Role of p300/CBP in SV40 Large T Antigen Transformation

描述(申请人提供)：这项拟议的项目将研究p300和CBP的活性是否有助于SV40大T抗原(LT)的转化功能。我们将使用人类致癌模型来研究p300/CBP与LT相关的需求。在这个多步骤系统中，LT和活性端粒酶亚基的表达促进了永生化。然后通过表达H-RAS和SV40小T抗原实现完全致瘤转化。由于LT活性在转化的初始步骤中是必需的，这提供了一个极好的模型来扩展当前对LT靶向的细胞通路的理解。我们已经确定了LT和p300/CBP转录共激活因子之间的相互作用，并暗示了P53的桥梁功能。我们的第一个目标是确定我们在细胞条件下发现的LT的乙酰化是否可以归因于p300/CBP结合，以及这是否需要P53的中介作用。这将使用P53-/-MEF细胞或纯化的蛋白质来解决。然后我们将确定p300/CBP活性是否对LT的整体转化性质有贡献。使用竞争性p300片段来破坏LT与内源性p300/CBP的结合，无论有没有融合的乙酰基转移酶结构域，都将被用来确定LT结合是招募p300/CBP乙酰基转移酶活性还是从正常细胞靶点隔离p300/CBP。最后，我们将确定非活性p53的稳定是否可以通过功能的获得进一步促进转化过程，从而将p300/CBP从正常的细胞功能中隔离出来。

DESCRIPTION (provided by applicant): This proposed project will examine whether activities of p300 and CBP contribute to the transforming function of SV40 large T antigen (LT). A requirement for p300/CBP association with LT will be studied using a model of human carcinogenesis. In this multi-step system, expression of LT and the active telomerase subunit promotes immortalization. Full tumorigenic conversion is then conferred through expression of H-Ras and SV40 small T antigen. Since LT activity is required for the initial steps of transformation, this provides an excellent model to expand current understanding of cellular pathways targeted by LT. We have identified an interaction between LT and the p300/CBP transcriptional coactivators and implicate a bridging function of p53. Our first goal is to determine whether the acetylation of LT that we have found under cellular conditions can be attributed to p300/CBP binding and whether this requires an intermediary role of p53. This will be addressed using p53-/- MEF cells or purified proteins. We will then determine whether p300/CBP activity contributes to the overall transforming properties of LT. The use of competitive p300 fragments to disrupt LT binding to endogenous p300/CBP, with or without a fused acetyltransferase domain, will be used to determine whether LT binding recruits p300/CBP acetyltransferase activity or sequesters p300/CBP from normal cellular targets. Finally, we will determine whether the stabilization of inactive p53 can further contribute to the transformation process through a gain of function that serves to sequester p300/CBP from normal cellular functioning.