Marshall M S, Hettich L A
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Oncogene. 1993 Feb;8(2):425-31.
The GTPase activating proteins Ras GAP and the neurofibromatosis-type 1 (NF1) gene product have been implicated as both potential mediators and regulators of the mitogenic effects of the ras proteins. In this study, the interactions of selected Ras effector mutants with the NF1-GAP related domain (NF1-GRD) were investigated. The NF1-GRD was unable to stimulate the GTPase of Ras[Asn33], Ras[Ser35] or Ras[Asn38], all transformation defective mutants. Each of these mutants had reduced but detectable binding to the NF1-GRD (apparent KD of 9 microM, 4 microM and 2 microM respectively, vs 0.5 microM for normal Ha-ras). The NF1-GRD was able to fully stimulate the intrinsic GTPase of the transformation-defective Ras[Gly26Ile27] and Ras[Glu45] mutants, each of which bound the NF1-GRD with wild type affinity or better (KD = 0.13 microM and 0.4 microM respectively). The transforming Ras[Glu30Lys31] protein showed no GTPase stimulation and bound most poorly to the NF1-GRD (apparent KD of 16 microM). The interaction of the NF1-GRD with these specific Ras effector mutations is similar to that observed for Ras GAP. When the relative transforming activity of the valine 12 form of each Ras mutant was plotted against the apparent KD for NF1-GRD binding, little correlation was observed. These results support the hypothesis that the NF1 gene product does not function as a downstream effector of Ras in the mitogenic pathway.
GTP酶激活蛋白Ras GAP和神经纤维瘤病1型（NF1）基因产物被认为是ras蛋白促有丝分裂作用的潜在介质和调节因子。在本研究中，研究了选定的Ras效应突变体与NF1-GAP相关结构域（NF1-GRD）的相互作用。NF1-GRD无法刺激Ras[Asn33]、Ras[Ser35]或Ras[Asn38]的GTP酶，这些都是转化缺陷型突变体。这些突变体与NF1-GRD的结合均有所减少但仍可检测到（表观解离常数分别为9 microM、4 microM和2 microM，而正常Ha-ras为0.5 microM）。NF1-GRD能够完全刺激转化缺陷型Ras[Gly26Ile27]和Ras[Glu45]突变体的内在GTP酶，这两种突变体与NF1-GRD的结合亲和力与野生型相当或更好（解离常数分别为0.13 microM和0.4 microM）。具有转化能力的Ras[Glu30Lys31]蛋白未表现出GTP酶刺激作用，与NF1-GRD的结合最差（表观解离常数为16 microM）。NF1-GRD与这些特定Ras效应突变体的相互作用与Ras GAP所观察到的相似。当将每个Ras突变体缬氨酸12形式的相对转化活性与NF1-GRD结合的表观解离常数作图时，未观察到明显的相关性。这些结果支持了NF1基因产物在促有丝分裂途径中不作为Ras下游效应器发挥作用的假说。
