Updated on 2021/03/30

写真a

 
AMANO, Mutsuki
 
Organization
Graduate School of Medicine Center for Neural Disease and Cancer Division Associate professor
Title
Associate professor
Contact information
メールアドレス

Degree 2

  1. 博士(バイオサイエンス)

  2. 修士(理学)

Research Interests 3

  1. kinase

  2. phosphorylation

  3. Cytoskeleton

Research Areas 2

  1. Others / Others  / Cell Biology

  2. Others / Others  / Biochemistry

Current Research Project and SDGs 2

  1. Regulation of cytoskeleton by small GTPase Rho and its targets.

  2. タンパク質リン酸化酵素とリン酸化シグナルネットワークの解析

Education 3

  1. Nara Institute of Science and Technology   Graduate School, Division of Biological Science

    - 1998.3

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    Country: Japan

  2. Kobe University   Graduate School, Division of Natural Science

    1990.4 - 1992.3

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    Country: Japan

  3. Kobe University   Faculty of Science

    - 1990

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    Country: Japan

Professional Memberships 6

  1. The Japanese Pharmacological Society

  2. 日本分子生物学会

  3. 日本生化学会

  4. 日本細胞生物学会

  5. 日本癌学会

  6. Japan Neuroscience Society

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Papers 71

  1. Dynamic subcellular localization and transcription activity of the SRF cofactor MKL2 in the striatum are regulated by MAPK

    Ariza Anthony, Funahashi Yasuhiro, Kozawa Sachi, Faruk Md Omar, Nagai Taku, Amano Mutsuki, Kaibuchi Kozo

    JOURNAL OF NEUROCHEMISTRY     2021.2

  2. ARHGAP10, which encodes Rho GTPase-activating protein 10, is a novel gene for schizophrenia risk

    Sekiguchi Mariko, Sobue Akira, Kushima Itaru, Wang Chenyao, Arioka Yuko, Kato Hidekazu, Kodama Akiko, Kubo Hisako, Ito Norimichi, Sawahata Masahito, Hada Kazuhiro, Ikeda Ryosuke, Shinno Mio, Mizukoshi Chikara, Tsujimura Keita, Yoshimi Akira, Ishizuka Kanako, Takasaki Yuto, Kimura Hiroki, Xing Jingrui, Yu Yanjie, Yamamoto Maeri, Okada Takashi, Shishido Emiko, Inada Toshiya, Nakatochi Masahiro, Takano Tetsuya, Kuroda Keisuke, Amano Mutsuki, Aleksic Branko, Yamomoto Takashi, Sakuma Tetsushi, Aida Tomomi, Tanaka Kohichi, Hashimoto Ryota, Arai Makoto, Ikeda Masashi, Iwata Nakao, Shimamura Teppei, Nagai Taku, Nabeshima Toshitaka, Kaibuchi Kozo, Yamada Kiyofumi, Mori Daisuke, Ozaki Norio

    TRANSLATIONAL PSYCHIATRY   Vol. 10 ( 1 ) page: 247   2020.7

  3. Dopamine Receptor Dop1R2 Stabilizes Appetitive Olfactory Memory through the Raf/MAPK Pathway in Drosophila

    Sun Huan, Nishioka Tomoki, Hiramatsu Shun, Kondo Shu, Amano Mutsuki, Kaibuchi Kozo, Ichinose Toshiharu, Tanimoto Hiromu

    JOURNAL OF NEUROSCIENCE   Vol. 40 ( 14 ) page: 2935 - 2942   2020.4

  4. Protein kinases phosphorylate long disordered regions in intrinsically disordered proteins

    Koike Ryotaro, Amano Mutsuki, Kaibuchi Kozo, Ota Motonori

    PROTEIN SCIENCE   Vol. 29 ( 2 ) page: 564 - 571   2020.2

  5. Phosphorylation of Npas4 by MAPK Regulates Reward-Related Gene Expression and Behaviors

    Funahashi Yasuhiro, Ariza Anthony, Emi Ryosuke, Xu Yifan, Shan Wei, Suzuki Ko, Kozawa Sachi, Ahammad Rijwan Uddin, Wu Mengya, Takano Tetsuya, Yura Yoshimitsu, Kuroda Keisuke, Nagai Taku, Amano Mutsuki, Yamada Kiyofumi, Kaibuchi Kozo

    CELL REPORTS   Vol. 29 ( 10 ) page: 3235 - +   2019.12

  6. Protein Kinase N Promotes Stress-Induced Cardiac Dysfunction Through Phosphorylation of Myocardin-Related Transcription Factor A and Disruption of Its Interaction With Actin

    Sakaguchi Teruhiro, Takefuji Mikito, Wettschureck Nina, Hamaguchi Tomonari, Amano Mutsuki, Kato Katsuhiro, Tsuda Takuma, Eguchi Shunsuke, Ishihama Sohta, Mori Yu, Yura Yoshimitsu, Yoshida Tatsuya, Unno Kazumasa, Okumura Takahiro, Ishii Hideki, Shimizu Yuuki, Bando Yasuko K., Ohashi Koji, Ouchi Noriyuki, Enomoto Atsushi, Offermanns Stefan, Kaibuchi Kozo, Murohara Toyoaki

    CIRCULATION   Vol. 140 ( 21 ) page: 1737 - 1752   2019.11

  7. Pathological Progression Induced by the Frontotemporal Dementia-Associated R406W Tau Mutation in Patient-Derived iPSCs

    Nakamura Mari, Shiozawa Seiji, Tsuboi Daisuke, Amano Mutsuki, Watanabe Hirotaka, Maeda Sumihiro, Kimura Taeko, Yoshimatsu Sho, Kisa Fumihiko, Karch Celeste M., Miyasaka Tomohiro, Takashima Akihiko, Sahara Naruhiko, Hisanaga Shin-ichi, Ikeuchi Takeshi, Kaibuchi Kozo, Okano Hideyuki

    STEM CELL REPORTS   Vol. 13 ( 4 ) page: 684 - 699   2019.10

  8. Discovery of a key missing signaling between RHOA/RHO-kinase and ras underlying spine enlargement and LTP

    Wu M., Funahashi Y., Takano T., Tsuboi D., Ahammad R., Amano M., Kaibuchi K.

