Regulation of Phospholipid Synthesis

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DESCRIPTION (provided by applicant): The yeast Saccharomyces cerevisiae serves as a model eukaryotic organism to study the regulation of lipid synthesis. The PAH1-encoded phosphatidic acid phosphatase (PAP1) catalyzes the Mg2+dependent dephosphorylation of phosphatidic acid to yield diacylglycerol and Pi. This enzyme generates the diacylglycerol used for the synthesis of triacylglycerol and for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the Kennedy pathway. PAP1 activity also controls the cellular concentration of its substrate phosphatidic acid, which is the precursor for phospholipids that are synthesized via the CDP- diacylglycerol pathway. The analysis of yeast pah1 mutants defective in PAP1 activity has shown that the PAP1-mediated control of phosphatidic acid content plays a major role in the transcriptional regulation of UASINO-containing phospholipid synthesis genes and the growth of the nuclear/ endoplasmic reticulum membrane. The importance of PAP1 activity in lipid metabolism is further emphasized by the fact that overexpression of the enzyme (i.e., lipin 1) in mice leads to obesity while its loss leads to lipodystrophy and peripheral neuropathy. Moreover, unregulated lipid synthesis, as mediated by the PAP1 enzyme, may cause metabolic disorders such as insulin resistance, diabetes, and heart disease. The PAH1-encoded PAP1 has been identified as a phosphoprotein, and sixteen phosphorylation sites have been identified from the enzyme isolated from yeast. PAP1 is a target for several protein kinases (e.g., cyclin-dependent protein kinase, protein kinases A and C, casein kinases I and II, Mpk1, and Dbf2-Mob1) that regulate cell physiology. The phosphorylation of PAP1 governs its enzymatic activity and its cellular functions. In the next grant period, we propose biochemical and molecular genetic studies to elucidate the complex phosphorylation that regulates the PAH1-encoded PAP1 enzyme. In specific aim 1, we will address the hypothesis that PAP1 is a substrate for protein kinases predicted to phosphorylate the identified sites in the enzyme, and to examine the interdependencies of these phosphorylations. In aim 2, we will examine the effects of the phosphorylations on PAP1 activity and its cellular location, and examine the physiological relevance of these phosphorylations. The results of the proposed studies should be of great interest to, and complement the work of other investigators who study PAP1 and lipid metabolism in higher eukaryotic cells. PUBLIC HEALTH RELEVANCE: The PAH1-encoded phosphatidic acid phosphatase (PAP1) enzyme catalyzes the dephosphorylation of phosphatidic acid to yield diacylglycerol and Pi. The diacylglycerol is used for the synthesis of triacylglycerol and for the synthesis of the major phospholipids phosphatidylethanolamine and phosphatidylcholine. The PAP1-mediated control of phosphatidic acid content plays a major role in the transcriptional regulation of phospholipid synthesis genes and the growth of the nuclear/endoplasmic reticulum membrane. The importance of PAP1 activity in lipid metabolism is further emphasized by the fact that overexpression of the enzyme (i.e., lipin 1) in mice leads to obesity while its loss leads to lipodystrophy and peripheral neuropathy. Moreover, unregulated lipid synthesis, as mediated by the PAP1 enzyme, may cause metabolic disorders such as insulin resistance, diabetes, and heart disease.