Cotsiki, Marina; (2001) Identification of novel protein interactors of the SV40 large T antigen using the yeast two-hybrid system. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The SV40 large T antigen is a viral oncogene able to immortalize mammalian cells in culture, and to occasionally transform them to tumourigenicity. It can also cause genomic instability by inducing chromosome aberrations and aneuploidy. T antigen is a multifunctional protein which interacts with a number of cellular proteins. Studies have previously shown that the critical activities required for these functions map to the N-terminus of the protein. The N-terminus carries the pRb binding activity and the J domain, but data suggests that there may be additional proteins binding in this region. To isolate such proteins, the yeast two-hybrid approach was chosen, and the N-terminus of T antigen fused to the LexA DNA binding domain was used as a bait. In order to optimize the screen, different versions of the system were setup, by cloning T antigen into an N-terminal fusion vector, two C-terminal fusion vectors and an inducible C-terminal fusion vector. A HeLa library and two mouse embryonic libraries carrying cDNAs fused either to the B42 or the GAL4 activation domains were screened. A number of proteins were identified as potential interactors with the N-terminus of T antigen in yeast. One of these proteins was the Bub1 mitotic checkpoint protein, a member of the family of proteins that monitor the assembly of the mitotic spindle. The interaction of Bub1 with T antigen was investigated in mammalian cells. T antigen was found to co-immunoprecipitate with endogenous Bub1 from extracts of rat embryo fibroblasts conditionally immortalized with T antigen, as well as from NIH 3T3 cells ectopically expressing T antigen. The interaction of T antigen with Bub3, another checkpoint protein of the same family and a binding partner of Bub1, was also demonstrated. In addition, T antigen was found to co-localize with Bub1 in the nuclei of early prophase cells. To investigate the functional significance of this interaction, FACS analysis was used to show that expression of T antigen in cells treated with nocodazole (a microtubule disrupting agent) makes them more refractory to the spindle assembly checkpoint. Moreover, kinase assays indicated that T antigen expression affects the kinase activity of Bub1. The T antigen/Bub1 interaction suggests a novel role for T antigen and provides a new insight into its ability to cause aneuploidy. Finally, a preliminary investigation of two other proteins isolated from the yeast two-hybrid screens, Hsp40 and β-tubulin, indicated that these proteins may also co-localize with T antigen in mammalian cells.

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