Biochemical studies have shown that the enzymatic activity of PKC isoforms can be regulated by diacylglycerol (DG), free fatty acids (FFA), ceramide and Ca2+. At the same time, these messengers control the translocation of PKC to isoform specific cellular sites. The precise cellular localization of PKC is important for the specificity of substrate phosphorylation. The key structural determinants for Ca2+ and lipid messenger mediated PKC translocation are thought to reside in Cys- and C2-domains. More recently, these domains were also proposed to be essential for the function of a large number of signaling proteins including Raf, GAP, PLC, Vav, myosin, Unc-13 and synaptotagmin. The goal of the proposed research is to understand Cys- and C2-domains in the cellular context. This study involves the hypothesis that Cys-domains target signaling proteins to different sites of action by binding DG, FFA or ceramide and C2-domains by binding Ca2+. It has been demonstrated that individual Cys-domains tagged with green fluorescent protein (GFP) can be used to study receptor-mediated translocation processes and to measure binding parameters in intact cells. These measurements were made possible by (1) the development of an efficient RNA transfection method for mammalian cells, (2) identification of a GFP mutant that is ideally suited for use as a fusion tag, and (3) analysis procedures for measuring diffusion and binding parameters of GFP-tagged protein domains in intact cells. The use of the preceding in vivo approaches to understand distinct roles of Cys- and C2-domains from different proteins is proposed. The localization and binding parameters of individual Cys- and C2-domains in response to different stimuli will be investigated. The role of CysCysC2 domain motifs of PKC in isoform specific localization will be examined in parallel experiments. The results from these measurements will be integrated into a model for the role of lipid messengers and Ca2+ in Cys- and C2-domain function.
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