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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.
Molecular and Cellular Biology
|August 1, 1991
Summary
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.