    JOURNAL OF NEUROCHEMISTRY   Vol. 150   page: 121 - 121   2019.7

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  9. Comprehensive analysis of kinase-oriented phospho-signaling pathways.

    Amano M, Nishioka T, Tsuboi D, Kuroda K, Funahashi Y, Yamahashi Y, Kaibuchi K

    Journal of biochemistry   Vol. 165 ( 4 ) page: 301 - 307   2019.4

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1093/jb/mvy115

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  10. In Vivo Identification of Protein Kinase Substrates by Kinase-Oriented Substrate Screening (KIOSS).

    Nishioka T, Amano M, Funahashi Y, Tsuboi D, Yamahashi Y, Kaibuchi K

    Current protocols in chemical biology   Vol. 11 ( 1 ) page: e60   2019.3

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1002/cpch.60

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  11. Balance between dopamine and adenosine signals regulates the PKA/Rap1 pathway in striatal medium spiny neurons.

    Zhang X, Nagai T, Ahammad RU, Kuroda K, Nakamuta S, Nakano T, Yukinawa N, Funahashi Y, Yamahashi Y, Amano M, Yoshimoto J, Yamada K, Kaibuchi K

    Neurochemistry international   Vol. 122   page: 8 - 18   2019.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.neuint.2018.10.008

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  12. Targeting Tyro3 ameliorates a model of PGRN-mutant FTLD-TDP via tau-mediated synaptic pathology

    Fujita Kyota, Chen Xigui, Homma Hidenori, Tagawa Kazuhiko, Amano Mutsuki, Saito Ayumu, Imoto Seiya, Akatsu Hiroyasu, Hashizume Yoshio, Kaibuchi Kozo, Miyano Satoru, Okazawa Hitoshi

    NATURE COMMUNICATIONS   Vol. 9 ( 1 ) page: 433   2018.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1038/s41467-018-02821-z

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  13. Mechanism of dopamine signaling for membrane excitability

    Tsuboi D., Shimomura T., Nakano T., Nagai T., Amano M., Yoshimoto J., Kubo Y., Kaibuchi K.

    JOURNAL OF NEUROCHEMISTRY   Vol. 142   page: 135 - 135   2017.8

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  14. Discovery of long-range inhibitory signaling to ensure single axon formation Reviewed

    Takano T, Wu M, Nakamuta S, Naoki H, Ishizawa N, Namba T, Watanabe T, Xu C, Hamaguchi T, Yura Y, Amano M, Hahn KM, and Kaibuchi K.

    Nat Commun   Vol. 8 ( 1 ) page: 33   2017.6

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1038/s41467-017-00044-2

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  15. KANPHOS (Kinase-Associated Phospho-Signaling) Platform - A database for neural phosphoproteomics with quality control

    Kuroda Keisuke, Nagai Taku, Amano Mutsuki, Yoshimoto Junichiro, Kannon Takayuki, Nishioka Tomoki, Usui Shiro, Kaibuchi Kozo

    JOURNAL OF PHARMACOLOGICAL SCIENCES   Vol. 133 ( 3 ) page: S263 - S263   2017.3

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  16. A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology Reviewed

    Li C, Imanishi A, Komatsu N, Terai K, Amano M, Kaibuchi K, and Matsuda M.

    Cell Struct Funct   Vol. 42 ( 1 ) page: 1 - 13   2017.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1247/csf.16016

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  17. A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology

    Li Chunjie, Imanishi Ayako, Komatsu Naoki, Terai Kenta, Amano Mutsuki, Kaibuchi Kozo, Matsuda Michiyuki

    CELL STRUCTURE AND FUNCTION   Vol. 42 ( 1 ) page: 1 - 13   2017

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  18. Identification of Protein Kinase Substrates by the Kinase-Interacting Substrate Screening (KISS) Approach Invited Reviewed

    Amano, M. Nishioka, T. Yura, Y. Kaibuchi, K.

    Curr Protoc Cell Biol   Vol. 72   page: 14 16 1-14 16 12   2016.9

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    Authorship:Lead author   Language:English  

    DOI: 10.1002/cpcb.8

  19. Focused Proteomics Revealed a Novel Rho-kinase Signaling Pathway in the Heart Reviewed

    Yura, Y. Amano, M. Takefuji, M. Bando, T. Suzuki, K. Kato, K. Hamaguchi, T. Hasanuzzaman Shohag, M. Takano, T. Funahashi, Y. Nakamuta, S. Kuroda, K. Nishioka, T. Murohara, T. Kaibuchi, K.

    Cell Struct Funct   Vol. 41 ( 2 ) page: 105-120   2016.8

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1247/csf.16011

  20. Phosphoproteomics of the Dopamine Pathway Enables Discovery of Rap1 Activation as a Reward Signal In Vivo Reviewed

    Nagai, T. Nakamuta, S. Kuroda, K. Nakauchi, S. Nishioka, T. Takano, T. Zhang, X. Tsuboi, D. Funahashi, Y. Nakano, T. Yoshimoto, J. Kobayashi, K. Uchigashima, M. Watanabe, M. Miura, M. Nishi, A. Kobayashi, K. Yamada, K. Amano, M. Kaibuchi, K.

    Neuron   Vol. 89 ( 3 ) page: 550-565   2016.2

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    DOI: 10.1016/j.neuron.2015.12.019

  21. Single-Cell Memory Regulates a Neural Circuit for Sensory Behavior Reviewed

    Kobayashi, K. Nakano, S. Amano, M. Tsuboi, D. Nishioka, T. Ikeda, S. Yokoyama, G. Kaibuchi, K. Mori, I.

