Phosphorylation of Yeast Phosphatidate Phosphatase by Protein Kinase C

Abstract

The yeast PAH1‐encoded phosphatidate (PA) phosphatase catalyzes the Mg2+‐dependent dephosphorylation of PA to yield diacylglycerol and Pi. This enzyme plays a major role in the regulation of lipid metabolism. PA phosphatase is a phosphoprotein in vivo, and contains putative phosphorylation sites for multiple protein kinases. In this study, we examined the phosphorylation of the enzyme by protein kinase C using recombinant PA phosphatase expressed in E. coli. Purified His6‐tagged PA phosphatase was active, and was phosphorylated by protein kinase C in a time‐ and dose‐dependent manner. In addition, protein kinase C activity was dependent on the concentrations of PA phosphatase (28 μg/ml) and ATP (4.2 μM). Phosphoamino acid analysis indicated that phosphorylation by protein kinase C occurs primarily at the serine residue. Mass spectrometry of phosphorylated enzyme identified Ser773 and Ser788 as phosphorylation sites. However, phosphopeptide mapping analysis indicated additional sites of phosphorylation. Phosphorylation analysis of truncated forms of PA phosphatase indicated that the additional phosphorylation sites were located at the C‐terminal and central regions of the protein. Supported by NIH grant GM 50679.