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.
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Regulator | Target | Direction | Regulation Of | Happens During | Method | Evidence |
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PHO80-PHO85 kinase complex | PAH1 | protein activity | high-throughput | experimental evidence | ||
CLB1-CDC28 kinase complex | PAH1 | protein activity | high-throughput | experimental evidence | ||
cAMP-dependent protein kinase complex variant 1 | PAH1 | protein activity | high-throughput | experimental evidence | ||
Casein kinase II complex, CKA1-CKA2 variant | PAH1 | protein activity | high-throughput | experimental evidence | ||
PKC1 | PAH1 | protein activity | high-throughput | experimental evidence |
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Protein | Site | Modification | Modifier |
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PAH1 | S110 | phosphorylated residue | PHO80-PHO85 kinase complex |
PAH1 | S677 | phosphorylated residue | PKC1 |
PAH1 | S769 | phosphorylated residue | PKC1 |
PAH1 | S773 | phosphorylated residue | PKC1 |
PAH1 | S788 | phosphorylated residue | PKC1 |
PAH1 | S10 | phosphorylated residue | cAMP-dependent protein kinase complex variant 1 |
PAH1 | S114 | phosphorylated residue | PHO80-PHO85 kinase complex |
PAH1 | S168 | phosphorylated residue | PHO80-PHO85 kinase complex |
PAH1 | T170 | phosphorylated residue | Casein kinase II complex, CKA1-CKA2 variant |
PAH1 | S250 | phosphorylated residue | Casein kinase II complex, CKA1-CKA2 variant |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Interactor | Interactor | Assay | Annotation | Action | Modification | |
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NEM1 | PAH1 | Biochemical Activity | manually curated | Bait-Hit | Dephosphorylation | |
PAH1 | CKA2 | Biochemical Activity | manually curated | Hit-Bait | phosphorylated residue | |
PAH1 | PHO85 | Biochemical Activity | manually curated | Hit-Bait | phosphorylated residue | |
PAH1 | TPK2 | Biochemical Activity | manually curated | Hit-Bait | phosphorylated residue | |
PKC1 | PAH1 | Biochemical Activity | manually curated | Bait-Hit | phosphorylated residue |