Updated on 2021/12/03

写真a

 
TOKUMITSU Hiroshi
 
Organization
Interdisciplinary Science and Engineering in Health Systems Professor
Position
Professor
External link

Degree

  • Ph.D. ( 1991.3   Nagoya University )

Research Interests

  • 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

  • カルモデュリン

  • 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

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

    2021.4

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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)

    2000 - 2003

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  • Nagoya University   School of Medicine   Research Assistant

    1991 - 1992

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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

  • Oligomerization of Ca²⁺/calmodulin-dependent protein kinase kinase. Reviewed

    Biochemical and Biophysical Research Communications   in press   2021.12

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

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

    American Journal of Physiology-Cell Physiology   321 ( 3 )   C549 - C557   2021.9

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    DOI: 10.1152/ajpcell.0013

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

    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 )   2021.6

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    DOI: 10.1016/j.ceca.2021.

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

    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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  • 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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    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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    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

    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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    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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    The Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)/5-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKK that exhibits higher basal activity (autonomous activity), activation of the CaMKK/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKK appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKK activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKK at multiple residues by CaMKK-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKK activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKK indicated that Thr(144) phosphorylation by activated AMPK converts CaMKK into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKK mutant. CaMKK mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr(144) phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr(144) antibody revealed phosphorylation of Thr(144) in CaMKK in transfected COS-7 cells that was further enhanced by exogenous expression of AMPK. These results indicate that AMPK-mediated feedback phosphorylation of CaMKK regulates the CaMKK/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKK.

    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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    S100A6 is a Ca2+-signal transducer that interacts with numerous proteins and regulates their biochemical functions. Here we identified a centrosomal protein, FOR20 (FOP-related protein of 20 kDa) as a novel S100A6 target by screening protein microarrays carrying 19,676 recombinant GST-fused human proteins. Binding experiments revealed that S100A6 interacts with the N-terminal region (residues 1-30) of FOR20 in a Ca2+-dependent manner in vitro and in living cells. Several S100 proteins including S100A1, A2, A4, All, B also exhibited Ca2+-dependent interactions with FOR20 as well as S100A6. We found that two distantly related centrosomal proteins, FOP and OFD1, also possess N-terminal regions with a significant sequence similarity to the putative S100A6-binding site (residues 1-30) in FOR20 and are capable of binding to S100A6 in a Ca2+-dependent manner. Taken together, these results may indicate that S100A6 interacts with FOR20 and related centrosomal proteins through a conserved N-terminal domain, suggesting a novel Ca2+-dependent regulation of centrosomal function. (C) 2017 Elsevier Inc. All rights reserved.

    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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    Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSFI-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSFI-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, over expression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSFI-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C terminus-dependent manner. (C) 2017 Elsevier Inc. All rights reserved.

    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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    Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis.

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

    To search for novel target(s) of the Ca2+-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca2+/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca2+-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles. (C) 2016 Elsevier Ltd. All rights reserved.

    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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    Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKK phosphorylates Thr(172) in the AMPK subunit more efficiently than CaMKK, with a lower K-m (approximate to 2 m) for AMPK, whereas the CaMKI phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKK/CaMKK chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKK/Ile(322) in CaMKK confer, at least in part, a distinct recognition of AMPK but not of CaMKI.

    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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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a particular fate whereas the other retains the multipotent characteristics of its parent. The NUMB, a tumor suppressor, in conjunction with another tumor-suppressor protein, p53, preserves this property and acts as a barrier against deregulated expansion of tumor-associated stem cells. In this context, NUMB-p53 interaction plays a crucial role to maintain the proper homeostasis of both stem cells, as well as differentiated cells. Because the molecular mechanism governing the assembly and stability of the NUMB-p53 interaction/complex are poorly understood, we tried to identify the molecule(s) that govern this process. Using cancer cell lines, tumor-initiating cells (TICs) of liver, the mouse model, and clinical samples, we identified that phosphorylations of NUMB destabilize p53 and promote self-renewal of TICs in a pluripotency-associated transcription factor NANOG-dependent manner. NANOG phosphorylates NUMB by atypical protein kinase C zeta (aPKC), through the direct induction of Aurora A kinase (AURKA) and the repression of an aPKC inhibitor, lethal (2) giant larvae. By radioactivity-based kinase activity assays, we showed that NANOG enhances kinase activities of both AURKA and aPKC, an important upstream process for NUMB phosphorylation. Phosphorylation of NUMB by aPKC destabilizes the NUMB-p53 interaction and p53 proteolysis and deregulates self-renewal in TICs. Conclusion: Post-translational modification of NUMB by the NANOG-AURKA-aPKC pathway is an important event in TIC self-renewal and tumorigenesis. Hence, the NANOG-NUMB-p53 signaling axis is an important regulatory pathway for TIC events in TIC self-renewal and liver tumorigenesis, suggesting a therapeutic strategy by targeting NUMB phosphorylation. Further in-depth in vivo and clinical studies are warranted to verify this suggestion. (Hepatology 2015;62:1466-1479)

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

    To assess the isoform specificity of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKK alpha mutant (Ala292Thr/Leu233Phe) and a CaMKK beta mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wildtype or the STO-609-resistant mutant of CaMKK alpha was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKK beta mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKK beta is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK. isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

    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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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

    AMP-activated protein kinase (AMPK), whose activity is a critical determinant of cell health, serves a fundamental role in integrating extracellular and intracellular nutrient information into signals that regulate various metabolic processes. Despite the importance of AMPK, its specific roles within the different intracellular spaces remain unresolved, largely due to the lack of real-time, organelle-specific AMPK activity probes. Here, we present a series of molecular tools that allows for the measurement of AMPK activity at the different subcellular localizations and that allows for the rapid induction of AMPK inhibition. We discovered that AMPK alpha 1, not AMPK alpha 2, was the subunit that preferentially conferred spatial specificity to AMPK, and that inhibition of AMPK activity at the mitochondria was sufficient for triggering cytosolic ATP increase. These findings suggest that genetically encoded molecular probes represent a powerful approach for revealing the basic principles of the spatiotemporal nature of AMPK regulation.

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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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    S100 proteins bind to numerous target proteins, as well as other S100 proteins and activate signaling cascades. S100 proteins can be modified by various post-translational modifications, such as phosphorylation, methylation and acetylation. In addition, oxidation is important for modulating their activities. Previous studies have shown that S100A1 interacts with S100A4 in vitro and in vivo. Due to this potential cross-talk among the S100 proteins, the aim of the present study was to examine whether S100A4 modulates the activity of S100A1. S100A4 was readily oxidized and formed disulfide-linked dimers and oligomers. Although non-oxidized S100A4 bound to protein phosphatase 5 (PP5), the Cu-oxidized S100A4 failed to bind PP5. Instead, the Cu-oxidized S100A4 directly interacted with S100A1 and prevented PP5 activation. Hydrogen peroxide induced S100A4 oxidation in MKN-45 gastric adenocarcinoma cells and decreased S100A1-PP5 interaction, resulted in the inhibition of PP5 activation by S100A1. These data indicate that oxidized S100A4 regulates PP5 activity in a unique manner under oxidative stress conditions.

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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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    FKBP38 (FK506-binding protein 38), a membrane-anchored TPR (tetratricopeptide repeat)-containing immunophilin, regulates signalling pathways such as cell survival, apoptosis, proliferation and metastasis. However, the mechanisms that regulate the activity of FKBP38 are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hop [Hsp70 (heat-shock protein of 70 kDa)/Hsp90-organizing protein], kinesin-light chain, Tom70 (translocase of outer mitochondrial membrane 70), FKBP52, CyP40 (cyclophilin 40), CHIP (C-terminus of Hsc70-interacting protein) and PP5 (protein phosphatase 5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore we have hypothesized that Ca2+/S100 proteins can interact with FKBP38 and regulate its function. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, S100B and S100P specifically interact with FKBP38 and inhibit the interaction of FKBP38 with Bcl-2 and Hsp90. Overexpression of permanently active S100P in Huh-7 cells inhibited the interaction of FKBP38 with Bcl-2, resulting in the suppression of Bcl-2 stability. The association of the S100 proteins with FKBP38 provides a Ca2+-dependent regulatory mechanism of the FKBP38-mediated signalling pathways.

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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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    Suramin is an activator of ryanodine receptors and competitively binds to the calmodulin-binding site. In addition, S100A1 and calmodulin compete for the same binding site on ryanodine receptors. We therefore studied the effects of suramin on protein phosphatase 5 (PP5) and S100-activated PP5. In the absence of S100 proteins, suramin bound to the tetratricopeptide repeat (TPR) domain of PP5 and activated the enzyme in a dose-dependent manner. In the presence of S100A2/Ca2+, lower concentrations of suramin dose-dependently inhibited PP5 activity as an S100 antagonist, whereas higher concentrations of suramin reactivated PP5. Although the C-terminal fragment of heat shock protein 90 (HspC90) also weakly activated PP5, the binding site of suramin and HspC90 may be different, and addition of suramin showed no clear effect on the phosphatase activity of PP5. Similar biphasic effects of suramin were observed with S100A1-, S100B- or S100P-activated PP5. However, the inhibitory effects of lower concentrations of suramin on S100A6-activated PP5 are weak and high concentrations of suramin further activated PP5. SPR and the cross-linking study showed inhibition of the interaction between S100 protein and PP5 by suramin. Our results revealed that suramin is a novel PP5 activator and modulates S100-activated PP5 activity by competitively binding to the TPR domain.

