The rate of de novo pyrimidine biosynthesis in mammalian cells is regulated by CAD, a large multifunctional protein that catalyzes the first three steps in the pathway. The flux through the pathway is precisely controlled and increases in tumors and in cells induced to proliferate. Carbamoyl phosphate synthetase, the CAD component that catalyzes the initial, rate limiting step, the locus of regulation, is controlled by UTP inhibition, PRPP activation, by protein kinase A (PKA) mediated phosphorylation and by phosphorylation of CAD via the MAP kinase cascade in response to EGF stimulation. The pathway is up-regulated just prior to S phase by MAP kinase mediated phosphorylation and subsequently down-regulated as the cells exit the S phase by dephosphorylation of the MAP kinase site and phosphorylation by PKA. The first Specific Aim is to elucidate the mechanism and physiological significance of three newly discovered, putative regulatory mechanisms; 1) autophosphorylation of CAD that results in large changes in catalytic activity and regulation, 2) protein kinase C that acts in concert with MAP kinase to activate CAD and 3) the regulated signaling complexes of CAD, MAP kinase, PKA and PP1 that may regulate the timing of the sequential phosphorylations. The synergistic and antagonist interactions between the individual control mechanisms that allow them to work in concert to respond to varying demands for pyrimidine nucleotides are of special interest. The second Specific Aim is to assess the physiological significance of the growth state dependent changes in CAD intracellular dynamics. Microscopic and biochemical approaches have shown that an appreciable fraction of the CAD phosphorylated by MAP kinase is localized in the nucleus and that a fraction of the CAD in the cytosol is associated with the microtubules. The postulated direct transfer of dihydroorotate to the mitochondrial dehydrogenase, catalyzing the next step in the pathway, will be investigated in vivo using a histochemical assay. Fluorescence microscopy of live cells in conjunction with mutants and constructs that target CAD to different cellular compartments will be used to elucidate the functional significance of CAD nucleocytoplasmic dynamics. The overall objective is to develop a comprehensive model for the regulation of the pathway in normal and neoplastic cells taking into consideration the integration of signals and the interplay of these diverse regulatory mechanisms. ? ?
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