    Cell Rep   Vol. 14 ( 1 ) page: 11-21   2016.1

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    DOI: 10.1016/j.celrep.2015.11.064

  22. Developing novel methods to search for substrates of protein kinases such as Rho-kinase Invited Reviewed

    Nishioka, T. Shohag, M. H. Amano, M. Kaibuchi, K.

    Biochim Biophys Acta   Vol. 1854 ( 10 ) page: 1663-1666   2015.10

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    DOI: 10.1016/j.bbapap.2015.03.001

  23. Phosphoproteomic Analysis Using the WW and FHA Domains as Biological Filters Reviewed

    Hasanuzzaman Shohag, M. Nishioka, T. Uddin Ahammad, R. Nakamuta, S. Yura, Y. Hamaguchi, T. Kaibuchi, K. Amano, M.

    Cell Struct Funct   Vol. 40 ( 2 ) page: 95-104   2015.8

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    DOI: 10.1247/csf.15004

  24. Kinase-interacting substrate screening is a novel method to identify kinase substrates Reviewed

    Amano, M., Hamaguchi, T., Shohag, M. H., Kozawa, K., Kato, K., Zhang, X., Yura, Y., Matsuura, Y., Kataoka, C., Nishioka, T., and Kaibuchi, K.

    J Cell Biol   Vol. 209 ( 6 ) page: 895-912   2015.6

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    DOI: 10.1083/jcb.201412008

  25. In vivo Screening for Substrates of Protein Kinase A Using a Combination of Proteomic Approaches and Pharmacological Modulation of Kinase Activity. Reviewed

    Hamaguchi, T., Nakamuta, S., Funahashi, Y., Takano, T., Nishioka, T., Shohag, M. H., Yura, Y., Kaibuchi, K., and Amano, M.

    Cell Struct Funct   Vol. 40   page: 1-12   2015.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1247/csf.14014

  26. Preferential targeting of p39-activated Cdk5 to Rac1-induced lamellipodia. Reviewed

    Ito, Y., Asada, A., Kobayashi, H., Takano, T., Sharma, G., Saito, T., Ohta, Y., Amano, M., Kaibuchi, K., and Hisanaga, S.

    Mol Cell Neurosci   Vol. 61   page: 34-45   2014.6

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    DOI: 10.1016/j.mcn.2014.05.006

  27. Distinct distribution and localization of Rho-kinase in mouse epithelial, muscle and neural tissues Reviewed

    Iizuka, M., Kimura, K., Wang, S., Kato, K., Amano, M., Kaibuchi, K., Mizoguchi, A.

    Cell Struct Funct   Vol. 37 ( 2 ) page: 155-175   2012.9

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    The small GTP-binding protein Rho plays a crucial role in a wide variety of cellular functions through various effector proteins. Rho-kinase is a key effector protein of Rho, which is composed of two isoforms, ROCK1 and ROCK2. To clarify the site of action of ROCK1 and ROCK2, we performed immunofluorescence and immunoelectron microscopic analyses using isoform-specific antibodies in mouse tissues. In the large and small intestines, ROCK1 immunoreactivity was predominantly identified in epithelial cells, and ROCK2 immunoreactivity was negligible. In these epithelial cells, ROCK1 immunoreactivity was distributed on plasma membranes, while ROCK1 immunogold signals were localized at cell-cell contacts and cell adhesion sites, especially at the adherens junctions at the ultrastructural level. In the bladder epithelium, however, ROCK1 and ROCK2 signals were identified at intermediate filaments, and ROCK2 signals were also observed in nuclei. In the three types of muscular cells - smooth, cardiac, and skeletal muscle cells - ROCK1 and ROCK2 also showed differential distribution. ROCK1 signals were localized at actin filaments, plasma membranes, and vesicles near plasma membranes in smooth muscle cells; at the lysosomes in skeletal muscle cells; and were undetectable in cardiac muscle cells. ROCK2 signals were localized at actin filaments and centrosomes in smooth muscle cells, at intercalated discs in cardiac muscle cells, and at Z-discs and sarcoplasmic reticulum in skeletal muscle cells. In the brain, ROCK1 immunoreactivity was distributed in glia, whereas ROCK2 immunoreactivity was observed in neurons. These results indicate that the two isoforms of Rho-kinase distribute differentially to accomplish their specific functions.

  28. The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration Reviewed

    Kato, K., Yazawa, T., Taki, K., Mori, K., Wang, S., Nishioka, T., Hamaguchi, T., Itoh, T., Takenawa, T., Kataoka, C., Matsuura, Y., Amano, M., Murohara, T., Kaibuchi, K.

    Mol Biol Cell   Vol. 23 ( 13 ) page: 2593-2604   2012.5

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    Cell migration is essential for various physiological and pathological processes. Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. Although RhoA participates in a front-rear polarization in migrating cells, little is known about the functional interaction between RhoA and lipid turnover. We find here that src-homology 2-containing inositol-5-phosphatase 2 (SHIP2) interacts with RhoA in a GTP-dependent manner. The association between SHIP2 and RhoA is observed in spreading and migrating U251 glioma cells. The depletion of SHIP2 attenuates cell polarization and migration, which is rescued by wild-type SHIP2 but not by a mutant defective in RhoA binding. In addition, the depletion of SHIP2 impairs the proper localization of phosphatidylinositol 3,4,5-trisphosphate, which is not restored by a mutant defective in RhoA binding. These results suggest that RhoA associates with SHIP2 to regulate cell polarization and migration.

    DOI: 10.1091/mbc.E11-11-0958

  29. Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3zeta affinity chromatography Reviewed

    Nishioka, T., Nakayama, M., Amano, M., Kaibuchi, K.