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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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    Background & Aim: FKBP8/FKBP38 is a unique FK506-binding protein with a C-terminal membrane anchor and localizes at the outer membranes of mitochondria and the endoplasmic reticulum. Similar to some immunophilins, such as FKBP51, FKBP52 and Cyclophilin 40, FKBP8/FKBP38 contain a putative Calmodulin-binding domain and a tetratricopeptide-repeat (TPR) domain for the binding of Hsp90. Both Hsp90 and the non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) interact specifically with FKBP8/FKBP38 through its TPR domain, and the ternary complex formation plays a critical role in HCV RNA replication. The goal of this study is to evaluate that the host factor inhibits the ternary complex formation and the replication of HCV in vitro and in vivo. Methods: S100 proteins, FKBP38, FKBP8, HCV NS5A, Hsp90, and calmodulin were expressed in E.coli and purified. In vitro binding studies were performed by GST pull-down, S-tag pull-down and surface plasmon resonance analyses. The effect of S100 proteins on HCV replication was analysed by Western blotting using an HCV NS3 antibody following transfection of S100 proteins into the HCV replicon harbouring cell line (sO cells). Results: In vitro binding studies showed that S100A1, S100A2, S100A6, S100B and S100P directly interacted with FKBP8/FKBP38 in a Ca2+-dependent manner and inhibited the FKBP8/FKBP38-Hsp90 and FKBP8/FKBP38-NS5A interactions. Furthermore, overexpression of S100A1, S100A2 and S100A6 in sO cells resulted in the efficient inhibition of HCV replication. Conclusion: The association of the S100 proteins with FKBP8/FKBP38 provides a novel Ca2+-dependent regulatory role in HCV replication through the NS5A-host protein interaction.

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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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    The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca2+/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca2+/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca2+/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca2+-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.

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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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    Background: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases -including CaMKI, CaMKIV, and AMPK- to stimulate multiple Ca2+-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK.
    Results: Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKI alpha (Thr(177)) and of AMPK (Thr(172)) in vitro. Kinetic analysis indicated that the K-m values of CaMKK isoforms for GTP (400-500 mu M) were significantly higher than those for ATP (similar to 15 mu M), and a 2- to 4-fold decrease in V-max was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of similar to 45 kDa and similar to 35 kDa whose Ca2+/CaM-induced phosphorylation was inhibited by STO-609.
    Conclusions: These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.

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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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    PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca2+/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca2+-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.

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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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    Neurons are highly polarized cells that have structurally distinct processes-the axons and dendrites-that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals contribute to axon specification; however, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF or NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in calcium ions (Ca2+) in an inositol 1,4,5-trisphosphate (IP3)-dependent fashion as well as local activation of the Ca2+ effector Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3-induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP3-Ca2+-CaMKK signaling in axon specification.

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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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    Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5'-AMP-activated protein kinase, which mediates a variety of Ca2+ signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKK alpha and beta isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca2+/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography tandem mass spectrometry analysis identified Thr(482) in the autoinhibitory domain as one of the autophosphorylation sites in GaMKK beta, but phosphorylation of the equivalent Thr residue (Thr(446)) in the alpha isoforrn was not observed. Unlike CaMKK alpha that has high Ca2+/CaM-dependent activity, wild-type CaMKK beta displays enhanced autonomous activity (Ca2+/CaM-independent activity, 71% of total activity). This activity was significantly reduced (to 37%) by substitution of Thr(482) with a nonphosphorylatable Ala, without significant changes in Ca2+/CaM binding. In addition, a CaMKK alpha mutant containing the CaMKK beta regulatory domain was shown to be partially phosphorylated at Thr(446), resulting in a modest elevation of its autonomous activity. The combined results indicate that, in contrast to the alpha isoform, CaMKK beta exhibited increased autonomous activity, which was caused, at least in part, by autophosphorylation at Thr(482), resulting in partial disruption of the autoinhibitory mechanism.

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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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    A change in intracellular free calcium is a common signaling mechanism that modulates a wide array of physiological processes in most cells. Responses to increased intracellular Ca(2+) are often mediated by the ubiquitous protein calmodulin (CaM) that upon binding Ca(2+) can interact with and alter the functionality of numerous proteins including a family of protein kinases referred to as CaM-kinases (CaMKs). Of particular interest are multifunctional CaMKs, such as CaMKI, CaMKII, CaMKIV and CaMKK, that can phosphorylate multiple downstream targets. This review will outline several protocols we have used to identify which members and/or isoforms of this CaMK family mediate specific cellular responses with a focus on studies in neurons. Many previous studies have relied on a single approach such as pharmacological inhibitors or transfected dominant-negative kinase constructs. Since each of these protocols has its limitations, that will be discussed, we emphasize the necessity to use multiple, independent approaches in mapping out cellular signaling pathways. Published by Elsevier Ltd.

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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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    Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates specific downstream protein kinases including CaMKI, CaMKIV and 5'-AMP-activated protein kinase. In order to examine the variety of CaMKK-mediated signaling pathways, we searched for novel CaMKK substrate(s) using N(6)-(1-methylbutyl)-ATP and genetically engineered CaMKK alpha mutant, CaMKK alpha (Phe(230)Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKK alpha (Phe(230)Gly), but not with wild-type kinase, in the presence of N(6)-(1-methylbutyl)-ATP and Ca(2+)/CaM, induced significant threonine phosphorylation of a 50 kDa protein as well as CaMKI phosphorylation at Thr(177). The 50 kDa CaMKK substrate was partially purified by using serial column chromatography, and was identified as Syndapin I by LC-MS/MS analysis. We confirmed that recombinant Syndapin I was phosphorylated by CaMKK alpha and beta isoforms at Thr(355) in vitro. Phosphorylation of HA-Syndapin I at Thr(355) in transfected HeLa cells was significantly induced by co-expression of constitutively active mutants of CaMKK isoforms. This is the first report that CaMKK is capable of phosphorylating a non-kinase substrate suggesting the possibility of CaMKK-mediated novel Ca(2+)-signaling pathways that are independent of downstream protein kinases. (C) 2011 Elsevier Inc. All rights reserved.

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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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    The GLUT2 glucose transporter plays an important role in glucose-induced insulin secretion in pancreatic beta-cells by catalyzing the uptake of glucose into the cell. In this study, we investigated whether exendin-4, a long-acting agonist of glucagon-like peptide-1, mediates stimulatory effects on GLUT2 gene expression through the Ca(2+)/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. GLUT2 expression was examined by real-time polymerase chain reaction, Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. An increased expression level of GLUT2 protein was noted in response to increasing concentrations of exendin-4, with maximal induction at 10 nmol/L. Real-time polymerase chain reaction analysis similarly revealed a significant increase in the amount of GLUT2 messenger RNA by 10 nmol/L exendin-4. Exendin-4 also stimulated GLUT2 promoter activity in response to increasing exendin-4 concentrations, but failed to do so in the presence of STO-609, a CaMKK inhibitor. We also investigated the effect of the constitutively active form of CaMKK (CaMKKc) on GLUT2 promoter activity. The result is consistent with the observations that CaMKKc/CaMKIV enhanced or up-regulated GLUT2 promoter activity in INS-1 cells. Furthermore, exendin-4 induction of GLUT2 protein expression was significantly suppressed in the cells knocking down the CaMKIV. In summary, activation of the CaMKK/CaMKIV cascade might be required for exendin-4-induced GLUT2 gene transcription, indicating that exendin-4 plays an important role in insulin secretion in pancreatic beta-cells. (C) 2011 Elsevier Inc. All rights reserved.

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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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    Although the precise intracellular roles of S100 proteins are not fully understood, these proteins are thought to be involved in Ca2+-dependent diverse signal transduction pathways. In this report, we identified importin alpha as a novel target of S100A6. Importin alpha contains armadillo repeats, essential for binding to nuclear localization signals. Based on the results from GST pull-down assay, gel-shift assay, and co-immunoprecipitation, we demonstrated that S100A6 specifically interacts with the armadillo repeats of importin alpha in a Ca2+-dependent manner, resulting in inhibition of the nuclear localization signal (NLS)-importin alpha complex formation in vitro and in vivo. These results indicate S100A6 may regulate the nuclear transport of NLS-cargos in response to increasing concentrations of intracellular Ca2+.
    Structured summary:
    MINT-8045244: Importin alpha (uniprotkb:P52292) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by pull down (MI: 0096)
    MINT-8044928: Importin alpha (uniprotkb: P52292) binds (MI: 0407) to S100A6 (uniprotkb:P06703) by pull down (MI: 0096)
    MINT-8044941: Importin alpha (uniprotkb: P52292) and S100A6 (uniprotkb: P06703) bind (MI: 0407) by electrophoretic mobility supershift assay (MI: 0412)
    MINT-8044997: Importin alpha (uniprotkb: P52292) physically interacts (MI: 0915) with S100A6 (uniprotkb: P06703) by anti bait coimmunoprecipitation (MI: 0006)
    MINT-8045031: Importin beta (uniprotkb: Q14974) physically interacts (MI: 0915) with importin alpha (uniprotkb: P52293) and S100A6 (uniprotkb: P06703) by pull down (MI: 0096)
    MINT-8044917: Importin alpha (uniprotkb: P52292) binds (MI: 0407) to S100A2 (uniprotkb: P29034) by pull down (MI: 0096)
    MINT-8045257: Importin alpha (uniprotkb: P52292) physically interacts (MI: 0915) with S100A6 (uniprotkb: P06703) by pull down (MI: 0096)
    MINT-8045015: Importin beta (uniprotkb: Q14974) physically interacts (MI: 0915) with importin alpha (uniprotkb: P52293) and S100A2 (uniprotkb: P29034) by pull down (MI: 0096)
    MINT-8045267: Importin alpha (uniprotkb: P52292) physically interacts (MI: 0915) with S100A2 (uniprotkb: P29034) and npm2 (uniprotkb: Q6GQG6) by pull down (MI: 0096)
    MINT-8045316: Importin beta (uniprotkb: Q14974) physically interacts (MI: 0915) with importin alpha (uniprotkb: P52293) by pull down (MI: 0096)
    MINT-8045302: Importin alpha (uniprotkb: P52292) physically interacts (MI: 0915) with NPM1 (uniprotkb:P06748) and S100A2 (uniprotkb: P29034) by pull down (MI: 0096)
    MINT-8045290: Importin alpha (uniprotkb: P52292) physically interacts (MI: 0915) with npm2 (uniprotkb: Q6GQG6) by pull down (MI: 0096)
    MINT-8044963, MINT-8044985: importin alpha (uniprotkb: P52292) physically interacts (MI: 0915) with S100A2 (uniprotkb: P29034) by anti bait coimmunoprecipitation (MI: 0006)
    MINT-8044951: Importin alpha (uniprotkb: P52292) and S100A2 (uniprotkb: P29034) bind (MI: 0407) by electrophoretic mobility supershift assay (MI: 0412) (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

