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1998 Fiscal Year Final Research Report Summary

Characterization of Novel Ras Effectors and Gene Knockout of All Effectors in Nemadote Caenorhabditis elegans

Research Project

Project/Area Number 09670128
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General medical chemistry
Research InstitutionKobe University

Principal Investigator

KARIYA Ken-ichi  Kobe Univ.Sch.Med., Dept.Physiology II,Associate Professor, 医学部, 助教授 (40263371)

Project Period (FY) 1997 – 1998
KeywordsC.elegans / Ras / two-hybrid screening / gene knockout / RalGDS / AF6 / Cdc25 / phospholipase C
Research Abstract

1. In nematode Caenorhabditis elegans (C.elegans), EGF receptor and Ras regulate differentiation of vulva, an egg-laying organ, through Raf, an only known Ras effector in this organism. On the other hand, mammalian Ras possesses multiple effectors/effector candidates such as Raf, RalGDS and AF6. By a yeast two-hybrid screening of a C.elegans cDNA library, we identified two novel Ras-binding proteins, a phospholipase C (Ce-PLC210) and a GDP/GTP exchange factor (Ce-Cdc25), in addition to Raf, RalGDS and AF-6. Further, we isolated human orthologs of Ce-PLC210 and Ce-Cdc25 (Hu-PLC210 and Hu-Cdc25).
2. All these C.elegans proteins bound to wild-type Ras and RaplA (Ras homolog) but not to effector region munants or other small G proteins. Like Ce-PLC2 10, Hu-PLC210 bound to Ras and Rap IA in a GTP-dependent manner and hydrolyzed PIP2 in vitro. Studies on regulation by Ras/RaplA is underway. Hu-Cdc25 bound only to RaplA in a GTP-dependent manner and showed a strong GDP/GTP exchange activity on … More Rap LA, although it had a weak activity on Ras as well (Ce-Cdc25 had similar activity on both Ras and Rap lA). Hu-Cdc25 may be recruited to the membrane through binding to a GTP-RaplA and activate other RaplA molecules present in the membrane, thereby amplifying effects of extracellular signals.
3. Transposon-insertion alleles and deletion alleles (except for Ce-Cdc25) were isolated for all effector genes in C.elegans. Ce-PLC210 gene deletion mutants exhibited a sterile phenotype (similar to that found in EGF receptor mutants) and an egg-laying defective phenotype (similar to that found in Ras mutants), and these phenotypes were rescued by transgenic expression of the wild-type gene. In contrast to mutations of Raf, those of other effector/effector candidates exhibited no abnormality in vulval differentiation and did not suppress the super-induction of vulval differentiation by an activated Ras mutation. Vulva development may be regulated by Raf alone.
4. A novel Ras-binding protein (Ce-FLI-1) was found in C.elegans (not through two-hybrid screening) and its Ras-binding activity and actin-binding and -severing activities were characterized. Less

  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Kariya,K.,et al.: "C.elegans homologs of ralGDS,AF-6,Cdc25 and phospholipase Cβ interact with Let-60" Worm Breeder's Gazette. 14・5. 34-35 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hu,C.-D.,et al.: "Coassociation of Rap 1A and Ha-Ras with Raf-1 N-terminal region interferes with Ras-dependent activation of Raf- 1" J.Biol.Chem.272・18. 11702-11705 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shibatohge,M.,et al.: "Identification of PLC210,a Caenorhabditis elegans phospholipase C,as a putative effector of Ras" J.Biol.Chem.273・11. 6218-6222 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Watari,Y.,et al.: "Identification of Ce-AF-6,a novel Caenorhabditis elegans protein,as a putative Ras effector" Gene. 224・1-2. 53-58 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hu,C.-D.,et al.: "Effect of phosphorylation on activities of Rap 1A to interact with Raf-1 and to suppress Ras-dependent Raf- 1 activation" J.Biol.Chem.274・1. 48-51 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Goshima,M.,et al.: "Characterization of a novel Ras-binding protein Ce-FLI-1 comprising leucine-rich repeats and gelsolin-like domains" Biochem.Biophys.Res.Commun.(in press).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kariya, K.et al.: "C.elegans homologs of ralGDS,AF-6, Cdc25 and Phospholipase Cbeta interact with Let-60." Worm Breeder's Gazette. 14-5. 34-35 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hu, C.-D.et al.: "Coassociation of RaplA and Ha-Ras with Raf-1 N-terminal region interferes with Ras-dependent activation of Raf-1." J.Biol.Chem.272-18. 11702-11705 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Shibatohge, M.et al.: "Identification of PLC210, a Caenorhabditis elegans phospholipase C,as a putative effector of Ras." J.Biol.Chem.273-11. 6218-6222 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Watari, Y.et al.: "Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector." Gene. 224-1-2. 53-58 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hu, C.-D.et al.: "Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation." J.Biol.Chem.274-1. 48-51 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Goshima, M.et al.: "Characterization of a novel Ras-binding protein Ce-FLI-1 comprising leucine-rich repeats and gelsolin-like domains" Biochem.Biophys.Res.Commun.(in press). (1999)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-12-08  

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