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Oncogenic insertional mutations in the P-loop of Ras are overactive in MAP kinase signaling
B Klockow1, M R Ahmadian, C Block
1Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Abstract:
Mutations of Ras with three extra amino acids inserted into the phosphate-binding (P) loop have been investigated both in vitro and in vivo. Such mutants have originally been detected as oncogenes both in the ras and the TC21 genes. Biochemical experiments reveal the molecular basis of their oncogenic potential: the mutants show a strongly attenuated binding affinity for nucleotides, most notably for GDP, leading to a preference for GTP binding. Furthermore, both the intrinsic as well as the GAP-stimulated GTP hydrolysis are drastically diminished. The binding interaction with GAP is reduced, whereas binding to the Ras-binding domain of the downstream effector c-Raf1 is not altered appreciably. Microinjection into PC12 cells shows the mutants to be as potent to induce neurite outgrowth as conventional oncogenic Ras mutants. Unexpectedly, their ability to stimulate the MAP kinase pathway as measured by a reporter gene assay in RK13 cells is much higher than that of the normal oncogenic mutant G12V. This characteristic was attributed to an increased stimulation of c-Raf1 kinase activity by the insertional Ras mutants.
Insights
Ras mutations with inserted amino acids in the phosphate-binding loop act as oncogenes. These mutants exhibit impaired nucleotide binding and GTPase activity, yet potently induce neurite outgrowth and hyperactivate the MAP kinase pathway.
Area of Science:
- Molecular Biology
- Oncology
- Cell Signaling
Background:
- Ras proteins are key regulators of cell signaling pathways.
- Mutations in Ras genes are frequently found in human cancers.
- Specific mutations, such as insertions in the phosphate-binding loop, can alter Ras function and contribute to oncogenesis.
Purpose of the Study:
- To investigate the in vitro and in vivo effects of Ras mutations with three extra amino acids inserted into the phosphate-binding (P) loop.
- To elucidate the molecular mechanisms underlying the oncogenic potential of these insertional Ras mutants.
- To compare their biological activity with conventional oncogenic Ras mutants.
Main Methods:
- Biochemical assays to assess nucleotide binding affinity and GTP hydrolysis rates.
- Analysis of interactions with Guanine nucleotide Dissociation Inhibitor (GDI) and c-Raf1.
- Microinjection into PC12 cells to evaluate neurite outgrowth.
- Reporter gene assays in RK13 cells to measure MAP kinase pathway activation.
Main Results:
- Insertional Ras mutants displayed significantly reduced binding affinity for GDP and diminished intrinsic and GAP-stimulated GTP hydrolysis.
- Binding to Guanine nucleotide Dissociation Inhibitor (GDI) was reduced, while binding to c-Raf1 remained largely unaffected.
- Microinjection demonstrated potent induction of neurite outgrowth, comparable to conventional oncogenic Ras mutants.
- These mutants showed unexpectedly higher stimulation of the MAP kinase pathway compared to the G12V mutant, attributed to increased c-Raf1 kinase activity.
Conclusions:
- Ras mutants with insertions in the P-loop possess oncogenic potential due to altered nucleotide binding and impaired GTPase activity.
- These mutants are potent inducers of cellular differentiation (neurite outgrowth) and exhibit enhanced activation of the MAP kinase pathway.
- The findings highlight a novel mechanism of Ras-mediated oncogenesis and pathway dysregulation.