MANIPULATION OF KINASE ACTIVITY BY KSHV LANA

KSHV LANA 对激酶活性的调控

• 批准号: 7619329
• 负责人: S DIANE HAYWARD
• 金额: $ 21.65万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7619329
• 关键词: Acquired Immunodeficiency Syndrome; Address; Apoptosis; Apoptotic; B lymphoid malignancy; B-Lymphocytes; Binding; Binding Proteins; Biological Assay; Cell Line; Cells; Chemicals; Complement; Cultured Cells; Custom; Data; Data Set; EMSA; Embryo; Endothelial Cells; Enzymes; Fibroblasts; Frequencies; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Glycogen Synthase Kinase 3; Growth; Hematopoietic Neoplasms; Human; Human Herpesvirus 8; Hyperplasia; Immune system; In Vitro; Incidence; Individual; Kaposi Sarcoma; Laboratories; Lead; Lesion; Link; Longevity; Luciferases; Lymphoma; MAPK1 gene; MAPK3 gene; Malignant Neoplasms; Mediating; Modeling; Multicentric Angiofollicular Lymphoid Hyperplasia; Nuclear; Outcome; Pathway interactions; Phase; Phosphorylation; Phosphotransferases; Process; Protein Array; Protein Microchips; Protein-Serine-Threonine Kinases; Proteins; Proteomics; Rattus; Reagent; Regulation; Reporter; Reverse Transcriptase Polymerase Chain Reaction; S-Phase Fraction; Skin Cancer; Source; Testing; Tetanus Helper Peptide; Time; Transgenic Mice; Transplant Recipients; Up-Regulation; Viral; c-myc Genes; cell growth; data integration; effusion; immortalized cell; in vivo; inhibitor/antagonist; insight; interest; kinase inhibitor; mutant; novel; oncology; prevent; promoter; public health relevance; scaffold; transcription factor

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.

描述（由申请人提供）：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。在免疫缺陷个体（包括移植患者和艾滋病患者）中，这些癌症的发病率大大增加。 LANA 是 KSHV 编码的潜伏蛋白，在所有 KSHV 相关恶性肿瘤中表达。 LANA 是一种多功能蛋白，具有抗凋亡和细胞增殖活性，还负责 KSHV 感染细胞中发生的一些细胞基因表达重编程。 LANA 多效性功能的一个潜在重要来源是 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。我们建议通过获取和整合两组数据，从机制上解决 LANA 对这两种激酶的操纵的贡献：GSK-3 和 ERK1/2 的细胞蛋白底物的全局鉴定，以及依赖于 GSK-3 和 ERK1/2 的 LANA 介导的细胞基因表达变化。该应用程序有两个目标。目标 1 旨在使用蛋白质组学方法鉴定 ERK1/2 磷酸化和 ERK1/2 引发的 GSK-3 磷酸化细胞蛋白的整体概况。 ERK1/2 激酶测定将在 Co-PI 生成的 5,000 个蛋白质定制人类蛋白质阵列上进行。 GSK-3 需要事先引发磷酸化才能发挥活性。 ERK1/2 引发的 GSK-3 底物也将在使用人类蛋白质芯片的激酶测定中进行鉴定。在这些测定中鉴定出的新型 ERK1/2 和 ERK1/2 引发的 GSK-3 底物将使用体外激酶测定并通过检查细胞中的蛋白质磷酸化进行验证，其中 ERK1/2 和 GSK-3 激酶活性已被化学抑制剂下调或通过使用短沉默 miRNA 修饰激酶的表达。目标 2 将检查 LANA 介导的转录重编程通过 ERK1/2 激活和 GSK-3 失活介导的程度。基因阵列分析将在具有 tet 诱导表达 LANA 或不与 GSK-3 相互作用的突变 LANA 的细胞系上进行，以及在 tet 诱导表达 LANA 表达细胞（ERK1/2 表达下调）上进行。然后将寻找 LANA 调节依赖于 ERK1/2 和/或 GSK-3 的基因与目标 1 中鉴定为 ERK1/2 或 GS​​K-3 底物的蛋白质之间的联系。整合本研究中获得的数据将加深对 LANA 对 KSHV 相关恶性肿瘤的贡献的理解，还将提供对 Ras/MAPK/ERK1/2 和 Wnt 通路失调驱动的其他人类癌症中发生的过程的机制见解。公众健康相关性：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，这些癌症的发生频率更高。 LANA 蛋白是由 KSHV 在这些癌症中产生的。我们正在测试这样的假设：LANA 会改变称为激酶的细胞酶的活性，从而使细胞走上一条可能导致癌症的道路。