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Identification of amino acid residues required for Ras p21 target activation
M S Marshall1, L J Davis, R D Keys
1Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Abstract:
The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.
Insights
Ras and Krev-1 proteins share effector domains but differ in flanking residues. Altering these specific residues in Krev-1 grants it Ras function, impacting Ras-GTPase activating protein interactions.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Ras proteins are key regulators of cell signaling pathways.
- Ras-mediated transformation is a critical process in cancer development.
- Krev-1 protein acts as a suppressor of Ras-mediated transformation.
- Ras and Krev-1 proteins share identical effector domains (residues 32-40) essential for biological activity and Ras GTPase-activating protein (GAP) interaction.
Purpose of the Study:
- To investigate the role of amino acid residues flanking the Ras effector domain in protein-protein interactions and biological activity.
- To determine if altering non-conserved residues can confer Ras function to Krev-1.
- To assess the impact of Krev-1-like substitutions on Ras protein function and GAP binding.
Main Methods:
- Site-directed mutagenesis was used to create mutant Krev-1 and Ras proteins.
- Mammalian and yeast biological assays were employed to evaluate protein function.
- In vitro binding assays were performed to measure Ras p21-GAP affinity.
Main Results:
- Substitution of Krev-1 residues 26, 27, 30, 31, and 45 with corresponding Ras residues resulted in a Krev-1 protein exhibiting Ras function in both mammalian and yeast systems.
- Conversely, replacing Ras residues with Krev-1 counterparts led to Ras proteins with impaired biological activity or reduced GAP binding affinity.
- These findings highlight the critical role of specific flanking residues in modulating Ras protein function and interactions.
Conclusions:
- Five specific amino acid residues flanking the effector domain are crucial for normal Ras protein-protein interactions and biological activity.
- These residues dictate the functional differences between Ras and Krev-1 proteins.
- Understanding these residue-specific interactions provides insights into Ras p21-GAP interactions in vivo and has implications for therapeutic strategies targeting Ras signaling.