The Role of Bub1 in SV40 Large T Mediated Transformation

我们发现了三种蛋白质之间一组有趣的功能相互作用：SV40大T 抗原(LT)、P53肿瘤抑制蛋白和有丝分裂酶Bub1。Bub1是这个家庭的一员 监控有丝分裂纺锤体组装的检查点蛋白，并已被发现在 某些以非整倍体为特征的人类癌症。LT抗原还可以通过以下方式导致基因组不稳定 诱发染色体异常和非整倍体。遗传分析表明，T的相互作用 带有Bub1的抗原不是永生所必需的，但T抗原是驱动病毒复制所必需的 并进行改造。值得注意的是，LT似乎是一个支架，将Bub1带到P53上，然后P53被磷酸化 在Ser37上直接通过Bub1，在Ser15上间接通过第二个激酶，导致P53的稳定。 初步数据表明，这种对p53的稳定对于LT的转化是必要的，至少在 在某些情况下。同样值得注意的是，发现通过LT表达或通过 在缺乏LT的情况下，通过针对Bub1的RNAi，导致P53依赖的细胞衰老，并伴随 Ser37的磷酸化。最近，另一个研究小组证明了ras诱导衰老，这是 我们可以阻断Bub1的显性负等位基因，也依赖于丝氨酸上P53的磷酸化 37.在这笔赠款的背景下，关键问题是Bub1如何促进LT介导的复制和 在没有LT的情况下，通过衰老抑制肿瘤的形成。我们可以想象 几种可能的机制：它可能使用Bub1通过其直接的 P53的磷酸化，通过Bub1介导的LT复合体或更多其他靶标的磷酸化 间接地通过颠覆Bub1在调节基因组稳定性中的作用。这些机制不需要 相互排斥。显然，类似的机制可能也在发挥作用，因为Bub1保护“正常”细胞 通过促进衰老来转化。在本应用程序中，我们建议探索其中的每一个 机制依次发生变化。

We have discovered an interesting set of functional interactions among three proteins: SV40 Large T antigen (LT), the p53 tumor suppressor protein and the mitotic kinase Bub1. Bub1 is a member of the family of checkpoint proteins that monitor the assembly of the mitotic spindle, and has been found to be mutated in certain human cancers characterized by aneuploidy. LT antigen can also cause genomic instability by inducing chromosomal aberrations and aneuploidy. Genetic analysis demonstrates that interaction of T antigen with Bub1 is not required for immortalization but is necessary for T antigen to drive viral replication and transform. Notably LT appears to act as a scaffold bringing Bub1 to p53, which then is phosphorylated on ser37 directly by Bub1 and on ser15, indirectly by a second kinase, leading to the stabilization of p53. Preliminary data suggests that this stabilization fo p53 is necessary for transformation by LT at least in certain circumstances. Also of note is finding that downregulating Bub1 function either by LT expression or by RNAi against Bub1 in the absence of LT, results in p53 dependent cellular senescence, accompanied by phosphorylation of ser37. Recently another group has demonstrated that ras induced senescence, which we can block with a dominant negative allele of Bub1, is also dependent on phosphorylation of p53 at serine 37. The key questions in the context of this grant are how Bub1 contributes to LT mediated replication and transformation and to suppressing tumor formation via senescence in the absence of LT. We can imagine several possible mechanisms: LT may use Bub1 to regulate replication and transformation via its direct phosphorylation of p53, via Bub1 mediated phosphorylation of other targets in the LT complex or more indirectly via by perverting Bubl's role in regulating genome stability. These mechanisms need not be mutually exclusive. Obviously similar mechanisms may be in play as Bub1 guards "normal" cells from transformation by promoting senescence. In this application we propose to probe each of these mechanisms in turn.