Schmidt G, Lenzen C, Simon I, Deuter R, Cool R H, Goody R S, Wittinghofer A
Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Dortmund, Germany.
Oncogene. 1996 Jan 4;12(1):87-96.
The D119N mutation of p21ras was prepared by site-directed mutagenesis. Its nucleotide binding properties were investigated using fluorescently labelled guanosine and xanthosine nucleotides. Its affinity for guanosine nucleotides is severely reduced, with a concomitant increase in the affinity for xanthosine nucleotides, which leads to an almost complete reversal of base specificity. The protein is a GTPase as well as a XTPase and the hydrolysis reaction can be efficiently stimulated by GAP. Dissociation of XDP from the mutant is stimulated by the guanine nucleotide exchange factor Cdc25Mm in a similar manner to that of GDP from wildtype. The interaction of the mutant with the effector domain of c-Raf kinase or Ral-GEF is normal. In microinjection experiments in PC12 and NIH3T3 cells the protein behaves as an oncogenic mutant due to its high dissociation rate for GDP. However, when the protein is loaded with XDP before microinjection the onset of the oncogenic signal can be efficiently retarded. Thus, the protein behaves initially as wildtype and later as an oncogenic protein.
通过定点诱变制备了p21ras的D119N突变体。使用荧光标记的鸟苷和黄苷核苷酸研究了其核苷酸结合特性。其对鸟苷核苷酸的亲和力严重降低，同时对黄苷核苷酸的亲和力增加，这导致碱基特异性几乎完全逆转。该蛋白既是一种GTP酶也是一种XTP酶，水解反应可被GAP有效刺激。XDP从突变体上的解离受到鸟嘌呤核苷酸交换因子Cdc25Mm的刺激，其方式与野生型中GDP的解离相似。突变体与c-Raf激酶或Ral-GEF的效应结构域的相互作用正常。在PC12和NIH3T3细胞的显微注射实验中，由于该蛋白对GDP的高解离速率，其表现为致癌突变体。然而，当在显微注射前该蛋白加载有XDP时，致癌信号的起始可被有效延迟。因此，该蛋白最初表现为野生型，随后表现为致癌蛋白。
