Analysis of cellular signaling pathways activated by polyomavirus middle T antigen.

摘要

In its host organism, polyomavirus expresses a total of only 6 viral proteins that cause the deregulation of multiple cellular processes for oncogenic transformation. Middle T (mT) antigen is considered the primary transforming agent of polyomavirus, since deletion of mT from the polyornavirus genome results in a nontransforming virus. This thesis focuses on mT and attempts to characterize the cellular signaling pathways utilized by mT in stable mT transformation of cells.;mT requires SHC binding for transformation. If the SHC binding site within mT is mutated, reinsertion of a minimal NPTY sequence can rescue SHC binding and mT transformation. Other sequences were tested for transformation rescue; however none of the new mutants could function in transformation. Other mutants tested, including mutations made adjacent to the original Y250 at T249, indicate that Src in a middle T complex has a distinct preference to phosphorylate only an NPTY sequence for SHC binding and mT transformation.;Phosphatidylinositol 3 kinase (P13K) is also required for mT transformation. A screen was designed to uncover novel mutants of mT that can transform despite mutations in the P13K binding sequence. Four new mutants are described in this study that suggest there may exist multiple ways to compensate for P13K binding to mT for transformation.;As a consequence of phosphatidylinositol-3 kinase (P13K) activation, mT stably activates the Akt serine/threonine kinase. Using mT mutants, other signaling pathways were examined for their potential contribution to Akt activation in mT cells. Data presented here suggests that SHC positively contributes to mT mediated activation of Akt, and that a constitutively active PLC-γ may negatively regulate Akt in mT cells. Akt in both wild type and mT mutant cell lines were also examined by immunofluorescence since Akt localization has been shown to be an integral part of transient Akt activation by growth factors. Wild-type mT's constitutive activation of P13K may contribute to the relocalization of activated Akt in stable mT cell lines. These results suggest that not only the P13K pathway but also crosstalk from the SHC and PLC-γ pathways contribute to the mT activation and regulation of Akt.