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Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity
Abstract:
Middle T antigen of polyoma virus is associated principally with the plasma membrane. Comparison of the trypsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [gamma-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32PO4 were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.
Insights
Polyoma virus middle T antigen is located inside the cell membrane and is associated with the cell framework. Its kinase activity suggests it may interact with a cellular tyrosine kinase.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- The middle T antigen of polyoma virus is a key protein involved in viral oncogenesis.
- Its precise localization and function within the cell are crucial for understanding its role.
Purpose of the Study:
- To determine the cellular orientation and localization of polyoma virus middle T antigen.
- To investigate the kinase activity associated with middle T antigen and its potential interaction partners.
Main Methods:
- Differential trypsin sensitivity assays on intact cells and membrane preparations.
- Cell permeabilization and radiolabeling with [gamma-32P]ATP.
- Analysis of middle T antigen distribution in cell frameworks using Triton buffers.
- In vitro kinase assays and inhibition studies with ATP affinity reagents.
Main Results:
- Middle T antigen is oriented towards the intracellular side of the plasma membrane.
- Kinase-active middle T antigen fractions are resistant to trypsin and associated with the cell framework.
- Phosphorylation of middle T antigen in vivo also localizes it to the cell framework.
- ATP affinity reagents failed to label middle T antigen directly but could inhibit kinase activity.
Conclusions:
- Middle T antigen's intracellular orientation and association with the cell framework are established.
- The kinase activity associated with middle T antigen is distinct from the bulk protein.
- Evidence suggests middle T antigen may associate with a cellular tyrosine kinase rather than possessing intrinsic activity.