Determining the Role of Bub1 in T Antigen Mediated Transformation

我们已经检测到SV 40大T抗原和有丝分裂调节因子Bub 1之间的功能性相互作用。蛋白激酶Bub 1是监测有丝分裂纺锤体组装的检查点蛋白家族的成员，已发现在某些以非整倍性为特征的人类癌症中发生突变。值得注意的是，T抗原还可以通过诱导染色体畸变和非整倍性而引起基因组不稳定性。T抗原与内源性Bub 1以及Bub 3（动粒处检查点复合物的另一种组分）共免疫沉淀。虽然我们认为T抗原是其靶蛋白的灭活剂， 与p53和pRb的情况一样，T实际上增强Bub 1激酶活性。Bub 1磷酸化p53的ser 37，以前确定为一个网站的磷酸化DNA-PK。遗传分析表明，T抗原与Bub 1的相互作用不是永生化所必需的，但似乎是T抗原解除纺锤体组装检查点和转化所必需的。这种与Bub 1的相互作用表明T抗原的新作用，这可能为其调节p53，引起染色体畸变和转化的能力提供新的见解。我们建议详细研究相互作用。我们的目的是确定： (1)Bub 1介导的p53磷酸化的机制后果是什么？(2)除了p53，T抗原是否指导Bub 1朝向其他底物？（3）T抗原是否需要与Bub 1相互作用才能使靶细胞的染色体不稳定？

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?