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A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants
1Department of Molecular Biology, Cetus Corporation, Emeryville, CA 94608.
Abstract:
The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.
Insights
Oncogenic ras p21 mutants bind guanosine triphosphate (GTP) and activate cell maturation. Normal ras p21 binds guanosine diphosphate (GDP) due to a cytoplasmic protein that enhances GTPase activity.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Ras p21 proteins are key regulators of cellular processes.
- Guanine nucleotide binding (GTP or GDP) dictates ras p21 activity.
- Mutations in ras p21 are implicated in oncogenesis.
Purpose of the Study:
- To investigate the role of guanine nucleotides in ras p21 function.
- To characterize the mechanism by which oncogenic ras p21 mutants gain activity.
- To identify factors regulating ras p21 guanine nucleotide binding.
Main Methods:
- Xenopus oocyte maturation assay to measure ras p21 biological activity.
- In vivo and in vitro assays to determine ras p21 guanine nucleotide binding.
- Biochemical analysis of cytoplasmic factors influencing ras p21 GTPase activity.
Main Results:
- Oncogenic Asp12 and Val12 N-ras p21 mutants induced oocyte maturation, unlike normal Gly12 p21.
- Mutant proteins were GTP-bound, while normal Gly12 p21 was GDP-bound.
- A cytoplasmic protein significantly stimulated GTP hydrolysis by Gly12 p21, but not mutants.
- This regulatory protein was also detected in mammalian cell extracts.
Conclusions:
- A cytoplasmic protein maintains normal ras p21 in an inactive GDP-bound state via GTPase activity.
- Position 12 mutations in ras p21 prevent this protein's regulatory effect, leading to an active GTP-bound state.
- This mechanism highlights how ras p21 mutations contribute to oncogenesis by disrupting regulatory pathways.