Reference: Hsieh LS, et al. (2016) Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism. J Biol Chem 291(19):9974-90

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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.

Reference Type
Journal Article | Research Support, N.I.H., Extramural
Authors
Hsieh LS, Su WM, Han GS, Carman GM
Primary Lit For
SPO7 | CKB1 | CKA1 | NEM1 | CKA2 | CKB2 | PAH1 | Nem1-Spo7 phosphatase complex
Additional Lit For
pah1-S705D/7A

Phenotype Annotations


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Gene Phenotype Experiment Type Mutant Information Strain Background Chemical Details Reference

Regulation Annotations 5 entries for 6 genes


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RegulatorTargetDirectionRegulation OfHappens DuringMethodEvidence
PHO80-PHO85 kinase complexPAH1protein activityhigh-throughputexperimental evidence
CLB1-CDC28 kinase complexPAH1protein activityhigh-throughputexperimental evidence
cAMP-dependent protein kinase complex variant 1PAH1protein activityhigh-throughputexperimental evidence
Casein kinase II complex, CKA1-CKA2 variantPAH1protein activityhigh-throughputexperimental evidence
PKC1PAH1protein activityhigh-throughputexperimental evidence
Showing 1 to 5 of 5 entries

Post-translational Modifications25 entries for 19 sites


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ProteinSiteModificationModifier
PAH1S110phosphorylated residuePHO80-PHO85 kinase complex
PAH1S677phosphorylated residuePKC1
PAH1S769phosphorylated residuePKC1
PAH1S773phosphorylated residuePKC1
PAH1S788phosphorylated residuePKC1
PAH1S10phosphorylated residuecAMP-dependent protein kinase complex variant 1
PAH1S114phosphorylated residuePHO80-PHO85 kinase complex
PAH1S168phosphorylated residuePHO80-PHO85 kinase complex
PAH1T170phosphorylated residueCasein kinase II complex, CKA1-CKA2 variant
PAH1S250phosphorylated residueCasein kinase II complex, CKA1-CKA2 variant
Showing 1 to 10 of 25 entries

Interaction Annotations


Genetic Interactions

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Interactor Interactor Allele Assay Annotation Action Phenotype SGA score P-value Source Reference

Physical Interactions 5 entries for 6 genes

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InteractorInteractorAssayAnnotationActionModification
NEM1PAH1Biochemical Activitymanually curatedBait-HitDephosphorylation
PAH1CKA2Biochemical Activitymanually curatedHit-Baitphosphorylated residue
PAH1PHO85Biochemical Activitymanually curatedHit-Baitphosphorylated residue
PAH1TPK2Biochemical Activitymanually curatedHit-Baitphosphorylated residue
PKC1PAH1Biochemical Activitymanually curatedBait-Hitphosphorylated residue
Showing 1 to 5 of 5 entries