We have found that PDGF and TNFa results in translocation of cPLA2 from cytosol into the nucleus and, using yeast two-hybrid cloning methods, have identified two novel nuclear proteins, PLIP-1 (for cPLA2-interacting protein-1), and PLIP-2, one of which (PLIP-1) colocalizes with cPLA2 in the nucleus. PLIP-2 localizes to both the cytosol and nucleus. Our hypothesis is: nuclear translocation and docking of cPLA2 is finely controlled by interactions with cPLA2 binding proteins. This translocation results in a nuclear lipid signal transduction pathway consisting of cPLA2, prostaglandin synthases, and lipoxygenases, producing arachidonate metabolites and reactive oxygen spices which mediate nuclear transduction events important for mitogenesis, cell differentiation and inflammation. The specific goals of this project are to determine the roles of PLIP-1 and PLIP-2 in the regulation and nuclear translocation of cPLA2 activity and determine the role of cPLA2-interacting proteins in signal transduction effector pathways of growth factors and TNFa. We will: obtain a full lenght cDNA for PLIP-2 and characterize the protein sequence; characterize cellular expression of PLIPs in vitro; generate antibodies, and immunolocalize these proteins in the kidney. We will determine which amino acids of the calcium lipid binding (CaLB) region of cPLA2 are responsible for translocation to the nucleus and interaction with PLIP-1 and 2.We will determine the functional significance of the structural features of PLIPs important to the interaction with cPLA2, generate dominant negative forms of PLIPs and evaluate their effects on nuclear localization of cPLA2. We will evaluate whether PLIPs bind to other proteins that contain the CaLB domain. The role of phosphorylation of PLIPs in translocation and interaction with cPLA2 will be defined and a potential role for the p38/RK kinase in activation of cPLA2 explored. A number of effector pathways will be examined to evaluate the function al significance of the PLIP-cPLA2 interactions. The cytoplasm/nuclear distribution of cPLA2 and the dependence of this distribution on PLIPs and their state of phosphorylation during Go to G1 transition or S phase of the cell cycle will be determined. The effects of addition of a CAAX box to PLIP on intracellular targeting of cPLA2 will be determined. Agonist-inducted arachidonic acid release and its dependence on nuclear localization of cPLA2 and PLIPs will be compared in quiescent and mitotic cells. We will evaluate whether NF-kappebeta activation in response to cytokines is regulated by cPLA2 activity and subsequent metabolism by cyclooxygenase and/or lipoxygenase.
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