PP2A AND POLYOMAVIRUS TUMOR ANTIGEN FUNCTIONS

特异性结合的细胞蛋白质的研究 事实证明，罂粟病毒的肿瘤(T)抗原对 我们对人类癌症的理解。这些研究给出了重要的 对c-src和anti-rc等原癌基因的作用机制的洞察 致癌基因，如视网膜母细胞瘤基因。这项提案试图填补 在我们关于最近发现的细胞的知识空白中 与T抗原、蛋白磷酸酶2A(PP2A)形成复合体的酶。 该提案有两个主要目标：1)确定一个或多个角色 PP2A/T抗原复合体的形成在多种小T抗原(ST)和 中T抗原(MT)的功能；2)研究正常和T 抗原干扰对PP2A功能的调节。方法将是 平行分析正常55 kDa PP2A调控蛋白的功能 亚基和多瘤病毒替代品，ST和MT，在调节 36/63 kDa PP2A杂二聚体。首先，PP2A plus的所有三个亚基 多瘤病毒ST将被过度生产。过量生产的亚基将是 用于体外生化研究以及生产 需要免疫印迹和免疫沉淀抗血清来研究 T抗原与PP2A的体内外相互作用同时，我们 将对36/63的相互作用进行完整的遗传分析 KDA PP2A与ST和55 kDa蛋白的异源二聚体。除了……之外 确定每种蛋白质形成复合体所必需的区域和 活动，这种方法将允许我们确定是否关联 ST和55 kDa蛋白与36/63 kDa异二聚体的相互作用受 36 kDa和63 kDa蛋白的结构变化相似。A精选 将对一组得到的ST突变体进行功能分析，以 确定PP2A/ST关联对个体功能的重要性 多瘤病毒ST.某些ST突变体的平行中T版本将是 构造和分析以更彻底地调查 中间T与PP2A结合以进行中间T介导的转化，以及 各种中间T相关蛋白相互依赖的结合。这个 中T复合体各组分结合的相互依赖性将 也可以通过尝试将中间的T复合体从 纯化杆状病毒蛋白的体外和混合感染 杆状病毒表达每一种成分。我们会用我们的抗血清 分析了PP2在中间T介导的转化中的作用，以及PP2在转化过程中的作用 各种中间T相关蛋白相互依赖的结合。这个 中T复合体各组分结合的相互依赖性将 也可以通过尝试将中间的T复合体从 纯化杆状病毒蛋白的体外和混合感染 杆状病毒表达每一种成分。我们会用我们的抗血清 从生化角度分析PP2A在细胞周期调控中的作用 PP2A亚基的修饰及其形成的复合体的性质 在使用哺乳动物、青蛙卵和酵母的细胞周期过程中 系统。我们将测试我们的各种蛋白质在体内发生突变的能力 调节细胞功能和磷酸化的相互作用位点 Events.es

The study of those cellular proteins which are specifically bound by the tumor (T) antigens of papova viruses is proving increasingly important to our understanding of human cancer. These studies have given important insights into the workings of proto-oncogenes such as c-src and anti- oncogenes such as the retinoblastoma gene. This proposal attempts to fill in gaps in our knowledge concerning the most recently identified cellular enzyme found in complex with T antigens, protein phosphatase 2A (PP2A). This proposal has two major goals: 1) to determine what role (or roles) PP2A/T antigen complex formation plays in various small T antigen (ST) and middle T antigen (MT) functions and 2) to investigate both normal and T antigen-perturbed regulation of PP2A function. The approach will be to analyze in parallel the functioning of the normal 55 kDa PP2A regulatory subunit and the polyomavirus substitutes, ST and MT, in modulating the 36/63 kDa PP2A heterodimer. First, all three subunits of PP2A plus polyomavirus ST will be overproduced. The overproduced subunits will be used for biochemical studies in vitro as well as for production of immunoblotting and immunoprecipitating antisera needed to study the interactions of T antigens and PP2A in vitro and in vivo. In parallel we will perform a complete genetic analysis of the interaction of the 36/63 kDa PP2A heterodimer with ST and with the 55 kDa protein. In addition to determining the regions of each protein necessary for complex formation and activity, this approach will allow us to determine whether association of ST and of the 55 kDa protein with the 36/63 kDa heterodimer are affected by similar structural changes in the 36 kDa and 63 kDa proteins. A select group of the resulting ST mutants will be analyzed in functional assays to determine the importance of PP2A/ST association for individual function of polyomavirus ST. Parallel middle T versions of certain ST mutants will be constructed and analyzed to investigate more thoroughly the importance of middle T binding to PP2A for middle T-mediated transformation, and the interdependent binding of various middle T-associated proteins. The interdependency of binding of the components of middle T complexes will also be studied by attempting to reassemble the middle T complex from purified baculoviral proteins in vitro and in mixed infections of baculoviruses expressing each component. We will use our antisera to analyze of the role of PP2 for middle T-mediated transformation, and the interdependent binding of various middle T-associated proteins. The interdependency of binding of the components of middle T complexes will also be studied by attempting to reassemble the middle T complex from purified baculoviral proteins in vitro and in mixed infections of baculoviruses expressing each component. We will use our antisera to analyze of the role of PP2A in cell cycle regulation by probing biochemical modifications of PP2A subunits and the nature of the complexes they form over the course of the cell cycle using mammalian, frog egg, and yeast systems. We will test the abilities of our various proteins mutated in the interaction sites to modulate cellular function and phosphorylation events.es