Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.
The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.
Stone
J C
JC
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Colleton
M
M
Bottorff
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020690:GTPase-Activating Proteins; D005786:Gene Expression Regulation; D011905:Genes, ras; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025542:Neurofibromin 1; D017354:Point Mutation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D020703:ras GTPase-Activating Proteins
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D025542:Neurofibromin 1; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras)