In mammalian cells, the rate of de novo pyrimidine biosynthesis is regulated by the multi-functional protein CAD. The protein, which catalyzes the first half of the pathway, consists of six copies of a 243 kDa polypeptide folded into separate structural domains that carry glutamine dependent carbamoyl phosphate synthetase (CPSase), aspartate transcarbomoylase (ATCase) and dihydrooortase (DHOase) activities. The activity of the pathway is precisely controlled and increases when cells are induced to proliferate. Studies of the purified protein have shown that CPSase, which catalyzes the initial, rate limiting step in the pathway is the major locus of regulation and is allosterically controlled by UTP, an inhibitor and PRPP, an activator. In addition, the activity of the complex is regulated by protein kinase A mediated phosphorylation, although its role in regulating the growth state and cell cycle changes in the activity of the pyrimidine biosynthetic pathway is not at all clear. The recent discovery that EGF stimulation results in MAP kinase mediated phosphorylation and activation of CAD makes its possible to resolve this ambiguity. The objective of this research is to decipher the interrelationships and assess the importance of all these control mechanisms on the regulation of pyrimidine biosynthesis in vivo. The rate of de no pyrimidine biosynthesis, the size of the allosteric effector pools and the phosphorylation state of the protein will be examined in mammalian cells grown in culture and in transfectants that express CAD mutants in which one or more of the regulatory mechanisms have been disabled. The approach will be to extrapolate the extensive information developed on the regulation of purified CAD to develop a comprehensive model that can account for the in vivo regulation of de novo pyrimidine biosynthesis in different growth states, phases of the cycle and in cells stimulated by growth factors.
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