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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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    Intracellular Ca(2+)-dependent cellular responses are often mediated by the ubiquitous protein CaM (calmodulin), which, upon binding Ca(2+), can interact with and alter the function of numerous proteins. In the present study, using a newly developed functional proteomic screen of rat brain extracts, we identified PRO-1 (plasticity-related gene-1) as a novel CaM target. A CaM-overlay and an immunoprecipitation assay revealed that PRO-1 is capable of binding the Ca(2+)/CaM complex in vitro and in transfected cells. Surface plasmon resonance and zero-length cross-linking showed that the C-terminal putative cytoplasmic domain (residues 466-766) of PRG-1 binds equimolar amounts of CaM in a Ca(2+)-dependent manner, with a relatively high affinity (a K(d) value for Ca(2+)/CaM of 8 nM). Various PRO-1 mutants indicated that the Ca(2+)/CaM-binding region of PRG-1 is located between residues Ser(554) and Gin(588), and that Trp(559) and Ile(578) potentially anchor PRO-1 to CaM. This is supported by pronounced changes in the fluorescence emission spectrum of Trp(559) in the PRO-1 peptide (residues 554-588) upon binding to Ca(2+)/CaM, showing the stoichiometrical binding of the PRO-1 peptide with Ca(2+)/CaM. Immunoblot analyses revealed that the PRO-1 protein is abundant in brain, but is weakly expressed in the testes. Immunohistochemical analysis revealed that PRO-1 is highly expressed in forebrain structures and in the cerebellar cortex. Furthermore, PRO-1 localizes at the postsynaptic compartment of excitatory synapses and dendritic shafts of hippocampal neurons, but is not present in presynaptic nerve terminals. The combined observations suggest that PRO-1 may be involved in postsynaptic functions regulated by intracellular Ca(2+)-signalling.

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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.

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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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    ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and glucose homeostasis. This study investigates whether the long-acting agonist of the glucagon-like peptide 1, namely exendin-4, which mediates stimulatory effects on ABCA1 gene expression, could interfere with the Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) cascade. ABCA1 promoter activity was examined by reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. CaMKIV activity was assessed by detection of activation-loop phosphorylation (Thr196) of CaMKIV. We investigated the influence of the constitutively active form (CaMKIVc) or CaMKIV knockdown on ABCA1 expression. Increased abundance of ABCA1 protein was noted in response to rising concentrations of exendin-4 with maximum induction at 10 nM. Exendin-4 also stimulated ABCA1 promoter activity, but failed to do so in the presence of STO-609, a CaMKK inhibitor. Up-regulation of CaMKIV phosphorylation (at Thr196) peaked after 10 min. of exposure to exendin-4. CaMKIVc enhanced or up-regulated ABCA1 promoter activity in INS-1 cells. Furthermore, exendin-4 induction of ABCA1 protein expression was significantly suppressed in cells treated with CaMKIV-siRNA. Activation of the CaMKK/CaMKIV cascade by exendin-4 stimulated ABCA1 gene transcription, indicating that exendin-4 plays an important role in insulin secretion and cholesterol ester content in pancreatic beta cells.

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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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    S100 proteins are a subfamily of the EF-hand type calcium sensing proteins, the exact biological functions of which have not been clarified yet. In this work, we have identified Cyclophilin 40 (CyP40) and FKBP52 ( called immunophilins) as novel targets of S100 proteins. These immunophilins contain a tetratricopeptide repeat (TPR) domain for Hsp90 binding. Using glutathione-S transferase pull-down assays and immunoprecipitation, we have demonstrated that S100A1 and S100A2 specifically interact with the TPR domains of FKBP52 and CyP40 in a Ca(2+)-dependent manner, and lead to inhibition of the CyP40-Hsp90 and FKBP52-Hsp90 interactions. These findings have suggested that the Ca(2+)/S100 proteins are TPR-targeting regulators of the immunophilins-Hsp90 complex formations.
    Structured summary:
    MINT-7710442: FKBP52 (uniprotkb: Q02790) physically interacts (MI: 0915) with S100A6 ( uniprotkb: P06703) by competition binding ( MI: 0405)
    MINT-7710192: Cyp40 ( uniprotkb: P26882) binds ( MI: 0407) to S100A1 ( uniprotkb: P35467) by pull down ( MI: 0096)
    MINT-7710412: Cyp40 ( uniprotkb: P26882) physically interacts ( MI: 0915) with S100A2 ( uniprotkb: P29034) by competition binding ( MI: 0405)
    MINT-7710374: FKBP52 ( uniprotkb: Q02790) binds ( MI: 0407) to S100A2 ( uniprotkb: P29034) by pull down ( MI: 0096)
    MINT-7710452: Cyp40 ( uniprotkb: P26882) physically interacts ( MI: 0914) with S100A2 ( uniprotkb: P29034) and Hsp90 ( uniprotkb: P07900) by anti tag coimmunoprecipitation ( MI: 0007)
    MINT-7710387: FKBP52 ( uniprotkb: Q02790) binds ( MI: 0407) to S100A6 ( uniprotkb: P06703) by pull down ( MI: 0096)
    MINT-7710279: FKBP52 ( uniprotkb: Q02790) physically interacts ( MI: 0915) with S100A1 ( uniprotkb: P35467) by competition binding ( MI: 0405)
    MINT-7710224: FKBP52 ( uniprotkb: Q02790) binds ( MI: 0407) to Hsp90 ( uniprotkb: P07900) by pull down ( MI: 0096)
    MINT-7710464: Cyp40 ( uniprotkb: P26882) physically interacts ( MI: 0914) with S100A6 ( uniprotkb: P06703) and Hsp90 ( uniprotkb: P07900) by anti tag coimmunoprecipitation ( MI: 0007)
    MINT-7710249: Cyp40 ( uniprotkb: P26882) binds ( MI: 0407) to Hsp90 ( uniprotkb: P07900) by pull down ( MI: 0096)
    MINT-7710422: Cyp40 ( uniprotkb: P26882) physically interacts ( MI: 0915) with S100A6 ( uniprotkb: P06703) by competition binding ( MI: 0405)
    MINT-7710348: Cyp40 ( uniprotkb: P26882) binds ( MI: 0407) to S100A2 ( uniprotkb: P29034) by pull down ( MI: 0096)
    MINT-7710208: FKBP52 ( uniprotkb: Q02790) binds ( MI: 0407) to S100A1 ( uniprotkb: P35467) by pull down ( MI: 0096)
    MINT-7710265: Cyp40 ( uniprotkb: P26882) physically interacts ( MI: 0915) with S100A1 ( uniprotkb: P35467) by competition binding ( MI: 0405)
    MINT-7710361: Cyp40 (uniprotkb: P26882) binds (MI: 0407) to S100A6 ( uniprotkb: P06703) by pull down ( MI: 0096)
    MINT-7710476: FKBP52 ( uniprotkb: Q02790) physically interacts ( MI: 0914) with S100A2 ( uniprotkb: P29034) and Hsp90 ( uniprotkb: P07900) by anti tag coimmunoprecipitation ( MI: 0007)
    MINT-7710316: FKBP52 ( uniprotkb: Q02790) physically interacts ( MI: 0914) with S100A1 ( uniprotkb: P35467) and Hsp90 ( uniprotkb: P07900) by anti tag coimmunoprecipitation ( MI: 0007)
    MINT-7710432: FKBP52 ( uniprotkb: Q02790) physically interacts ( MI: 0915) with S100A2 ( uniprotkb: P29034) by competition binding ( MI: 0405)
    MINT-7710488: FKBP52 ( uniprotkb: Q02790) physically interacts ( MI: 0914) with S100A6 ( uniprotkb: P06703) and Hsp90 ( uniprotkb: P07900) by anti tag coimmunoprecipitation ( MI: 0007)
    MINT-7710329: S100A6 ( uniprotkb: P14069) physically interacts ( MI: 0914) with FKBP52 ( uniprotkb: P30416) and Cyp40 ( uniprotkb: Q08752) by anti bait coimmunoprecipitation ( MI: 0006)
    MINT-7710295: Cyp40 ( uniprotkb: P26882) physically interacts ( MI: 0914) with Hsp90 ( uniprotkb: P07900) and S100A1 ( uniprotkb: P35467) by anti tag coimmunoprecipitation ( MI: 0007) (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

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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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    Cancer stem cells (CSC) are resistant to chemo- and radiotherapy. To eliminate cells with phenotypic markers of CSC-like we characterized: (1) expression of CD44, CD24, CD133 and MIC-A/B (NKG2 receptors) in breast (MCF7) and ovarian (SK-OV-3) cells resistant to gemcitabine (GEM), paclitaxel (PTX) and 5-fluorouracil (5-FU) and (2) their elimination by Numb- and Notch-peptide activated CTL. The number of cells in all populations with the luminal CSC phenotype [epithelial specific antigen+ (ESA) CD44hi CD24lo, CD44hi CD133+, and CD133+ CD24lo] increased in drug-resistant MCF7 and SK-OV-3 cells. Similarly, the number of cells with expressed MIC-A/B increased 4 times in drug-resistant tumor cells compared with drug-sensitive cells. GEMRes MCF7 cells had lower levels of the Notch-1-extracellular domain (NECD) and Notch trans-membrane intracellular domain (TMIC) than GEMSens MCF7. The levels of Numb, and Numb-L-[P]-Ser265 were similar in GEM Res and GEMSens MCF7 cells. Only the levels of Numb-L (long)-Ser295 decreased slightly. This finding suggests that Notch-1 cleavage to TMIC is inhibited in GEMRes MCF7 cells. PBMC activated by natural immunogenic peptides Notch-1 (2112-2120) and Numb-1 (87-95) eliminated NICDpositive, CD24hi CD24lo MCF7 cells. It is likely that the immunogenic Numb-1 peptide in MCF7 cells originated from Numb, [P]-lated by an unknown kinase, because staurosporine but not wortmannin and MAPK-inhibitors decreased peptide presentation. Numb and Notch are antagonistic proteins which degrade each other to stop and activate cell proliferation, respectively. Their peptides are presented alternatively. Targeting both antagonistic proteins should be useful to prevent metastases in patients whose tumors are resistant to conventional treatments. © 2008 Springer-Verlag.

