PROJECT 3: Pathways Controlled by PP2A A-Beta in Normal, Transformed and Tumor Cells

负责高达50%的细胞磷酸丝氨酸/苏氨酸磷酸酶活性，蛋白磷酸酶2A （PP 2A）调节几乎所有的细胞信号通路。PP 2A有80多种异源三聚体， 一个催化C亚基和一个调节B亚基与Aα（90%丰度）或（10%丰度）结合 丰富）Aβ亚基支架。我们的前提是，使用Aβ支架的蛋白磷酸酶2A（PP 2A） 对于控制正常细胞和癌细胞的表型至关重要。此外，研究 多瘤病毒，一再给予新的见解，生长控制，将是无价的， 了解Aβ功能。我们对鼠多瘤病毒（MuPyV）ST/MT的研究已经证明了 Aβ对存活、分化和转化的重要性。我们的sh/siRNA Aβ敲除证实了其 重要性，即使在没有病毒的情况下，对癌症的重要途径。Akt和c-Src信号传导都是 由Aβ调节。此外，人类肺癌、乳腺癌和结肠癌显示出Aβ的改变，这表明， β-PP 2A介导的信号传导与癌症相关。关于Aβ的研究很少，所以有一个 迫切需要研究其功能。在目标1中，我们将在功能调查中使用基础广泛的技术 并整合这些方法来鉴定正常和转化细胞中Aβ改变的途径。 通过RNA-seq、磷酸化蛋白质组学分析和NMR代谢组学进行的表达分析将确定途径 PP 2A Aβ特异性靶向。对照与表达MuPyV MT或ST的细胞的比较将提供信息。 我们可以确定癌蛋白是否抑制Aβ活性和/或将其重定向到新的靶点。目标二，聚焦 Aβ，将决定Aβ/ST结构。将测定结合Aβ的PP 2 A B亚基和其他靶标。 Aβ的遗传分析将揭示导致其独特表型的序列。In Aim 3 Aβ 酪氨酸激酶信号的调节将被检查，以了解c-Src控制的机制， 确定Aβ如何广泛控制酪氨酸磷酸化，可能通过酪氨酸磷酸酶。St 将鉴定Aβ结合缺陷的突变体以测试Aβ在控制细胞表型中的作用。最后， 我们将利用条件性基因敲除技术证实Aβ在Her 2/neu和MT肿瘤发生中的作用。 !

Responsible for up to 50% of cellular phosphoserine/threonine phosphatase activity, protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. PP2A comes as > 80 kinds of heterotrimers, consisting of a catalytic C subunit and one of many regulatory B subunits bound to an Aα (90% abundant) or (10% abundant) Aβ subunit scaffold. Our premise is that protein phosphatase 2A (PP2A) using the Aβ scaffold is fundamentally important for controlling phenotypes of both normal and cancer cells. Moreover, study of polyomaviruses, which have repeatedly given novel insights into growth control, will be invaluable to understand Aβ function. Our studies of murine polyomavirus (MuPyV) ST/MT already demonstrate the importance of Aβ to survival, differentiation and transformation. Our sh/siRNA Aβ knockdowns of confirm its importance, even in the absence of virus, to pathways important in cancer. Both Akt and c-Src signaling are regulated by Aβ. In addition, human lung, breast and colon cancers show alterations in Aβ, suggesting that Aβ-PP2A-mediated signaling is relevant to cancer. Very little work has been done on Aβ, so there is a pressing need to study its function. In Aim 1 we will use broad-based technologies in a survey of functions and integrate these approaches to identify pathways altered by Aβ in normal and transformed cells. Expression analysis by RNA-seq, phosphoproteomic analysis, and NMR metabolomics will identify pathways specifically targeted by PP2A Aβ. Comparisons of controls with cells expressing MuPyV MT or ST will inform us whether the oncoproteins are inhibiting Aβ activity and/or redirecting it to new targets. Aim 2, focusing on Aβ, will determine the Aβ/ST structure. PP2A B subunits and other targets that bind Aβ will be determined. Genetic analysis of Aβ will uncover sequences responsible for its unique phenotype(s). In Aim 3 Aβ regulation of tyrosine kinase signaling will be examined to learn the mechanism of c-Src control and to determine how Aβ broadly controls tyrosine phosphorylation, perhaps via tyrosine phosphatases. ST mutants defective for Aβ binding will be identified to test the role of Aβ in controlling cell phenotype. Finally, we will confirm the role of Aβ in Her2/neu and MT tumorigenesis using conditional knockout technology. !