Regulation of phospholipid synthesis

磷脂合成的调节

• 批准号: 8490796
• 负责人: GEORGE M. CARMAN
• 金额: $ 30.71万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8490796
• 关键词: 1,2-diacylglycerol; Acute; Attenuated; Binding; Biochemistry; Cell Nucleus; Complex; Coupled; Cyclic AMP-Dependent Protein Kinases; Cyclin B; Cyclin-Dependent Kinases; Cytidine Diphosphate Diglycerides; Cytosol; Defect; Diglycerides; Diphosphates; Divalent Cations; Endoplasmic Reticulum; Enzymatic Biochemistry; Enzymes; Exhibits; Fatty Acids; Genes; Genetic Transcription; Grant; Growth; Homeostasis; Human; Inflammation; Inositol; Insulin Resistance; Ions; Lecithin; Light; Lipids; Lipodystrophy; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane; Metabolic Diseases; Metabolic Pathway; Molecular; Molecular Genetics; Mus; Mutation; Myoglobinuria; Nuclear; Obesity; Orthologous Gene; Peripheral Nervous System Diseases; Phosphatidate Phosphatase; Phosphatidylethanolamine; Phosphatidylinositols; Phosphatidylserines; Phospholipids; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Protein Dephosphorylation; Protein Kinase C; Proteins; Reaction; Regulation; Role; Saccharomyces cerevisiae; Site; Specificity; Techniques; Toxic effect; Transcription Coactivator; Transcriptional Regulation; Triglycerides; Vacuole; Work; Yeasts; base; casein kinase II; gene synthesis; haloacid dehalogenase; in vivo; insulin sensitivity; lipid metabolism; lipid phosphate phosphatase; lipine; mutant; overexpression; phosphatidate; prevent; public health relevance

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.

描述（由申请人提供）：在酿酒酵母中，PAH 1基因编码磷脂酸磷酸酶（Pah 1 p PAP），其已成为脂质代谢中最重要和高度调节的酶之一。该酶催化磷脂酸（PA）的脱磷酸化，产生二酰基甘油（DAG）和Pi，该反应依赖于Mg 2+离子，并且基于蛋白质中卤酸脱卤酶样结构域内的DXDX（T/V）催化基序。由Pah 1 p 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Pah 1 p的直系同源物，其作为PAP酶的分子功能已通过发现酵母PAH 1编码的PAP而被揭示。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含UASINO的磷脂合成基因的诱导表达、磷脂质量的增加、核/ER膜的扩张、脂滴形成和空泡稳态的缺陷以及对脂肪酸诱导的毒性的急性敏感性。小鼠中lipin 1的缺失导致脂肪营养不良、胰岛素抵抗和周围神经病变，而lipin 1的过度表达导致肥胖和胰岛素敏感性。因此，对于脂质稳态，必须控制膜上的PAP活性，并且这种调节是由酶的磷酸化/去磷酸化介导的。在本研究期间，我们发现细胞周期蛋白依赖性蛋白激酶Pho 85 p-Pho 80 p和Cdc 28 p-cyclin B通过磷酸化Ser/Thr-Pro基序中的7个位点来调节Pah 1 p。这些磷酸化导致Pah 1 p的胞质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Pah 1 p PAP通过激酶A、蛋白激酶C和酪蛋白激酶II磷酸化的调节。我们将确认Pah 1 p是这些激酶的底物，确定其磷酸化位点，并研究磷酸化的生理相关性。还将检查磷酸化的相互依赖性。Pah 1 p与底物PA所在的膜的关联对于Pah 1 p在体内的功能是必不可少的。胞质溶胶中磷酸化的Pah 1 p被转运到核/ER膜，这是由Nem 1 p-Spo 7 p磷酸酶复合物控制的。去磷酸化导致Pah 1 p锚定到膜上，允许PAP反应和脂质合成。在具体目标2中，我们将检查Nem 1 p-Spo 7 p磷酸酶复合物相对于由Pho 85 p-Pho 80 p、Cdc 28 p-cyclin B、蛋白激酶A、蛋白激酶C和酪蛋白激酶II磷酸化的位点的酶学和特异性。在这些目标中，我们提出的方法，联合收割机生物化学和分子遗传学，再加上质谱技术，以确定磷酸化位点，并分析所带来的磷酸化/去磷酸化的Pah 1 p的脂质组成的变化。拟议的工作将阐明PAP活性是如何调节的，并为理解PA产生的会聚和发散脂质代谢途径的控制和整合开辟了新的途径。