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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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    To search for calmodulin (CaM) targets, we performed affinity chromatography purification of a rat brain extract using CaM fused with GST as the affinity ligand. Proteomic analysis was then carried out to identify CaM-binding proteins. In addition to identifying 36 known CaM-binding proteins, including CaM kinases, calcineurin, nNOS, the IP(3) receptor, and Ca(2+)-ATPase, we identified an ER transmembrane protein, wolframin [the product of the Wolfram syndrome gene (WFS1)] as interacting. A CaM overlay and an immunoprecipitation assay revealed that wolframin is capable of binding the Ca(2+)/CaM complex in vitro and in transfected cells. Surface plasmon resonance analysis and zero-length cross-linking showed that the N-terminal cytoplasmic domain (residues 2-285) of wolframin binds to an equimolar unit of CaM in a Ca(2+)-dependent manner with a K(D) for CaM of 0.15 mu M. Various truncation and deletion mutants showed that the Ca(2+)/CaM binding region in wolframin is located from Glu90 to Trp186. Furthermore, we demonstrated that three mutations (Ala127Thr, Ala134Thr, and Arg178Pro) associated with Wolfram syndrome completely abolished CaM binding of wolframin. This observation may indicate that CaM binding is important for wolframin function and that impairment of this interaction by mutation contributes to the pathology seen in Wolfram syndrome.

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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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    S100A2 and S100A6 interact with several target proteins in a Ca2+-regulated manner. However, the exact intracellular roles of the S100 proteins are unclear. In this study we identified Hsp70/Hsp90-organizing protein (Hop) and kinesin light chain (KLC) as novel targets of S100A2 and S100A6. Hop directly associates with Hsp70 and Hsp90 through the tetratricopeptide (TPR) domains andregulates Hop-Hsp70 and Hop-Hsp90 complex formation. We have found that S100A2 and S100A6 bind to the TPR domain of Hop, resulting in inhibition of the Hop-Hsp70 and Hop-Hsp90 interactions in vitro. Although endogenous Hsp70 and Hsp90 interact with Hop in resting Cos-7 cells, but not with S100A6, stimulation of these cells with ionomycin caused a Hop-S100A6 interaction, resulting in the dissociation of Hsp70 and Hsp90 from Hop. Similarly, glutathione S-transferase pulldown and co-immunoprecipitation experiments revealed that S100A6 binds to the TPR domain of KLC, resulting in inhibition of the KLC-c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1) interaction in vitro. The transiently expressed JIP-1 interacts with KLC in resting Cos-7 cells but not with S100A6. Stimulation of these cells with ionomycin also caused a KLC-S100A6 interaction, resulting in dissociation of JIP- 1 from KLC. These results strongly suggest that the S100 proteins modulate Hsp70-Hop-Hsp90 multichaperone complex formation and KLC-cargo interaction via Ca2+-dependent S100 protein-TPR protein complex formation in vivo as well as in vitro. Moreover, we have shown that S100A2 and S100A6 interact with another TPR protein Tom70 and regulate the Tom70-ligand interaction in vitro. Thus, our findings suggest a new intracellular Ca2+-signaling pathway via S100 proteins-TPR motif interactions.

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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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    In the nervous system, many intracellular responses to elevated calcium are mediated by CaM kinases (CaMKs), a family of protein kinases whose activities are initially modulated by binding Ca2+/calmodulin and subsequently by protein phosphorylation. One member of this family, CaMKII, is well-established for its effects on modulating synaptic plasticity and learning and memory. However, recent studies indicate that some actions on neuronal development and function attributed to CaMKII may instead or in addition be mediated by other members of the CaMK cascade, such as CaMKK, CaMKI, and CaMKIV. This review summarizes key neuronal functions of the CaMK cascade in signal transduction, gene transcription, synaptic development and plasticity, and behavior. The technical challenges of mapping cellular protein kinase signaling pathways are also discussed.

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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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    Objective. Adult T-cell leukemia (ATL) is a mature CD4(+) T-cell malignancy caused by infection with human T-lymphotrophic virus type-1 and is associated with a marked hypercalcemia in many patients. Recently, it has been proposed that macrophage inflammatory protein-1 alpha (MIP-1 alpha) is the clinical hallmark of hypercalcemia in ATL. In this study, we investigated the effect of extracellular calcium on MIP-1 alpha secretion in ATL cells and the role of Ca2+/cal-modulin (CaM)-dependent protein kinase (CaM-K) cascade in transcriptional activation of MIP-1 alpha.Materials and Methods. MIP-1 alpha protein levels in the culture supernatant collected from ATL cells were measured by enzyme-linked immunosorbent assay. Reporter plasmid containing the MIP-1 alpha promoter was transfected to ATL cells, and the promoter activity was measured by luciferase assay.Results. The addition of calcium to the culture medium enhanced the secretion of MIP-1 alpha from ATL cells, which was inhibited by the CaM-KK inhibitor. The transfection of CaM-KIV stimulated MIP-1 alpha promoter activity, and the upstream kinase CaM-KK enhanced the stimulatory effect of CaM-KIV on the promoter activity. Mutation in the cyclic adenosine 5' monophosphate response element (CRE) within the MIP-1 alpha promoter significantly reduced the effect of CaM-KIV, and CRE mutant promoter activity was not significantly enhanced by the addition of calcium to the culture medium as compared to wild-type promoter activity.Conclusion. Hypercalcemia enhances MIP-1 alpha secretion in ATL cells, and this mechanism requires the involvement of CaM-KK/CaM-KIV cascade through the CRE. These findings raise a possibility that the inhibitory effect of CaM-KK/CaM-KIV cascade may be a potential therapeutic target for ATL. (C) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

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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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    To search for the downstream target protein kinases of Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKK alpha catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5'-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaNIKK at Thr(189) in the activation loop. Phosphorylation of Thr189 by CaMKK alpha induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr(189) phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKK(x (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKK(x is capable of activating SAD-B through phosphorylation of Thr(189) both in vitro and in vivo and demonstrate for the first time-that CaMKK may be an alternative activating kinase for SAD-B.

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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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    Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKK alpha) plays critical roles in the modulation of neuronal cell survival as well as many other cellular activities. Here we show that 14-3-3 proteins directly regulate CaMKK alpha when the enzyme is phosphorylated by protein kinase A on either Ser74 or Ser475. Mutational analysis revealed that these two serines are both functional: the CaMKK alpha mutant with a mutation at either of these residues, but not the double mutant, was inhibited significantly by 14-3-3. The mode of regulation described herein differs the recently described mode of 14-3-3 regulation of CaMKK alpha. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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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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    Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) are upstream protein kinases that phosphorylate and activate CaMKI and CaMKIV, both of which are involved in a variety of neuronal functions. Here, we first demonstrated that the two isoforms of CaMKK were differentially expressed during neural development by in situ hybridization. We also demonstrated that both dominant negative and pharmacological interference with CaMKK inhibitor, STO-609 resulted in a significant decrease in the number of primary dendrites of cultured hippocampal neurons. Our present findings provide the detailed anatomical information on the developmental expression of CaMKKs and the functional involvement of CaMKK in the formation of primary dendrites. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

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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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    Among multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs), CaMKI has been shown to comprise a family of four structurally related isoforms (alpha, beta, gamma, and delta) encoded by separate genes with abundant expression in mature brain. In this study, we first examined the developmental gene expression of the four isoforms of CaMKI in mouse brain with special attention to the hippocampal formation by in situ hybridization analysis. The four isoforms of CaMKI were found to exhibit distinct spatiotemporal expression during neuronal development. We also examined the functional involvement of CaMKI in the dendritic formation of cultured hippocampal neurons. The overexpression of kinase-dead mutants of CaMKI reduced the average dendritic length of the transfected neurons without any significant effects on the number of primary dendrites and the branching index. Our present findings provide the detailed anatomical information on the developmental expression of the four isoforms of CaMKI in mouse brain, which represents the possible functional involvement of CaMKI in the basal dendritic growth of hippocampal neurons. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

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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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    Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates triultifunctional Ca2+/Calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebratish whole genome Shotgun database, we isolated a cDNA clone For zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in Mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. lonomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKP-N in ionomycin-stimulated cells. Whole mount in siru hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebratish. (c) 2006 Elsevier Inc. All rights reserved.

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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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    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. Numb is also known to be phosphorylated at Ser(264) in vitro and in vivo. Here, we found that Numb is phosphorylated in vitro by Ca2+/calmodulin-dependent protein kinase I on Ser(283). This phosphorylation was also observed in transfected COS-7 cells, indicating its physiological relevance. 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. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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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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    To search for the substrates of Ca2+/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, K49E) 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 Numb1) 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 Numb1 were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser(264) in Numb and Ser(304) in Numb1) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numb1 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 Numb1-blocked dephosphorylation of Ser(304). Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein.

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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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    TRH binds to a membrane receptor that activates several intracellular signaling pathways and increases transcription of the TSH and prolactin (PRL) genes. Although TRH induces TSH and PRL gene expression, the underlying mechanism is not clear. In this report we examined the role of the Ca2+/calmodulin-dependent protein (CaM) kinase cascade in mediating TRH-stimulated transcription of TSH and PRL. RTPCR and Western blot analysis were used to show that CaM kinase kinase (CaM-KK) and CaM IV (CaM-KIV) were present in rat anterior pituitary and its cell line GH(3). Next, the effects of constitutively active CaM-KIV (CaM-KIVc) or its dominant negative mutant (CaM-KIVdn) on TSH and PRL promoter activity were tested in GH(3) cells. The results showed that either CaM-KIVc alone or an upstream kinase, CaM-KK, induced the activity of both TSH and PRL promoters. Exposure of GH(3) cells to 100 muM TRH induced CaM-KIV activity within 5 min and, as expected, also increased both TSH and PRL promoter activity. In contrast, cells carrying the CaM-KIVdn isoform had suppressed TRH induction of both TSH and PRL promoter activity. These results indicate that the CaM-KK-CaMK-IV cascade probably plays an important role in TRH induction of TSH and PRL transcriptional activity in pituitary cells.

