abca1 gene in their beta cells had markedly impaired glucose tolerance and defective insulin secretion, but normal insulin sensitivity. Pancreatic islets isolated from these mice demonstrated altered cholesterol homeostasis and impaired insulin secretion in vitro. These results establish a new role for the ABCA1 gene in beta cell cholesterol homeostasis and insulin secretion, indicating that cholesterol accumulation may contribute to beta cell dysfunction in type 2 diabetes, so called “pancreatic lipotoxicity”.

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Comprehensive identification and signal transduction analysis of calmodulin-targets by functional proteomics

Grant number：21570143 2009 - 2011

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, KOBAYASHI Ryouji, HATANO Naoya

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Grant amount：\4940000 （ Direct expense: \3800000 、 Indirect expense：\1140000 ）

We have identified Wolframin and PRG-1 from rat brain as novel calmodulin binding proteins by functional proteomics using calmodulin-GST fused protein and LC-MS/ MS technique. According to the biochemical study, we have demonstrated that some mutations causing Wolfram syndrome disrupt calmodulin-binding ability of wolframin.

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Mechanisms of insulin gene expression and regeneration of pancreatic beta-cell

Grant number：19591054 2007 - 2008

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)

ISHIDA Toshihiko, MURAO Koji, TOKUMITSU Hiroshi, IMACHI Hitomi

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Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）

糖尿病の病因はインスリンの作用不足であり、進行した病態では膵β細胞からのインスリン合成/分泌不全が生じる。インスリン遺伝子転写の生理的刺激は血中糖濃度の変化であり、インスリン遺伝子プロモーター内にグルコース応答領域が数カ所存在している。我々は新たな転写因子PREBがグルコース応答領域に結合してインスリン遺伝子転写を促進することを明らかにした。さらにグルコース応答に関与するglucokinaseを調節することで、膵β細胞の機能改善をおこなうことを明らかにした。

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Identification and characterization of a novel target for CaM-kinase cascade

Grant number：19570134 2007 - 2008

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, KOBAYASHI Ryoji

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Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）

機能プロテオミクス法によりCaM-キナーゼの新規標的リン酸化酵素を探索した。その結果、神経特異的リン酸化酵素であるSAD-キナーゼがCaMKKの標的酵素であることを見いだした。試験管内においてSAD-キナーゼはCaMKKによってそのThr189がリン酸化されることにより、約60倍の活性化を示した。このSAD-キナーゼの活性化は培養細胞系においても確認され、CaMKK/SAD-キナーゼという新しい細胞内カルシウムシグナル伝達経路が示唆された

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The development of the molecular target drugs for intracellular calcium signal pathway

Grant number：18300123 2006 - 2008

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)

KOBAYASHI Ryoji, TOKUMITSU Hiorshi

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Grant amount：\8520000 （ Direct expense: \7200000 、 Indirect expense：\1320000 ）

S100タンパク質 (S100)、Neuronal calcium sensor (NCS)、CaMKKの分子標的薬を開発し、新しいCaシグナル機構の生理学的意義を明らかにした。新しいS100活性測定法を案出し、S100拮抗薬を見いだした。NCS分子標的薬のスクリーニング法としてPpCaMKが有用であることを見いだし、スクリーニング法を確立し、NCS拮抗薬を発見した。更に、CaMKK阻害薬(STO609)を利用し、新しいCaMKKカスケードの標的分子を発見した。

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Analysis of calmodulin-kinase cascade by using functional proteomics.

Grant number：17570115 2005 - 2006

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, KOBAYASHI Ryoji, HATANO Naoya

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Grant amount：\3500000 （ Direct expense: \3500000 ）

To search for the substrates of Ca^<2+>/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr^<177>) GST-fused CaM-KI catalytic domain (residues 1-293, Lys^<49>Glu) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr^<177>-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser^<264> in Numb and Ser^<304> in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phosphoNumb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser^<264> in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl blocked dephosphorylation of Ser^<304>. Numb is thought to participate in clathrin-dependent endocytosis by directly interacting with the clathrin-associated adaptor complex AP-2, although the underlying mechanisms are unknown. Pull-down experiments showed that the phosphorylation of Numb impaired its binding to the AP-2 complex and simultaneously recruited 14-3-3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser^<264> and the subsequent phosphorylation of Ser^<283> are sufficient to abolish the binding of Numb to AP-2 and to promote the interaction with 14-3-3 protein. These findings suggest a novel mechanism for the regulation of Numb-mediated endocytosis, namely through direct phosphorylation.