    Cell Struct Funct   Vol. 37 ( 1 ) page: 39-48   2012.1

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    The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.

  30. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase Reviewed

    Amano, M., Tsumura, Y., Taki, K., Harada, H., Mori, K., Nishioka, T., Kato, K., Suzuki, T., Nishioka, Y., Iwamatsu, A.,Kaibuchi, K.

    PLoS One   Vol. 5 ( 1 ) page: e8704   2010

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    BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  31. Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity Invited Reviewed

    Amano, M., Nakayama, M., Kaibuchi, K.

    Cytoskeleton   Vol. 67 ( 9 ) page: 545-54   2010

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    Rho-associated kinase (Rho-kinase/ROCK/ROK) is an effector of the small GTPase Rho and belongs to the AGC family of kinases. Rho-kinase has pleiotropic functions including the regulation of cellular contraction, motility, morphology, polarity, cell division, and gene expression. Pharmacological analyses have revealed that Rho-kinase is involved in a wide range of diseases such as vasospasm, pulmonary hypertension, nerve injury, and glaucoma, and is therefore considered to be a potential therapeutic target. This review focuses on the structure, function, and modes of activation and action of Rho-kinase.

  32. *Rho-kinase contributes to sustained RhoA activation through phosphorylation of p190A RhoGAP Reviewed

    Mori, K.Amano, M.Takefuji, M.Kato, K.Morita, Y.Nishioka, T.Matsuura, Y.Murohara, T.Kaibuchi, K.

    J Biol Chem   Vol. 284 ( 8 ) page: 5067-5076   2009

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    RhoA is transiently activated by specific extracellular signals such as endothelin-1 (ET-1) in vascular smooth muscle cells. RhoGAP negatively regulates RhoA activity: thus, RhoA becomes the GDP-bound inactive form afterward. Sustained activation of RhoA is induced with high doses of the extracellular signals and is implicated in certain diseases such as vasospasms. However, it remains largely unknown how prolonged activation of RhoA is induced. Here we show that Rho-kinase, an effector of RhoA, phosphorylated p190A RhoGAP at Ser(1150) and attenuated p190A RhoGAP activity in COS7 cells. Binding of Rnd to p190A RhoGAP is thought to enhance its activation. Phosphorylation of p190A RhoGAP by Rho-kinase impaired Rnd binding. Stimulation of vascular smooth muscle cells with a high dose of ET-1 provoked sustained RhoA activation and p190A RhoGAP phosphorylation, both of which were prohibited by a Rho-kinase inhibitor. The phosphomimic mutation of p190A RhoGAP weakened Rnd binding and RhoGAP activities. Taken together, these results suggest that ET-1 induces Rho-kinase activation and subsequent phosphorylation of p190A RhoGAP, leading to prolonged RhoA activation.

  33. Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation Reviewed

    Nakayama, M.Goto, T. M.Sugimoto, M.Nishimura, T.Shinagawa, T.Ohno, S.Amano, M.Kaibuchi, K.

    Dev Cell   Vol. 14 ( 2 ) page: 205-215   2008

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    A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.

  34. Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation Reviewed

    Takefuji, M., Mori, K., Morita, Y., Arimura, N., Nishimura, T., Nakayama, M., Hoshino, M., Iwamatsu, A., Murohara, T., Kaibuchi, K., Amano, M.

    Biochem Biophys Res Commun   Vol. 355 ( 3 ) page: 788   2007.4

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    Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rac GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF.

  35. Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase Reviewed

    J NeurochemJ Biol Chem   Vol. 281 ( 22 ) page: 15320-15329   2006

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  36. Molecular mechanism for the regulation of rho-kinase by dimerization and its inhibition by fasudil Reviewed

    Structure   Vol. 14 ( 3 ) page: 589-600   2006

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  37. Regulatory machinery of UNC-33 Ce-CRMP localization in neurites during neuronal development in Caenorhabditis elegans Reviewed

    J Neurochem   Vol. 95 ( 6 ) page: 1629-1641   2005

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  38. Rho-kinase and myosin II activities are required for cell type and environment specific migration Reviewed

    Genes Cells   Vol. 10 ( 2 ) page: 107-117   2005

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  39. Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase Reviewed

    Genes Cells   Vol. 10 ( 10 ) page: 973-987   2005

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  40. Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones Reviewed

    Mol Cell Biol   Vol. 25 ( 22 ) page: 9973-9984   2005

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  41. Interaction of Rho-kinase with myosin II at stress fibres Reviewed

    Genes Cells   Vol. 9 ( 7 ) page: 653-660   2004

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  42. Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells Reviewed

    J Mol Cell Cardiol   Vol. 37 ( 2 ) page: 537-546   2004

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  43. Design and synthesis of Rho kinase inhibitors (I) Reviewed

    Bioorg Med Chem   Vol. 12 ( 9 ) page: 2115-2137   2004

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  44. Entrapment of Rho ADP-ribosylated by Clostridium botulinum C3 exoenzyme in the Rho-GDI-1 complex

    J Biol Chem   Vol. 278 ( 31 ) page: 28523-28527   2003

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  45. Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase Reviewed

    J Neurochem   Vol. 87 ( 3 ) page: 780-790   2003

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  46. Parallel coiled-coil association of the RhoA-binding domain in Rho-kinase Reviewed

    J Biol Chem   Vol. 278 ( 41 ) page: 46046-46051   2003

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  47. Isolation of the interacting molecules with GEX-3 by a novel functional screening Reviewed

    Biochem Biophys Res Commun   Vol. 292 ( 3 ) page: 697-701   2002

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  48. Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells Reviewed

    Biochem Biophys Res Commun   Vol. 297 ( 5 ) page: 1231-1237   2002

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  49. Rho-kinase-mediated contraction of isolated stress fibers Reviewed

