van den Berghe N, Cool R H, Horn G, Wittinghofer A
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Oncogene. 1997 Aug 14;15(7):845-50. doi: 10.1038/sj.onc.1201407.
A catalytically active fragment of the Rap-specific guanine-nucleotide exchange factor C3G was expressed in E coli. It was purified and its interaction with GTP-binding proteins was investigated using fluorescence spectroscopy. C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily. Like the corresponding fragment from CDC25Mm, the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A x GDP up to 100 microM, indicating an apparent K(M) higher than 100 microM. Unlike the Ras-CDC25Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro. These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative effects.
Rap特异性鸟嘌呤核苷酸交换因子C3G的一个具有催化活性的片段在大肠杆菌中表达。对其进行了纯化，并使用荧光光谱法研究了它与GTP结合蛋白的相互作用。C3G可刺激GDP从Rap1上解离，但不能刺激GDP从Rap2上解离，也不能刺激GDP从Bud1（被认为是Rap1的酵母同源物）或人类Ras亚家族的所有其他蛋白质上解离。与来自CDC25Mm的相应片段一样，GDP解离速率的增加与Rap1A x GDP浓度增加呈线性关系，直至100微摩尔，这表明其表观K(M)高于100微摩尔。与Ras-CDC25Mm系统不同，Rap1A(SIIN)突变体在体外并不抑制C3G激活的鸟嘌呤核苷酸从野生型Rap1A上解离。这些数据表明，Rap1A(S17N)在体内不太可能通过滴定作用消耗C3G，而这是S17N突变体发挥其显性负效应的一种推测机制。
