title: Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism.
abstract: Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex.
authors.affiliation: From the Department of Food Science and the Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, New Jersey 08901.
authors.forename: Lu-Sheng
authors.initials: LS
authors.lastname: Hsieh
authors.affiliation: From the Department of Food Science and the Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, New Jersey 08901.
authors.forename: Wen-Min
authors.initials: WM
authors.lastname: Su
authors.affiliation: From the Department of Food Science and the Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, New Jersey 08901.
authors.forename: Gil-Soo
authors.initials: GS
authors.lastname: Han
authors.affiliation: From the Department of Food Science and the Rutgers Center for Lipid Research, New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, New Jersey 08901 carman@aesop.rutgers.edu.
authors.forename: George M
authors.initials: GM
authors.lastname: Carman
mesh_terms: D019943:Amino Acid Substitution; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010711:Phosphatidate Phosphatase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014280:Triglycerides / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D014280:Triglycerides; D047390:Casein Kinase II; C079831:PAH1 protein, S cerevisiae; D010711:Phosphatidate Phosphatase
keywords: diacylglycerol; lipid; phosphatidate; phospholipid; phosphorylation; triacylglycerol; yeast