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rap1A基因的过表达诱导ras转化细胞的逆转。这些基因编码类似的小gtp结合蛋白Ras和Rap1A。我们从线虫中鉴定出两种新的Ras/ rap1a结合蛋白，一种是磷脂酶C (Ce-PLC-ε)，另一种是GDP/GTP交换因子（Ce-RA-GEF）。这两种分子都具有Ras/ rap1a - associate （RA）结构域。我们还分离了它们的人类同源物的部分cdna （Hs-PLC-ε, Hs-RA-GEF）。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gtp依赖的方式与Ras相关。活化的Ras突变体与Hs-PLC-ε共表达诱导其从细胞质溶胶转移到质膜。在EGF刺激下，观察到类似的易位，这种易位被显性负Ras的共同表达所抑制。在脂质体重构实验中，Ras以gtp依赖的方式刺激Hs-PLC-ε的体外4,5-二磷酸水解活性。这些结果表明Ras通过Hs-PLC-ε的膜靶向直接调控磷酸肌苷的分解。Ce-RA-GEF和Hs-RA-GEF除具有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结合域同源的区域，该区域不能结合cAMP或cGMP。虽然REM结构域和GEF结构域与其他作用于Ras家族蛋白的GEF结构域是保守的，但RA和PDZ结构域在它们中都是不可见的。Hs-RA-GEF不仅表现出对Rap1A的GTP依赖性结合活性，而且在体外和体内都表现出刺激Rap1A的GDP/GTP交换的活性。另一方面，Ce-RA-GEF与Ras和Rap1A的GDP/GTP交换相关并刺激了两者的交换。这些结果表明，Ce-RA-GEF和Hs-RA-GEF定义了一类新的Rap1A 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