The yeast, Saccharomyces cerevisiae serves as a model eukaryote to study the regulation of phospholipid synthesis. The major hypothesis of the work proposed in this application is that phospholipid synthesis is regulated by phosphorylation of key enzymes. CTP synthetase is essential for the synthesis of all membrane phospholipids, whereas choline kinase catalyzes the committed step of phospholipid synthesis via the CDP-choline pathway. CTP synthetase is phosphorylated on serine residues by protein kinase A and by protein kinase C, and the latter kinase phosphorylates CTP synthetase on multiple sites. Mutant CTP synthetase enzymes defective in phosphorylation by these kinases will be used to examine the regulation of CTP synthetase activity by phosphorylation. Hierarchical phosphorylation (i.e., effect of phosphorylation on one site by the phosphorylation at another site) of the PKA and PKC sites will be examined. Phosphorylation site mutants of CTP synthetase will be used to identify sites of phosphorylation by additional protein kinase(s). Choline kinase is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. The protein kinase A phosphorylation site(s) in choline kinase will be identified. A mutant(s) choline kinase that is defective in phosphorylation by protein kinase A will be constructed and used to examine the regulation of choline kinase activity by phosphorylation. Sites of phosphorylation in choline kinase by additional protein kinase(s) will be identified. The phosphorylation site mutants in CTP synthetase and in choline kinase will be used to examine the physiological relevance of the phosphorylation of these enzymes. We will examine the hypothesis that there is a direct connection between the phosphorylations of CTP synthetase and choline kinase and the regulation of phospholipid synthesis. The regulation of phospho lipid synthesis by phosphorylation will be examined in a CTP synthetase mutant defective in CTP product inhibition and in mutants defective in the RASIcAMP pathway.
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