迅速Rab-GAPスクリーニング法の開発

• Project/Area Number: 18057026
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Tohoku University
• Principal Investigator: 福田 光則 Tohoku University, 大学院・生命科学研究科, 教授 (50311361)
• Project Period (FY): 2006 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥6,200,000 (Direct Cost: ¥6,200,000); Fiscal Year 2007: ¥3,100,000 (Direct Cost: ¥3,100,000); Fiscal Year 2006: ¥3,100,000 (Direct Cost: ¥3,100,000)
• Keywords: Rab-GAP / Rab27A / Rabエフェクター / メラノソーム輸送 / Griscelli症候群 / スクリーニング / Slac2-a / melanophilin / 膜輸送

Research Abstract

低分子量G蛋白質Rabはヒトにおいて60種類以上もの異なるアイソフォームが存在し、個々のRabはオルガネラ特異的あるいは細胞種特異的に膜輸送を制御すると考えられている。RabはGTPと結合している活性化型、GDPと結合している不活性化型の二つのフォームをとり、活性化型のGTP-Rabが特定の標的因子と結合することで膜輸送を促進する。適切なRabの不活性化はRabの正常な機能発現に不可欠と考えられており、Rabの不活性化酵素としてGAP(GTPase-activating protein)の存在が提唱されている。しかしながら、ヒトに数多く存在するRabのうち特定のGAPが同定されているものはほとんどなく、Rab不活性化の分子メカニズムは未だ謎に包まれている。そこで本研究では、Rabに特異的な不活性化酵素GAPを迅速にスクリーニングする手法の確立に取り組んだ。これまでの研究でメラノソーム輸送を司るRab27Aに着目し、Rab27Aエフェクタードメイン(Slac2-aのSHD領域)による活性化型GTP-Rab27Aの測定法を開発するとともに、メラノソーム輸送阻害活性を指標とてTBCドメインを有する二種類の蛋白質Rab27A-GAPα及びβを同定することに成功した。本年度はさらに、インスリン刺激依存的に細胞膜に輸送されるグルコース輸送体4の制御に関わるAS160という分子が、Rab10-GAPとして機能することを突き止めた(Cell Metab.(2007)5,293-303)。

Research Products

• [Journal Article] Synaptotagmin VII splice variants α, β, and δ are expressed in pancreatic β-cells and regulate insulin exocytosis. (2008)
  • Author(s): Gauthier, B. R., Duhamel, D. L., lezzi, M., Theander, S., Saltel, F., Fukuda, M., Wehrle-Haller, B. & Wollheim, C. B.
  • Journal Title: FASEB J. 22 Pages: 194-206
  • Peer Reviewed
• [Journal Article] JNK mediates Syt 4 localization to mature secretory vesicles and enhances Ca^<2+>-evoked release. (2008)
  • Author(s): Mori, Y., Higuchi, M., Hirabayashi Y., Fukuda, M. & Gotoh, Y.
  • Journal Title: EMBO J. 27 Pages: 76-87
  • Peer Reviewed
• [Journal Article] Confusion in the nomenclature of synaptotagmins V and IX: which is which? (2008)
  • Author(s): Fukuda, M. & Sagi-Eisenberg, R.
  • Journal Title: Calcium Binding Proteins 3 Pages: 1-3
  • Peer Reviewed
• [Journal Article] Synaptotagmin VII modulates the kinetics of dense-core vesicle exocytosis in PC12 cells. (2007)
  • Author(s): Tsuboi, T. & Fukuda, M.
  • Journal Title: Genes Cells 12 Pages: 511-519
  • Peer Reviewed
• [Journal Article] Up-regulation of ras-GAP genes is reversed by a MEK inhibitor and doxorubicin in ν-Ki-ras transformed NIH/3T3 fibroblasts. (2007)
  • Author(s): Hashii, M., Fukuda M., Nomura, H., Ito, N., Takahashi, H., Hattori, S., Mikoshiba, K., Noda, M. & Higuchi, Y.
