MANIPULATION OF KINASE ACTIVITY BY KSHV LANA

描述(申请人提供)：Kaposi肉瘤相关疱疹病毒(KSHV)与内皮病变Kaposi肉瘤、B细胞恶性肿瘤、原发渗出性淋巴瘤和多中心Castleman病有关。在包括移植患者和艾滋病患者在内的免疫缺陷个体中，这些癌症的发病率大大增加。LANA是一种KSHV编码的潜伏蛋白，在所有与KSHV相关的恶性肿瘤中都有表达。LANA是一种多功能蛋白，具有抗凋亡和细胞增殖活性，也负责KSHV感染细胞中发生的一些细胞基因表达的重新编程。LANA多效性功能的一个潜在的重要来源是LANA介导的丝氨酸/苏氨酸激酶GSK-3的失活和丝氨酸/苏氨酸激酶ERK1/2的激活。我们建议通过获得和整合两组数据从机制上解决LANA对这两个激酶的操纵的贡献：GSK-3和ERK1/2细胞蛋白底物的全球鉴定以及LANA介导的依赖于GSK-3和ERK1/2的细胞基因表达的变化。该应用有两个目的。目的1试图用蛋白质组学方法鉴定ERK1/2磷酸化和ERK1/2启动的、GSK-3磷酸化的细胞蛋白的全球图谱。ERK1/2激酶分析将在Co-PI产生的5,000个蛋白质定制的人类蛋白质阵列上进行。GSK-3需要事先启动磷酸化才能激活。被ERK1/2激活的GSK-3底物也将在使用人类蛋白芯片的激酶分析中得到鉴定。在这些检测中确定的新的ERK1/2和ERK1/2激活的GSK-3底物将通过体外激酶试验和检测ERK1/2和GSK-3激酶活性已被化学抑制剂下调的细胞中的蛋白质磷酸化来验证，或者通过用短沉默miRNAs改变这些激酶的表达来验证。目的2将研究LANA介导的转录重编程在多大程度上通过ERK1/2激活和GSK-3失活而介导。基因阵列分析将在具有tet诱导的LANA表达的细胞系或与GSK-3不相互作用的突变的LANA，以及在tet诱导的表达ERK1/2下调的LANA的细胞上进行。然后将寻找LANA调控依赖ERK1/2和/或GSK-3的基因与目标1中确定为ERK1/2或GSK-3底物的蛋白质之间的联系。整合这项研究中获得的数据将增加对LANA在KSHV相关恶性肿瘤中所做贡献的理解，并将提供对其他人类癌症中发生的过程的机械性洞察，这些过程是由RAS/MAPK/ERK1/2和Wnt通路的失调驱动的。公共卫生相关性：Kaposi肉瘤相关疱疹病毒(KSHV)存在于皮肤癌(Kaposi肉瘤)和血癌(淋巴瘤)中，这些癌症在免疫系统受损的个人中发生的频率更高。在这些癌症中，LANA蛋白是由KSHV产生的。我们正在测试一种假设，即LANA改变了称为激酶的细胞酶的活性，并使细胞走上了可能导致癌症的道路。

DESCRIPTION (provided by applicant): Kaposi's sarcoma associated herpesvirus (KSHV) is associated with the endothelial lesion Kaposi's sarcoma and the B cell malignancies primary effusion lymphoma and multicentric Castleman's disease. The incidence of these cancers is greatly increased in immuno-deficient individuals including transplant patients and those with AIDS. LANA is a KSHV encoded latency protein that is expressed in all KSHV associated malignancies. LANA is a multi-functional protein that has anti-apoptotic and cell proliferative activities and is also responsible for some of the reprogramming of cell gene expression that occurs in KSHV infected cells. A potentially significant source of LANA's pleiotropic functioning is LANA mediated inactivation of the serine/threonine kinase GSK-3 and activation of the serine/threonine kinase ERK1/2. We propose to address, mechanistically, the contribution of LANA's manipulation of these two kinases by obtaining and integrating two sets of data: Global identification of cell protein substrates of GSK-3 and ERK1/2 and LANA mediated changes in cell gene expression that are dependent upon GSK-3 and ERK1/2. The application has two Aims. Aim 1 seeks to identify the global profile of ERK1/2 phosphorylated and ERK1/2 primed, GSK-3 phosphorylated cell proteins using a proteomic approach. ERK1/2 kinase assays will be performed on a 5,000 protein custom human protein array generated by the Co-PI. GSK-3 requires prior priming phosphorylation for activity. GSK-3 substrates that are ERK1/2 primed will also be identified in kinase assays using the human protein chips. Novel ERK1/2 and ERK1/2 primed, GSK-3 substrates identified in these assays will be validated using in vitro kinase assays and by examining protein phosphorylation in cells in which ERK1/2 and GSK-3 kinase activity has been down-regulated by chemical inhibitors or by modifying expression of the kinases with short silencing miRNAs. Aim 2 will examine the extent to which LANA mediated transcriptional reprogramming is mediated through ERK1/2 activation and GSK-3 inactivation. Gene array analyses will be performed on cell lines with tet-inducible expression of LANA or mutant LANA that does not interact with GSK-3 and on tet-induced LANA expressing cells that have down-regulated ERK1/2 expression. Linkages will then be sought between genes whose regulation by LANA is ERK1/2 and/or GSK-3 dependent and the proteins identified in Aim 1 as being ERK1/2 or GSK-3 substrates. Integration of the data obtained in this study will increase understanding of the contributions made by LANA to KSHV associated malignancies and will also provide mechanistic insight into the processes that occur in other human cancers that are driven by dysregulation of the Ras/MAPK/ERK1/2 and Wnt pathways. PUBLIC HEALTH RELEVANCE: Kaposi's sarcoma associated herpesvirus (KSHV) is present in a skin cancer (Kaposi's sarcoma) and in blood cancers (lymphomas) that occur with increased frequency in individuals whose immune systems are impaired. The LANA protein is produced by KSHV in these cancers. We are testing the hypothesis that LANA changes the activity of cell enzymes called kinases and that this sets the cell on a path that can lead to cancer.