• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Screening of suppressor mutations of aetivated Ras using a yenst system

Research Project

Project/Area Number 06557011
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section試験
Research Field Pathological medical chemistry
Research InstitutionTokyo Institute of Technology

Principal Investigator

KAJIRO Yoshito  Tokyo Institute of Technology, Fac.Biosci.Biotech., Professor, 生命理工学部, 寄附講座客員教授 (90012690)

Co-Investigator(Kenkyū-buntansha) KOIDE Hiroshi  Tokyo Institute of Technology, Fac.Biosci.Biotech., Assistant, 生命理工学部, 寄附講座教員 (70260536)
SATO Takaya  Tokyo Institute of Technology, Fac.Biosci.Biotech., Assistant, 生命理工学部, 寄附講座教員 (20251655)
Project Period (FY) 1994 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1995: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1994: ¥2,200,000 (Direct Cost: ¥2,200,000)
KeywordsRas / NF-1 / Rat / signal tramduction / GTP-binding protein / Rat
Research Abstract

The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, containing GTPase-activating protein-related domain (GRD) that stimulates intrinsic GTPase activity of Ras protein. By screening a randomly mutagenized NF1-GRD library in Saccharomyces cerevisiae, we isolated two NF1-GRD mutants (NF201 and NF204) with single amino acid substitutions, which suppress the heat shock-sensitive phenotype of the RAS2 (G19V) mutant. The NF1-GRD mutants also suppress the oncogenic Ras-induced transformation of NIH 3T3 mouse fibroblasts (Nakafuku, M., Nagamine, M., Ohtoshi, A., Tanaka, K., Toh-e, A., and Kaziro, Y.(1993) Proc.Natl.Acad.Sci.U.S.A.90,6706-6710). In this paper, we investigated the molecular mechanism of inhibition of the transforming Ras-specific function by the NF1-GRD mutants in mammalia cells. In human embryonic kidney (HEK) 293 cells, the mutant NF1-GRDs attenuated the stimulation of mitogen-activated protein kinase by Ras (G12V), but not by platelet-derived growth factor. In cell-free systems, purified recombinant NF1-GRD mutants showed an inhibitory effect on the association of Ras・guanosine 5'-O- (3-thiotriphosphate) (GTPgammaS) with Raf at several times lower concentrations than the wild type. Furthermore, it was revealed that the binding affinity of the mutant NF1-GRDs toward Ras-GTPgammaS is approximately 5-10 times higher than the wild type.These results suggest that the mutant NF1-GRDs tightly bind to an oncogenic Ras in its GTP-bound active conformation and block the interaction between Ras and its effector, Raf.

