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Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine
L A Quilliam1, K Kato, K M Rabun
1Department of Pharmacology, University of North Carolina at Chapel Hill School of Medicine 27599.
Abstract:
The Ras(17N) dominant negative antagonizes endogenous Ras function by forming stable, inactive complexes with Ras guanine nucleotide exchange factors (GEFs; e.g., SOS1). We have used the growth-inhibitory phenotype of Ras(17N) to characterize two aspects of Ras interaction with GEFs. First, we used a nonprenylated version of Ras(17N), designated Ras(17N/186S), which no longer associates with the plasma membrane and lacks the growth-inhibitory phenotype, to address the importance of Ras subcellular location and posttranslational modification for its interaction with GEFs. We observed that addition of an N-terminal myristylation signal to Ras(17N/186S) restored the growth-inhibitory activity of nonprenylated Ras(17N). Thus, membrane association, rather than prenylation, is critical for Ras interaction with Ras GEFs. Second, we used a biological selection approach to identify Ras residues which are critical for Ras(17N) growth inhibition and hence for interaction with Ras GEFs. We identified mutations at residues 75, 76, and 78 that abolished the growth-inhibitory activity of Ras(17N). Since GEF interaction is dispensable for oncogenic but not normal Ras function, our demonstration that single-amino-acid substitutions at these three positions impaired the transforming activity of normal but not oncogenic Ras provides further support for the role of these residues in Ras-GEF interactions. Finally, Ras(WT) proteins with mutations at these residues were no longer activated by mammalian SOS1. Altogether, these results suggest that the Ras intracellular location and Ras residues 75 to 78 are critical for Ras-GEF interaction.
Insights
Ras protein interaction with guanine nucleotide exchange factors (GEFs) is crucial for cell growth. Membrane association, not prenylation, and specific residues (75-78) are critical for this Ras-GEF interaction.
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein-Protein Interactions
Background:
- Ras proteins are key regulators of cell signaling pathways.
- Ras guanine nucleotide exchange factors (GEFs) activate Ras proteins.
- Dominant-negative Ras mutants can inhibit endogenous Ras function.
Purpose of the Study:
- To investigate the role of Ras subcellular localization and specific residues in Ras-GEF interactions.
- To determine the importance of membrane association versus prenylation for Ras-GEF binding.
- To identify critical Ras residues involved in GEF interaction and downstream signaling.
Main Methods:
- Utilized a nonprenylated Ras(17N/186S) mutant to assess the role of membrane association.
- Introduced an N-terminal myristylation signal to restore membrane localization.
- Employed biological selection to identify critical Ras residues for GEF interaction.
- Assessed the impact of mutations on Ras transforming activity and activation by SOS1.
Main Results:
- Membrane association, facilitated by myristylation, restored growth-inhibitory activity to nonprenylated Ras(17N).
- Mutations at Ras residues 75, 76, and 78 abolished growth inhibition and impaired Ras-GEF interaction.
- These mutations affected normal Ras transforming activity but not oncogenic Ras.
- Mutated Ras(WT) proteins were not activated by mammalian SOS1.
Conclusions:
- Ras membrane localization is critical for interaction with GEFs.
- Ras residues 75-78 are essential for mediating Ras-GEF interactions.
- These findings provide insights into the regulation of Ras signaling pathways.