Imaging of Ras-MAP kinase signal transduction cascade

• Project/Area Number: 16025201
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Osaka University
• Principal Investigator: MATSUDA Michiyuki Osaka University, Research Institute for Microbial Diseases, Professor, 微生物病研究所, 教授 (10199812)
• Co-Investigator(Kenkyū-buntansha): NAKAMURA Takeshi Osaka University, The Research Institute for Microbial Diseases, Assistant Professor, 微生物病研究所, 講師 (60362604) ; KUROKAWA Kazuo Osaka University, The Research Institute for Microbial Diseases, Research Associate, 微生物病研究所, 助手 (40333504)
• Project Period (FY): 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥30,000,000 (Direct Cost: ¥30,000,000); Fiscal Year 2004: ¥30,000,000 (Direct Cost: ¥30,000,000)
• Keywords: Ras / MAPK / Raf / Simulation

Research Abstract

The recent development of a technology based on GFP and fluorescence resonance energy transfer (FRET) has enabled us to monitor conformational changes in signaling molecules as they occur in living cells. We have successfully developed probes to monitor the activity change of Ras-family GTPases and shown the dynamic change of Ras activity in living cells. A key signaling molecule, c-Raf, is situated downstream from Ras and upstream from the mitogen-activated protein kinase (MAPK) kinase (MEK). Here, we studied the mechanism underlying the signal transduction from Ras to MEK by using probes based on the principle of fluorescence resonance energy transfer (FRET). In agreement with previous models, it was found that c-Raf adopted two conformations: open active and closed inactive. Ras binding induced the c-Raf transition from closed to open conformation, which enabled c-Raf to bind to MEK. In the presence of a cytosolic Ras mutant, c-Raf bound to, but failed to phosphorylate, MEK in the cytoplasm. In contrast, the cytosolic Ras mutant significantly enhanced MEK phosphorylation by a membrane-targeted c-Raf. These results demonstrated the essential role of Ras-induced conformational change in MEK activation by c-Raf.

