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Cross-talk phosphorylations by protein kinase C and Pho85p-Pho80p protein kinase regulate Pah1p phosphatidate phosphatase abundance in Saccharomyces cerevisiae.

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      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.

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      Affiliations
      [1 ] 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.
      [2 ] 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.
      Journal
      J. Biol. Chem.
      The Journal of biological chemistry
      American Society for Biochemistry & Molecular Biology (ASBMB)
      1083-351X
      0021-9258
      Jul 04 2014
      : 289
      : 27
      24876385
      M114.581462
      10.1074/jbc.M114.581462
      4081924

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