Functional Characterization of Novel Ras/Rap1A Effector Candidates Conserved between Nematode and Humans

ras基因的显性活性突变诱导恶性细胞转化，而rap 1A基因的过表达诱导ras转化细胞的逆转。这些基因编码相似的小GTP结合蛋白Ras和Rap 1A。我们从线虫中鉴定了两个新的Ras/Rap 1A结合蛋白：磷脂酶C（Ce-PLC-ε）和GDP/GTP交换因子（Ce-RA-GEF）。这两种分子都具有Ras/Rap 1A结合（RA）结构域。我们还分离了它们的人类同源物（Hs-PLC-ε，Hs-RA-GEF）的部分cDNA。在本研究中，我们获得了全长cDNA并检测了它们的功能。Hs-PLC-ε在体外以GT依赖性方式与Ras结合。激活的Ras突变体与Hs-PLC-ε的共表达诱导其从细胞质移位到质膜。在用EGF刺激后，观察到类似的易位，其被显性负性Ras的共表达抑制。在基于脂质体的重建试验中，磷酸化 关于我们 Ras对Hs-PLC-ε的4，5-二磷酸肌醇水解活性具有GTP依赖性。这些结果表明Ras通过Hs-PLC-ε的膜靶向作用直接调控磷酸肌醇的降解。Ce-RA-GEF和Hs-RA-GEF除了具有RA和GEF结构域外，还具有PDZ结构域和Ras交换基序（REM）结构域。它们还含有一个与cAMP/cGMP结合结构域同源的区域，该区域被证明不能结合cAMP或cGMP。尽管REM结构域和GEF结构域与作用于Ras家族蛋白的其他GEF保守，但RA和PDZ结构域在它们中的任何一个中都看不到。Hs-RA-GEF不仅表现出GTP依赖性的Rap 1A结合活性，而且在体外和体内都表现出刺激Rap 1A的GDP/GTP交换活性。这些结果表明，Ce-RA-GEF和Hs-RA-GEF定义了一类新的Rap 1A GEF分子，它们在进化过程中是保守的。少

Dominant active mutations of the ras gene induce malignant cellular transformation, while overexpression of the rap1A gene induces reversion of the ras-transformed cells. These genes encode similar small GTP-binding proteins, Ras and Rap1A.We had identified two novel Ras/Rap1A-binding proteins, a phospholipase C (Ce-PLC-ε) and a GDP/GTP exchange factor (Ce-RA-GEF), from nematode C.elegans. Both of these molecules possessed the Ras/Rap1A-associating (RA) domain (s). We had also isolated partial cDNAs for their human homologs (Hs-PLC-ε, Hs-RA-GEF). In the present study, we obtained full-length cDNAs and examined their functions.Hs-PLC-ε associated with Ras in a GTP-dependent manner in vitro. Co-expression of an activated Ras mutant with Hs-PLC-ε induced its translocation from the cytosol to the plasma membrane. Upon stimulation with EGF, similar translocation was observed, which is inhibited by co-expression of dominant negative Ras. In a liposomebased reconstitution assay, the phosphati … More dylinositol 4,5-bisphosphate-hydrolyzing activity of Hs-PLC-ε was stimulated in vitro by Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of Hs-PLC-ε.Ce-RA-GEF and Hs-RA-GEF possessed a PDZ domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cAMP/cGMP-binding domain, which turned out to be incapable of binding cAMP or cGMP.Although the REM domain and GEF domains are conserved with other GEFs acting on Ras family proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A.These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution. Less