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Polyoma virus middle T antigen-pp60c-src complex associates with purified phosphatidylinositol 3-kinase in vitro
K R Auger1, C L Carpenter, S E Shoelson
1Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Abstract:
Reconstitution of the polyoma virus middle T antigen (mT)-pp60-src complex and phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been accomplished in vitro with immunopurified baculovirus-expressed mT-pp60c-src and PtdIns 3-kinase purified from rat liver. Both the 110- and 85-kDa subunits of the PtdIns 3-kinase associated with the mT-pp60c-src complex. The association of PtdIns 3-kinase with the mT-pp60c-src complex was dependent on the protein-tyrosine kinase activity of pp60c-src as a kinase-inactive mutant (pp60(295c-src)) still complexed with mT, but the mT-pp60(295c-src)) complex was unable to bind PtdIns 3-kinase. The mT-pp60c-src complex phosphorylated both subunits of PtdIns 3-kinase on tyrosine residues. The immunopurified mT-pp60c-src complex also associated with PtdIns 3-kinase activity from whole cell lysates, and this association was dependent upon the protein-tyrosine kinase activity of pp60c-src. Comparison of 35S-labeled proteins from whole cell lysates which associated with immunopurified mT-pp60c-src and mT-pp60(295c-src) revealed proteins of 110 and 85 kDa as the major peptides dependent on protein-tyrosine kinase activity for association with the complex. In addition, a synthetic phosphopeptide (13-mer) containing sequences conserved between the major tyrosine phosphorylation site of murine polyoma virus mT, hamster polyoma virus mT, and the insulin receptor substrate (IRS-1) specifically blocked the association of the 85- and 110-kDa polypeptides with the mT-pp60c-src complex. The ability to block the association was dependent on the tyrosine phosphorylation of the peptide. Association of PtdIns 3-kinase activity was blocked concurrently. This is the first demonstration that the 110-kDa subunit of PtdIns 3-kinase can associate with mT-pp60c-src. This association in vitro is a step toward understanding protein-protein interactions important in the signal transduction pathway of oncogenic proteins.
Insights
This study demonstrates that polyoma virus middle T antigen (mT)-pp60c-src complex binds phosphatidylinositol 3-kinase (PtdIns 3-kinase) in a protein-tyrosine kinase-dependent manner. This interaction is crucial for oncogenic signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Virology
Background:
- The polyoma virus middle T antigen (mT) is a potent oncogene.
- mT forms a complex with cellular pp60c-src kinase, activating its protein-tyrosine kinase activity.
- Phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a key enzyme in signal transduction pathways.
Purpose of the Study:
- To reconstitute and characterize the interaction between the mT-pp60c-src complex and PtdIns 3-kinase in vitro.
- To elucidate the role of pp60c-src kinase activity in this association.
- To identify the subunits of PtdIns 3-kinase involved in the complex formation.
Main Methods:
- In vitro reconstitution using immunopurified mT-pp60c-src and PtdIns 3-kinase.
- Kinase-inactive pp60c-src mutant was used to assess the role of kinase activity.
- Analysis of protein association using labeled proteins and synthetic phosphopeptides.
Main Results:
- The mT-pp60c-src complex associated with both 110- and 85-kDa subunits of PtdIns 3-kinase.
- This association was dependent on the protein-tyrosine kinase activity of pp60c-src.
- A synthetic phosphopeptide mimicking mT and IRS-1 phosphorylation sites blocked PtdIns 3-kinase association.
Conclusions:
- This study provides the first evidence for the association of the 110-kDa PtdIns 3-kinase subunit with the mT-pp60c-src complex.
- The interaction is mediated by the kinase activity of pp60c-src.
- Understanding this protein-protein interaction is vital for deciphering oncogenic signaling pathways.