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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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    Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) is phosphorylated at Thr(196) by Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), resulting in induction of both autonomous activity and a high level of Ca2+/CaM-dependent activity. We have shown that the kinetics of Thr(196) phosphorylation of CaM-KIV by CaM-KK is well correlated with the generation of its autonomous activity, although Thr(177) phosphorylation of CaM-KI does not induce its autonomous activity. The activities of CaM-KI chimera mutants fused with C-terminal regions ( residues 296 - 469 and 296 - 350) of CaM-KIV are completely dependent on Ca2+/CaM, which is also the case for CaM-KI. Unlike wild-type CaM-KI, however, phosphorylation of Thr177 in the chimera mutants by CaM-KK resulted in generation of significant autonomous activities, indicating that the phosphorylation of Thr in the activation loop is sufficient to partially release the autoinhibitory region of CaM-KIV from the catalytic core. Indeed, the CaM-KIV peptide ( residues 304 - 325) containing minimum autoinhibitory sequences ( residues 314 - 321) suppressed the activity of non-phosphorylated CaM-KIV with an IC50 of similar to50 muM, and this suppression was competitive with respect to the peptide substrate; however, the CaM-KIV peptide was not capable of inhibiting Thr(196)-phosphorylated CaM-KIV. Taken together, these results indicated that the Thr(196) phosphorylation of CaM-KIV by CaM-KK reduced the interaction of the catalytic core with the autoinhibitory region, resulting in generation of the autonomous activity.

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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.

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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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    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 Ca2+/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.

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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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    Calcium and calmodulin (CaM) are important signaling molecules that regulate axonal or dendritic extension and branching. The Ca2+-dependent stimulation of neurite elongation has generally been assumed to be mediated by CaM-kinase II (CaMKII), although other members of the CaMK family are highly expressed in developing neurons. We have examined this assumption using a combination of dominant-negative CaMKs (dnCaMKs) and other specific CaMK inhibitors. Here we report that inhibition of cytosolic CaMKI, but not CaMKII or nuclear CaMKIV, dramatically decreases axonal outgrowth and branching in cultured neonatal hippocampal and postnatal cerebellar granule neurons. CaMKI is found throughout the cell cytosol, including the growth cone. Growth cones of neurons expressing dnCaMI or dnCaMKK, the upstream activator of CaMKI, exhibit collapsed morphology with a prominent reduction in lamellipodia. Live-cell imaging confirms that these morphological changes are associated with a dramatic decrease in growth cone motility. Treatment of neurons with 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), an inhibitor of CaMKK, causes a similar change in morphology and reduction in growth cone motility, and this inhibition can be rescued by transfection with an STO-609-insensitive mutant of CaMKK or by transfection with constitutively active CaMKI. These results identify CaMKI as a positive transducer of growth cone motility and axon outgrowth and provide a new physiological role for the CaMKK-CaMKI pathway.

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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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    Although calmodulin is known to be a component of the Hsp70/Hsp90 multichaperone complex, the functional role of the protein remains uncertain. In this study, we have identified S100A1, but not calmodulin or other S100 proteins, as a potent molecular chaperone and a new member of the multichaperone complex. Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments indicated the formation of stable complexes between S100A1 and Hsp90, Hsp70, FKBP52, and CyP40 both in vitro and in mammalian cells. S100A1 potently protected citrate synthase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and rhodanese from heat-induced aggregation and suppressed the aggregation of chemically denatured rhodanese and citrate synthase during the refolding pathway. In addition, S100A1 suppressed the heat-induced inactivation of citrate synthase activity, similar to that for Hsp90 and p23. The chaperone activity of S100A1 was antagonized by calmodulin antagonists, such as fluphenazine and prenylamine, that is, indeed an intrinsic function of the protein. The overexpression of S100A1 in COS-7 cells protected transiently expressed firefly luciferase and Escherichia coli beta-galactosidase from inactivation during heat shock. The results demonstrate a novel physiological function for S100A1 and bring us closer to a comprehensive understanding of the molecular mechanisms of the Hsp70/Hsp90 multichaperone complex.

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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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    In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr(166) by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an similar to 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr(289) and that, subsequent to Thr(166) phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr(289) is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283 - 288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr(166) phosphorylation. Simple removal of this region resulted in a significant increase in the k(cat) of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr(166) phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.

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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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    Hsp90 (beat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but hi vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists 41 might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly Suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5-3.0 muM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, Such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both hi vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90.

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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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    In this report, we cloned a novel calmodulin-kinase (CaM-KIdelta) from HeLa cells and characterized its activation mechanism. CaM-KIdelta exhibits Ca2+/CaM-dependent activity that is enhanced (similar to30-fold) in vitro by phosphorylation of its Thr180 by CaM-K kinase (CaM-KK)alpha consistent with detection of CaM-KIdelta-activating activity in HeLa cells. We also identified a novel CaM-KKbeta isoform (CaM-KKbeta-3) in HeLa cells whose activity was highly Ca2+/CaM-independent. Transiently expressed CaM-KIdelta exhibited enhanced protein kinase activity in HeLa cells without ionomycin stimulation. This sustained activation of CaM-KIdelta was completely abolished by Thr180Ala mutation and inhibited by CaM-KK inhibitor, STO-609, indicating a functional CaM-KK/CaM-KIdelta cascade in HeLa cells. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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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.

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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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    We recently developed STO-609, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), and we demonstrated that CaM-KKbeta is more sensitive to STO-609 than the CaM-KKalpha isoform (Tokumitsu H., Inuzuka H., Ishikawa Y., Ikeda M., Saji I., and Kobayashi R. (2002) J. Biol. Chem. 277,15813-15818). By using catalytic chimera and point mutants of both isoforms, we demonstrated that Val(269) in CaM-KKbeta/Leu(233) in CaM-KKalpha confers a distinct sensitivity (similar to10fold) to STO-609 on CaM-KK isoforms. Various mutations of Val(269) in CaM-KKbeta indicate that substitution by hydrophobic residues with bulky side chains significantly decreases drug sensitivity and that the V269F mutant is the most effective drug-resistant enzyme (similar to80-fold higher IC50 value). These findings are consistent with a result obtained with a full-length mutant expressed in COS-7 cells. Furthermore, suppression of CaM-KK-mediated CaM-KIV activation in transfected HeLa cells by STO-609 treatment was completely abolished by the co-expression of the CaM-KKbeta V269F mutant. Based on the results that the distinct sensitivity of CaM-KK isoforms to STO-609 is because of 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.

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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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    Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM-KI), which is a member of the multifunctional CaM-K family, is thought to be involved in various Ca2+-signalling pathways. In this report, we demonstrate that CaM-KI activated by an upstream kinase (CaM-K kinase), but not unactivated CaM-KI, phosphorylates myosin 11 regulatory light chain (MRLC) efficiently (K-eat, 1.7 s(-1)) and stoichiometrically (approximate to 0.8 mol of phosphate/mol) in a Ca2+/CaM-dependent manner in vitro. One-dimensional phosphopeptide mapping and mutational analysis of MRLC revealed that the activated CaM-KI mono-phosphorylates only Ser-19 in MRLC. Transient expression of the Ca2+/CaM-independent form of CaM-KI (CaM-KI1-293) in HeLa cells induced Ser-19 phosphorylation of myosin, 11 accompanied by reorganization of actin filaments in the peripheral region of the cells. CaM-KI-induced reorganization of actin filaments was suppressed by co-expression of non-phosphorylatable MRLC mutants (S19A and T18AS19A). Furthermore, a kinase-negative form of CaM-KI (CaM-KI1-298,K49E) significantly reduced reorganization of actin filaments, indicating a dominant negative effect. This is the first demonstration that the activation of the CaM-KI cascade induces myosin 11 phosphorylation, resulting in regulation of actin filament organization in mammalian cells.

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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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    Calcium (Ca2+) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+/CaM-dependent protein kinases I and IV (CaMKs) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP response element binding protein (CREB) has been proposed as a target for a CaMK cascade involving not only CaMKI or CaMKIV, but also an upstream kinase kinase that is also CaM regulated (CaMKK). Here, we report that all components of this pathway are coexpressed in head neurons of Caenorhabditis elegans. Utilizing a transgenic approach to visualize CREB-dependent transcription in vivo, we show that this CaMK cascade regulates CRE-mediated transcription in a subset of head neurons in living nematodes.

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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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    We found that transcription of NOR-1 (neuron-derived orphan receptor-1) is induced by the Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV) cascade by differential hybridization screening of a cDNA array using probes prepared from SH-SY5Y cells infected with recombinant adenoviruses expressing Ca2+/CaM-independent mutants of CaM-KIV and CaM-K kinase (CaM-KK). Induction of NOR-1 mRNA expression by the CaM-KIV cascade was confirmed by RT-PCR. Promoter analyses indicate that CaM-KIV cascade response element is located between -162 bp and -42 bp in the 1.7 kb NOR-1 promoter containing triple cAMP response elements. Disruption of each element significantly reduced the promoter activity, indicating the direct transcriptional regulation of NOR-1 by CaM-KIV cascade. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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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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    STO-609, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) was synthesized, and its inhibitory properties were investigated both in vitro and in vivo. STO-609 inhibits the activities of recombinant CaM-KKalpha and CaM-KKbeta isoforms, with K-i values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activities. Comparison of the inhibitory potency of the compound against various protein kinases revealed that STO-609 is highly selective for CaM-KK without any significant effect on the downstream CaM kinases (CaM-KI and -IV), and the IC50 value of the compound against CaM-KII is similar to10 mug/ ml. STO-609 inhibits constitutively active CaM-KKa (glutathione S-transferase (GST)-CaM-KK-(84-434)) as well as the wild-type enzyme. Kinetic analysis indicates that the compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 suppresses the Ca2+- induced activation of CaM-KIV in a dose-dependent manner. In agreement with this observation, the inhibitor significantly reduces the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 mug/ml (similar to80% inhibitory rate). Taken together, these results indicate that STO-609 is a selective and cell-permeable inhibitor of CaM-KK and that it may be a useful tool for evaluating the physiological significance of the CaM-KK-mediated pathway in vivo as well as in vitro.

