Protein kinase C mediates the phosphorylation of the Nem1-Spo7 protein phosphatase complex in yeast.
The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase required for the nuclear/endoplasmic reticulum membrane localization of Pah1, a phosphatidate phosphatase that produces diacylglycerol for triacylglycerol synthesis at the expense of phospholipid synthesis. In a previous study, we showed that the protein phosphatase is subject to phosphorylation by protein kinase A (PKA). Here, we demonstrate that Nem1-Spo7 is regulated through its phosphorylation by protein kinase C (PKC), which plays multiple roles, including the regulation of lipid synthesis and cell wall integrity. Phosphorylation analyses of Nem1-Spo7 and its synthetic peptides indicate that both subunits of the complex are bona fide PKC substrates. Site-directed mutagenesis of NEM1 and SPO7, coupled with phosphopeptide mapping and immunoblotting with a phosphoserine-specific PKC substrate antibody, revealed that Ser-201 in Nem1 and Ser-22/Ser-28 in Spo7 are major PKC target sites of phosphorylation. Activity analysis of mutant Nem1-Spo7 complexes indicates that the PKC phosphorylation of Nem1 exerts a stimulatory effect, but the phosphorylation of Spo7 has no effect. Lipid-labeling analysis of cells expressing the phosphorylation-deficient alleles of NEM1 and SPO7 indicates that the stimulation of the Nem1-Spo7 activity has the effect of increasing triacylglycerol synthesis. Prephosphorylation of Nem1-Spo7 by PKC inhibits the PKA phosphorylation of Nem1, whereas prephosphorylation of the phosphatase complex by PKA inhibits the PKC phosphorylation of Spo7. Collectively, this work advances the understanding of the Nem1-Spo7 regulation by phosphorylation and its impact on lipid synthesis.
['Dey|Prabuddha|P|', 'Su|Wen-Min|WM|', 'Mirheydari|Mona|M|', 'Han|Gil-Soo|GS|0000-0002-8170-854X', 'Carman|George M|GM|0000-0003-4951-8233']
D050356:Lipid Metabolism D008565:Membrane Proteins D009687:Nuclear Proteins D010766:Phosphorylation D011493:Protein Kinase C D012441:Saccharomyces cerevisiae D029701:Saccharomyces cerevisiae Proteins
protein phosphatase phosphatidate protein kinase C (PKC) lipid lipid synthesis PA phosphatase lipid metabolism membrane lipid