Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism

• Published in: The Journal of biological chemistry Vol. 291; no. 19; pp. 9974 - 9990
• Main Authors: Hsieh, Lu-Sheng, Su, Wen-Min, Han, Gil-Soo, Carman, George M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.05.2016; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Casein Kinase II - genetics; Casein Kinase II - metabolism; diacylglycerol; lipid; Lipids; Mass Spectrometry; Mutagenesis, Site-Directed; Mutation, Missense; phosphatidate; Phosphatidate Phosphatase - genetics; Phosphatidate Phosphatase - metabolism; phospholipid; phosphorylation; Phosphorylation - physiology; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; triacylglycerol; Triglycerides - genetics; Triglycerides - metabolism; yeast; triacylglycerol; phosphatidate; phospholipid; lipid; phosphorylation; diacylglycerol; yeast
• DOI: 10.1074/jbc.M116.726588
• ISSN: 0021-9258

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.