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Delineation of functional determinants in the transforming protein of Fujinami sarcoma virus
1Jackson Laboratory, Bar Harbor, Maine 04609.
Abstract:
We analyzed linker insertion mutations throughout the 3' region of the v-fps gene of Fujinami sarcoma virus to identify tyrosine kinase transforming protein (P130gag-fps) determinants that are important for catalysis and transforming activity and, in particular, to define residues that participate in substrate selection. Mutations that encode kinase-active, transformation-defective v-fps alleles were recovered, defining sites in the transforming protein that may normally facilitate kinase-substrate interaction. Additionally, one region within the catalytic domain of the transforming protein (amino acid residues 1012 to 1020) that tolerates peptide insertions without loss of transforming activity was discovered, although the insertion mutations in this region of v-fps exhibited qualitatively abnormal transforming function. Transformed rat cell lines that express these mutations displayed unusual phenotypes, including giant cells and cells with an extremely fusiform shape. Furthermore, the insertion mutations in this region were temperature sensitive, transformed cells assumed a flat morphology, cellular protein phosphotyrosine was reduced, and the kinase activity of the transforming protein was decreased when cells were incubated at 40.5 degrees C. Point mutations that specify the ancestral chicken c-fps sequence in the insertion-tolerant region were also introduced into v-fps. These back mutations led to a modest decrease in kinase activity, decreased tumorigenic potential in chickens, and an unexpected increase in transforming activity in rat cells. These results indicate that the insertion-tolerant region of P130gag-fps influences the biologic activity and thermostability of the kinase.
Insights
Analyzing mutations in the v-fps gene revealed key regions of the P130gag-fps tyrosine kinase. Specific mutations impact substrate selection, cell transformation, and kinase thermostability, offering insights into oncogenic activity.
Area of Science:
- Molecular Biology
- Virology
- Cancer Research
Background:
- The Fujinami sarcoma virus (FSV) v-fps gene encodes the P130gag-fps tyrosine kinase, a potent oncogene.
- Understanding the structure-function relationship of P130gag-fps is crucial for elucidating its transforming capabilities and substrate interactions.
Purpose of the Study:
- To identify specific residues within P130gag-fps critical for kinase catalysis and cellular transformation.
- To investigate the role of the v-fps gene's 3' region in substrate selection and oncogenic activity.
- To characterize the impact of linker insertion mutations on P130gag-fps function and cell phenotype.
Main Methods:
- Generated and analyzed linker insertion mutations throughout the 3' region of the v-fps gene.
- Assessed kinase activity, transforming potential, and substrate interaction of mutant P130gag-fps proteins.
- Examined cellular phenotypes and protein phosphotyrosine levels in transformed rat cell lines expressing mutant v-fps.
- Introduced point mutations to revert v-fps to the ancestral c-fps sequence in specific regions.
Main Results:
- Identified kinase-active, transformation-defective v-fps alleles, suggesting sites involved in kinase-substrate interaction.
- Discovered a peptide insertion-tolerant region (residues 1012-1020) within the catalytic domain, though mutations here altered transforming function.
- Observed unusual cell morphologies (giant, fusiform) in transformed cells with insertion mutations.
- Demonstrated temperature sensitivity of insertion mutations, leading to reduced kinase activity and phosphotyrosine levels at elevated temperatures.
- Showed that reverting v-fps to c-fps sequence in the tolerant region decreased kinase activity and tumorigenicity but increased transforming activity in rat cells.
Conclusions:
- The insertion-tolerant region of P130gag-fps significantly influences its biological activity, including substrate interaction and thermostability.
- Mutations within this region can decouple kinase activity from potent transforming potential, leading to altered cellular phenotypes.
- These findings provide critical insights into the molecular mechanisms underlying v-fps oncogenesis and kinase regulation.
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