Differential interaction of the Ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor
Differential interaction of the Ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor
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DOI：
10.1074/jbc.271.12.6794
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发表时间：
1996-03-22
影响因子：
4.8
通讯作者：
Wittinghofer, A
中科院分区：
文献类型：
--
作者：
Herrmann, C;Horn, G;Wittinghofer, A
在溶液中生物化学研究了H-Ras、R-Ras和Rap1A与c-Raf激酶ras结合域（RED）和Ral鸟嘌呤核苷酸交换因子（RGF）的相互作用，通过缺失克隆确定RGF- rbd为人类RGF c端一个97个氨基酸长的片段，是一个具有高稳定性的独立折叠结构域。有趣的是，虽然H-Ras与Raf-RBD具有高亲和力（K-D = 20 nM），与RGF-RBD具有低亲和力（K-D = 1 μ M），但Rap1A表现出相反的行为，两种rbd与R-Ras的结合都很弱，没有特异性。Rap1A与RGF-RBD的相互作用具有与Ras-Raf相互作用相似的特征，因为它被效应区（D38A）突变阻断，并抑制鸟嘌呤核苷酸的解离，这是本工作定量测量的基础。此外，RGF-RBD的结合抑制了Rap1A与Rap-GAP的相互作用。只要不同蛋白的细胞定位及其生物学功能不明确，这些生化数据似乎表明，ral -鸟嘌呤核苷酸交换因子是Rap1A而不是H-Ras的效应分子。
The interactions of H-Ras, R-Ras, and Rap1A with the Ras-binding domains (RED) of the c-Raf kinase and of the Ral guanine nucleotide exchange factor (RGF) was studied biochemically in solution, From deletion cloning the RGF-RBD was defined as a 97-amino acid-long fragment from the C-terminal end of the human RGF, which is an independent folding domain with high stability. Interestingly, whereas H-Ras binds with high affinity (K-D = 20 nM) to Raf-RBD and with low affinity (K-D = 1 mu M) to RGF-RBD, Rap1A shows the opposite behavior, The binding of both RBDs to R-Ras is weak and shows no specificity. The interaction between Rap1A and RGF-RBD shows similar characteristics to the Ras-Raf interaction because it is blocked by mutations in the effector region (D38A) and it inhibits the dissociation of guanine nucleotide, which is the basis for the quantitative measurements in this work, Furthermore, the binding of RGF-RBD inhibits the interaction between Rap1A and Rap-GAP. As long as the cellular localizations of the different proteins and their biological functions are not clarified, these biochemical data seem to indicate that Ral-guanine nucleotide exchange factors is an effector molecule of Rap1A rather than of H-Ras.