Protein kinase C mediates the phosphorylation of the Nem1–Spo7 protein phosphatase complex in yeast

Prabuddha Dey,Wen-Min Su,Mona Mirheydari,Gil-Soo Han,George M Carman

doi:10.1074/jbc.ra119.010592

Prabuddha Dey, Wen-Min Su + Show 3 more

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https://doi.org/10.1074/jbc.ra119.010592

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Abstract

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.

Highlights

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
Elevated PA phosphatase activity is associated with the production of DAG that is used for the synthesis of the storage lipid TAG, whereas reduced enzyme activity is associated with the production of CDP-DAG that is used for the synthesis of membrane phospholipids [3,4,5,6,7]
We characterized the phosphorylation of Nem1–Spo7 complex by protein kinase C (PKC), a PS/DAG-dependent protein kinase in yeast [39] that is required for cell cycle progression [41] and plays a role in regulating lipid synthesis [42,43,44,45,46,47] and in maintaining cell wall integrity [41, 48, 49]

Summary

To whom correspondence should be addressed

Emerged as a key regulatory enzyme in yeast that controls the bifurcation of PA between DAG and CDP-DAG [3,4,5,6,7] (see Fig. 1A). The function of such an important regulatory enzyme is controlled by genetic and biochemical mechanisms, with perhaps phosphorylation and dephosphorylation being the most important [3, 5,6,7] These posttranslational modifications control the location and stability of Pah as well as its PA phosphatase activity [23,24,25,26,27,28,29,30,31,32,33]. We characterized the phosphorylation of Nem1–Spo complex by PKC, a PS/DAG-dependent protein kinase in yeast [39] that is required for cell cycle progression [41] and plays a role in regulating lipid synthesis [42,43,44,45,46,47] and in maintaining cell wall integrity [41, 48, 49]. Lipid composition analysis of cells bearing phosphorylation-site mutations indicated that PKC has a positive

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Discussion

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