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Cytoplasmic interaction between pp60c-src and a truncated polyoma virus middle T antigen
S A Courtneidge1, M Read, J B Wilson
1European Molecular Biology Laboratory, Heidelberg, West Germany.
Abstract:
We have investigated the subcellular localization, transforming capacity and cellular protein binding properties of a mutant middle T antigen, 82JF3. This mutant lacks the carboxyterminal 86 amino acids of middle T and, as expected, failed to associate with cellular membranes. Like other cytoplasmic mutants, it also failed to transform. Nevertheless, 82JF3 middle T antigen associated with pp60c-src and increased its tyrosine kinase activity. The associated pp60c-src was also cytoplasmic, raising interesting questions about the nature and formation of the complex. This soluble complex also contained very reduced levels both of the p81 protein and of associated phosphatidylinositol (PI) kinase activity. These results are consistent with the hypothesis that p81 is a component of PI kinase.
Insights
A mutant middle T antigen, 82JF3, lacking membrane association, still bound and activated pp60c-src tyrosine kinase in the cytoplasm. This suggests p81 protein is a component of phosphatidylinositol (PI) kinase activity.
Area of Science:
- Molecular and Cellular Biology
- Oncogenesis
- Protein Kinase Signaling
Background:
- Middle T antigen is a viral oncoprotein known to associate with cellular membranes and activate signaling pathways.
- Mutations in middle T antigen can disrupt its localization and function, providing insights into its biological activity.
Purpose of the Study:
- To investigate the subcellular localization, transforming capacity, and protein binding properties of a specific middle T antigen mutant (82JF3).
- To elucidate the role of the carboxyterminal domain of middle T antigen in its interactions with cellular proteins and its transforming ability.
Main Methods:
- Site-directed mutagenesis to create the 82JF3 middle T antigen mutant.
- Analysis of subcellular localization using cell fractionation techniques.
- Assessment of transforming capacity in cell-based assays.
- Co-immunoprecipitation assays to study protein-protein interactions.
- In vitro kinase assays to measure tyrosine kinase activity.
Main Results:
- The 82JF3 mutant, lacking the C-terminal 86 amino acids, failed to associate with cellular membranes and did not transform cells.
- Despite its cytoplasmic localization, 82JF3 associated with pp60c-src and increased its tyrosine kinase activity.
- The soluble pp60c-src/82JF3 complex showed reduced levels of p81 protein and phosphatidylinositol (PI) kinase activity.
Conclusions:
- The carboxyterminal domain of middle T antigen is essential for membrane association and cellular transformation.
- Middle T antigen can associate with and activate pp60c-src in the cytoplasm.
- The findings support the hypothesis that p81 protein is a component of PI kinase activity.