Characterization of Ras effector mutant interactions with the NF1-GAP related domain.
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.
Marshall
M S
MS
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Hettich
L A
LA
D000595:Amino Acid Sequence; D002471:Cell Transformation, Neoplastic; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000032:analysis; D011905:Genes, ras; D008969:Molecular Sequence Data; D009154:Mutation; D025542:Neurofibromin 1; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism
D025542:Neurofibromin 1; D011506:Proteins; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)