Research Products

• [Journal Article] Ras binding opens c-Raf to expose the docking site for MEK. (2005)
  • Author(s): Terai, K., Matsuda, M.
  • Journal Title: EMBO Rep. 6 Pages: 251-255
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Local PIP3 accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor -stimulated PC12 cells. (2005)
  • Author(s): Aoki, K., Nakamura, T., Fujikawa, K., Matsuda, M.
  • Journal Title: Mol. Biol. Cell (In press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes. (2005)
  • Author(s): Itoh, R.E., Kurokawa, K., Fujioka, A., Sharma, A., Mayer, B.J., Matsuda, M.
  • Journal Title: Experimental Cell Research. (In press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Ras binding opens c-Raf to expose the docking site for MEK. (2005)
  • Author(s): Terai, K., Matsuda, M.
  • Journal Title: EMBO Rep. (In press)
• [Journal Article] Visualizing the signal transduction pathways in living cells with GFP-based FRET probes. (2005)
  • Author(s): Kurokawa, K., Takaya, A., Terai, K., Fujioka, A., Matsuda, M.
  • Journal Title: Acta Histochemica et Cytochemica (In press)
  • NAID: 110003161214
• [Journal Article] Local PIP3 accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor -stimulated PC12 cells. (2005)
  • Author(s): Aoki, K., Nakamura, T., Fujikawa, K., Matsuda, M.
  • Journal Title: Mol.Biol.Cell (In press)
• [Journal Article] Cell type-specific regulation of RhoA activity during cytokinesis. (2004)
  • Author(s): Yoshizaki, H., Ohba, Y., Parrini, M.C., Dulyaninova, N.G., Bresnick, A.R., Mochizuki, N., Matsuda,M.
  • Journal Title: J. Biol. Chem. 279 Pages: 44759-44762
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion. (2004)
  • Author(s): Yano, H., Mazaki, Y., Kurokawa, K., Hanks, S.K., Matsuda, M., Sabe,H.
  • Journal Title: J. Cell Biol. 166
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] RalA activation at nascent lamellipodia of EGF-stimulated Cos7 cells and migrating MDCK cells. (2004)
  • Author(s): Takaya, A., Ohba, Y., Kurokawa, K., Matsuda,M.
  • Journal Title: Mol. Biol. Cell 15 Pages: 2549-2557
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] STAT3 regulates cell polarity during zebrafish convergence and extension through the Wnt-PCP pathway. (2004)
  • Author(s): Miyagi, C., Yamashita, S., Ohba, Y., Yoshizaki, H., Matsuda, M., Hirano,T.
  • Journal Title: J. Cell Biol. 166 Pages: 975-981
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor. (2004)
  • Author(s): Kurokawa, K., Itoh, R.E., Yoshizaki, H., Ohba, Y., Nakamura, T., Matsuda,M.
  • Journal Title: Mol. Biol. Cell 15 Pages: 1003-1010
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Spatio-temporal regulation of Rac1 and Cdc42 activity during nerve growth, factor-induced neurite outgrowth in PC12 cells. (2004)
  • Author(s): Aoki, K., Nakamura, T., Matsuda, M.
  • Journal Title: J. Biol. Chem. 279 Pages: 713-719
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. (2004)
  • Author(s): Kawai, T., Sato, S., Ishii, K.J., Coban, C., Hemmi, H., Yamamoto, M., Terai, K., Matsuda, M., Inoue, J.I., Uematsu, S., Takeuchi, O., Akira,S.
  • Journal Title: Nat. Immunol. 5 Pages: 1061-1068
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis. (2004)
  • Author(s): Kamioka, Y., Fukuhara, S., Sawa, H., Nagashima, K., Masuda, M., Mochizuki,N.
  • Journal Title: J. Biol. Chem. 279 Pages: 40091-40099
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A novel role of nectins in inhibition of the E-cadherin-induced activation of Rac and formation of cell-cell adherens junctions. (2004)
  • Author(s): Hoshino, T., Shimizu, K., Honda, T., Kawakatsu, T., Fukuyama, T., Nakamura, T., Matsuda, M., Takai,Y.
  • Journal Title: Mol. Biol. Cell 15 Pages: 1077-1088
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Visualizing the signal transduction pathways in living cells with GFP-based FRET probes. Acta Histochemica et Cytochemica. (2004)
  • Author(s): Kurokawa, K., Takaya, A., Terai, K., Fujioka, A., Matsuda, M.
  • Journal Title: Acta Histochemica et Cytochemica 37 Pages: 347-355
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Cell type-specific regulation of RhoA activity during cytokinesis. (2004)
  • Author(s): Yoshizaki, H., Ohba, Y., Parrini, M.C., Dulyaninova, N.G., Bresnick, A.R., Mochizuki, N., Matsuda, M.
  • Journal Title: J.Biol.Chem. 279 Pages: 44759-44762
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion. (2004)
  • Author(s): Yano, H., Mazaki, Y., Kurokawa, K., Hanks, S.K., Matsuda, M., Sabe, H.
  • Journal Title: J. Cell Biol. 166 Pages: 2549-2557
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Ra1A activation at nascent lamellipodia of EGF-stimulated Cos7 cells and migrating MDCK cells. (2004)