    J Cell Biol   Vol. 153 ( 3 ) page: 569-584   2001

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  50. Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase Reviewed

    Pflugers Arch   Vol. 441 ( 5 ) page: 596-603   2001

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  51. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells

    Trends Pharmacol Sci   Vol. 22 ( 1 ) page: 32-39   2001

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  52. CRMP-2 induces axons in cultured hippocampal neurons Reviewed

    Nat Neurosci   Vol. 4 ( 8 ) page: 781-782   2001

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  53. Phosphorylation of ERM proteins at filopodia induced by Cdc42 Reviewed

    Genes Cells   Vol. 5 ( 7 ) page: 571-581   2000

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  54. Regulation and functions of Rho-associated kinase

    Exp Cell Res   Vol. 261 ( 1 ) page: 44-51   2000

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  55. Purification and in vitro activity of Rho-associated kinase Reviewed

    Methods Enzymol   Vol. 325   page: 149-155   2000

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  56. Phosphorylation of Collapsin Response Mediator Protein-2 by Rho-kinase: Evidence for Two Separate Signaling Pathways for Growth Cone Collapse Reviewed

    J Biol Chem   Vol. 275 ( 31 ) page: 23973-23980   2000

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  57. Identification of Calponin as a Novel Substrate of Rho-Kinase Reviewed

    Biochem Biophys Res Commun   Vol. 273 ( 1 ) page: 110-116   2000

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  58. *The COOH terminus of Rho-kinase negatively regulates Rho-kinase activity. Reviewed

    J. Biol. Chem.   Vol. 274   page: 32418-32424   1999

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  59. Phosphorylation of myosin-binding subunit(MBS) of myosin phosphatase by Rho-kinase in vivo.

    J. Cell Biol.   Vol. 147   page: 1023-1038   1999

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  60. Rho-associated kinase of chicken gizzard smooth muscle.

    J. Biol. Chem.   Vol. 274   page: 3744-3752   1999

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  61. Regulation of Cytoskeleton and cell adhesions by the small GTPase Rho and its targets.

    Trends in Cardiovascular Medicine   Vol. 8   page: 162-168   1998

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  62. Rho-kinase phosphorylates COOH-terminal threonines of Ezrin/Radixin/Moesin(ERM)proteins and regulates their Head-to-tail association.

    J Cell Biol   Vol. 140   page: 647-657   1998

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  63. Myosin II activation promotes neurite retraction during the action of Rho and Rho-kinase.

    Genes Cells   Vol. 3   page: 177-188   1998

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  64. Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase.

    J Biol Chem   Vol. 272   page: 26121-25127   1997

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  65. *Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Reviewed

    Science   Vol. 275   page: 1308-1311   1997

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  66. Phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and Rho-associated kinase.

    J Biol Chem   Vol. 272   page: 10333-10336   1997

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  67. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation.

    J Biol Chem   Vol. 272   page: 12257-12260   1997

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  68. Regulation of myosin phosphatase by Rho and Rho-associated kinase(Rho-kinase). Reviewed

    Science   Vol. 273   page: 245-248   1996

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  69. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for the small GTP-binding protein Rho.

    EMBO J   Vol. 15   page: 2208-2216   1996

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  70. *Phosphorylation and activation of myosin by Rho-associated kinase(Rho-kinase).

    J Biol Chem   Vol. 271   page: 20246-20249   1996

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  71. Identification of a putative target for Rho as a serine-threonine kinase protein Kinase N. Reviewed

    Science   Vol. 271   page: 648-650   1996

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Research Project for Joint Research, Competitive Funding, etc. 1

  1. Rhoファミリーシグナル伝達系分子を対象とした疾患関連遺伝子の探索

    2006

KAKENHI (Grants-in-Aid for Scientific Research) 11

  1. がん幹細胞のリン酸化シグナルの解析による治療標的分子の探索

    Grant number:20K07600  2020.04 - 2023.03

    渡辺 崇

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    がん幹細胞は自己複製能と多分化能、強い造腫瘍性、治療抵抗性といった特徴を有し、がんの再発や転移の原因とされている。申請者の所属する研究室では、乳がんの手術検体を移植した異種移植(PDX)マウスを作製することで、肝臓に自然転移するがん幹細胞を解析できる実験系を世界に先駆けて樹立した。申請者らは本PDXマウスのがん幹細胞では足場タンパク質S100が高発現しており、転移がん幹細胞ではS100の発現がさらに10倍以上誘導されることを見出した。本研究では、S100による乳がん幹細胞の特性を制御する分子機構、特に機能的リン酸化シグナルに焦点を絞り、乳がん幹細胞を標的とする治療ターゲットを提案する。

  2. 心臓の硬化を制御するG蛋白質共役受容体の機能解明と心不全治療薬シーズの探索

    Grant number:20H03674  2020.04 - 2023.03

    竹藤 幹人

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    近年、心臓が硬くなり拡がりにくいために症状を呈する「収縮機能が保たれた心不全(HFpEF)」が注目されている。従来の心収縮機能低下を標的とする治療法ではHFpEFの治療効果は乏しく、HFpEFに対する新たな治療法の開発が求められている。adhesion-GPCRファミリーは物理的刺激により活性化されるGPCRとして新たに分類され、メカノストレスに対する生体反応を制御する分子として注目されている。adhesion-GPCR X がHFpEFに関わっていることを明らかにし、その下流で活性化するリン酸化シグナルを解析する。本研究では、HFpEF治療薬の開発シーズを得ることを目指す。

