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.
The Pah1p phosphatidate phosphatase enzyme catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol and Pi. 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 contents regulate lipid droplet formation, vacuole homeostasis, and fatty acid- induced toxicity. 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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