Report

(4 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • 1994 Annual Research Report
  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] Tomokazu Ohtsuka: "Analysis of the T-cell activation signaling pathway mediated by tyrosine kinases, protein kinase C, and Ras protein, which is modulated by intracellular cyclic AMP" Biochim. Biophys, Acta. 1310. 223-232 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takuya Satoh: "Tyrosine phosphorylation of ACK in response to temperature shift-down, hyperosmotic shock, and epidermal growth factor stimulation" FEBS Lett.386. 230-234 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Koji Terada: "Ras-dependent activation of c-Jun N-terminal kinase/stress-activated protein kinase in response to interleukin-3 stimulation in hematopoietic BaF 3 cells" J.Biol.Chem.272. 4544-4548 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Mika Miyake: "Unfarnesylated transforming Ras mutant inhibits the Ras-signaling pathway by forming a stable Ras・Raf complex in the cytosol" FEBS Lett.378. 15-18 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hironori Edamatsu: "Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1 α promoter and lac operator" Gene. (印刷中). (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Junji Yamauchi: "C-teminal mutation of G protein β subunit affects differentially extracellular signal-kinase and c-Jun N-terminal kinase pathways in HEK 293 cells" J.Biol.Chem.272(印刷中). (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tomokazu Ohtsuka et al.: "Analysis of the T-cell activation signaling pathway mediated by tyrosine kinases, protein kinase C,and Ras protein, which is modulated by intracellular cyclic AMP" Biochim.Biophys.Acta. 1310. 223-232 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takaya Satoh et al.: "Tyrosine phosphorylation of ACK in response to temperature shift-down, hyperosmotic shock, and epidermal growth factor stimulation" FEBS Letl.386. 230-234 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Koji Terada et al.: "Ras-dependent activation of c-Jun N-terminal kinase/stress-activated protein kinase in response to interleukin-3 stimulation in hematopoietic BaF3 cells" J.Biol.Chem.272. 4544-4548 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Mika Miyake et al.: "Unfarnesylated transforming Ras mutant inhibits the Ras-signaling pathway by forming a stable Ras・Raf complex in the cytosol" FEBS Letl.378. 15-18 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hironori Edamatsu et al.: "Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1alpha Promoter and lac operator" Gene. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Junji Yamauchi et al.: "C-teminal mutation of G protein beta subunit affects differentially extracellular signal-kinase and c-Jun N-terminal kinase pathways in HEK 293 cells" J.Biol.Chem.(in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tomokazu Ohtsuka et al.: "Analysis of the T-cell activation signaling pathway mediated by Tyroine kinase proteinkinase C,and Ris protein,mhick is modulated by intracellular cyclic AMP." Biochim.Biophys,Acta. 1310. 223-232 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Takaya Satoh et al.: "Tyroine phosghasylation of ACK in respnse to Tem perature shift-drwn,hyperosmotic shock,and epidermal growth factor stimulation" FEBS Lett.386. 230-342 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Koji Terada et al.: "Ras-dependent activation of c-Jun N-teminal kinase/stress-activated protein kenase in response to interleukin-3 stimulation in ulct in hematopoietic BaF3 cells." J.Biol,Chem.272. 4544-4548 (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Mika Miyake et al.: "Unfanesylated tromsforming Ras mufant inhibits the Ras-segnaling pathwas by farming a stable Ras・Rat compley in the cytoral" FEBS Lett.378. 15-18 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Edamatsu H.et al.: "Inclncible high-level expression vector for mammalian cells,PEF-LAC carrying human elong ation factor ld promoter and lac operator." Gene. (印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Yamauchi,J.et al.: "C-terminal mintation of G protein β Rafunit affects differentially extro cellular signal-kinase and c-Tun N-teminal kinase pathways in HEK293 cells" J,Biol.Chem.272(印刷中). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Akiko.Ito,et al.: "G protein Br subunit activates Ras. Rat. and MAP kinare in HEK 293 cells" FEBS Letl. 368. 183-187 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Koji.Terada,et al.: "Ras in not reguired for the interleukin 3‐inolnced probterilun of a more pro‐B cell line Ba F3" J.Bial.Chem.270. 27880-27886 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Sadao Man et al: "Inlibition of Rac/Rat interaction by anti‐onco‐genenic must ants ofnemafileromen the nemafibramatosis Type 1(NF1)gene poduit in a all‐free seplerne" J.Bial.Chem.270. 28834-28838 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Tamakazu Ohtsuka et al: "Analysis of the T‐cell activetien signalire Pathway mechated by Tyranibainiri protem kinare C and Ras protein mlnih in madulo‐til by intracellalin cycliic AMP" Biochem.Biophys.Acta. 印刷中. (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Junji Yamauchi et al: "Carboeyl terminal of G protein B subunit in required for association with r subunit" Biochem.Bioplys.Res. Communs. 214. 694-700 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Mika Miyake et al: "Unlarnesylated tram lamis Ras mutant inhibits the Ras‐signalns pathiving by formura stable Ras Rat complex in the cytosal" FEBS Lett. 378. 15-18 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Takaya Satoh,et.al.: "Platelef-deived growth-lactor receptor mediates activatron of Ras Throngh different sejnalns pathway in different cell Typer" Mal.Cell.Biol.13. 3706-3713 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] Masato Nakatuka,et al.: "Suppression of oncogbuic Ras by mutant neurofibiom atorir type1 gener with amini and snbtitntionr" Proc.Natl.Acad.Sai.USA. 90. 6706-6710 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] Hiroshi Koide ,et al.: "GTP-depondent associaton of Rat-1 with Ha-Ras identification of Rat as a target downstuam of Ras mammalsan clls" Proc.Natl.Acad.Sai.USA. 90. 8683-8686 (1993)

    • Related Report
      1994 Annual Research Report

URL: 

Published: 1994-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi