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Identification of amino acids in p21ras involved in exchange factor interaction
1Chester Beatty Laboratories, Institute of Cancer Research, London, England.
Abstract:
Through the genetic analysis of vulva development in C. elegans several different sites of mutation have been identified in the let-60 ras protein which have been postulated to affect the function of normal but not oncogenic p21ras (Beitel, G. J., S. G. Clark, and H. R. Horvitz. 1990. Nature 348: 503-509). We have introduced these mutations into mammalian Ha-ras and determined their effect on the function of cellular ras (c-ras), an oncogenically activated variant D12ras and the dominant negative N17ras. From these studies we conclude that two of the mutations S89-->F89 and delta 103-108 destabilise ras when it is in the GDP-bound form. However mutations at A66 and G75 lead to stable proteins on which a ras exchange factor SCD25 is unable to promote the formation of ras-GTP. The mutations at A66 show for the first time that helix alpha 2 of p21ras is involved in the stimulation of guanine nucleotide exchange by exchange factors.
Insights
Mutations in the ras protein affect its stability and function. Specific mutations destabilize GDP-bound ras, while others prevent activation by exchange factors, revealing helix alpha 2
Area of Science:
- Molecular Biology
- Genetics
- Cell Signaling
Background:
- Genetic analysis of vulva development in C. elegans identified mutations in the let-60 ras protein.
- These mutations were postulated to affect normal p21ras function but not oncogenic p21ras.
- Ras proteins are key regulators of cellular signaling pathways, and their function is tightly controlled by guanine nucleotide binding and exchange.
Purpose of the Study:
- To investigate the functional consequences of specific let-60 ras mutations in mammalian ras proteins.
- To determine the effect of these mutations on cellular ras (c-ras), oncogenic D12ras, and dominant-negative N17ras.
- To elucidate the role of specific ras protein regions, such as helix alpha 2, in guanine nucleotide exchange.
Main Methods:
- Introduction of C. elegans let-60 ras mutations into mammalian Ha-ras constructs.
- Functional analysis of mutated ras proteins in cellular contexts, including oncogenic and dominant-negative variants.
- Assays to assess ras protein stability, GDP-bound state, GTP formation, and interaction with ras exchange factors like SCD25.
Main Results:
- Mutations S89-->F89 and delta 103-108 were found to destabilize ras in its GDP-bound form.
- Mutations at A66 and G75 resulted in stable ras proteins that were resistant to activation by the ras exchange factor SCD25.
- Mutations at A66 provided the first evidence for the involvement of helix alpha 2 of p21ras in mediating stimulation of guanine nucleotide exchange.
Conclusions:
- Specific ras mutations can differentially affect ras protein stability and its response to guanine nucleotide exchange factors.
- The GDP-bound state of ras is particularly sensitive to destabilization by certain mutations.
- Helix alpha 2 of p21ras plays a critical role in the interaction with and activation by ras exchange factors.