|Budget Amount *help
¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
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
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