  • Journal Title: Biochem. Biophys. Res. Commun. 356 Pages: 374-380
  • Peer Reviewed
• [Journal Article] Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane. (2007)
  • Author(s): Sano, H., Eguez, L., Teruel, M. N., Fukuda, M., Chuang, T. D., Chavez, J. A., Lienhard, G. E. & McGraw, T. E.
  • Journal Title: Cell Metab. 5 Pages: 293-303
  • Peer Reviewed
• [Journal Article] Synaptotagmin (Syt) IX is an essential determinant for protein sorting to secretory granules in mast cells. (2007)
  • Author(s): Haberman, Y., Ziv, I., Gorzalczany, Y., Hirschberg, K., Mittleman, L., Fukuda, M. & Sagi-Eisenberg, R.
  • Journal Title: Blood 109 Pages: 3385-3392
  • Peer Reviewed
• [Journal Article] Synaptotagmin I binds intestinal epithelial NHE3 and mediates cyclic AMP-and Ca^<2+>-induced endocytosis by recruitment of AP2 and clathrin. (2007)
  • Author(s): Musch, M. W., Arvans, D. L., Walsh-Reitz, M. M., Uchiyama, K., Fukuda, M. & Chang, E. B.
  • Journal Title: Am. J. Physiol. Gastrointest. Liver Physiol. 292
  • Peer Reviewed
• [Journal Article] Proteomic analysis of insulin secretory granules. (2007)
  • Author(s): Brunner, Y., Coute, Y., Iezzi, M., Foti, M., Fukuda, M., Hochstrasser, D., Wollheim, C. B. & Sanchez, J.-C.
  • Journal Title: Mol. Cell. proteomics 6 Pages: 1007-1017
  • Peer Reviewed
• [Journal Article] Cholesterol controls lipid endocytosis through rabll. (2007)
  • Author(s): Takahashi, M., Murate, M., Fukuda, M., Sato, S. B., Ohta, A. & Kobayashi, T.
  • Journal Title: Mol. Biol. Cell 18 Pages: 2667-2677
  • Peer Reviewed
• [Journal Article] Spatial segregation of degradation-and recycling-trafficking pathways in COS-1 cells. (2007)
  • Author(s): Misaki, R., Nakagawa, T., Fukuda, M., Taniguchi, N. & Taguchi, T.
  • Journal Title: Biochem. Biophys. Res. Commun. 360 Pages: 580-585
  • Peer Reviewed
• [Journal Article] Myosin Vb is required for trafficking of cystic fibrosis transmembrane conductance regulator in Rablla-specific apical recycling endosomes in polarized human airway epithelial cells. (2007)
  • Author(s): Swiatecka-Urban, A., Talebian, L., Kanno, E., Moreau-Marquis, S., Coutermarsh, B., Hansen, K. el al.
  • Journal Title: J. Biol. Chem. 282 Pages: 23725-23736
  • Peer Reviewed
• [Journal Article] メラニン色素輸送の分子メカニズム (2007)
  • Author(s): 福田光則
  • Journal Title: 太陽紫外線防御研究委員会学術報告 17 Pages: 45-49
• [Journal Article] Rabファミリータンパク質による膜輸送制御の特異性と多様性 (2007)
  • Author(s): 福田光則
  • Journal Title: 生化学 79 Pages: 1046-1051
  • NAID: 10019995765
• [Journal Article] The polybasic sequence in the C2B domain of rabphilin is required for the vesicle docking step in PC12 cells. (2007)
  • Author(s): Tsuboi, T., Kanno, E., Fukuda, M.
  • Journal Title: J. Neurochem. 100 Pages: 770-779
• [Journal Article] Dvl regulates endo- and exocytotic processes through binding to synaptotagmin. (2007)
  • Author(s): Kishida, S., Hamao, K.Inoue, M., Hasegawa, M., Matsuura, Y., Mikoshiba, K., Fukuda, M., Kikuchi, A.
