The major goal of this proposal is to elucidate the fundamental mechanisms involved in regulation of copper transport in human cells. Copper is an essential nutrient required for functional activity of various enzymes; disruption of copper transport across cell membranes leads to severe multi-system disorders in humans. In the last several years, a number of proteins involved in distribution of copper in mammalian cells have been identified. Among them, the human copper-transporting ATPases mutated in Menkes disease and in WUson's disease (the Menkes disease and Wilson's disease proteins, respectively) were shown to play a key role in the export of copper out of the cell and in delivery of copper to some intracellular compartments. Recent studies indicate that copper regulates these transporters on several levels, however the molecular mechanisms of this regulation remain poorly understood. In this proposal, we will utilize the Wilson's disease protein (WNDP) as a model to elucidate how copper regulates its own transport and to identify the regulatory proteins involved in this process. We will employ modern biochemical and cell biological tools, including mass-spectroscopy, site-directed mutagenesis, confocal microscopy, two-dimensional electrophoresis and affinity chromatography to reach several specific goals. Firstly, the structural determinants for the copper-dependent phosphorylation in WNDP will be identified. Secondly, the physiological role of copper-dependent protein phosphorylation will be determined by testing our hypothesis that the regulated phosphorylation of WNDP serves as a signal for the intracellular trafficking of this transporter. Thirdly, we will characterize the kinase and phosphatase involved in ostranslational modification of WNDP. The proteins interacting with the copper-transporter in response to copper will also be identified. Finally, the effect of copper on protein phosphorylation will be compared for control cells and cells with abnormal copper metabolism. The results of this work will yield the basic and practical information important for understanding of human copper homeostasis and its regulation in both normal and diseased human cells.
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