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Regeneration of pancreatic beta-cells that secret insulin in response to glucose stimulation

Grant number：17590937 2005 - 2006

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)

ISHIDA Toshihiko, TOKUMITSU Hiroshi, MURAO Koji, OHNISHI Hiroaki, IMACHI Hitomi

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Grant amount：\3500000 （ Direct expense: \3500000 ）

PREB (prolactin regulatory element binding) protein has been identified as a factor that regulates prolactin promoter activity in rat anterior pituitary. PREB is located not only in the anterior pituitary but also in pancreas, however its role in pancreas is not known. Therefore, we examined the role of PREB in insulin gene expression. To analyze the effects of PREB on insulin gene transcription, we employed the reporter gene assay and EMSA. In the cells expressing or knocked down the PREB, insulin expression and secretion were determined. PREB was located mainly in nuclei of rat pancreatic β-cells and its cell line, INS-1. A nuclear extract of INS-1 cells contained material that was recognized by PREB antiserum. Furthermore, this nuclear extract contained insulin promoter binding activity that was super-shifted by PREB antiserum in EMSA studies. In the INS-1 cells, the co-expression of PREB and the insulin promoter induced activity of the latter. The addition of glucose to the cells increased PREB expression. Deletional analysis of the insulin promoter showed that A3, a glucose-responsive cis-element in the insulin promoter mediated the transcriptional effect of PREB. Additionally, synthesized PREB bound the A3 element by EMSA, while a mutant of this motif in the insulin promoter abrogated the effect of PREB. Furthermore, cells expressing or knock-down PREB exhibited increased or decreased insulin expression, respectively. These results demonstrate that PREB may contribute to the regulation of insulin gene transcription and insulin secretion in response to glucose stimulation.

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Determine the molecular mechanism of insulin gene expression for the regeneration of pancreatic islets

Grant number：15590944 2003 - 2004

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)

ISHIDA Toshihiko, MURAO Koji, TOKUMITSU Hiroshi, YOSHITAKA Sayo, OHNISHI Hiroaki

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Grant amount：\3500000 （ Direct expense: \3500000 ）

A number of factors have been reported to affect insulin synthesis in beta-cells. Although glucose is the most important regulator of insulin gene expression in pancreatic beta-cells, the mechanisms whereby glucose stimulates insulin gene transcription in response to changes in glucose concentration have not been clarified yet. In this study, we examined the role of the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaM-K) cascade in transcriptional activation of insulin. RT-PCR, Western blotting, and immunohistochemical staining analysis revealed that CaM-K kinase-alpha (CaM-KKalpha) and CaM-KIV were localized in rat pancreatic beta-cells and their cell line, INS-1. Exposure of INS-1 cells to 11.2 mmol/l glucose elicited an increase of insulin promoter activity as well as upregulation of CaM-KIV activity within 2 min after stimulation. We investigated the influence on insulin promoter activity of the constitutively active form (CaM-KIVc) or dominant-negative mutant (CaM-KIVdn) of CaM-KIV in transfected INS-1 cells. CaM-KIVc alone was sufficient, and the upstream kinase, CaM-KK, was enhanced to upregulate the insulin promoter activity in INS-1 cells. Furthermore, cotransfection of CaM-KIVdn suppressed to a significant degree the glucose-upregulated activity of the insulin promoter. Taken together, these results indicated that the CaM-KK/CaM-KIV cascade might play an important role in glucose-upregulated transcriptional activation of the insulin gene. We have also determined the role of PREB and IB 1 as a transcriptional factor for insulin gene transcription.

