Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras.
Two dominant inhibitory Ras mutant proteins were analyzed by microinjection. One, [Asn-17]Ras, had a substitution in the putative Mg(2+)-binding site of Ha-Ras. The other, RAST, had a mutation in a yeast RAS protein that impaired its GTPase activity and increased its affinity for GAP. RAST also had a mutation that blocked its localization to the plasma membrane. In NIH 3T3 cells [Asn-17]Ras inhibited the function of normal Ras much more efficiently than that of oncogenic Ras. In contrast, RAST interfered with the transforming activity of oncogenic Ras more efficiently than that of normal Ras. These conclusions were based on two separate types of analysis. The inhibitory Ras mutant proteins were first microinjected into cells stably transformed either by oncogenic Ras or by high levels of expression of cellular Ras. Results obtained in stably transformed cells were then verified by coinjection of the inhibitory Ras mutant proteins together with transforming concentrations of either oncogenic or normal Ras protein. Whereas RAST was active in soluble form. [Asn-17]Ras required membrane localization for activity. Furthermore, mutations in the GAP/effector-binding domain reduced or eliminated the inhibitory activity of RAST but had no detectable effect on [Asn-17]Ras. These results are consistent with the possibility that [Asn-17]Ras functions by blocking the activation of endogenous Ras proteins, while RAST functions by blocking the ability of activated Ras to stimulate a downstream target within the cells. The properties of RAST suggest that interference with the GAP/effector-binding function of RAS represents a strategy for the preferential inactivation of oncogenic Ras in cells.
Stacey
D W
DW
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44106.
Feig
L A
LA
Gibbs
J B
JB
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004261:DNA Replication; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D005799:Genes, Dominant; D011905:Genes, ras; D051379:Mice; D008845:Microinjections; D015689:Oncogene Protein p21(ras) / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
D011994:Recombinant Proteins; D020558:GTP Phosphohydrolases; D015689:Oncogene Protein p21(ras); D013936:Thymidine