Regulation of phospholipid synthesis

描述(申请人提供)：在酿酒酵母中，PAH1基因编码磷脂酸磷酸酶(Pah1p PAP)，它已成为脂代谢中最重要和最受调控的酶之一。该酶催化磷脂酸(PA)脱磷生成二酰甘油(DAG)和PI，该反应依赖于镁离子，并基于蛋白质中卤酸脱卤酶样域中的DXDX(T/V)催化基序。由Pah1p PAP活性产生的DAG用于合成三酰甘油(TAG)和主要的膜磷脂磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)。在哺乳动物细胞中，Lipin是Pah1p的同源基因，它作为PAP酶的分子功能已经通过发现酵母PAH1编码的PAP而被揭示。酵母中PAP活性的丧失会导致PA的积累和TAG的大量减少。因此，缺乏PAP活性的突变体表现出含有UASINO的磷脂合成基因的诱导表达，磷脂质量增加，核/ER膜扩张，脂滴形成和液泡稳态缺陷，以及对脂肪酸诱导的毒性的急性敏感性。在小鼠中，脂类1的缺失会导致脂肪营养不良、胰岛素抵抗和周围神经病变，而脂类1的过度表达会导致肥胖和胰岛素敏感性。因此，为了维持脂质平衡，必须控制膜上PAP的活性，而这种调节是通过酶的磷酸化/去磷酸化来介导的。在当前的授权期，我们发现周期蛋白依赖的蛋白激酶Pho85p-Pho80p和CDC28p-Cyclin B通过磷酸化Ser/Thr-Pro基序中的七个位点来调节Pah1p。这些磷酸化导致Pah1p的胞浆定位和其在脂质合成中的功能失活。在特定的ai 1中，我们将研究通过激酶A、蛋白激酶C和酪蛋白激酶II的磷酸化对Pah1p PAP的调节。我们将确认Pah1p是这些酶的底物，确定它们的磷酸化位置，并检查这些磷酸化的生理相关性。还将研究磷酸化的相互依存关系。Pah1p与底物PA所在的膜的结合是Pah1p在体内发挥作用所必需的。胞浆中的磷酸化的Pah1p被转移到核/ER膜上，该膜由Nem1p-Spo7p磷酸酶复合体控制。去磷酸化导致Pah1p锚定在膜上，从而允许PAP反应和脂质合成。在特定目标2中，我们将研究Nem1p-Spo7p磷酸酶复合体与Pho85p-Pho80p、CDC28p-Cyclin B、蛋白激酶A、蛋白激酶C和酪蛋白激酶II磷酸化位点的酶学和特异性。在这些目标中，我们提出了结合生物化学和分子遗传学以及质谱学技术来确定磷酸化位点和分析Pah1p磷酸化/去磷酸化引起的脂质组成变化的方法。这项拟议的工作将阐明PAP活性是如何调节的，并为理解PA产生的聚合和发散脂质代谢途径的控制和整合开辟了新的途径。

DESCRIPTION (provided by applicant): In the yeast Saccharomyces cerevisiae, the PAH1 gene encodes phosphatidate phosphatase (Pah1p PAP), which has emerged as one of the most important and highly regulated enzymes in lipid metabolism. The enzyme catalyzes the dephosphorylation of phosphatidate (PA) to yield diacylglycerol (DAG) and Pi, a reaction that is dependent on Mg2+ ions and is based on the DXDX(T/V) catalytic motif within a haloacid dehalogenase-like domain in the protein. The DAG produced by Pah1p PAP activity is used for the synthesis of triacylglycerol (TAG) and the major membrane phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In mammalian cells, lipin is the Pah1p ortholog, and its molecular function as a PAP enzyme has been revealed through the discovery of the yeast PAH1-encoded PAP. The loss of PAP activity in yeast causes the accumulation of PA and a massive reduction in TAG. Consequently, mutants lacking PAP activity exhibit induced expression of UASINO-containing phospholipid synthesis genes, an increase in phospholipid mass, expansion of the nuclear/ER membrane, defects in lipid droplet formation and vacuole homeostasis, and acute sensitivity to fatty acid-induced toxicity. The loss of lipin 1 in mice causes lipodystrophy, insulin resistance, and peripheral neuropathy, while overexpression of lipin 1 causes obesity and insulin sensitivity. Thus, for lipid homeostasis, PAP activity on the membrane must be controlled, and this regulation is mediated by phosphorylation/dephosphorylation of the enzyme. In the current grant period, we showed that cyclin-dependent protein kinases Pho85p-Pho80p and Cdc28p-cyclin B regulated Pah1p by phosphorylating seven sites within a Ser/Thr-Pro motif. These phosphorylations cause a cytosolic localization of Pah1p and inactivation of its function in lipid synthesis. In specific ai 1, we will examine the regulation of Pah1p PAP via phosphorylations by kinase A, protein kinase C, and casein kinase II. We will confirm that Pah1p is a substrate for these kinases, determine the sites of their phosphorylations, and examine the physiological relevance of the phosphorylations. The interdependencies of the phosphorylations will also be examined. The association of Pah1p with the membrane where the substrate PA resides is essential to Pah1p function in vivo. Phosphorylated Pah1p in the cytosol is translocated to the nuclear/ER membrane, which is controlled by the Nem1p-Spo7p phosphatase complex. The dephosphorylation leads to the anchoring of Pah1p to the membrane allowing for the PAP reaction and lipid synthesis. In specific aim 2, we will examine the enzymology and specificity of the Nem1p-Spo7p phosphatase complex with respect to the sites phosphorylated by Pho85p-Pho80p, Cdc28p-cyclin B, protein kinase A, protein kinase C, and casein kinase II. In these aims, we propose approaches that combine biochemistry and molecular genetics, coupled with mass spectrometry techniques, to determine sites of phosphorylation and to analyze changes in lipid composition that are brought about by phosphorylation/dephosphorylation of Pah1p. The proposed work will shed light on how PAP activity is regulated, and open new avenues for understanding the control and integration of convergent and divergent lipid metabolic pathways emanating from PA.