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Physiological function of calcium/calmodulin-dependent protein kinase cascade

Grant number：14580649 2002 - 2003

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, KOBAYASHI Ryoji, MURAO Koji, ISHIDA Toshihiko, SAJI Ikutaro

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Grant amount：\4000000 （ Direct expense: \4000000 ）

Ca^<2+>/calmodulin-dependent protein kinases (CaM-Ks) constitute a diverse group of enzymes, which are involved in many cellular responses mediated by an increase in the concentration of intracellular calcium. Previous studies have demonstrated that two multifunctional CaM-kinases, CaM-KI and IV, are activated by phosphorylation of an activation loop Thr residue by an upstream CaM-kinase kinase (CaM-KK) resulting in a large increase in catalytic efficiency. In order to evaluate the physiological functions of CaM-KK and of the CaM-kinase cascade, we attempted to synthesize a potent and specific inhibitor of CaM-KK, STO-609. In this study, we characterize the effects of the inhibitor STO-609 on CaM-KK activity both in vitro and in intact cells. Furthermore, based on the results that the distinct sensitivity of CaM-KK isoforms to STO-609 is due to a single amino acid substitution (Val/Leu) in the ATP-binding pocket, we have generated an STO-609-resistant CaM-KK mutant, which might be useful for validating the pharmacological effects and specificity of STO-609 in vivo. We also have examined the activation mechanism of Dictyostelium MLCK-A by using constitutively active Ca^<2+>/calmodulin-dependent protein kinase kinase (CaM-KKc) as a surrogate MLCK-A kinase, indicating that the protein kinase cascade regulates MLCK-A in Dictyostelium analogous to CaM-kinase cascade.

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Study on structure biology and molecular pathology of Clostridium perfringens epsilon-toxin

Grant number：13470060 2001 - 2002

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)

OKABE akinobu, KOBAYASHI ryoji, MIYATA shigeru, MATSUSHITA osamu, TOKUDA masaki, TOKUMITSU hiroshi

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Grant amount：\10300000 （ Direct expense: \10300000 ）

Clostridium perfringens epsilon-protoxin, in which His6 was N-terminally tagged and a factor Xa cleavage site was generated to cleave an N-terminal propeptide, was replaced with Se-methionine. The Se-methionine protoxin was purified, and then the N-terminal propeptide was cleaved off with factor Xa, followed by crystallization. Although the resulting crystal was shown to be twined, we are now attempting to solve the three-dimensional structure of the protoxin by computer analysis.
We showed that epsilon-toxin (e-toxin) assembles to a heptameric pore within the lipid rafts of the rat synaptosome and Madin-Darby canine kidney (MDCK) cell membranes. To assess how physicochemical properties of the lipid rafts affect e-toxin assembly, we change major lipid constituents, cholesterol and gangliosides of MDCK cells. The heptamerization of e-toxin and its cytotoxicity towards MDCKcells was decreased by depletion of cholesterol, and was adversely stimulated by inhibition of gangliosides synthesis, suggesting that alteration in a lipid rafts environment modulates the assembly and/or the insertion of the toxin therein.
In an attempt to study the molecular pathology of e-toxin enterotoxeamia, we examined the distribution of e-toxin by whole body autoradiography involving mice injected intravenously with 35S-labeled e-toxin. The toxin was most prominently distributed in the kidneys, and fairly abundantly in the brain, spinal cord, and nasal turbinates. Immunostaining of the kidneys showed that the toxin was detected mainly in the glomeruli and capillaries, and that it was also detectable in the distal tubules and collecting ducts. Although histological examination showed some pathological changes, e.g. shrinkage of glomeruli and degeneration of epithelial cells in the distal tubules and collecting ducts, they were not so severe as those found in the brain such as neuronal cell damage and perivascular edema. The biological relevance of the toxin accumulation in the kidneys was approached by examining an effect of nephrectomy on the lethal toxicity of e-toxin against mice. The nephrectomy shortened the time required for the toxin to kill mice. When mice was intoxicated with botulinus toxin or C. perfringens alpha-toxin, such an effect of nephrectomy was not observed. Based on these results, we propose that the kidneys contribute to the host defense by accumulating circulating e-toxin and thereby protecting the brain from a lethal damage.
A cDNA clone encoding for a portion of a putative e-toxin receptor has been isolated by a yeast two-hybrid system. Studies are currently under way to identify the corresponding whole receptor protein and also to characterize molecular mechanism of e-toxin cytotoxicity involving e-toxin-resistant clones isolated fromMDCK cells

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Regulation of gene expression mediated by Ca^<2+>/calmodulin-dependent protein kinase cascade

