PAH1-encoded Phosphatidate Phosphatase Research Articles

PAH1-encoded phosphatidate phosphatase activity plays an essential role in protecting yeast against fatty acid-induced toxicity

PAH1-encoded phosphatidate phosphatase activity plays an essential role in protecting yeast against fatty acid-induced toxicity

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

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.

DGK1-encoded Diacylglycerol Kinase Activity Is Required for Phospholipid Synthesis during Growth Resumption from Stationary Phase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, triacylglycerol mobilization for phospholipid synthesis occurs during growth resumption from stationary phase, and this metabolism is essential in the absence of de novo fatty acid synthesis. In this work, we provide evidence that DGK1-encoded diacylglycerol kinase activity is required to convert triacylglycerol-derived diacylglycerol to phosphatidate for phospholipid synthesis. Cells lacking diacylglycerol kinase activity (e.g. dgk1Δ mutation) failed to resume growth in the presence of the fatty acid synthesis inhibitor cerulenin. Lipid analysis data showed that dgk1Δ mutant cells did not mobilize triacylglycerol for membrane phospholipid synthesis and accumulated diacylglycerol. The dgk1Δ phenotypes were partially complemented by preventing the formation of diacylglycerol by the PAH1-encoded phosphatidate phosphatase and by channeling diacylglycerol to phosphatidylcholine via the Kennedy pathway. These observations, coupled to an inhibitory effect of dioctanoyl-diacylglycerol on the growth of wild type cells, indicated that diacylglycerol kinase also functions to alleviate diacylglycerol toxicity.

Identification of cyclin‐dependent kinase phosphorylation sites in yeast phosphatidate phosphatase

The PAH1‐encoded phosphatidate (PA) phosphatase (PAP) plays a major role in controlling the cellular content of PA in the yeast Saccharomyces cerevisiae. The enzyme is phosphorylated in vivo on multiple residues, and one of the protein kinases involved is cyclin‐dependent kinase (CDK). In vitro, CDK phosphorylates purified recombinant PAP on both serine and threonine residues. An analysis of purified truncated forms of PAP revealed that the sites of CDK phosphorylation were at the C‐terminal end of the enzyme. A site‐directed mutagenesis study showed that Ser602 and Thr723 were the major sites of phosphorylation. In vitro, the S723A mutation caused a 50 % reduction in the CDK phosphorylation of PAP, whereas the S602A mutation caused the complete loss of the CDK phosphorylation of the enzyme. This indicated that phosphorylation of Ser602 was required for the phosphorylation of Thr723. Yeast strains that express S602A, T723A, and S602A T723A mutant enzymes have been constructed to examine the effects of CDK phosphorylation on PAP function in lipid metabolism. Supported by NIH grant GM‐50679.

Phosphorylation of yeast phosphatidate phosphatase by protein kinase C

The yeast PAH1‐encoded phosphatidate (PA) phosphatase, which catalyzes the Mg2+‐dependent dephosphorylation of PA to yield diacylglycerol, plays a major role in the regulation of lipid metabolism. In fact, mutants defective in the enzyme show defects in phospholipid synthesis regulation, an abnormal expansion of the nuclear/ER membrane, and a 90 % reduction in triacylglycerol. PA phosphatase has been identified as a phosphoprotein in vivo. Studies with recombinant enzyme isolated from Escherichia coli have shown that PA phosphatase is a substrate for protein kinase C. Truncation analysis of PA phosphatase followed by site‐directed mutagenesis experiments revealed that Ser677, Ser769, and Ser788 are the major sites for protein kinase C phosphorylation in vitro. Yeast cells that carry a S677A S769A S788A triple mutation in PA phosphatase exhibit a 40 % reduction in enzyme phosphorylation. These results indicated that protein kinase C phosphorylates PA phosphatase in vivo. Supported by NIH grant GM 50679.

Cyclin‐dependent kinase‐mediated phosphorylation of yeast phosphatidate phosphatase

The yeast PAH1‐encoded phosphatidate (PA) phosphatase catalyzes the dephosphorylation of PA to yield diacylglycerol and Pi. The diacylglycerol generated in this reaction is used for the synthesis of the storage lipid triacylglycerol and for the synthesis of the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine via the Kennedy Pathway. PA phosphatase is phosphorylated by cyclin‐dependent kinase (Cdk). Data indicate that Cdk phosphorylation down‐regulates PA phosphatase function. PA phosphatase is also dephosphorylated by the Nem1‐Spo7 complex, which results in an up‐regulation of PA phosphatase function. In this work, we examined the effects of phosphorylation and dephosphorylation of PA phosphatase on its cellular location, and on the composition of phospholipids and neutral lipids. In wild type cells, PA phosphatase is about equally distributed between the cytosolic and membrane fractions of the cell. A defect in Cdk phosphorylation of PA phosphatase caused an increase in the amount of enzyme associated with membranes, whereas a defect in the dephosphorylation of PA phosphatase caused a decrease in membrane association. Cdk phosphorylation of PA phosphatase caused a decrease in triacylglycerol content and an increase in phospholipid content. Conversely, dephosphorylation of the enzyme had the opposite effects. Supported by NIH grant GM 28140.

Phosphorylation of Yeast Phosphatidate Phosphatase by Protein Kinase C

The yeast PAH1‐encoded phosphatidate (PA) phosphatase catalyzes the Mg2+‐dependent dephosphorylation of PA to yield diacylglycerol and Pi. This enzyme plays a major role in the regulation of lipid metabolism. PA phosphatase is a phosphoprotein in vivo, and contains putative phosphorylation sites for multiple protein kinases. In this study, we examined the phosphorylation of the enzyme by protein kinase C using recombinant PA phosphatase expressed in E. coli. Purified His6‐tagged PA phosphatase was active, and was phosphorylated by protein kinase C in a time‐ and dose‐dependent manner. In addition, protein kinase C activity was dependent on the concentrations of PA phosphatase (28 μg/ml) and ATP (4.2 μM). Phosphoamino acid analysis indicated that phosphorylation by protein kinase C occurs primarily at the serine residue. Mass spectrometry of phosphorylated enzyme identified Ser773 and Ser788 as phosphorylation sites. However, phosphopeptide mapping analysis indicated additional sites of phosphorylation. Phosphorylation analysis of truncated forms of PA phosphatase indicated that the additional phosphorylation sites were located at the C‐terminal and central regions of the protein. Supported by NIH grant GM 50679.