The Role of Bub1 in SV40 Large T Mediated Transformation

我们发现了三种蛋白质之间有趣的功能相互作用：SV40 Large T 抗原（LT）、p53肿瘤抑制蛋白和有丝分裂激酶Bub1。Bub1是家庭的一员 检查点蛋白的监测有丝分裂纺锤体的组装，并已被发现突变， 某些以非整倍体为特征的人类癌症。LT抗原还可通过以下方式引起基因组不稳定性： 诱导染色体畸变和非整倍性。遗传分析表明，T Bub1抗原不是永生化所必需的，但对于T抗原驱动病毒复制是必需的 和变形值得注意的是，LT似乎作为一个支架，将Bub1带到p53，然后磷酸化 通过Bub1直接作用于ser37，通过第二激酶间接作用于ser15，导致p53的稳定。 初步的数据表明，p53的这种稳定性对于LT的转化是必要的，至少在 某些情况下。同样值得注意的是，发现下调Bub1功能，无论是通过LT表达， 在没有LT的情况下，通过针对Bub1的RNAi，导致p53依赖性细胞衰老，伴随着 丝氨酸37的磷酸化。最近，另一个研究小组证明ras诱导衰老， 我们可以用Bub1的显性负性等位基因阻断，也依赖于p53丝氨酸磷酸化， 37.本研究的关键问题是Bub1如何促进LT介导的复制， 在没有LT的情况下，通过衰老来抑制肿瘤的形成。 几种可能的机制：LT可能通过Bub1直接调控复制和转化， 通过Bub1介导的LT复合物中其他靶点的磷酸化或更多， 间接地通过改变Bubl在调节基因组稳定性中的作用。这些机制不必 相互排斥。显然，类似的机制可能在起作用，因为Bub1保护“正常”细胞， 通过促进衰老进行转化。在本申请中，我们建议探测这些中的每一个 机制反过来。

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.