Grant number：12680637 2000 - 2001

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, KIMURA Yoshishige, KOBAYASHI Ryoji, MURAO Koji

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Grant amount：\3800000 （ Direct expense: \3800000 ）

Ca^<2+>/calmodulin-dependent protein kinases (CaM-Ks) constitute a diverse group of enzymes, which are involved in many cellular responses mediated by an increase in the concentration of intracellular calcium. Previous studies have demonstrated that two multifunctionla CaM-kinases, CaM-KI and IV, are activated byphosphorylation of an activation loop Thr residue by an upstream CaM-kinase (CaM-KK) resulting in a large increase in catalytic efficiency. In this study, we have found that Ile441 in CaM-KKα is essential for the autoinhibition of CaM-KK and the binding orientation of CaM to CaM-KKα is not critical for relief of the autoinhibition. The unique binding orientation of CaM to CaM-KKα which was originally discovered using NMR analysis, was also confirmed with C.elegans CaM-KK by using X-ray crystallography. In contrast to CaM-KKα, CaM-KKβ-isoform has shown to exhibit enhanced Ca^<2+>/CaM-independent activity which is due to the second regulatory domain (residues 129-151) located in N-terminal of the catalytic domain. This domain inhibits the autoinhibition of CaM-KKβ resulting in generation of its autonomous activity. We also have generated C.elegans carrying CRE-GFP reporter gene and cloned C.elegans CREB. C.elegans CREB-mediated gene expression was induced by C.elegans CaM-K cascade (CaM-KK/CaM-KI) in transfected cells. In living worm, GFP-expression was induced by overexpression of C.elegans CaM-KI 1-295 (constitutively active mutant) in some neurons, which was not observed in CREB-deficient worm. This indicates that CaM-K cascade mediats CREB-dependent transcriptional activation is conserved in C.elegans.

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卵巣癌細胞における薬剤耐性克服薬の新規開発とその機能

Grant number：04152058 1992

日本学術振興会科学研究費助成事業がん特別研究がん特別研究

日高弘義, 徳光浩, 渡辺正人

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Grant amount：\2600000 （ Direct expense: \2600000 ）

卵巣癌の化学療法では薬剤耐性が大きな障害となっており、P糖蛋白やグルタチオンSトランスフェラーゼ(GST)が耐性のメカニズムに関わるとされている。新規開発薬剤W-77がGSTと結合するという事実から我々は、W-77とアドリアマイシン(ADR)耐性との関わりを以下の方法を用いて研究した。ドラッグアフィニティーカラムクロマトグラフィーを用い、W-77とGSTとの結合について調べた。W-77のGST活性抑制効果を吸光度を用いて測定した。卵巣癌耐性株NOS2、NOS3およびADR耐性の細胞株NOS2AR、NOS3ARを確立し、MDR1とGSTの発現を調べた。MTTアッセイでW-77とベラパミリによる癌細胞のADRに対する感受性の変化を測定し比較した。^<14>C-ADRを用い、細胞内のADRの量への薬剤の影響を調べた。[結果]W-77は、直接GSTと強く結合した。W-77は、下拮抗的にGSTの活性を阻害し、1mMグルタチオン、1mMCDNB存在下300μMで47.3%に低下させた。P-糖蛋白とGSTは、耐性株で過剰発現した。ADR感受性試験ではNOS2に対し67.8倍の耐性を持つNOS2ARは10μMW-77の投与により10.6倍に低下するが、10μMベラパミルでは17.5倍であった。NOS3に対し18.8倍の耐性を持つNOS3ARは、10μMW-77の投与により6.0倍に低下するが、10μMベラパミルでは9.6倍であった。耐性株の細胞内ADR量は10μMW-77は10μMベラパミルと共にNOS2ARでは約1.5倍、NOS3ARでは約2.2倍に増加させた。[結語]以上より、W-77は、P-糖蛋白とGSTの両方の活性を阻害することにより耐性を克服することが判明し、それはP-糖蛋白の活性阻害剤であるベラパミルより有効であった。

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The Development of the Strategy for the Presumption of the Tertiary Structure of Protein Kinases by Specific Inhibitors

