REGULATION OF PHOSPHOLIPID SYNTHESIS

描述（改编自申请人的摘要）：酵母，Saccharomyces 酿酒酵母是研究磷脂调节的模式真核生物 合成.本申请中提出的工作的主要假设是 磷脂合成是由关键酶的磷酸化调节的。CTP 合成酶是合成所有膜磷脂所必需的， 而胆碱激酶催化磷脂合成的关键步骤 通过CDP-胆碱途径。CTP合成酶在丝氨酸上磷酸化 通过蛋白激酶A和蛋白激酶C，以及后一种激酶 在多个位点磷酸化CTP合成酶。突变CTP合成酶 这些激酶的磷酸化缺陷将用于检查 通过磷酸化调节CTP合成酶活性。分层 磷酸化（即，磷酸化对一个位点的影响， 将检查PKA和PKC位点的磷酸化）。 CTP合成酶的磷酸化位点突变体将用于鉴定 通过额外的蛋白激酶磷酸化。胆碱激酶是 在多个丝氨酸残基上磷酸化，并且其中一些磷酸化是 由蛋白激酶A介导。蛋白激酶A的磷酸化位点 将鉴定胆碱激酶。一种有缺陷的突变型胆碱激酶 在蛋白激酶A的磷酸化中，将构建并用于检查 通过磷酸化调节胆碱激酶活性。位点 另外的蛋白激酶引起的胆碱激酶的磷酸化将被 鉴定CTP合成酶和胆碱磷酸化位点突变体 激酶将被用来检查的生理相关性， 这些酶的磷酸化。我们将检验这样一个假设： CTP合成酶和胆碱的磷酸化之间的直接联系 激酶和磷脂合成的调节。磷酸调节 将在CTP合成酶突变体中检查通过磷酸化的脂质合成 CTP产物抑制缺陷和RASIcAMP缺陷突变体 通路

DESCRIPTION(adapted from applicant's abstract): 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.