H-ras mutants lacking the epitope for the neutralizing monoclonal antibody Y13-259 show decreased biological activity and are deficient in GTPase-activating protein interaction.
We have generated deletion mutants of the H-ras p21 protein which lack residues 58 to 63 or 64 to 68 and contain either the normal glycine or an activating mutation, arginine, at position 12. None of the deleted proteins were recognized by monoclonal antibody Y13-259, and those mutants with activating mutations showed at least a 100-fold reduction in their transforming activities compared with the activities of their nondeleted counterparts. Alterations observed in the in vitro GTPase or GTP interchange properties of the deletion mutants were not consistent with the decrease in their transforming activities. Moreover, each mutant showed normal membrane localization, which is essential for its biological activity. Recently, a newly identified protein, designated GTPase-activating protein (GAP), was found to markedly increase GTPase activity of the normal ras p21 but not of p21 mutants bearing activating lesions (H. Adari, D. R. Lowy, B. M. Willumsen, C. J. Der, and F. McCormick, Science 240:518-521, 1988). We showed that GAP had no effect on the in vitro GTPase activity of the deletion mutants of the normal p21 protein. Since similar deletions in mutants with activating lesions at position 12 or 59 or both showed decreased transforming activity, our results suggest that the recognition site for Y13-259 within the ras p21 molecule influences directly or indirectly the interaction of ras p21 with GAP and that this interaction is critical for biological activity of ras proteins.
Srivastava
S K
SK
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Di Donato
A
A
Lacal
J C
JC
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002872:Chromosome Deletion; D000939:Epitopes / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D009154:Mutation; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D020703:ras GTPase-Activating Proteins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)