  3. 血栓形成における12-リポキシゲナーゼの局在・活性制御メカニズムの解明

    Grant number:19K08853  2019.04 - 2022.03

    勝見 章

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    血小板におけるアゴニスト受容体からのシグナルは Gα13を介して低分子量GTPase RhoAを活性化する。その結果血小板の形態変化、濃染顆粒 内容物の分泌をおこす。我々はアフィニティクロマトグラフィーにより活性化RhoA がフォルミンDaam1を介して12-リポキシゲナーゼ(12-LOX)に結合することを見いだした。 12-LOXは主に血小板に発現し、アラキドン酸からの12-ヒドロキシエイコサテトラエン酸 (12-HETE)等の脂質メディエーター合成を介して血小板凝集を惹起する。本研究では活性化RhoA-Daam1が、12-LOXの細胞膜への局在を誘導し活性を制御することを証明する。
    血小板細胞膜から遊離したアラキドン酸は、プロスタグランジン(PG)、ロイコトルエン(LT)等の生理活性物質に代謝される。PG、TXA2を生成するシクロオキシゲナーゼ(COX)はよく研究されているが、血小板にはアラキドン酸を基質とするもう1つの酵素、リポキシゲナーゼ(LOX)が存在する。LOXには酸素添加部位の違いによって5-LOX、12-LOX、15-LOXが知られている。血小板には12-LOXのみが発現し12-HETE等の脂質メディエーターを産生する。12-HETEは血小板外に分泌され、血小板活性化、顆粒分泌、血餅退縮を惹起する。阻害剤を用いた研究で12-LOXはGPVIやFCγRIIAを介した血小板活性化に重要な役割を果たしている。また12-LOXのノックアウトマウスで血栓形成が阻害されることから12-LOX経路は向血栓性に働くことが示唆されている。我々は活性化RhoAに特異的にLOXが結合することを明らかにし、それを仲介する分子群の機能を解析中である。我々はRhoA, Rac1, Cdc42 をbait にした網羅的アフィニティクロマトグラフィーとそれに続くマススペクトロメトリー(LC/MS-MS)の実験系を確立した。この実験システムを用いて我々は活性型Rho family 蛋白の一部にLOX が結合することを明らかにした。さらに複数の活性化Rho GTPases と複数のLOX family の結合の証明を目的としている。次にGTP 結合型Rho familyとLOX をCOS7 細胞で共発現し、両者の結合を証明することができている。さらにRho familyのうち、LOXとの活性化依存性の結合が明らかになった分子につきそれぞれの阻害剤をCOS-7細胞に添加し、脂質メディエーター(ロイコトリエン、リポキシン、HETEなど)の産生が抑制されるかどうかを検討中である。
    我々は既にRhoA, Rac1, Cdc42 をbait にした網羅的アフィニティクロマトグラフィーとそれに続くマススペクトロメトリー(LC/MS-MS)の実験系を確立した。この実験システムを用いて我々は活性型Rho family 蛋白の一部にLOX が結合することを明らかにした。さらに複数の活性化Rho GTPases と複数のLOX family の結合の証明を目的としている。次にGTP 結合型Rho familyとLOX をCOS7 細胞で共発現し、両者の結合を証明することができている。さらにRho familyのうち、LOXとの活性化依存性の結合が明らかになった分子につきそれぞれの阻害剤をCOS-7細胞に添加し、脂質メディエーター(ロイコトリエン、リポキシン、HETEなど)の産生が抑制されるかどうかを検討中である。
    I 巨核球細胞株における活性型RhoAと12-LOXの共局在の証明
    巨核球細胞株MEG-O1、CMK-86にGFP-RhoAとRFP-12-LOXを安定発現し、両者の細胞内での共局在を共焦点顕微鏡を用いて観察する。必要に応じて免疫染色を併用する。既に発現ベクター構築と、遺伝子導入は確立している。RhoA阻害剤C3 toxin添加による12-LOXの局在変化と12-HETEの分泌を定量する。
    II 血小板シグナル伝達におけるRhoA-12-LOX経路の役割
    巨核球細胞株MEG-O1、CMK-86にL63RhoA(活性型)とN19RhoA(不活性型)を発現し、培養上清中の12-HETE産生がL63RhoAにより亢進することを確認する。さらに活性型RhoAによる12-HETE産生亢進がRhoA阻害薬であるC3 toxinにより抑制されるが、Rho-kinase阻害剤Y-27632では抑制されないことを証明する。
    III  血小板における12-LOX阻害のアクチン重合への影響
    洗浄血小板を用いたcytoplasmicアクチン重合アッセイとpyreneラベル精製アクチンアッセイの双方でアクチン重合の定量を行い、12-LOX阻害剤ML355によりアクチン重合が阻害されるかどうか検討する。これにより12-LOXが血小板のアクチン重合を制御していることを証明する。また、ML355の添加による血小板のアクチン形態変化をRhodamine Phalloidin染色で観察する。