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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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    S100 proteins are a multigenic family of low-molecular-weight Ca2+-binding proteins comprising 19 members. These proteins undergo a conformational change by Ca2+-binding and consequently interact with their target proteins. Recently, we reported that two antiallergic drugs, Amlexanox and Cromolyn, bind to S100A12 and S100A13 of the S100 protein family. In the present study, we used a newly developed antiallergic drug, Olopatadine, as a ligand for affinity chromatography and examined binding specificity of the drug to S100 protein family. Olopatadine binds specifically to S100 proteins, such as S100A1, S100B, S100L, S100A12, and S100A13, in a Ca2+-dependent manner but not to calmodulin. Mutagenesis study showed that amino acid residues 76-85 in S100A1 are necessary for its binding to Olopatadine. In contrast, residues 89-94 were identified as an Amlexanox-binding site in S100A1. Moreover, Olopatadine did not competitively inhibit S100A1-binding site of Amlexanox. Furthermore, we showed that Olopatadine inhibited the binding of S100A1 target protein's binding site peptides to S100A1 These results indicate that C-terminal region of S100A1 is important for antiallergic drug binding, although the drug binding sites are different according to each antiallergic drug. Differences in the binding sites of S100A1 to antiallergic drugs suggest that the regulatory functions of S100 proteins may exist in several regions. Therefore, these drugs may serve as useful tools for evaluating the physiological significance of S100 protein family. (C) 2002 Elsevier Science (USA).

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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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    We have previously demonstrated that the a isoform of Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK alpha) is strictly regulated by an autoinhibitory mechanism and activated by the binding of Ca2+/CaM [Tokumitsu, H., Muramatsu, M., Ikura, M., and Kobayashi, R. (2000) J. Biol. Chem. 275, 20090-20095]. In this study, we find that rat brain extract contains Ca2+/CaM-independent CaM-KK activity. This result is consistent with an enhanced Ca2+/CaM-independent activity (60-70% of total activity) observed with the recombinant CaM-KK beta isoform. By using various truncation mutants of CaM-KK beta, we have identified a region of 23 amino acids (residues 129-151) located at the N-terminus of the catalytic domain as an important regulatory element of the autonomous activity. A CaM-KK beta deletion mutant of this domain shows a significant increase of Ca2+/CaM dependency for the CaM-KK activity as well as for the autophosphorylation activity. The activities of CaM-KK alpha and CaM-KK beta chimera, in which autoinhibitory sequences were replaced by each other, were completely dependent on Ca2+/CaM, suggesting that the autoinhibitory regions of CaM-KK alpha and CaM-KK beta are functional. These results establish for the first time that residues 129-151 of CaM-KK beta participate in the release of the autoinhibitory domain from its catalytic core, resulting in generation of autonomous activity.

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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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    Calmodulin (CaM) is a ubiquitous calcium (Ca2+) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca2+-dependent manner. For this multi-functionality, conformational plasticity is essential
    however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 Å resolution crystal structure of Ca2+/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca2+/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156° to 196°. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM. © 2001 Academic Press.

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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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    Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of alpha CaM-KK (residues 438-463), which suppressed the activity of constitutively active CaM-KK (84-434) in the absence of Ca2+/CaM but competitively with ATP, Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile(441) is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu(440) exhibited enhanced Ca2+/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, IM. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol. 6, 819-824), the chimeric CaM-KKs containing Ile(441) remained Ca2+/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile(441) for autoinhibition, which is located at the -3 position from the N-terminal anchoring residue (Trp(444)) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.

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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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    To elucidate the molecular mechanism involved in the suppression of keloids and hypertrophic scars by tranilast, we investigated the target protein of tranilast in bovine skin and aorta. A specific tranilast-binding protein was isolated from both tissues by drug affinity chromatography and was identified as 36-kDa microfibril-associated glycoprotein (36-kDa MAGP). Binding of 36-kDa MAGP to tranilast seemed to be specific since 36-kDa MAGP could be eluted from the drug affinity column by tranilast itself and also binding of 36-kDa MAGP to other anti-allergy drugs (amlexanox and cromolyn) is significantly weaker than that to tranilast, Light and electron microscopic immunohistochemistry detected the protein at the periphery of elastic fibers in normal human skin. In hypertrophic scar tissue, however, 36-kDa MAGP was located on small bundles of micro fibrils. These findings provide support for the concept that elastogenesis occurs in scar tissue and 36-kDa MAGP might be one of the targets for tranilast. (C) 2000 Academic Press.

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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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    The structure of calcium-bound calmodulin (Ca2+/CaM) complexed with a 26-residue peptide, corresponding to the CaM-binding domain of rat Ca2+/CaM-dependent protein kinase kinase (CaMKK), has been determined by NMR spectroscopy. In this complex, the CaMKK peptide forms a fold comprising an alpha-helix and a hairpin-like loop whose C-terminus folds back on itself. The binding orientation of this CaMKK peptide by the two CaM domains is opposite to that observed in all other CaM-target complexes determined so far. The N- and C-terminal hydrophobic pockets of Ca2+/CaM anchor Trp 444 and Phe 459 of the CaMKK peptide, respectively. This 14-residue separation between two key hydrophobic groups is also unique among previously determined CaM complexes. The present structure represents a new and distinct class of Ca2+/CaM target recognition that may be shared by other Ca2+/CaM-stimulated proteins.

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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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    We have recently demonstrated that Caenorhabditis elegans Ca2+/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CecaM-KI), which has approximately 60% identity to mammalian CaM-KI. Ce-CaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by Ce-CaM-KK through phosphorylation of Thr(179) in a Ca2+/CaM-dependent manner, resulting in a 30-fold decrease in the K-m of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-RI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH2-terminal six residues ((PLFKRR7)-P-2) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CecaM-KI reconstituted a signaling pathway that mediates Ca2+-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of Ce-CaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca2+-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

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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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    Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Togumitsu, Il., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KH can also activate and phosphorylate protein kinase B (PKB), In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases, Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP-domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP-domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB, These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.

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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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    The protection against apoptosis provided by growth factors in several cell lines is due to stimulation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, which results in activation of protein kinase B-1,B-2 (PKB; also known as c-Akt and Rac) and phosphorylation and sequestration to protein 14-3-3 of the pro-apoptotic Bcl-2-family member BAD(3-7). A modest increase in intracellular Ca2+ concentration also promotes survival of some cultured neurons(8,9) through a pathway that requires calmodulin but is independent of PI(3)K and the MAP kinases(10,11), Here we report that Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) activates PKB directly, resulting in phosphorylation of BAD on serine residue 136 and the interaction of BAD with protein 14-3-3, Serum withdrawal induced a three- to fourfold increase in cell death of NG108 neuroblastoma cells, and this apoptosis was largely blocked by increasing the intracellular Ca2+ concentration with NMDA (N-methyl-D-aspartate) or KCl or by transfection with constitutively active CaM-KK. The effect of NMDA on cell survival was blocked by transfection with dominant-negative forms of CaM-KK or PKB. These results identify a Ca2+-triggered signalling cascade in which CaM-KK activates PKB, which in turn phosphorylates BAD and protects cells from apoptosis.

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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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    A novel protein kinase related to the C. elegans serine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization. PIA-tagged ULK1 is expressed as a protein of similar to 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes. (C) 1998 Academic Press.

    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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    The CD5 receptor on T lymphocytes is involved in T cell activation and T-B cell interactions. In the present study, we have characterized the signaling pathways induced by anti-CD5 stimulation in human T lymphocytes, In T lymphocytes, anti-CD5 co-stimulation enhances the phytohemagglutinin/anti-CD28-induced interleukin-2 (IL-2) mRNA accumulation 1.6-fold and IL-2 protein secretion 2.2-fold, whereby the up-regulation is mediated at both the transcriptional and post-transcriptional level. The CD5 signaling pathway up-regulates the IL-2 gene expression by increasing the DNA binding and transactivation activity of activator protein 1 but affects none of the other transcription factors like nuclear factor of activated T cells, nuclear factor kappa B, OCt, and CD28-responsive complex/nuclear factor of mitogen-activated T cells involved in the regulation of the IL-2 promoter activity. The CD5-induced increase of the activator protein 1 activity is mediated through the activation of calcium/calmodulin-dependent (CaM) kinase type IV, and is independent of the activation of mitogen-activated protein kinases Jun N-terminal kinase, extracellular signal-regulated kinase, and p38/Mpk2, and calcium/calmodul-independent kinase type IL The expression of a dominant negative mutant of CaM kinase IV in T Iymphocytes transfected with an IL-2 promoter-driven reporter construct completely abrogates the response to CD5 stimulation, indicating that CaM kinase IV is essential to the CD5 signaling pathway, In addition, it is demonstrated that calciuml/calmodulin-dependent kinase type IV is also involved in the stabilization of the IL-2 transcripts, which is observed after co-stimulation of phytohemagglutinin/anti-CD28 activated T lymphocytes with anti-CD5.

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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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    We recently cloned a calmodulin-dependent protein kinase kinase (CaM-KK) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324]. In the present study, we have identified its regulatory CaM-binding and autoinhibitory domains (CBD and AID, respectively) using a series of COOH-terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463 activated CaM-KIV and bound CaM similar to wild-type enzyme (CaM-KK1-505); CaM-KK1-448 did not bind CaM and was largely inactive; and CaM-KK1-434 also did not bind CaM but activated a CaM-independent mutant of CaM-KIV in the absence of Ca2+/CaM. Substitution of triple negative charges (Asp) at positions (RKR)-R-455, (ILV)-I-448, or (SWT)-S-443 blocked CaM binding and suppressed by 70-90% CaM-KK activities. Mutants (VKL)-V-438 and (KNS)-K-435 to DDD exhibited partial Ca2+/CaM-independent activities. These results identify overlapping AID and CBD between residues 430 and 460 in CaM-KK, similar to other CaM-Ks. Consistent with this assignment, the synthetic peptide corresponding to residues 438-463 bound CaM in a Ca2+-dependent manner with a K-d in the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser(458) at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., Tokumitsu, H., and Soderling, T. R. (1997) J. Biol. Chem. 272, 16073-16076], was blocked by prior binding of Ca2+/CaM to CaM-KK.

