Abstract
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.
Highlights
The PAP2 enzyme (EC 3.1.3.4) catalyzes the Mg2ϩ-dependent dephosphorylation of PA to produce DAG [1] (Fig. 1A)
Because PAP controls the cellular levels of its substrate PA, which is used for the synthesis of essential membrane phospholipids via CDP-DAG, and its product DAG, which is used for the synthesis of TAG and the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine, the enzyme is widely recognized for its key reg
We examined its phosphorylation using a purified preparation of yeast casein kinase II (CKII)
Summary
Reagents—Avanti Polar Lipids was the source of all lipids. Bradford [61] protein reagent, DNA size ladders, molecular mass protein standards, and reagents for electrophoresis and immunoblotting were obtained from Bio-Rad. Pah in the cytosol is phosphorylated by multiple protein kinases that include Pho85-Pho80 [37], Cdc28-cyclin B [35], PKA [38], PKC [42], and CKII (this study). B, diagram of Pah domain structure showing the positions of the amphipathic helix (AH) required for ER membrane interaction [36]; the NLIP and HAD-like domains containing the conserved glycine (G) and DIDGT catalytic sequence, respectively, that are required for PAP activity [16]; the acidic tail (AT) required for interaction with Nem1-Spo7 [39]; and the serine (S) and threonine (T) residues that are phosphorylated by Pho85-Pho80 [37], Cdc28-cyclin B [35], PKA [38], PKC [42], and CKII (this study). Thermo Scientific was the source of mouse anti-phosphoglycerate kinase antibodies (product 459250, lot 459250/ E1161), alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies (product 31340, lot NJ178812), alkaline phospha-
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