title: Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: identification of SER(602), THR(723), AND SER(744) as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase.
abstract: The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes. PAP is phosphorylated at multiple Ser and Thr residues and is dephosphorylated for in vivo function by the Nem1p-Spo7p protein phosphatase complex localized in the nuclear/endoplasmic reticulum membrane. In this work, we characterized seven previously identified phosphorylation sites of PAP that are within the Ser/Thr-Pro motif. When expressed on a low copy plasmid, wild type PAP could not complement the pah1Δ mutant in the absence of the Nem1p-Spo7p complex. However, phosphorylation-deficient PAP (PAP-7A) containing alanine substitutions for the seven phosphorylation sites bypassed the requirement of the phosphatase complex and complemented the pah1Δ nem1Δ mutant phenotypes, such as temperature sensitivity, nuclear/endoplasmic reticulum membrane expansion, decreased triacylglycerol synthesis, and derepression of INO1 expression. Subcellular fractionation coupled with immunoblot analysis showed that PAP-7A was highly enriched in the membrane fraction. In fluorescence spectroscopy analysis, the PAP-7A showed tighter association with phospholipid vesicles than wild type PAP. Using site-directed mutagenesis of PAP, we identified Ser(602), Thr(723), and Ser(744), which belong to the seven phosphorylation sites, as the sites phosphorylated by the CDC28 (CDK1)-encoded cyclin-dependent kinase. Compared with the dephosphorylation mimic of the seven phosphorylation sites, alanine substitution for Ser(602), Thr(723), and/or Ser(744) had a partial effect on circumventing the requirement for the Nem1p-Spo7p complex.
authors.affiliation: Department of Food Science and Rutgers Center for Lipid Research, Rutgers University, New Brunswick, New Jersey 08901, USA.
authors.forename: Hyeon-Son
authors.initials: HS
authors.lastname: Choi
authors.forename: Wen-Min
authors.initials: WM
authors.lastname: Su
authors.forename: Jeanelle M
authors.initials: JM
authors.lastname: Morgan
authors.forename: Gil-Soo
authors.initials: GS
authors.lastname: Han
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xu
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Karanasios
authors.forename: Symeon
authors.initials: S
authors.lastname: Siniossoglou
authors.forename: George M
authors.initials: GM
authors.lastname: Carman
mesh_terms: D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D007294:Inositol / Q000493:pharmacokinetics; D050356:Lipid Metabolism / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009685:Nuclear Envelope / Q000201:enzymology; D010641:Phenotype; D010711:Phosphatidate Phosphatase / Q000235:genetics / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014280:Triglycerides / Q000378:metabolism
chemical_list: D010712:Phosphatidic Acids; D029701:Saccharomyces cerevisiae Proteins; D014280:Triglycerides; D013912:Threonine; D012694:Serine; D007294:Inositol; D017489:CDC28 Protein Kinase, S cerevisiae; C079831:PAH1 protein, S cerevisiae; D010711:Phosphatidate Phosphatase