Regulation of phospholipid synthesis

描述（由申请人提供）：在酵母酿酒酵母中，PAH1基因编码磷脂酸磷酸酶（Pah1p PAP），该酶已成为脂质代谢中最重要和高度调控的酶之一。该酶催化磷脂酸（PA）的去磷酸化，生成二酰基甘油（DAG）和Pi，该反应依赖于Mg2+离子，并基于蛋白质中卤酸去卤酶样结构域内的DXDX（T/V）催化基元。Pah1p PAP活性产生的DAG用于合成三酰基甘油（TAG）和主要膜磷脂磷脂酰胆碱（PC）和磷脂酰乙醇胺（PE）。在哺乳动物细胞中，脂素是Pah1p的同源物，通过发现酵母pah1编码的PAP，揭示了其作为PAP酶的分子功能。酵母中PAP活性的丧失导致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诱导含有uasino的磷脂合成基因的表达，磷脂质量增加，核/内质网膜膨胀，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。小鼠体内脂素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，而脂素1的过度表达会导致肥胖和胰岛素敏感性。因此，为了维持脂质稳态，细胞膜上的PAP活性必须受到控制，而这种调节是由酶的磷酸化/去磷酸化介导的。在目前的资助期内，我们发现周期蛋白依赖性蛋白激酶Pho85p-Pho80p和Cdc28p-cyclin B通过磷酸化Ser/Thr-Pro基序中的7个位点来调节Pah1p。这些磷酸化导致Pah1p的胞质定位和它在脂质合成中的功能失活。在特定的ai 1中，我们将通过激酶A、蛋白激酶C和酪蛋白激酶II的磷酸化来研究Pah1p PAP的调控。我们将证实Pah1p是这些激酶的底物，确定它们磷酸化的位点，并检查磷酸化的生理相关性。磷酸化的相互依赖性也将被检查。Pah1p与底物PA所在的膜的结合对Pah1p在体内的功能至关重要。胞浆中磷酸化的Pah1p被转运到核/ER膜，这是由Nem1p-Spo7p磷酸酶复合物控制的。去磷酸化导致Pah1p锚定在膜上，允许PAP反应和脂质合成。在特定目标2中，我们将研究Nem1p-Spo7p磷酸酶复合物的酶学和特异性，以及Pho85p-Pho80p、cdc28p -细胞周期蛋白B、蛋白激酶A、蛋白激酶C和酪蛋白激酶II磷酸化的位点。在这些目标中，我们提出了结合生物化学和分子遗传学的方法，再加上质谱技术，来确定磷酸化位点，并分析由Pah1p的磷酸化/去磷酸化所带来的脂质组成的变化。所提出的工作将阐明PAP活性是如何被调节的，并为理解源自PA的趋同和发散脂质代谢途径的控制和整合开辟新的途径。

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.