MANIPULATION OF KINASE ACTIVITY BY KSHV LANA

描述（由申请方提供）：卡波西肉瘤相关疱疹病毒（KSHV）与内皮病变卡波西肉瘤和B细胞恶性肿瘤原发性渗出性淋巴瘤和多中心Castleman病相关。这些癌症的发病率在免疫缺陷个体中大大增加，包括移植患者和患有AIDS的患者。拉娜是KSHV编码的潜伏蛋白，在所有KSHV相关恶性肿瘤中表达。拉娜是一种多功能蛋白，具有抗凋亡和细胞增殖活性，并且还负责KSHV感染细胞中发生的细胞基因表达的一些重编程。拉娜的多效性功能的潜在重要来源是拉娜介导的丝氨酸/苏氨酸激酶GSK-3的失活和丝氨酸/苏氨酸激酶ERK 1/2的活化。我们建议解决，机械地，拉娜的操纵这两种激酶的贡献，通过获得和整合两组数据：GSK-3和ERK 1/2和拉娜介导的细胞基因表达的变化依赖于GSK-3和ERK 1/2的细胞蛋白底物的全球识别。申请有两个目的。目的1试图用蛋白质组学方法鉴定ERK 1/2磷酸化和ERK 1/2引发的GSK-3磷酸化细胞蛋白的全局谱。将在Co-PI生成的5，000蛋白定制人蛋白阵列上进行ERK 1/2激酶测定。GSK-3需要预先启动磷酸化以获得活性。ERK 1/2引发的GSK-3底物也将在使用人蛋白芯片的激酶测定中鉴定。将使用体外激酶试验并通过检查细胞中的蛋白磷酸化来验证这些试验中鉴定的新型ERK 1/2和ERK 1/2引发的GSK-3底物，其中ERK 1/2和GSK-3激酶活性已被化学抑制剂下调或通过用短沉默miRNA修饰激酶的表达。目的2将检测拉娜介导的转录重编程通过ERK 1/2激活和GSK-3失活介导的程度。将对tet诱导表达拉娜或不与GSK-3相互作用的突变体拉娜的细胞系以及tet诱导的表达ERK 1/2表达下调的拉娜的细胞进行基因阵列分析。然后将寻找由拉娜调节为ERK 1/2和/或GSK-3依赖性的基因与在Aim 1中鉴定为ERK 1/2或GSK-3底物的蛋白质之间的联系。本研究中获得的数据的整合将增加对拉娜对KSHV相关恶性肿瘤的贡献的理解，并且还将提供对发生在其他人类癌症中的过程的机制性见解，这些过程由Ras/MAPK/ERK 1/2和Wnt通路的失调驱动。公共卫生相关性：卡波西肉瘤相关疱疹病毒（KSHV）存在于皮肤癌（卡波西肉瘤）和血癌（淋巴瘤）中，在免疫系统受损的个体中发生频率增加。这些癌症中的拉娜蛋白由KSHV产生。我们正在测试一个假设，即拉娜改变了细胞酶（称为激酶）的活性，这使细胞走上了一条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.