Determining the Role of Bub1 in T Antigen Mediated Transformation

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We have detected a functional interaction between SV40 large T antigen and the mitotic regulator Bub 1. The protein kinase Bub1, a member of the family of checkpoint proteins that monitor the assembly of the mitotic spindle, has been found to be mutated in certain human cancers characterized by aneuploidy. Notably, T antigen can also cause genomic instability by inducing chromosomal aberrations and aneuploidy. T antigen co-immunoprecipitates with endogenous Bub1, as well as with Bub3, another component of the checkpoint complex at the kinetochore. While we have come to think of T antigen as an inactivator of its target proteins as is the case with p53 and pRb, T actually enhances Bub1 kinase activity. Bub1 phosphorylates p53 on ser37, previously identified as a site of phosphorylation by DNA-PK. Genetic analysis demonstrates that interaction of T antigen with Bub1 is not required for immortalization but appears to be necessary for T antigen to deregulate the spindle assembly checkpoint and transform. This interaction with Bub1 suggests a novel role for T antigen, which may provide new insight into its ability to regulate p53, cause chromosome aberrations, and transform. We propose to study the interaction in detail. It is our intent to determine: (1) What are the the mechanistic consequences of Bub1 mediated phosphorylation of p53? (2) Does T antigen direct Bub1 toward other substrates in addition to p53? And (3) Is T antigen's interaction with Bub1 required for T antigen to destabilize the chromosomes in its target cells?