Phosphorylation of the yeast lipin homolog Pah1p by Cdc28p kinase

Abstract

The yeast lipin homolog Pah1p is a Mg2+‐dependent phosphatidate phosphatase enzyme that catalyzes the dephosphorylation of phosphatidate to diacylglycerol and Pi. Pah1p is responsible for de novo lipid synthesis, its reaction being the committed step for the synthesis of the storage lipid triacylglycerol, which is formed from phosphatidate via diacylglycerol. The diacylglycerol generated in the phosphatidate phosphatase reaction is also utilized in the synthesis of the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine via the Kennedy Pathway. Previous studies indicate that Pah1p is phosphorylated by cyclin‐dependent Cdc28p kinase and dephosphorylated by the Nem1p‐Spo7p phosphatase complex. A phosphorylation‐deficient Pah1p mutant exhibits elevated phosphatidate phosphatase activity suggesting that phosphorylation inhibits activity. In this work, we examined the phosphorylation of Pah1p by Cdc28p kinase in vitro. Using purified Escherichia coli‐expressed Pah1p as a substrate, Cdc28p kinase activity was dependent on the time of phosphorylation and the concentration of the kinase. In addition, Cdc28p kinase activity was dependent on the concentration of Pah1p and ATP. Serine and threonine residues were targets for phosphorylation. The phosphorylation of Pah1p resulted in the loss of its phosphatidate phosphatase activity. Supported by NIH grant GM‐28140.