In the yeast Saccharomyces cerevisiae, the PAH1 gene encodes phosphatidate (PA) phosphatase (Pah1 PAP), which has emerged as one of the most important and highly regulated enzymes in lipid metabolism. The enzyme catalyzes the dephosphorylation of PA to yield diacylglycerol (DAG), 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 Pah1 PAP activity is used for the synthesis of triacylglycerol (TAG) and the major membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. In addition, PA and DAG are signaling molecules that influence transcription, membrane proliferation, vesicular trafficking, and cell growth. In mammalian cells, lipin is the Pah1 ortholog, and its molecular function as a PAP enzyme has been revealed through the discovery of yeast Pah1. The loss of Pah1 PAP activity in yeast causes the accumulation of PA and a massive reduction in TAG. Consequently, mutants lacking the enzyme activity exhibit induced expression of phospholipid synthesis genes, an increase in phospholipid mass, expansion of the nuclear/endoplasmic reticulum membrane, defects in lipid droplet formation and vacuole homeostasis, acute sensitivity to fatty acid- induced toxicity, and a reduction in chronological life span. In mammalian cells, loss of lipin PAP activity results in metabolic disorders that include lipodystrophy, insulin resistance, peripheral neuropathy, rhabdomyolysis, and inflammation. Pah1 is a peripheral membrane protein that must translocate from the cytosol to the nuclear/endoplasmic reticulum membrane in order to convert PA to DAG for the synthesis of TAG or phospholipids. The translocation of Pah1 requires its dephosphorylation at the membrane. In the cytosol, Pah1 is phosphorylated by Pho85 (CDK5), Cdc28 (CDK1), protein kinase A, protein kinase C, and casein kinase II; the sites of Pah1 phosphorylated by these protein kinases are dephosphorylated by the conserved (e.g., human CTDNEP1-NEP1-R1 complex) endoplasmic reticulum-localized Nem1-Spo7 protein phosphatase (e.g., Pah1 phosphate phosphatase). Besides its location, the phosphorylation/dephosphorylation of Pah1 controls its PAP activity and protein stability. Based on the premise that Nem1-Spo7 phosphatase is crucial for governing the phosphorylation state and function of Pah1 PAP at the membrane, it is important to know how the protein phosphatase complex is regulated. Preliminary studies indicate that like Pah1, both Nem1 and Spo7 are subject to phosphorylations by protein kinase A and protein kinase C. Work proposed in the next grant period will address the hypothesis that the phosphorylations of Nem1 and Spo7 control their function in the Nem1- Spo7/Pah1 phosphatase cascade to regulate lipid metabolism.
In aim 1, we will examine the phosphorylations of Nem1 and Spo7 by protein kinase A and protein kinase C, and determine the sites of phosphorylation.
In aim 2, we will examine the roles of the protein kinase A and protein kinase C phosphorylations in the regulation of Nem1-Spo7 phosphatase activity and specificity.
In aim 3, we will utilize phosphorylation-deficient and -mimicking mutants to examine the physiological effects of the Nem1-Spo7 phosphorylations on Pah1 location, stability, and function in lipid metabolism. We propose rigorous experimental approaches that combine biochemistry and molecular genetics along with mass spectrometry to determine sites of phosphorylation and to analyze changes in Pah1 function that are brought about by the phosphorylations of Nem1-Spo7. The proposed work will shed light on how the Nem1-Spo7/Pah1 phosphatase cascade is regulated, and open new avenues for understanding the control and integration of convergent and divergent lipid metabolic pathways emanating from PA and DAG. Based on the conserved nature of this phosphatase cascade, the information gained from these studies is expected to be relevant in humans.
The Nem1-Spo7/Pah1 phosphatase cascade controls phosphatidate phosphatase that catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. The diacylglycerol is used for the synthesis of triacylglycerol and for the synthesis of the major phospholipids phosphatidylcholine and phosphatidylethanolamine. The enzyme-mediated control of phosphatidate content plays a major role in the transcriptional regulation of phospholipid synthesis genes, membrane phospholipids, and growth of the nuclear/endoplasmic reticulum membrane. Enzyme control of diacylglycerol and triacylglycerol regulates lipid droplet formation, vacuole homeostasis, fatty acid-induced toxicity, and chronological life span. Genetic defects in yeast, mice, and humans are manifested in metabolic disorders that include lipodystrophy, obesity, peripheral neuropathy, myoglobinuria, and inflammation.
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