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Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant
Abstract:
Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.
Insights
Polyoma virus medium T antigen associates with pp60c-src, activating its kinase activity. This complex showed minimal phosphatidylinositol kinase activity, unlike related viral proteins, suggesting specific roles in cell transformation.
Area of Science:
- Molecular Biology
- Virology
- Cellular Biology
Background:
- Medium T antigen, a polyoma virus protein, interacts with pp60c-src, enhancing its tyrosine kinase activity.
- Certain viral transforming proteins, like pp60v-src and p68v-ros, exhibit phosphatidylinositol kinase activity.
- The role of the medium T-pp60c-src complex in phosphatidylinositol phosphorylation remains unclear.
Purpose of the Study:
- To investigate if the medium T-pp60c-src complex possesses phosphatidylinositol kinase activity.
- To compare the enzymatic activities of medium T from transforming and non-transforming polyoma virus mutants.
Main Methods:
- Purification of medium T antigen from polyoma virus-infected mouse fibroblasts using sequential affinity chromatography.
- Immune complex isolation utilizing antibodies against specific medium T peptides and pp60c-src.
- Assays for protein kinase and phosphatidylinositol kinase activities on purified complex fractions.
Main Results:
- Highly purified medium T-pp60c-src complexes exhibited very low phosphatidylinositol kinase activity.
- No significant difference in phosphatidylinositol kinase activity was observed between medium T from transforming viruses and hr-t mutants.
- Protein kinase activity was detected in medium T purified from transforming viruses but not from hr-t mutants.
Conclusions:
- The medium T-pp60c-src complex is not a significant phosphatidylinositol kinase.
- The protein kinase activity associated with medium T antigen is crucial for polyoma virus-mediated cell transformation, distinguishing it from phosphatidylinositol kinase activity observed in other viral oncoproteins.