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

秀丽隐杆线虫新型 Ras 效应子的表征和所有效应子的基因敲除

• 批准号: 09670128
• 负责人: KARIYA Ken-ichi
• 金额: $ 1.73万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670128/
• 关键词: C.elegans; Ras; two-hybrid screening; gene knockout; RalGDS; AF6; Cdc25; phospholipase C; Two-Hybrid法; Cds25

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

1。在线虫秀丽隐杆线虫（C.级别）中，EGF受体和RAS通过RAF调节外阴的分化，这是该生物体中唯一已知的RAS效应子。另一方面，哺乳动物的RAS潜力多种效应/效应子候选者，例如RAF，Ralgds和AF6。通过酵母的两杂化筛选，我们确定了两种新型的RAS结合蛋白，A磷脂酶C（CE-PLC210）和GDP/GTP交换因子（CE-CDC25），除了RAF，RALGD，RALGDS和AF-6外。此外，我们分离了CE-PLC210和CE-CDC25（HU-PLC210和HU-CDC25）的人类直系同源物。2。所有这些C.级蛋白质蛋白质与野生型Ras和Rapla（RAS同源物）结合，但不与有效的区域Munant或其他小G蛋白结合在一起。像CE-PLC2 10一样，Hu-Plc210以GTP依赖性方式与RAS结合，并在体外水解PIP2。 RAS/RAPLA对调节的研究正在进行中。 HU-CDC25仅以GTP依赖性方式绑定到RAPLA，并且在…更多的RAP LA上表现出强大的GDP/GTP交换活动，尽管它对RAS的活性也较弱（CE-CDC25在RAS和RAP LA上都具有相似的活性）。 HU-CDC25可以通过与GTP-Rapla结合并激活膜中存在的其他Rapla分子，从而扩增细胞外信号的作用3。分离c.Elegans中所有效应基因的转座子插入等位基因和缺失等位基因（CE-CDC25除外）。 CE-PLC210基因缺失突变体暴露了无菌表型（类似于EGF受体突变体中的表型）和卵形的缺陷表型（与Ras突变体中发现的表型相似），并且这些表型是通过野生型基因的转基因表达而挽救的。与RAF的突变相反，其他效应子/效应子候选者的突变在外阴分化中没有异常，也没有通过活化的RAS突变抑制外阴分化的超级诱导。外阴发育可能仅由RAF监管4。在C.Elegans（不是通过两个杂交筛选）中发现了一种新型的RAS结合蛋白（CE-FLI-1），其RAS结合活性以及肌动蛋白结合和 - 分散活性的表征。较少的

项目成果

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