低分子量G蛋白が細胞膜で情報を受け取るメカニズムの可視化

• Project/Area Number: 11242209
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Osaka University (2001-2003); Research Institute, International Medical Center of Japan (1999-2000)
• Principal Investigator: 松田 道行 大阪大学, 微生物病研究所, 教授 (10199812)
• Co-Investigator(Kenkyū-buntansha): 黒川 量雄 大阪大学, 助手 (40333504) ; 大場 雄介 大阪大学, 助手 (30333503) ; 中村 岳史 大阪大学, 講師 (60362604) ; 望月 直樹 国立国際医療センター, 臨床病理研究部・組織形態研究室, 室長 (30311426)
• Project Period (FY): 1999 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥65,400,000 (Direct Cost: ¥65,400,000); Fiscal Year 2003: ¥12,800,000 (Direct Cost: ¥12,800,000); Fiscal Year 2002: ¥12,800,000 (Direct Cost: ¥12,800,000); Fiscal Year 2001: ¥12,700,000 (Direct Cost: ¥12,700,000); Fiscal Year 2000: ¥12,700,000 (Direct Cost: ¥12,700,000); Fiscal Year 1999: ¥14,400,000 (Direct Cost: ¥14,400,000)
• Keywords: Ras / FRET / Rapl / G蛋白 / 低分子量G蛋白 / イメージング / Rap1 / GFP

Research Abstract

本研究の目的は、細胞内情報伝達の重要なキープレーヤーである低分子量G蛋白の活性化の様子を生細胞でリアルタイムにモニターするプローブを作成し、バーチャル細胞作成のために必要な、細胞内情報伝達の時空間パラメータを獲得することである。本年度は、K-Ras、N-Ras、R-Ras、RalA、RalBのプローブ開発を行った。K-Ras、N-Ras、RalAに関しては感度の高いプローブの開発に成功したが、R-RasおよびRalBのプローブは感度が低く、改良の余地を残している。また、既存のプローブの高感度化をYFPおよびCFPをより明るいものに置換することにより達成した。これにより、画像データのシグナルノイズ比を向上させることに成功した。さらに、共焦点レーザー顕微鏡を用いて、細胞内の膜画分と細胞膜画分とのシグナルを定量的に分ける手法を確立した。
一方、これら新規に開発したプローブを使って様々な生命現象の解明に取り組んだ。特に、これまで機能の不明であったRalAが、細胞増殖因子の刺激により葉状突起で限局して活性化されること、RalAの活性化が増殖因子依存性あるいは細胞運動時の葉状突起の形成に必要であることをさまざまな手法を用いて証明した。RalAは小胞の融合に関与するとうい他グループのデータとあわせて考えると、葉状突起の形成には、細胞骨格系と細胞膜系の双方のダイナミックな変化が必要であり、RhoファミリーG蛋白はその前者に、RalAはその後者に密接に関わることが示唆された。

