Related Experiment Videos
Deletions and insertions within an amino-terminal domain of pp60v-src inactivate transformation and modulate membrane
1Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
Abstract:
We previously showed (V. W. Raymond and J. T. Parsons, Virology 160:400-410, 1987) that variants of the Prague A strain of Rous sarcoma virus containing large deletions impinging on a region of the src gene encoding amino acid residues 143 to 169 were defective for transformation of chicken cells in culture. Here we report that introduction of small (tri-and tetrapeptide) deletions into a region of pp60v-src containing amino acid residues 155 to 175 was found to inactivate transformation. In addition, insertion of four, but not one, amino acid residues at position 161 also inhibited transformation. Biochemical analysis of the src proteins encoded by individual transformation-defective variants revealed that the structural alterations introduced into this domain had only marginal effects upon src tyrosine-specific protein kinase activity. However, the src proteins encoded by defective variants exhibited a significantly shorter half-life within the cell, although these proteins efficiently and rapidly associated with cellular membranes. Our results suggest that the structural domain encompassing residues 155 to 177 may influence the stability of pp60src in the cellular membrane, possibly via the interaction of src with a cellular membrane component(s) or substrate(s).
Insights
Small deletions in the Rous sarcoma virus src gene (pp60v-src) inactivate cell transformation. Altered src proteins show reduced stability but maintain kinase activity, suggesting a role in membrane association.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Previous studies indicated large deletions in the Rous sarcoma virus (RSV) src gene affect cell transformation.
- The specific domain responsible for transformation and protein stability was not fully elucidated.
Purpose of the Study:
- To investigate the role of specific amino acid residues within the pp60v-src protein domain (155-175) in chicken cell transformation.
- To analyze the impact of small deletions and insertions on pp60v-src kinase activity, stability, and membrane association.
Main Methods:
- Site-directed mutagenesis was used to introduce small deletions and insertions into the pp60v-src gene.
- Transformation assays were performed using chicken cells.
- Biochemical analyses assessed protein kinase activity, cellular half-life, and membrane association of mutant src proteins.
Main Results:
- Small deletions (tri- and tetrapeptide) within residues 155-175 of pp60v-src abolished cell transformation.
- Insertion of four amino acids at position 161 also inhibited transformation, while a single amino acid insertion did not.
- Mutant src proteins exhibited only marginal loss of tyrosine kinase activity but had significantly reduced cellular half-lives.
- These altered src proteins efficiently associated with cellular membranes.
Conclusions:
- The structural domain encompassing residues 155-177 of pp60v-src is critical for maintaining transformation.
- This domain may regulate pp60src stability at the cellular membrane, potentially through interactions with membrane components or substrates.
- Protein stability, rather than kinase activity, appears to be the primary factor affected by mutations in this region.