title: Cross-talk phosphorylations by protein kinase C and Pho85p-Pho80p protein kinase regulate Pah1p phosphatidate phosphatase abundance in Saccharomyces cerevisiae.
abstract: Yeast Pah1p is the phosphatidate phosphatase that catalyzes the penultimate step in triacylglycerol synthesis and plays a role in the transcriptional regulation of phospholipid synthesis genes. The enzyme is multiply phosphorylated, some of which is mediated by Pho85p-Pho80p, Cdc28p-cyclin B, and protein kinase A. Here, we showed that Pah1p is a bona fide substrate of protein kinase C; the phosphorylation reaction was time- and dose-dependent and dependent on the concentrations of ATP (Km = 4.5 μm) and Pah1p (Km = 0.75 μm). The stoichiometry of the reaction was 0.8 mol of phosphate/mol of Pah1p. By combining mass spectrometry, truncation analysis, site-directed mutagenesis, and phosphopeptide mapping, we identified Ser-677, Ser-769, Ser-773, and Ser-788 as major sites of phosphorylation. Analysis of Pah1p phosphorylations by different protein kinases showed that prephosphorylation with protein kinase C reduces its subsequent phosphorylation with protein kinase A and vice versa. Prephosphorylation with Pho85p-Pho80p had an inhibitory effect on its subsequent phosphorylation with protein kinase C; however, prephosphorylation with protein kinase C had no effect on the subsequent phosphorylation with Pho85p-Pho80p. Unlike its phosphorylations by Pho85p-Pho80p and protein kinase A, which cause a significant reduction in phosphatidate phosphatase activity, the phosphorylation of Pah1p by protein kinase C had a small stimulatory effect on the enzyme activity. Analysis of phosphorylation-deficient forms of Pah1p indicated that protein kinase C does not have a major effect on its location or its function in triacylglycerol synthesis, but instead, the phosphorylation favors loss of Pah1p abundance when it is not phosphorylated with Pho85p-Pho80p.
authors.affiliation: From the Department of Food Science, Rutgers Center for Lipid Research, and 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, Rutgers Center for Lipid Research, and 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, Rutgers Center for Lipid Research, and 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: D001665:Binding Sites; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D009154:Mutation; D010711:Phosphatidate Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
chemical_list: D016213:Cyclins; C066824:PHO80 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D011493:Protein Kinase C; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae; C079831:PAH1 protein, S cerevisiae; D010711:Phosphatidate Phosphatase
keywords: Diacylglycerol; Lipid Metabolism; Phosphatase; Phosphatidate; Protein Kinase C (PKC); Triacylglycerol; Yeast