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Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras
D W Stacey1, L A Feig, J B Gibbs
1Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44106.
Abstract:
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.
Insights
Two Ras mutant proteins, [Asn-17]Ras and RAST, were studied for their inhibitory effects on Ras function. [Asn-17]Ras inhibits normal Ras, while RAST preferentially inhibits oncogenic Ras by interfering with its effector function.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Ras proteins are key regulators of cell signaling pathways.
- Mutations in Ras are common in human cancers, leading to uncontrolled cell growth.
- Developing inhibitors that target oncogenic Ras is a significant therapeutic goal.
Purpose of the Study:
- To analyze the inhibitory mechanisms of two dominant Ras mutant proteins, [Asn-17]Ras and RAST.
- To determine the differential effects of these mutants on normal versus oncogenic Ras activity.
- To explore strategies for preferential inactivation of oncogenic Ras.
Main Methods:
- Microinjection of Ras mutant proteins ([Asn-17]Ras and RAST) into NIH 3T3 cells.
- Analysis of effects on cells expressing normal or oncogenic Ras.
- Coinjection experiments to verify inhibitory activities.
- Assessment of Ras localization and GTPase activity.
Main Results:
- [Asn-17]Ras inhibited normal Ras function more effectively than oncogenic Ras.
- RAST preferentially inhibited the transforming activity of oncogenic Ras over normal Ras.
- [Asn-17]Ras required membrane localization for activity, while RAST did not.
- Mutations in RAST's GAP/effector-binding domain reduced its inhibitory activity, unlike [Asn-17]Ras.
Conclusions:
- [Asn-17]Ras likely functions by blocking Ras activation.
- RAST appears to inhibit Ras by blocking downstream effector interaction.
- Targeting the GAP/effector-binding function of Ras offers a strategy for selectively inhibiting oncogenic Ras.