Grant number：02557009 1990 - 1992

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

HIDAKA Hiroyoshi, TOKUMITSU Hiroshi, WATANABE Masato, HAGIWARA Masatoshi, KOBAYASHI Ryoji

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Grant amount：\11600000 （ Direct expense: \11600000 ）

Protein kinases are enzymes which tansfer Pi into some proteins. These reactions have been revealed to be involved in the regulation of many cellular function. now that the number of protein kinases has reached to over 100, it is essential to study them with structural and functional approach to clarify the physiological function of each protein kinase. The aim of our study is, (1) to presume the tertiary structure of the functional domain of protein kinase, (2) to develop the new specific protein kinase inhibitors, and (3), it is final aim, to clarify the physiological function of protein kinases. Using specific inhibitor for Ca2+/calmodulin dependent protein kinase II(CaM kinase II), KN-62, we succeeded in revealing the involvement of CaM kinase II in smooth muscle contraction, the central regulation of systemic pressure, secretion of insulin or endotherin and so on. H-89, specific inhibitor for cAMP dependent protein kinase(A-kinase), revealed the involvement of the A-kinase in the regulation of transcription of c-fos gene, and in the regulation of induction of immediately early genes. We succeeded in synthesizing the novel specific inhibitor for CaM kinase II or for the novel protein kinase activated by MAP kinase, which is a key molecule for regulating the signals induced by various extracellular stimuli. KN-62 was revealed to inhibit CaM kinase V which was a novel Ca2+/calmodulin dependent protein kinase with respect with calmodulin. On the basis of this result, it will be suggested that the calmodulin binding domain of CaM kinase II is quite similar to that of CaM kinase V.
Taken together, we succeeded in clarifying the function of protein kinases, in developing the new specific inhibitors, and in clearing the similarity between the tertiary structure of calmodulin binding domain of CaM kinase II and CaM kinase V.

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Unification and reconstruction of myosin phosphorylation theory on contractile response of smooth muscle and nonmuscle cells.

Grant number：01044066 1989 - 1991

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for international Scientific Research Grant-in-Aid for international Scientific Research

HIDAKA Hiroyoshi, HARTSHORNE David J., TOKUMITSU Hiroshi, WATANABE Masato, KOBAYASHI Ryoji

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Grant amount：\7300000 （ Direct expense: \7300000 ）

Myosin phosphorylation-dephosphorylation is the primary Ca^2-mediated regulatory process in smooth muscle. However, recent physiological studies showed that the tension in intact smooth muscle fiber is maintained in spite of the dephosphorylation of myosin, and have suggested that other control mechanisms may exist which modulate the contractile state of the muscle. Can contraction be regulated by protein kinase (s) other than myosin light chain kinase (MLCK), and by Ca2^2-binding proteins other than calmodulin? In this international scientific research program, we have attempted to unify and reconstruct the myosin phosphorylation theory on contractile response of smooth muscle and non-muscle cells, and obtained the following results, according to the schedule.
1) We prepared monoclonal antibodies directed against chicken gizzard MLCK. One of the monoclonal antibody, MM-7 inhibited the kinase activity and the superprecipitation of bovine aortic smooth muscle actomyosin. We also demonstrated the existence of it least 4 subspecies of MLCK in chicken tissues and the heterogeneity of tissue- and species-specific isozyme forms.
2) Caldesmon, an actin and calmodulin binding protein, was phosphorylated by PK-C and calmodulin-dependent protein kinase.
3) The calmodulin-dependent caldesmon kinase was an isozyme of the brain-rich calmodulin-dependent protein kinase II (CaM KII).
4) CaM KII phosphorylated purified myosin light chain at same sites, as MLCK did. Our original CaM KII specific inhibitor, KN-62 inhibited the various agonist-induced contraction in rabbit common carotid arterial strips. CaM KII may be involved in smooth muscle contraction.
5) We detected and purified three new Ca^<2+> binding proteins, using our original compounds affinity chromatography. One was calcyclin and the others were novel Ca^<2+>-binding proteins (tentatively designated calgizzarin and calvasculin). The presence of these Ca^<2+>binding proteins in smooth muscle cells show that novel intracellular Ca^<2+> messenger system (s) may exist.

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日本学術振興会特別研究員等審査会専門委員

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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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日本生化学会評議員・代議員

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日本生化学会 2018

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