PROTEIN PHOSPHATASE 2A IN TRANSFORMATION BY POLYOMA/SV40

多瘤病毒中、小肿瘤抗原与肿瘤免疫反应的关系 SV 40小肿瘤抗原与蛋白磷酸酶2A形成复合物 病毒感染的细胞。 多瘤中T抗原突变体的研究 我已经表明，这种相互作用，沿着与其他几个 细胞蛋白质，是必要的，但不足以转化。 虽然SV 40小T本身不能引起转化， 增强大T抗原的转化。 目前的证据表明 SV 40小T的所有生物学效应都是由 与磷酸酶2A相互作用。 我们开发了一种重组的 由纯化的磷酸酶2A或其亚基组成的系统，和 重组小T.小T和磷酸酶2A在体外形成复合物， 这种相互作用以类似于 由通常存在于酶中的B亚基引起的。 我们目前 模型提出，小T的生物效应是由于变化 在磷酸酶2A对一组选定底物的活性中， 2A类活动的总体水平没有重大变化。 本项目将研究SV 40小T与 蛋白磷酸酶2A及其形成的生物化学效应 复杂. 我们还将尝试确定潜在的底物， 磷酸化受到这种相互作用的影响。 一系列小T 将制备改变生物化学和转化的突变体， helper函数 这些突变体将用于复溶试验， 确定小T-磷酸酶相互作用的分子基础。 小T（和具有改变的生物学特性的小T突变体）的影响 特性）对磷酸酶2A活性的影响将用一系列 模型基质 我们还将确定， 潜在的体内底物，包括SV 40大T，视网膜母细胞瘤 p53通过与小T的相互作用而改变。这些研究 应该可以让我们确定小T对转化的影响 是由于与磷酸酶2A的相互作用。 的参与 小T与磷酸酶相互作用中的细胞交换因子 2A将进行测试。 一种先前表征的生物化学活性， 小T是某些基因的反式激活。 我们将确定 参与的小T/磷酸酶2A复合物的潜在底物 腺病毒E2和T细胞受体γ链的反式激活 基因，使用体外转录测定。

The middle and small tumor antigens of polyoma virus and the small tumor antigen of SV40 form complexes with protein phosphatase 2A in virally infected cells. Studies with mutants of polyoma middle T antigen have shown that this interaction, along with those of several other cellular proteins, is necessary but not sufficient for transformation. Although not capable of causing transformation by itself, SV40 small T enhances transformation by large T antigen. Current evidence suggests that all of the biological effects of SV40 small T are mediated by interaction with phosphatase 2A. We have developed a reconstituted system consisting of purified phosphatase 2A, or its subunits, and recombinant small T. Small T and phosphatase 2A form complexes in vitro, and this interaction inhibits phosphatase activity in a manner similar to that caused by the B subunit normally present in the enzyme. Our current model proposes that the biological effects of small T are due to changes in the activity of phosphatase 2A toward a selected set of substrates, without a major change in the overall level of type 2A activity. This project will study the interaction of SV40 small T with protein phosphatase 2A and the biochemical effects of formation of this complex. We will also attempt to identify potential substrates whose phosphorylation are influenced by this interaction. A series of small T mutants will be prepared that have altered biochemical and transformation helper functions. These mutants will be used in reconstitution assays to determine the molecular basis for the small T-phosphatase interaction. The effects of small T (and small T mutants with altered biological properties) on phosphatase 2A activity will be tested with a series of model substrates. We will also determine if dephosphorylation of potential in vivo substrates including SV40 large T, the retinoblastoma protein, and p53 is altered by interaction with small T. These studies should allow us to determine if the effects of small T on transformation are due to interaction with phosphatase 2A. The involvement of a cellular exchange factor in the interaction of small T with phosphatase 2A will be tested. A previously characterized biochemical activity of small T is the transactivation of certain genes. We will identify potential substrates of the small T/phosphatase 2A comp involved in trans-activation of the adenovirus E2 and T cell receptor gamma-chain genes, using in vitro transcription assays.