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.
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.
