The Nem1‐Spo7/Pah1 Phosphatase Cascade is Negatively Regulated by Protein Kinase A

Yeast Pah1 phosphatidate phosphatase, which dephosphorylates phosphatidate to generate diacylglycerol, catalyzes the penultimate step in the synthesis of triacylglycerol. The translocation of Pah1 from cytosol to ER membrane is essential for its function and is strictly dependent on the nuclear/ER membrane‐associated Nem1‐Spo7 protein phosphatase complex. Dephosphorylation of Pah1 by Nem1‐Spo7 enables its membrane association, and also stimulates its phosphatidate phosphatase activity. Previous work has shown that protein kinase A phosphorylates Pah1 to inhibit its function in lipid metabolism. Here, we showed that Nem1, the catalytic subunit of the protein phosphatase complex, is also phosphorylated by protein kinase A. The kinase phosphorylated Nem1 on multiple serine residues and caused a 35% decrease in its protein phosphatase activity for phospho‐Pah1. The Nem1 synthetic peptides KRNRG S NASEN ( V max / K m = 2.2 μmol min −1 mg −1 mM −1 ) and RPRSY S KSELS ( V max / K m = 94.9 μmol min −1 mg −1 mM −1 ) that contain Ser‐140 and Ser‐210, respectively, were phosphorylated by protein kinase A. Site‐direct mutagenesis of Ser‐140 and Ser‐210 in Nem1 followed by phosphopeptide analysis showed that Ser‐210 was the major protein kinase A target site. The alanine mutation of Ser‐210 caused a 50% reduction in the in vivo phosphorylation state of Nem1. These data indicated that protein kinase A plays a negative regulatory role on the activity of the Nem1‐Spo7/Pah1 phosphatase cascade. Support or Funding Information Supported by NIH grant GM050679