Cool R H, Schmidt G, Lenzen C U, Prinz H, Vogt D, Wittinghofer A
Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund, Germany.
Mol Cell Biol. 1999 Sep;19(9):6297-305. doi: 10.1128/MCB.19.9.6297.
The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras-a strong decrease in nucleotide affinity-enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.
在各种类Ras蛋白的NKxD核苷酸结合基序中引入突变D119N（或其同源物）会产生组成型激活或显性负效应，具体取决于系统和检测方法。在此我们表明，Ras（D119N）在PC12和NIH 3T3细胞中在特定细胞浓度下具有抑制作用。生化数据有力地表明，Ras中突变D119N的主要作用——核苷酸亲和力大幅降低——使该突变体（i）能够隔离其鸟嘌呤核苷酸交换因子，以及（ii）独立于交换因子的调节作用快速结合GTP。由于突变D119N不影响Ras与效应分子之间的相互作用，后一种效应导致Ras（D119N）在高于交换因子浓度时充当激活的Ras蛋白。相比之下，同样显示核苷酸亲和力大幅降低的Ras（S17N）不与效应分子结合。这些结果指出了显性负Ras突变体的两个重要前提条件：突变Ras对交换因子的相对亲和力高于对核苷酸的相对亲和力，以及无法与一种或多种效应分子相互作用。值得注意的是，如Ras（E37G/D119N）对PC12细胞中神经生长因子介导的神经突生长的抑制作用以及Ras（T35S/D119N）对NIH 3T3细胞中胎牛血清介导的DNA合成的抑制作用所示，引入第二个功能部分丧失的突变甚至在高浓度下也会使Ras（D119N）变成强显性负突变体。本文讨论了这些结果的解释。
