REGULATION OF PHOSPHOLIPID SYNTHESIS

描述(改编自申请者摘要)：酵母菌 酿酒酵母作为真核生物研究磷脂调节的模式生物 综合。本申请中提出的工作的主要假设是 磷脂的合成是由关键酶的磷酸化来调节的。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合成酶在丝氨酸上被磷酸化 蛋白激酶A和蛋白激酶C的残基，以及后者的激酶 使多个位点上的CTP合成酶磷酸化。突变型CTP合成酶 这些激酶的磷酸化缺陷将被用来检查 磷酸化对CTP合成酶活性的调节。分层次 磷酸化(即，一个位点上的磷酸化的影响 将检测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识别位点 额外的蛋白激酶的磷酸化作用(S)。胆碱激酶是 在多个丝氨酸残基上磷酸化，其中一些磷酸化是 蛋白激酶A介导的蛋白激酶A磷酸化位点(S) 胆碱激酶将被鉴定出来。一种有缺陷的突变(S)胆碱激酶 蛋白激酶A的磷酸化将被构建并用于检测 通过磷酸化来调节胆碱激酶的活性。网站： 额外的蛋白激酶(S)对胆碱激酶的磷酸化作用 已确认身份。CTP合成酶和胆碱中的磷酸化位点突变 激酶将被用来检查生理上的相关性 这些酶的磷酸化。我们将检验这一假设，即存在 CTP合成酶的磷酸化与胆碱的直接联系 激酶和磷脂合成的调节。磷的调节 磷酸化的脂肪合成将在CTP合成酶突变体中进行检测 CTP产物抑制缺陷和RASIcAMP缺陷突变体 路径。

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.