Products of the Transforming Genes of Polyomavirus

描述（申请人提供）：小鼠多瘤病毒为肿瘤转化提供了重要的模型。这种病毒会引起广泛的肿瘤。在实验室得出的结论可以很容易地在动物身上进行试验。对多瘤的研究一再提供了对细胞生长调节的基本机制的见解。酪氨酸激酶和磷脂酰肌醇3-激酶（PI3K）信号传导是多瘤研究发现的两个基本机制。转化由三种病毒基因产物的作用产生：大T （LT）、中T （MT）和小T （ST）抗原。有四个特定的目标将为它们如何工作提供新的见解：1)第一个特定的目标涉及大T，宿主细胞RNA表达的模式将由不同的大T途径决定。将建立LT调控含有CREB/ATF位点的基因的机制。由于乙酰化与转录调控有关，因此将建立LT上的乙酰化位点，并通过位点定向诱变测试其功能。最近的研究结果表明，LT导致G2/M阻滞。将进行实验以确定LT引起这种阻滞的机制。我们已经确定了两个新的lt结合伙伴，Pinl和Bubl，它们是G2/M的已知调节因子。我们将探讨它们在LT功能中的作用。2)第二个具体目标涉及小t，在多瘤和SV40小t之间已经发现了功能上的重要差异，表达谱将用于确定它们之间差异的程度和基础。将通过串联亲和纯化和质谱技术寻找新的多瘤小T伴侣。蛋白磷酸酶2A是一个关键的小T靶点。实验将区分小T靶向PP2A的不同机制。3)人类乳腺上皮细胞提供了一个有用的人类癌症模型。第三个具体目标将使用该模型探索中T和小T的功能，确定转化所需的MT信号通路。结合新伴侣的小T突变体将在这些细胞中进行测试。4)我们的第四个具体目标涉及使用核磁共振进行结构研究。将确定LT的n端结构域（NT）的结构。该结构域足以促进细胞生长、引起细胞凋亡和调节细胞RNA转录。我们还将开展多瘤小T的结构测定研究。

DESCRIPTION (provided by applicant): Murine polyomavirus provides an important model for neoplastic transformation. The virus causes a broad spectrum of tumors. Conclusions reached in the laboratory can be readily tested in animals. Studies on polyoma have repeatedly provided insight into basic mechanisms of cellular growth regulation. Tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) signaling are two fundamental mechanisms uncovered from studies on polyoma. Transformation results from the action of three viral gene products: large T (LT), middle T (MT) and small T (ST) antigens. There are four specific aims that will provide new insight into how they work: 1) the first specific aim concerns large T. Patterns of host cellular RNA expression that result from different large T pathways will be determined. The mechanism will be established by which LT regulates genes containing CREB/ATF sites. Because acetylation has been connected to transcriptional regulation, the sites of acetylation on LT will be established and their function tested by site-directed mutagenesis. Recent results show that LT causes G2/M arrest. Experiments will be carried out to determine the mechanism by which LT causes this arrest. We have identified two new LT-binding partners, Pinl and Bubl, which are known regulators of G2/M. Their role in LT function will be probed. 2) The second specific aim concerns small T. Important differences in function have been detected between polyoma and SV40 small T. Expression profiling will be used to identify the extent of, and the basis for, the differences between them. New polyoma small T partners will be sought by tandem affinity purification and mass spec. Protein phosphatase 2A is a critical small T target. Experiments will be performed to distinguish between different mechanisms by which small T targets PP2A. 3) Human mammary epithelial cells provide a useful model of human cancer. The third specific aim will use the model to probe the function of middle T and small T. The MT signaling pathways required for transformation will be determined. Small T mutant in binding novel partners will be tested in these cells. 4) Our fourth specific aim involves structural studies using NMR. The structure of the N-terminal domain (NT) of LT will be determined. This domain is sufficient to promote cell growth, to cause apoptosis and to regulate cellular RNA transcription. We will also initiate studies to carry out structure determination of polyoma small T.