  • Journal Title: Genes Cells 12 Pages: 49-61
• [Journal Article] Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development. (2007)
  • Author(s): Iwashita, S., Kobayashi, K., Kubo, Y., Hinohara, Y., Sezaki, M., Nakamura, K., Suzuki-Migishima, R., Yokoyama, M., Satoh, S., Fukuda, M., Ohba, M., Kato, C., Adachi, E., Song, S.-Y.
  • Journal Title: J. Biol. Chem. 282 Pages: 3413-3417
• [Journal Article] Live cell imagingによる細胞活動の可視化解析 (2007)
  • Author(s): 坪井貴司, 福田光則
  • Journal Title: 日本比較内分泌学会ニュース 124(2) Pages: 45-53
  • NAID: 10018293579
• [Journal Article] Distinct Rab27A binding affinities of Slp2-a and Slac2-a/melanophilin : Hierarchy of Rab27A effectors. (2006)
  • Author(s): Fukuda, M.
  • Journal Title: Biochem. Biophys. Res. Commun. 343 Pages: 666-674
• [Journal Article] Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Muncl3-4 effector mechanism. (2006)
  • Author(s): Saxena, S.K., Horiuchi, H., Fukuda, M.
  • Journal Title: Biochem. Biophys. Res. Commun. 344 Pages: 651-657
• [Journal Article] The Slp4-a linker domain controls exocytosis through interaction with Munc18-1・syntaxin-la complex. (2006)
  • Author(s): Tsuboi, T., Fukuda, M.
  • Journal Title: Mol. Biol. Cell 17 Pages: 2101-2112
• [Journal Article] Rab3A and Rab27A cooperatively regulate the docking step of densecore vesicle exocytosis in PC12 cells. (2006)
  • Author(s): Tsuboi, T., Fukuda, M.
  • Journal Title: J. Cell Sci. 119 Pages: 2196-2203
• [Journal Article] Decreased basal mucus secretion by Slp2-a-deficient gastric surface mucous cells. (2006)
  • Author(s): Saegusa, C., Tanaka, T., Tani, S., Itohara, S., Mikoshiba, K., Fukuda, M.
  • Journal Title: Genes Cells 11 Pages: 623-631
• [Journal Article] Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans. (2006)
  • Author(s): Mahoney, T.R., Liu, Q., Itoh, T., Luo, S., Hadwiger, G., Vincent, R., Wang, Z.-W., Fukuda, M., Nonet, M.L.
  • Journal Title: Mol. Biol. Cell 17 Pages: 2617-2625
• [Journal Article] Chronic olanzapine treatment causes differential expression of genes in frontal cortex of rats as revealed by DNA microarray technique. (2006)
  • Author(s): Fatemi, S.H., Reutiman, T.J., Folsom, T.D., Bell, C., Nos, L., Fried, P., Pearce, D.A., Singh, S., Siderovski, D.P., Willard, F.S., Fukuda, M.
  • Journal Title: Neuropsychopharmacol 31 Pages: 1888-1899
• [Journal Article] Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activity. (2006)
  • Author(s): Itoh, T., Satoh, M., Kanno, E., Fukuda, M.
  • Journal Title: Genes Cells 11 Pages: 1023-1037
• [Journal Article] Neuronal calcium sensor-1 (NCS-1) and PI4Kβ stimulate ERK1/2 signaling by accelerating recycling through endocytic recycling compartment (ERC). (2006)
  • Author(s): Kapp-Barnea, Y., Ninio-Many, L., Hirschberg, K., Fukuda, M., Jeromin, A., Sagi-Eisenberg, R.
  • Journal Title: Mol. Biol. Cell 17 Pages: 4130-4141
• [Journal Article] Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets. (2006)
  • Author(s): Kondo, H., Shirakawa, R., Higashi, T., Kawato, M., Fukuda, M., Kita, T., Horiuchi, H.
