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

1.在线虫秀丽隐杆线虫（C.elegans）中，EGF受体和Ras通过Raf（该生物体中唯一已知的Ras效应子）调节外阴（产卵器官）的分化。另一方面，哺乳动物Ras具有多种效应子/效应子候选物，例如Raf、RalGDS和AF 6。通过酵母双杂交筛选秀丽隐杆线虫cDNA文库，我们鉴定了两个新的Ras结合蛋白，磷脂酶C（Ce-PLC 210）和GDP/GTP交换因子（Ce-Cdc 25），以及Raf、RalGDS和AF-6。此外，我们分离了Ce-PLC 210和Ce-Cdc 25的人直系同源物（Hu-PLC 210和Hu-Cdc 25）。所有这些秀丽隐杆线虫蛋白结合野生型Ras和RaplA（Ras同源物），但不结合效应子区突变体或其他小G蛋白。与Ce-PLC 210一样，Hu-PLC 210以GTP依赖性方式与Ras和Rap IA结合，并在体外水解PIP 2。Ras/RaplA调控的研究正在进行中。Hu-Cdc 25仅以GTP依赖性方式与RaplA结合，并且显示出强烈的GDP/GTP交换活性。 ...更多信息 Rap LA，尽管其对Ras也具有弱活性（Ce-Cdc 25对Ras和Rap 1A两者具有相似的活性）。Hu-Cdc 25可以通过与GTP-RaplA结合而被募集到膜上，并激活膜上存在的其他RaplA分子，从而放大细胞外信号的作用。转座子插入等位基因和缺失等位基因（除了Ce-Cdc 25）被分离的所有效应基因在秀丽隐杆线虫。Ce-PLC 210基因缺失突变体表现出不育表型（类似于EGF受体突变体中发现的表型）和产卵缺陷表型（类似于Ras突变体中发现的表型），这些表型通过野生型基因的转基因表达得到拯救。与Raf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表现出异常，并且没有抑制激活的Ras突变对外阴分化的超诱导。外阴发育可能仅受Raf的调节。在秀丽隐杆线虫中发现了一种新的Ras结合蛋白（Ce-FLI-1），并对其Ras结合活性、肌动蛋白结合和切割活性进行了表征。少

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