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Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety
Published on: July 24, 2011
Abstract:
The transforming protein of polyoma virus, middle T antigen, is a membrane-associated phosphoprotein. Middle T forms a complex with, and is phosphorylated by, a cellular tyrosine kinase pp60c-src. Mutant analysis suggests that formation of this complex is critical to transformation by polyoma. Middle T binding causes pp60c-src to autophosphorylate at novel sites in its amino terminus, and increases the specific activity of the enzyme. However, at least one non-transforming mutant of middle T, dl1015, can also activate pp60c-src in these ways. This suggests that properties of middle T other than the ability to activate pp60c-src are also necessary for transformation. These properties may include the ability to associate with a phosphatidylinositol kinase, and/or with a protein of 61 kDa. The mechanism by which middle T activates pp60c-src may involve its ability to alter the phosphorylation state of the enzyme, and thus interfere with the regulation of pp60c-src activity in vivo. Polyoma virus transformed cells might then be a good model system for investigating the control of pp60c-src activity as well as defining substrates of the enzyme.
Insights
Polyoma virus middle T antigen activates cellular tyrosine kinase pp60c-src, but this activation alone doesn't cause transformation. Other middle T interactions are crucial for polyoma virus-induced cell changes.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Polyoma virus middle T antigen is a key viral oncoprotein.
- Middle T antigen is a membrane-associated phosphoprotein involved in cell transformation.
- Middle T antigen interacts with cellular tyrosine kinase pp60c-src.
Purpose of the Study:
- To investigate the role of middle T antigen-pp60c-src complex formation in polyoma virus transformation.
- To identify other essential interactions of middle T antigen for cellular transformation.
- To elucidate the mechanism by which middle T antigen activates pp60c-src.
Main Methods:
- Mutant analysis of middle T antigen.
- Biochemical assays to study protein complex formation and phosphorylation.
- Enzyme activity measurements of pp60c-src.
Main Results:
- Middle T antigen forms a complex with and is phosphorylated by pp60c-src.
- Middle T binding induces pp60c-src autophosphorylation and increases its specific activity.
- A non-transforming middle T mutant (dl1015) activates pp60c-src, indicating other factors are necessary for transformation.
- Middle T may transform cells by associating with phosphatidylinositol kinase and/or a 61 kDa protein.
- Middle T might interfere with pp60c-src regulation by altering its phosphorylation state.
Conclusions:
- Middle T antigen-induced pp60c-src activation is necessary but not sufficient for polyoma virus transformation.
- Additional interactions of middle T antigen, possibly with phosphatidylinositol kinase or a 61 kDa protein, are critical for oncogenic transformation.
- Polyoma virus-transformed cells serve as a model for studying pp60c-src regulation and identifying its substrates.