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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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    The calmodulin-dependent kinase (CaM-K) cascade, a Ca2+-triggered system involving phosphorylation and activation of CaM-KI and CaM-KIV by CaM kinase kinase (CaM-KK), regulates transcription through direct phosphorylation of transcription factors such as cAMP response element-binding protein, We have shown previously that activated CaM-KIV can activate the mitogen-activated protein kinases (Enslen, H., Tokumitsu, H., Stork, P. J. S., Davis, R. J., and Soderling, T. R. (1996) Proc. Natl., Acad. Sci. U. S. A. 93, 10803-10808), and the present paper describes a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gave rapid phosphorylation in vitro and in cells of recombinant CaM-KK, resulting in 50-75% inhibition of CaM-KK activity, part of which was due to suppression of CaM-binding by phosphorylation of Ser(458) in the CaM-binding domain, However, the Ser(458) --> Ala mutant, or a truncation mutant in which the CaM-binding and autoinhibitory domains were deleted, was still partially suppressed by PKA-mediated phosphorylation, The second inhibitory site was identified as Thr(108) by site-specific mutagenesis, Treatments of COS-7, PC12, hippocampal, or Jurkat cells with the PKA activators forskolin or isoproterenol gave 30-90% inhibition of either endogenous or transfected CaM-KK and/or CaM-KIV activities. These results demonstrate that the CaM kinase cascade is negatively regulated in cells by the cAMP/PKA pathway.

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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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    Membrane depolarization of NG108 cells gives rapid (<5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK), To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively), In the absence of depolarization, CaM-KK, transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were cotransfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38), The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays, We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases, Mutation of the phosphorylation site (Thr(196)) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade, These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription.

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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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    We have previously purified and cloned rat brain Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324), In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr(196) by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr(196)) but not the autoinhibitory domain or the CaM-binding domain, still required Ca2+/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2+/CaM-independent manner, but this constitutively active CaM-KK1-434 required Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV, These results demonstrate that binding of Ca2+/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade, Both CaM-KK and CaM-K IV appear to have similar Ca2+/CaM requirements with EC(50) values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca2+/CaM-independent activities of wild-type CaM-K IV, but not the Thr(196) --> Ala mutant, upon ionomycin stimulation.

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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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    Recent studies have demonstrated that Ca2+/calmodulin dependent protein kinase IV (CaM-kinase IV) can mediate Ca2+-dependent regulation of gene expression through the phosphorylation of transcriptional activating proteins, We have previously identified and purified a 68-kDa rat brain CaM-kinase kinase that phosphorylates and increases total and Ca2+-independent activities of CaM-kinase IV (Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. Biol. Chem, 269, 28640-28647), Using a partial amino acid sequence of the purified brain kinase, a CaM-kinase kinase cDNA was cloned from a rat brain cDNA library, Northern blot analysis showed that CaM-kinase kinase mRNA (3.4 kilobases) was expressed in rat brain, thymus, and spleen. Sequence analyses revealed that the cDNA encoded a 505-amino acid protein, which contained consensus protein kinase motifs and was 30-40% homologous with members of the CaM-kinase family, Expression of the cDNA in COS-7 cells yielded an apparent 68-kDa CaM-binding protein, which catalyzed in vitro activation in the presence of Mg2+/ATP and Ca2+/CaM of CaM-kinases I and IV but not of CaM-kinase II. Co-expression of CaM-kinase kinase with CaM-kinase IV gave a 14-fold enhancement of cAMP-response element-binding protein-dependent gene expression compared with CaM-kinase IV alone. These results are consistent with the hypothesis that CaM-kinases I and IV are regulated through a unique signal transduction cascade involving CaM-kinase kinase.

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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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    Annexin XI is a newly identified annexin which localizes mainly in the nucleus of rat embryonic fibroblasts. There are no typical nuclear localization signals (NLS) in the molecule. To define the region responsible for its nuclear localization, a series of mutants and chimeric cDNA were constructed. These were transiently expressed in COS-7 cells, and the subcellular distributions of the mutants and chimeric proteins were determined by indirect immunofluorescence microscopy. Wild-type annexin XI was located predominantly within the nucleus. Deletion of the N-terminal tail domain (residues 3-196) changed the distribution of the protein from the nucleus to the cytoplasm whereas deletion of the C-terminal core domain (residues 208-504) still kept the protein sorting to the nucleus. Three other mutants lacking 60-80 amino acids in the N-terminal region (residues 3-61, 61-115, and 115-197, respectively) no longer efficiently imported into the nucleus. Furthermore, Escherichia coli beta-galactosidase polypeptide was efficiently localized to the nucleus only when fused with the whole N-terminal region of annexin XI (residues 1-207), not with part of the N-terminal region. In primary cultured rat hepatocytes, annexin XI was distributed in the cytoplasm but not in the nucleus. These results suggest that the whole N-terminal tail domain of annexin XI is necessary and sufficient for its nuclear localization, and may function as NLS in a cell-type specific manner. (C) 1995 Academic Press, Inc.

    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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    Previous reports have shown that CaM-kinase IV can phosphorylate the transcription factor CREB in vitro on Ser133. Furthermore, transfected CaM-kinase IV can activate CREB-dependent transcription, but at a lower efficiency than the cAMP-kinase. In this paper we examine the kinetics and site-specificity of CREB phosphorylation in vitro by CaM-kinase IV after its phosphorylation and activation by a newly discovered brain CaM-kinase IV kinase. Our results show that activated CaM-kinase IV has the same Km (1-5 mu M) for CREB phosphorylation, but the Vmax is about 30-fold higher than with non-activated CaM-kinase IV. Activated CaM-kinase IV still shows specificity for phosphorylation of Ser133, the site necessary for transactivation by CREB. It is likely that the lower efficiency of transcriptional activation by transfected CaM-kinase IV in previous studies was due to the fact that the CaM-kinase IV was not activated by CaM-kinase IV kinase. (C) 1995 Academic Press, Inc.

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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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    The nuclear factor of activated T cells (NFAT) regulates cytokine gene expression in T cells through cis-acting elements located in the promoters of cytokine genes. Here, we report the cDNA cloning, chromosomal localization, and initial characterization of a transcription factor related to NFATp and NFATc. The novel molecule, designated NFATx, exhibits in its middle a region very similar to the Rel homology domain in NFATc and NFATp. The amino- terminal region of NFATx also shows significant similarities to corresponding sequences in NFATc and NFATp and contains three copies of a conspicuous 17- residue motif of unknown function. We provide evidence showing that NFATx can reconstitute binding to the NFAT-binding site from the interleukin 2 promoter when combined with AP1 (c-Fos/c-Jun) polypeptides and that NFATx is capable of activating transcription of the interleukin 2 promoter in COS-7 cells when stimulated with phorbol ester and calcium ionophore. NFATx mRNA is preferentially and remarkably found in the thymus and at lower levels in peripheral blood leukocytes. The expression pattern of NFATx, together with its functional activity, strongly suggests that NFATx plays a role in the regulation of gene expression in T cells and immature thymocytes.

    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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    This manuscript examines the mechanisms by which Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated through the binding of Ca2+/CaM and by phosphorylation. Studies with the synthetic autoinhibitory domain peptides of CaM-kinase II indicate that CaM-kinase IV has a similarly located autoinhibitory domain, and this was confirmed since site-directed mutagenesis of this region (HMDT(308) to DEDD and FN317 to DD) generated fully active Ca2+/CaM-independent kinases. Total activities of purified, baculovirus-expressed wild type and mutant kinases were increased 2-fold by intramolecular autophosphorylation, but this reaction was extremely slow (1-2 h) and probably not physiological. However, CaM-kinase IV can be activated by brain CaM-kinase IV kinase resulting in large increases in both total (5-7-fold) and Ca2+/CaM-independent (>20-fold) CaM-kinase IV activities. This activation reaction required Mg2+/ATP and Ca2+/CaM, was intermolecularly catalyzed, and was reversed by protein phosphatase 2A. Activation of CaM-kinase IV resulted in a 10-fold decrease in K-m for syntide-2 with little effect on K-m for ATP or V-max. CaM-kinase IV kinase was highly purified from rat brain extract and was shown to be a 68-kDa monomer.
    The results of this study demonstrate that CaM-kinase IV does have an autoinhibitory domain within residues His(305)-Lys(321) that suppresses kinase activity in the absence of Ca2+/CaM. CaM-kinase IV is not significantly activated by autophosphorylation, but it can be activated 10 fold by a CaM-kinase IV kinase. This kinase cascade activation mechanism may be important for the physiological function of CaM-kinase IV such as transcriptional regulation through phosphorylation of cAMP responsive element binding protein (Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T. R. (1994) J. Biol. Chem. 269, 15520-15527).

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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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    Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation.

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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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    Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLEO, is a target for the induction signals. We observed induction with templates containing intact CLEO but not with templates with deleted or mutated CLEO. We also observed that two distinct signals were required for the stimulation through CLEO, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLEO-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor A.Pl. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation.

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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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    Annexin XI (CAP-50) is a probable target protein of calcyclin. Being different from other annexins, annexin XI localizes mainly in nuclei of cultured fibroblasts. In rat embryonic fibroblasts transformed by Rous sarcoma virus oncogene, SR-3Y1 cells, phosphorylation of annexin XI was increased on both serine and threonine residues (Ser < Thr), compared with findings in control 3Y1 cells. The amount of phosphorylated annexin XI was approximately 8.5% of the total cellular annexin XI and the phosphorylated annexin XI migrated slightly slower on SDS-polyacrylamide gel electrophoresis than did the non-phosphorylated form of annexin XI. Phosphorylated annexin XI was recovered in the cytoplasmic fraction and did not bind to phosphatidylserine vesicle in the presence of high Ca2+ (over 1 mM).
    Annexin XI was phosphorylated by mitogen-activated protein (MAP) kinase, which was reported to be activated in v-src-transformed fibroblast (Gupta, S. K., Gallego, C. Johnson, G. L. and Heasley, L. E. (1992) J. Biol. Chem. 267, 7987-7990), on both serine and threonine residues (Ser >> Thr) in vitro. Comparative phosphopeptide mappings analyzed by reverse-phase high performance liquid chromatography suggested that the sites phosphorylated in situ in SR-3Y1 cells are distinct from the sites by MAP kinase. Annexin XI phosphorylated by MAP kinase still possessed the ability to bind to phosphatidylserine vesicle.
    These results suggest that annexin XI is a substrate for some Ser/Thr kinase(s) which is activated in v-src-transformed cells and that the phosphorylation may regulate the function of annexin XI in living cells.

