Phosphorylation of phosphatidate phosphatase regulates its membrane association and physiological functions in Saccharomyces cerevisiae: Identification of SER602, THR723, and SER744 as the sites phosphorylated by CDC28 (CDK1)-encoded cyclin-dependent kinase

Hyeon Son Choi, Wen Min Su, Jeanelle M. Morgan, Gil Soo Han, Zhi Xu, Eleftherios Karanasios, Symeon Siniossoglou, George M. Carman

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86 Scopus citations

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, Thr723, and Ser744, 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, Thr723, and/or Ser744 had a partial effect on circumventing the requirement for the Nem1p-Spo7p complex.

Original languageEnglish (US)
Pages (from-to)1486-1498
Number of pages13
JournalJournal of Biological Chemistry
Volume286
Issue number2
DOIs
StatePublished - Jan 14 2011

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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