PP2A AND POLYOMAVIRUS TUMOR ANTIGEN FUNCTIONS

PP2A 和多瘤病毒肿瘤抗原功能

• 批准号: 2098058
• 负责人: DAVID C PALLAS
• 金额: $ 27.49万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-16 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2098058
• 关键词: Polyomavirus; antibody formation; binding proteins; cell growth regulation; cell transformation; immunoprecipitation; laboratory mouse; laboratory rabbit; mutant; phosphoprotein phosphatase; phosphorylation; protooncogene; tumor antigens; western blottings

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

蛋白质组学是对那些特异性结合于细胞内的蛋白质的研究， 乳多空病毒的肿瘤（T）抗原被证明越来越重要， 我们对人类癌症的理解。 这些研究提供了重要的 深入了解原癌基因的运作，如c-src和抗- 致癌基因如视网膜母细胞瘤基因。 该提案试图填补 在我们对最近发现的 与T抗原复合的蛋白磷酸酶2A（PP 2A）。 该提案有两个主要目标：1）确定什么角色（或角色） PP 2A/T抗原复合物的形成在各种小T抗原（ST）和 中T抗原（MT）功能和2）调查正常和T PP 2A功能的抗原干扰调节。 方法将是 平行分析正常55 kDa PP 2A调节蛋白的功能， 亚基和多瘤病毒的替代品，ST和MT，在调节 36/63 kDa PP 2A异二聚体。 首先，PP 2A+的所有三个亚基 多瘤病毒ST将过度产生。 生产过剩的亚基将 用于体外生物化学研究以及生产 免疫印迹和免疫沉淀抗血清需要研究 T抗原和PP 2A在体外和体内的相互作用。 同时，我们 将对36/63的相互作用进行完整的遗传分析。 kDa PP 2A异二聚体与ST和与55 kDa蛋白质。 除了 确定复合物形成所必需的每种蛋白质的区域， 活动，这种方法将使我们能够确定是否关联 ST和具有36/63 kDa异二聚体的55 kDa蛋白质受 在36 kDa和63 kDa蛋白质中存在类似的结构变化。 选择 将在功能测定中分析一组所得ST突变体， 确定PP 2A/ST关联对个体功能的重要性， 多瘤病毒ST。某些ST突变体的平行中间T版本将是 构建和分析，以更彻底地调查 中T结合PP 2A用于中T介导的转化， 各种中间T相关蛋白的相互依赖结合。 的 中间T复合物组分结合的相互依赖性将 也可以通过尝试重新组装中间的T复合体来研究， 纯化的杆状病毒蛋白在体外和混合感染的 表达每种组分的杆状病毒。 我们会用抗血清 分析PP 2在中间T介导的转化中的作用， 各种中间T相关蛋白的相互依赖结合。 的 中间T复合物组分结合的相互依赖性将 也可以通过尝试重新组装中间的T复合体来研究， 纯化的杆状病毒蛋白在体外和混合感染的 表达每种组分的杆状病毒。 我们会用抗血清 通过生物化学探针分析PP 2A在细胞周期调控中的作用 PP 2A亚基的修饰及其形成的复合物的性质 在细胞周期中使用哺乳动物、青蛙卵和酵母 系统. 我们将测试我们的各种蛋白质突变的能力， 调节细胞功能和磷酸化的相互作用位点 events.es