  4. 細胞内シグナルによる神経活動と情動行動・学習の制御機構の解明

    Grant number:17H01380  2017.04 - 2021.03

    貝淵 弘三

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    (1)リン酸化プロテオミクスによる神経伝達物質のリン酸化シグナル解析:マウス線条体スライスへのアデノシンA2A受容体作用薬による刺激やマウス個体への選択的セロトニンの再取り込み阻害剤の連続投与によりリン酸化が変動するタンパク質とそのリン酸化部位を同定した。
    (2)リン酸化シグナル伝達の時空間的モニタリング法の開発と応用: MYPT1上のRho-Kinaseのドッキングモチーフ(DM)として推定された配列がRho-Kinaseによるリン酸化に寄与した。また、DM配列のペプチドがリン酸化抑制効果を示した。
    (3)リン酸化シグナル分子の分子操作法の開発と応用:cre依存的にRho-Kinase-DNやPAK-AIDを発現するアデノ随伴ウイルス(AAV)ベクターをAdora2a-creマウスの側坐核に注入し、ドーパミンD2受容体を発現する中型有棘神経細胞(D2R-MSN)においてRho-KinaseやPAKの機能を抑制したところ、嫌悪学習・記憶が障害された。
    (4)神経細胞の膜興奮性を制御する機構の解析:D1Rシグナル伝達により制御されるKCNQ2チャネルのリン酸化修飾の役割をin vivoで明らかにするため、KCNQ2リン酸化部位(S404 & S448)を欠損させた遺伝子変異マウスを作製した。リン酸化部位欠損マウスの線条体スライスにおいてD1R作動薬によるKCNQ2のリン酸化が認められないことを確認した。
    (5)シナプス可塑性の制御機構の解析:線条体スライスにおいてNMDA受容体刺激がARHGEF2、ARHGAP21及びARHGAP39のリン酸化を亢進した。これらのリン酸化の亢進はCaMKⅡ阻害剤やNMDA受容体阻害剤の処置により抑制された。
    (6)神経可塑性に関与する遺伝子発現機構の解析: D1R-MSN特異的にNPAS4を欠損あるいは機能抑制させたところ、報酬学習・記憶が障害された。
    (1)リン酸化プロテオミクスによる神経伝達物質のリン酸化シグナル解析:セロトニンやアデノシン受容体刺激によりリン酸化が変動するタンパク質とそのリン酸化部位を同定し、KANPHOSデータベースに内部データとして登録した。
    (2)リン酸化シグナル伝達の時空間的モニタリング法の開発と応用:推定したMYPT1上のRho-KinaseのDM配列がRho-Kinaseによるリン酸化に影響すること、DM配列のペプチドがリン酸化抑制効果を示すことを見出した。
    (3)リン酸化シグナル分子の分子操作法の開発と応用: cre依存的にPKA やPKC、CaMKⅡ、Rho-Kinase、PAKの機能を抑制するAAVベクターの作製を完了した。作製したAAVを用いD2R-MSNにおいてRho-KinaseやPAKの機能を抑制したマウスを作製した結果、嫌悪学習・記憶が障害されることを見出した。
    (4)神経細胞の膜興奮性を制御する機構の解析:CRISPR/Cas9遺伝子編集技術を用いてMAPKによるKCNQ2リン酸化部位を欠損させた遺伝子変異マウスを作製した。リン酸化部位の欠損をシーケンス解析で確認するとともに、リン酸化部位欠損マウスの線条体スライスにおいてD1R作動薬によるKCNQ2のリン酸化が認められないことを見出した。
    (5)シナプス可塑性の制御機構の解析:新たにリン酸化抗体を作製し、NMDA受容体刺激がCaMKⅡ依存的にARHGEF2、ARHGAP21及びARHGAP39のリン酸化を亢進することを見出した。
    (6)神経可塑性に関与する遺伝子発現機構の解析:MAPKが転写因子NPAS4をリン酸化することで、NPAS4とCBPとの結合が増加し転写活性が上昇すること、D1R-MSNにおいてNPAS4が報酬学習・記憶を制御することを明らかにした (Funahashi et al. Cell Rep. 2019)。
    (1)リン酸化プロテオミクスによる神経伝達物質のリン酸化シグナル解析:引き続き、神経伝達物質の受容体作動薬によってリン酸化が変動するタンパク質を同定し、パスウェイ解析により各受容体の刺激に応答するシグナル経路を特定する。
    (2) リン酸化シグナル伝達の時空間的モニタリング法の開発と応用:MYPT1のDM配列を基に、Rho-kinaseの機能抑制ペプチドや活性モニターツールの作製・評価を試みる。
    (3)リン酸化シグナル分子の分子操作法の開発と応用:cre依存的にPKAやPKC、CaMKⅡの偽基質配列を発現するAAVベクターを神経細胞種特異的に発現させ、PKAやPKC、CaMKⅡの活性を抑制し、情動行動・学習への影響を評価する。また、LOV-Trap法を用い光刺激依存的にin vivoでPKAやPKC、CaMKⅡの活性を制御する分子ツールの開発を進める。
    (4)神経細胞の膜興奮性を制御する機構の解析:KCNQ2チャネルのリン酸化修飾の役割をin vivoで明らかにするため、リン酸化部位欠損マウスを用いて神経膜興奮性やKCNQ感受性電流を電気生理学的に評価する。
    (5) シナプス可塑性の制御機構の解析:Rho-KinaseやSHANK3のコンディショナルノックアウトマウスを用い、側坐核のD1R-MSNやD2R-MSN特異的にRho-KinaseやSHANK3を欠損させ、情動行動・学習への影響を評価する。SHANK3のリン酸化部位変異体を発現するAAVベクターを用い、樹状突起スパインの形態やPSD95などの足場タンパク質との相互作用、AMPA型受容体やNMDA型受容体の膜上への局在を解析する。
    (6)神経可塑性に関与する遺伝子発現機構の解析:MKL2-DNを発現するAAVを用い、D1R-MSN特異的にMKL2の機能を抑制したマウスを作製し、情動行動・学習への影響を評価する。

  5. The role of protein kinase N in cardiomyocyte

    Grant number:17K09571  2017.04 - 2020.03

    TAKEFUJI MIKITO

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    Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 protected mice from pressure overload induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.