Research Products

• [Publications] Aoki, K., T.Nakamura., M.Matsuda: "Spatio-temporal regulation of Rac1 and Cdc42 activity during nerve growth factor-induced neurite outgrowth in PC12 cells."J.Biol.Chem.. 279(1). 713-719 (2004)
• [Publications] Hoshino T, Shimizu K, Honda T, Kawakatsu T, Fukuyama T, Nakamura T, Matsuda M. Takai Y.: "A Novel Role of Nectins in Inhibition of the E-Cadherin-induced Activation of Rac and Formation of Cell-Cell Adherens Junctions."Mol Biol Cell.. In press.
• [Publications] Kurokawa K, Itoh RE, Yoshizaki H, Ohba Y, Nakamura T, Matsuda M.: "Co-activation of Rac1 and Cdc42 at Lamellipodia and Membrane Ruffles Induced by Epidermal Growth Factor."Mol Biol Cell.. In press.
• [Publications] Yohizaki, H., Y.Ohba, K.Kurokawa, R.E.Itoh, T.Nakamura, N.Mochizuki, K.Nagashima, M.Matsuda: "Activity of Rho-family GTPases during cell division as visualized with FRET-based probes."J.Cell Biol.. 162. 223-232 (2003)
• [Publications] T.Honda., K.Shimizu., T.Kawakatsu., A.Fukuhara., K.Irie., T.Nakamura., M.Matsuda., Y.Takai.: "Cdc42 and Rac small G proteins activated by transinteractions of nectins are involved in activation of c-Jun N-terminal kinase, but not in association of nectins and cadherin to form adherens junctions, in fibroblasts."Genes to Cells.. 8. 481-491 (2003)
• [Publications] Takaya, A., Y.Ohba, M.Matsuda: "RalA Activation at Nascent Lamellipodia in Cells Stimulated by Epidermal Growth Factor."Mol Biol Cell.. In press.
• [Publications] Endo, A., K.Nagashima, H.Kurose, S.Mochizuki, M.Matsuda, N.Mochizuki: "Sphingosine 1-phosphate induces membrane ruffling and increases motility of human umbilical vein endothelial cells via vascular endothelial growth factor receptor and CrkII"J. Biol. Chem.. 277. 23747-23754 (2002)
• [Publications] Itoh, R.E., K.Kurokawa, Y.Ohba, H.Yoshizaki, M.Mochizuki, M.Matsuda: "Activation of Rac and Cdc42 video-imaged by FRET-based single-molecule probes in the membrane of living cells"Mol. Cell. Biol.. 22. 6582-6591 (2002)
• [Publications] Nagashima, K., A.Endo, H.Ogita, A.Kawana, A.Yamagishi, A.Kitakabe, M.Matsuda, N.Mochizuki: "Adaptor protein Crk is required for Ephrin-B1-induced membrane ruffling and focal complex assembly of human aortic endothelial cells"Mol. Biol. Cell. 13. 4231-4242 (2002)
• [Publications] Sakakibara, A., Y.Ohba, K.Kurokawa, M.Matsuda, S.Hattori: "Novel function of Chat in controlling cell adhesion via Cas-Crk-C3G-pathway-mediated Rap1 activation"J. Cell Sci.. 115. 4915-4924 (2002)
• [Publications] Sawano, A., S.Takayama, M.Matsuda, A.Miyawaki: "Lateral propagation of EGF signalling after local stimulation depends on receptor density"Dev. Cell. 3. 245-257 (2002)
• [Publications] Ohba, Y., K.Kurokawa, M.Matsuda: "Mechanism of the spatio-temporal regulation of Ras and Rap1"EMBO J.. (In press). (2003)
• [Publications] N.Mochizuki, et al.: "Spacio-temporal Images of Growth Factor-induced Activation of Ras and Rap1"Nature. 411. 1065-1068 (2001)
• [Publications] Mochizuki N, et al.: "Crk Activation of JNK via C3G and R-Ras."J Biol Chem. 275. 12667-12671 (2000)
• [Publications] Yamashita S, et al.: "CalDAG-GEFIII activation of Ras, R-Ras, and Rap1."J Biol Chem. 275. 25488-25493 (2000)
• [Publications] Ohba Y, et al.: "Rap2 as a Slowly Responding Molecular Switch in the Rap1 Signaling Cascade."Mol Cell Biol. 20. 6074-6083 (2000)
• [Publications] Ohba Y, et al.: "Regulatory proteins of R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3."J Biol Chem. 275. 20020-20026 (2000)
• [Publications] Mochizuki N, et al.: "Activation of ERK/MAPK pathway by an isoform of rap1GAP associated with Gai."Nature. 400. 891-894 (1999)
• [Publications] Mochizuki N, et al.: "Crk Activation of JNK via C3G and R-Ras"J Biol Chem. (in press). (2000)
• [Publications] Mochizuki N, et al.: "Activation of ERK/MAPK pathway by an isoform of rap1GAP associated with Gαi"Nature. 400. 891-894 (1999)
• [Publications] Nishihara H, et al.: "Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins"Biochim Biophys Acta. 1452. 179-187 (1999)
• [Publications] Katayama H, et al.: "EGF-dependent dissociation of Crk proto-oncogene product from the epidermal growth factor receptor in human glioma cells"Jpn J Can Res. 90. 1096-1103 (1999)
• [Publications] Ichiba T, et al.: "Activation of C3G Guanine Nucleotide Exchange Factor for Rap1 by Phosphorylation of Tyrosine 504"J. Biol. Chem.. 274. 14376-14381 (1999)