Phosphorylation of yeast phosphatidate phosphatase by cyclin‐dependent kinase Pho85p

The PAH1 ‐encoded phosphatidate (PA) phosphatase, which catalyzes the Mg 2+ ‐dependent dephosphorylation of PA to produce diacylglycerol, plays a major role in the production of the storage lipid TAG as well as in the control of phospholipid synthesis. The function of PA phosphatase requires its membrane localization, which is controlled by phosphorylation and by interaction with its phosphatase complex Nem1p‐Spo7p. The phosphorylation of PA phosphatase occurs at multiple Ser and Thr residues, some of which are target sites for the cyclin‐dependent kinase Pho85p. In this study, we examined the Pho85p phosphorylation by using recombinant PA phosphatase expressed in E. coli . Purified His 6 ‐tagged PA phosphatase, which was catalytically active, was phosphorylated by Pho85p in a time‐ and dose‐dependent manner. Mass spectrometry analysis showed that PA phosphatase was phosphorylated by Pho85p at multiple sites (e.g., Ser 168 , Ser 602 , Ser 748 , Ser 773 , Ser 774 , and Thr 796 ). The roles of these phosphorylations on the physiological functions of PA phosphatase will be presented. Supported by NIH grant GM 50679.