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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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    Unlike human, rat and mouse calcyclin, purified rabbit calcyclin did not form a dimer on Tricine SDS-PAGE under non-reduced conditions. Based on the internal peptide sequence of rabbit calcyclin, we isolated and sequenced a cDNA clone encoding calcyclin. The sequence of this clone (pCalC) is 629 bp long and codes 90 amino acid residues of a protein with a molecular mass of 10,153 Da. By Northern blot analysis, a major band of 0.9 kbp and a minor band of 2.6 kbp were detected in the lung. The recombinant calcyclin mutated serine at the third position to cysteine was expressed in E. coli and made dimer formation under non-reduced conditions on SDS-PAGE. Whether or not this type of mutation which prevents dimer formation of calcyclin plays a physiological role in the rabbit lung is the subject of an ongoing study.

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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 50-kDa protein, which binds to the growth-regulated gene (2A9) product, calcyclin in a calcium-dependent manner, was purified from bovine lung. Partial amino acid sequencing of the protein revealed it to be the bovine equivalent of rabbit lung CAP-50 (calcyclin-associated protein, 50 kDa), which is a member of the annexin family and binds to calcyclin in a calcium-dependent manner. Specific polyclonal antibodies to bovine lung CAP-50 were prepared. Comparative studies between CAP-50 and synexin (annexin VII) on the immunoreactivity against anti-CAP-50 antibodies and the ability of binding to calcyclin revealed that CAP-50 was a distinct molecule from synexin. Using specific polyclonal antibodies to bovine lung CAP-50, tissue distribution and subcellular distribution of CAP-50 were investigated. In most rat tissues, except those in the central nervous systems and kidney, CAP-50 is expressed at a high or moderate level. Both studies by subcellular fractionation and by indirect immunofluorescence staining of the rat embryonic fibroblast cell line, 3Y1, revealed that CAP-50 mainly localized in nuclei. Moreover, between the cells at interphase and at mitotic phase, different distributions of CAP-50 were observed. That is, in the cells at interphase, CAP-50 seemed to localize throughout the nucleoplasm. On the other hand, in the cells during mitosis, CAP-50 was concentrated at the loop-like structure around the mitotic apparatus. CAP-50 was found in isolated 3Y1 nuclei lacking outer nuclear membranes, and approximately 50% of CAP-50 was extracted from the nuclei by chelating calcium. Thus, CAP-50, a unique annexin, localizes in nuclei.

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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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    A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endopeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidyl-ethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca2+-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent K(d) value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2-mu-M), however, calcyclin did not alter the inhibitory effect. With the I-125-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca2+-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane.

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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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    Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.

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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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  • 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)

    公益財団法人 日本生化学会  2018.8 

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MISC

  • BCRシグナル伝達を介したAID発現制御の解析

    石橋朋之, 梶浦雄也, 久原亜弓, 大柳翔, 秋山美咲, 曲正樹, 徳光浩, 金山直樹

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

  • 新規S100A6標的分子(HMG20A)の同定と相互作用解析

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

    日本生化学会大会(Web)   92nd   2019

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

    赤木魁, 田中啓之, 金山直樹, 曲正樹, 波多野直哉, 徳光浩

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

  • CaMKKβのリン酸化/脱リン酸化による動的制御機構の解明

    高畠翔太, 福本侑世, 金山直樹, 曲正樹, 波多野直哉, 徳光浩

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

  • 濾胞樹状細胞表面に発現するIL-34がB細胞活性化能力を有する単球系細胞の分化に関与する

    曲正樹, 小川紗也香, 松岡由希子, 高田美帆, 金山直樹, 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

  • 濾胞樹状細胞の活性化に伴い高発現する分子の探索とその機能解析

    西岡美玖, 西岡美穂, 小川紗也香, 波多野直哉, 金山直樹, 徳光浩, 曲正樹

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

  • 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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  • GRP78により発現制御される濾胞樹状細胞表面のIL-34が単球系細胞の分化に関与する

    小川紗也香, 松岡由希子, 高田美帆, 松井一恵, 山根文寛, 安原詩織, 金山直樹, 波多野直哉, 徳光浩, 曲正樹

    日本分子生物学会年会プログラム・要旨集(Web)   41st   2018

  • Analysis of a role for SRSF1-3 in a transcription-coupled process during IgV hypermutation

    Y. Kawaguchi, K. Yokoyama, N. Kawamoto, H. Nariki, Y. Matsuyama, Y. Kanehiro, T. Hikasa, M. Magari, H. Ohmori, H. Tokumitsu, N. Kanayama

    EUROPEAN JOURNAL OF IMMUNOLOGY   46   1267 - 1268   2016.8

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  • SRSF1-3 has a role in nuclear localization of AID by regulating its nuclear export

    N. Kanayama, Y. Kawaguchi, N. Kawamoto, H. Nariki, S. Miyazaki, K. Yokoyama, M. Magari, H. Ohmori, H. Tokumitsu

    EUROPEAN JOURNAL OF IMMUNOLOGY   46   1142 - 1142   2016.8

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  • IL-34-dependent differentiation of monocytic cell with B cell stimulating activity

    M. Magari, S. Ogawa, Y. Toya, K. Hieda, F. Yamane, N. Kanayama, H. Tokumitsu, H. Ohmori

    EUROPEAN JOURNAL OF IMMUNOLOGY   46   1139 - 1139   2016.8

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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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    J-GLOBAL

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  • Ca<sup>2+</sup>/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)   2015

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    J-GLOBAL

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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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    J-GLOBAL

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  • 3P-032 Engineering of antibodies using the hypermutating B cell line

    Kanayama Naoki, Watanabe Koji, Koga Mai, Kawakami Kanae, Uetsuki Hidetomo, Tokumitsu Hiroshi, Magari Masaki, Ohmori Hitoshi

    66   202 - 202   2014

  • 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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  • 3P-002 High-affinity antibody generation using the chicken B cell line DT40-SW by regulating B cell receptor-mediated apoptosis

    Koga Mai, Ikeda Mika, Kojima Hiroyuki, Kawakami Kanae, Watanabe Koji, Tokumitsu Hiroshi, Magari Masaki, Ohmori Hitoshi, Kanayama Naoki

    65   188 - 188   2013

  • 3P-003 Protein evolution in a protein display system using the hypermutating B cell line DT40-SW

    Watanabe Koji, Hikasa Takuya, Uetsuki Hidetomo, Tokumitsu Hiroshi, Magari Masaki, Ohmori Hitoshi, Kanayama Naoki

    65   189 - 189   2013

  • [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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    Aim
    Glucokinase (GK) in pancreatic beta cells is thought to be involved in insulin secretion and glucose homeostasis. This study investigates whether the long-acting agonist of the glucagon-like peptide 1, namely exendin-4, mediates stimulatory effects on GK gene expression through the Ca&lt;SU2+&lt;/SU/calmodulin (CaM)-dependent protein kinase (CaMK) cascade.
    Methods
    GK expression was examined by real-time PCR, western blot analysis and reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. CaMKIV activity was assessed by detection of activation loop phosphorylation (Thr&lt;SU196&lt;/SU) of CaMKIV. We investigated the effect of the constitutively active form (CaMKIVc) of CaMKIV on GK promoter activity.
    Results
    Increased expression level of GK protein was noted in response to rising concentrations of exendin-4 with maximum induction at 10 nM. Real-time PCR analysis showed a significant increase in the amount of GK mRNA in response to rising concentrations of exendin-4. Exendin-4 also stimulated GK promoter activity but failed to do so in the presence of STO-609, a CaMKK inhibitor. This result is consistent with the observations that the upregulation of CaMKIV phosphorylation (at Thr&lt;SU196&lt;/SU) peaked after 15 min of exposure to exendin-4 and that CaMKIVc enhanced or upregulated GK promoter activity in INS-1 cells. Furthermore, STO-609 significantly suppressed the exendin-4 - upregulated the expression of the GK protein.
    Conclusion
    Activation of the CaMKK/CaMKIV cascade might be required for exendin-4-induced GK gene transcription, indicating that exendin-4 plays an important role in insulin secretion in pancreatic beta cells.

    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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    The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants. lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK (Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists. we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S 100 proteins, in a Ca2+-dependent manner. Furthermore. the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 muM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 muM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked K-ATP channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists.

    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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  • 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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  • 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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    Language:English   Publishing type:Book review, literature introduction, etc.   Publisher:LIPPINCOTT WILLIAMS & WILKINS  

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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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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 )

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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)

    水津 太, 徳光 浩, 平田 徳幸, 松岡 達臣

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

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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)

    徳光 浩, 曲 正樹

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

    Ca2+/Calmodulin(CaM)依存性プロテインキナーゼ活性化キナーゼβ(CaMKKβ)は、CaMキナーゼⅠやCaMキナーゼⅣ、5’-AMP活性化キナーゼ(AMPK)をリン酸化依存的に活性化し、遺伝子発現や神経発生、代謝調節やがん細胞増殖に至る多様な細胞内Ca2+に応答した生理機能を調節する。これまでの研究より、cAMP/PKA経路がCaMKKβ(Thr144)のリン酸化に関与しており、細胞内Ca2+とcAMP経路がCaMKKβ分子上においてクロストークしていることが明らかとなった。
    そこで本研究では、細胞内における主要な脱リン酸化酵素であるProtein Phosphatase 2A/1 (PP2A/1)の選択的阻害剤(オカダ酸:OA)を用いた解析により、CaMKKβ(Thr144)の脱リン酸化を検証した。その結果、未刺激の状態においてCaMKKβ(Thr144)は、PP2A/PP1により脱リン酸化状態を保つことが分かった。さらに、細胞内CaMKKβキナーゼ活性の測定の結果、OAの処理時間に依存してCaMKKβキナーゼ活性が上昇し、脱リン酸化酵素阻害により活性化するCaMKKβ(Thr144)キナーゼの存在が示唆された。しかしこのキナーゼは、阻害剤による薬理学的実験から、これまでに示されたAMPK やPKAとは異なると考えられる。以上の結果より、CaMKKβは細胞内において、PKAを含めたリン酸化酵素および脱リン酸化酵素によりThr144残基のリン酸化/脱リン酸化制御が動的に調節されていることが明らかとなった。

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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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Academic Activities

  • 日本生化学会評議員・代議員

    Role(s):Planning, management, etc.

    日本生化学会  2018

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