  • Author(s): Takaya, A., Ohba, Y., Kurokawa, K., Matsuda, M.
  • Journal Title: Mol. Biol. Cell 15 Pages: 2549-2557
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] STAT3 regulates cell polarity during zebrafish convergence and extension through the Wnt-PCP pathway. (2004)
  • Author(s): Miyagi, C., Yamashita, S., Ohba, Y., Yoshizaki, H., Matsuda, M., Hirano, T.
  • Journal Title: J. Cell Biol. 166 Pages: 975-981
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor. (2004)
  • Author(s): Kurokawa, K., Itoh, R.E., Yoshizaki, H., Ohba, Y., Nakamura, T., Matsuda, M.
  • Journal Title: Mol. Biol. Cell 15 Pages: 1003-1010
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Spatio-temporal regulation of Rac1 and Cdc42 activity during nerve growth factor-induced neurite outgrowth in PC12 cells. (2004)
  • Author(s): Aoki, K., Nakamura, T., Matsuda, M.
  • Journal Title: J. Biol. Chem. 279 Pages: 713-719
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. (2004)
  • Author(s): Kawai, T., Sato, S., Ishii, K.J., Coban, C., Hemmi, H., Yamamoto, M., Terai, K., Matsuda, M., Inoue, J.I., Uematsu, S., Takeuchi, O., Akira, S.
  • Journal Title: Nat. Immunol. 5 Pages: 1061-1068
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis. (2004)
  • Author(s): Kamioka, Y., Fukuhara, S., Sawa, H., Nagashima, K., Masuda, M., Mochizuki, N.
  • Journal Title: J. Biol. Chem. 279 Pages: 40091-40099
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A novel role of nectins in inhibition of the E-cadherin-induced activation of Rac and formation of cell-cell adherens junctions. (2004)
  • Author(s): Hoshino, T., Shimizu, K., Honda, T., Kawakatsu, T., Fukuyama, T., Nakamura, T., Matsuda, M., Takai, Y.
  • Journal Title: Mol. Biol. Cell 15 Pages: 1077-1088
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Visualizing the signal transduction pathways in living cells with GFP-based FRET probes. Acta Histochemica et Cytochemica. (2004)
  • Author(s): Kurokawa, K., Takaya, A., Terai, K., Fujioka, A., Matsuda, M.
  • Journal Title: Acta Histochem. Cytochem. 37 Pages: 347-355
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Local PIP3 accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells. (2004)
  • Author(s): Aoki, K., Nakamura, T., Fujikawa, K., Matsuda, M.
  • Journal Title: Mol. Biol. Cell (In press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion. (2004)
  • Author(s): Yano, H., Mazaki, Y., Kurokawa, K., Hanks, S.K., Matsuda, M., Sabe, H.
  • Journal Title: J.Cell Biol. 166 Pages: 283-295
• [Journal Article] RalA activation at nascent lamellipodia of EGF-stimulated Cos7 cells and migrating MDCK cells. (2004)
  • Author(s): Takaya, A., Ohba, Y., Kurokawa, K., Matsuda, M.
  • Journal Title: Mol.Biol.Cell 15 Pages: 2549-2557
• [Journal Article] STAT3 regulates cell polarity during zebrafish convergence and extension through the Wnt-PCP pathway. (2004)
  • Author(s): Miyagi, C., Yamashita, S., Ohba, Y., Yoshizaki, H., Matsuda, M., Hirano, T.
  • Journal Title: J.Cell Biol. 166 Pages: 975-981
• [Journal Article] Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor. (2004)
  • Author(s): Kurokawa, K., Itoh, R.E., Yoshizaki, H., Ohba, Y., Nakamura, T., Matsuda, M.
  • Journal Title: Mol.Biol.Cell 15 Pages: 1003-1010
• [Journal Article] Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. (2004)
  • Author(s): Kawai, T., Sato, S., Ishii, K.J., Coban, C., Hemmi, H., Yamamoto, M., Terai, K., Matsuda, M., Inoue, J.I., Uematsu, S., Takeuchi, O., Akira, S.
  • Journal Title: Nat.Immunol. 5 Pages: 1061-1068
• [Journal Article] A novel dynamin-associating molecule, formin-binding protein 17, induces tubular membrane invaginations and participates in endocytosis. (2004)
  • Author(s): Kamioka, Y., Fukuhara, S., Sawa, H., Nagashima, K., Masuda, M., Matsuda, M., Mochizuki, N.
  • Journal Title: J.Biol.Chem. 279 Pages: 40091-40099
• [Journal Article] A novel role of nectins in inhibition of the E-cadherin-induced activation of Rac and formation of cell-cell adherens junctions. (2004)
  • Author(s): Hoshino, T., Shimizu, K., Honda, T., Kawakatsu, T., Fukuyama, T., Nakamura, T., Matsuda, M., Takai, Y.
  • Journal Title: Mol.Biol.Cell 15 Pages: 1077-1088
• [Journal Article] Spatio-temporal regulation of Rac1 and Cdc42 activity during nerve growth factor-induced neurite outgrowth in PC12 cells. (2004)
  • Author(s): Aoki, K., Nakamura, T., Matsuda, M.
  • Journal Title: J.Biol.Chem. 279 Pages: 713-719
• [Journal Article] A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes.
  • Author(s): Itoh, R.E., Kurokawa, K., Fujioka, A., Sharma, A., Mayer, B.J., Matsuda, M.
  • Journal Title: Experimental Cell Research (In press)
  • Description: 「研究成果報告書概要(欧文)」より