  6. 蛋白質リン酸化酵素の基質認識・活性調節機構とリン酸化シグナルネットワークの解析

    Grant number:17K07383  2017.04 - 2020.03

    天野 睦紀

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    Authorship:Principal investigator 

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

    本研究課題では、プロテインキナーゼの基質認識機構の解析およびキナーゼの活性調節・モニタリングツールの開発を行う。昨年度は、Rho-kinaseとその基質であるMYPT1のキナーゼ・基質間相互作用の解析を行い、MYPT1の2箇所のリン酸化部位それぞれの近傍の十数アミノ酸程の領域がRho-kinaseとの相互作用に重要であることを見出し、ドッキングモチーフ1、2 (DM1, DM2) と名付けた。本年度はこれらドッキングモチーフについてさらに詳細な検討を行った。DM2を欠くとその近傍のリン酸化は大きく低下したが、DM1を欠いても近傍のリン酸化に対する影響は限定的であった。また、ドッキングモチーフや偽基質 (リン酸化部位を含む周囲の配列でリン酸化部位はAlaに置換、PS1, PS2と名付ける) ペプチドを作製し、in vitroでRho-kinaseに対して阻害効果があるかを検討したところ、PS2-DM2ペプチドはキナーゼ活性を阻害したが、それ以外のPSのみ、DMのみ、およびPS1-DM1ペプチドには阻害効果はほとんど無かった。このことより、DM2は近傍のリン酸化部位のリン酸化に必要十分であるが、DM1については必要であるが十分ではないと思われた。そこでDM1以外の領域を探索したところ、リン酸化部位を挟んでDM1と逆側の9アミノ酸の領域もRho-kinaseとの相互作用に重要であることが分かり、DM3と名付けた。DM1とDM3の両方を欠くと中央のリン酸化部位のリン酸化が大きく低下し、またPS1-DM3ペプチドがRho-kinase活性を抑制することを見出した(いずれもDM3のみでは効果が無かった)。以上のことより、Rho-kinaseがMYPT1を認識しリン酸化するためには、リン酸化部位に加えてドッキングモチーフ (DM1/DM3, DM2) が必要であることが示された。
    本年度は、Rho-kinaseがその基質であるMYPT1をどのようにして認識しているか、基質認識機構の解析を行った。その結果、1箇所のリン酸化部位についてはN末側、C末側の両方に、もう一方のリン酸化部位はN末側にドッキングモチーフを持ち、ドッキングモチーフはそれぞれの近傍のリン酸化部位に対してRho-kinaseとの相互作用およびリン酸化部位のリン酸化に寄与していることを見出した。DM2とDM3は塩基性アミノ酸を多く含んでいたが、DM1とは配列類似性が無く、Rho-kinaseは少なくとも2種類の基質認識インターフェイスを持つことが示唆された。また、PS1-DM3ペプチド、PS2-DM2ペプチドがin vitroでRho-kinaseの活性を抑制したことから、新規のRho-kinase阻害薬として利用出来る可能性が示された。上述のように、Rho-kinaseとMYPT1のキナーゼ・基質相互作用機構の理解が大きく進み、また新規の活性調節ツールの候補も得られたことから、本研究はおおむね順調に進展していると考える。
    引き続き、キナーゼの基質認識機構の解析、キナーゼの活性調節・モニターツールの開発、およびシグナルネットワークの解析を行う。MYPT1については、Rho-kinaseとの相互作用インターフェースである3個のドッキングモチーフを同定・評価出来たため、KISS法で同定した他の基質について類似のドッキングモチーフが存在するか、検討する。また、Rho-kinase以外のキナーゼについても比較検討を行う。

  7. Analysis of genes that affect individuality

    Grant number:16H06528  2016.06 - 2021.03

    Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

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    Authorship:Coinvestigator(s) 

  8. The role of cellular localization of ALOX proteins in vascular aging

    Grant number:15K09451  2015.04 - 2019.03

    KATSUMI Akira

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    Authorship:Coinvestigator(s) 

    Rho family proteins play a role in organelle development, cytoskeletal dynamics, cell movement, and other common cellular functions.To identify novel downstream effectors of RhoA, we performed GST-RhoA affinity column chromatography by using platelet lysates. Cytosol fraction of platelet was loaded onto affinity columns on which GST, GST-RhoA-N19, or GST-RhoA-L63 was immobilized. Using triple quadrupole liquid chromatography tandem mass spectrometer, several proteins associated with active RhoA were identified. Arachidonate lipoxygenase (ALOX) binds to GST-RhoA-L63, but not to GST-RhoA-N19 along with the known RhoA effectors. ALOX does not directly bind to RhoA-L63, although Daam1 is shown to bind to ALOX. The second LH2 domain of ALOX directly binds to N-terminal of Daam1, suggesting that Daam1 functions as a scaffolding protein for the assembly of ALOX. We further investigate the roles of Daam1 on ALOX activity and subcellular localization of these molecules.

  9. 低分子量G蛋白質Rhoシグナルが関わる疾患の分子基盤の解明

    2011.04 - 2013.03

    科学研究費補助金  基盤研究(C)

    天野睦紀

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    Authorship:Principal investigator 

  10. Rhoファミリーシグナル伝達系分子を対象とした疾患関連遺伝子の探索と解析    

    2008

    科学研究費補助金  基盤研究(C),課題番号:20590308

    天野 睦紀

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    Authorship:Principal investigator 

  11. 細胞増殖・分化に関わる蛋白質リン酸化酵素の新規基質スクリーニング法の開発

    2008

    科学研究費補助金  特定領域研究,課題番号:20058012

    天野 睦紀

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    Authorship:Principal investigator 

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Teaching Experience (On-campus) 23

  1. 生体と薬物

    2020

  2. 生物学基礎II

    2020

  3. 生体と薬物

    2019

  4. 生物学基礎II

    2019

  5. 生体と薬物

    2018

  6. 生物学基礎II

    2018

  7. 生物学基礎II

    2017

  8. 生体と薬物

    2017

  9. 生物学基礎II

    2016

  10. 生体と薬物

    2016

  11. 生物学基礎II

    2015

  12. 生物学基礎II

    2014

  13. 生体と薬物

    2014

  14. 生物学基礎II

    2013

  15. 生体と薬物

    2013

  16. 生物学基礎II

    2012

  17. 生体と薬物

    2012

  18. 生物学基礎II

    2011

  19. 生体と薬物

    2011

  20. 生体と薬物

    2010

  21. 生体と薬物

    2009

  22. 生体と薬物

    2008

  23. 生体と薬物

    015

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