  • Journal Title: J. Biol. Chem. 281 Pages: 28657-28665
• [Journal Article] Identification of EPI64 as a GTPase-activating protein specific for Rab27A. (2006)
  • Author(s): Itoh, T., Fukuda, M.
  • Journal Title: J. Biol. Chem. 281 Pages: 31823-31831
• [Journal Article] Functional involvement of Noc2, a Rab27 effector, in rat parotid acinar cells. (2006)
  • Author(s): Imai, A., Yoshie, S., Nashida, T., Shimomura, H., Fukuda, M.
  • Journal Title: Arch. Biochem. Biophys. 455 Pages: 127-135
• [Journal Article] Rab27 and its effectors in secretory granule exocytosis : A novel docking machinery composed of a Rab27・effector complex. (2006)
  • Author(s): Fukuda, M.
  • Journal Title: Biochem. Soc. Trans. 11 Pages: 691-695
• [Journal Article] シナプトタグミンの分子進化 (2006)
  • Author(s): 福田光則
  • Journal Title: 生体の科学 57(5) Pages: 458-459
• [Presentation] Role of small GTPase Rab27A and its effectors in regulated exocytosis. (2008)
  • Author(s): Fukuda, M.
  • Organizer: The 1st International Conference of Tohoku Neuroscience GCOE"from Genes to Development and Behavior"
  • Place of Presentation: Zao
  • Year and Date: 2008-01-23
• [Presentation] TBCドメインを含む新規Rab3A-GAPのスクリーニング (2007)
  • Author(s): 石橋弘太郎、菅野栄子、伊藤敬、福田光則
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会大会 合同大会
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-13
• [Presentation] 低分子量Gタンパク質Rab27Aによる膜輸送制御の分子基盤の解明 (2007)
  • Author(s): 福田光則
  • Organizer: 第30回日本分子生物学会三菱化学奨励賞授賞講演
  • Place of Presentation: 横浜
  • Year and Date: 2007-12-12
• [Presentation] Regulation of secretory vesicle exocytosis by Rab small GTPases. (2007)
  • Author(s): Fukuda, M.
  • Organizer: International Symposium on Membrane Traffic
  • Place of Presentation: Awaji
  • Year and Date: 2007-11-29
• [Presentation] Rab4 negatively regulates CFTR expression in the plasma membrane in human airway epithelial cells. (2007)
  • Author(s): Fukuda, M. & Swiatecka-Urban, A.
  • Organizer: The 20th Annual North American Cystic Fibrosis Conference
  • Place of Presentation: Anaheim, USA
  • Year and Date: 2007-10-05
• [Presentation] シナプトタグミン7による調節性分泌制御機構の可視化解析 (2007)
  • Author(s): 坪井貴司、福田光則
  • Organizer: 第30回日本神経科学大会
  • Place of Presentation: 横浜
  • Year and Date: 2007-09-11
• [Presentation] 低分子量Gタンパク質Rab27とエフェクター分子による分泌小胞輸送の制御機構 (2007)
  • Author(s): 福田光則
  • Organizer: 第40回日本発生生物学会・第59回日本細胞生物学会合同大会・ミニシンポジウム
  • Place of Presentation: 福岡
  • Year and Date: 2007-05-28
• [Presentation] 肌や髪の毛が黒くなる仕組みーメラニン色素は動く! (2007)
  • Author(s): 福田光則
  • Organizer: 東北大学大学院生命科学研究科春期講習会
  • Place of Presentation: 弘前
  • Year and Date: 2007-04-21
• [Book] "Rab27による分泌小胞の輸送制御メカニズム", ブレインサイエンスレビュー2008 (2008)
  • Author(s): 福田光則
  • Publisher: 財団法人 ブレインサイエンス振興財団
• [Book] Molecular Mechanisms of Exocytosis (2006)
  • Author(s): Fukuda, M.
  • Publisher: The role of synaptotagmin and synaptotagmin-like protein (Slp) in regulated exocytosis.
• [Remarks]
  • URL: http://www.lifesci.tohoku.ac.jp/teacher/neuro/t_fukuda.html