| Pah1p, which functions as phosphatidate phosphatase (PAP) in the yeast Saccharomyces cerevisiae, plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The diacylglycerol produced by PAP is used for the synthesis of triacylglycerol as well as for the synthesis of phospholipids via the Kennedy pathway. Pah1p is a highly phosphorylated protein in vivo, and has been previously shown to be phosphorylated by the protein kinases Pho85p-Pho80p and Cdc28p-cyclin B. In this work, we showed that Pah1p was a bona fide substrate for protein kinase A (PKA) and protein kinase C (PKC). PKA phosphorylated Pah1p on Ser-10, Ser-677, Ser-773, Ser-774, and Ser-788, whereas Ser-677 and Ser-788 are also the PKC target sites in addition to Ser-769. PKA-mediated phosphorylation of Pah1p inhibited its PAP activity by decreasing catalytic efficiency, and the inhibitory effect was primarily conferred by phosphorylation at Ser-10. On the other hand, the phosphorylation of Pah1p by PKC caused a slight increase in PAP activity. The pre-phosphorylation of Pah1p with PKA caused a reduction on the phosphorylation by PKC and vice versa. The phosphorylation of Pho85p-Pho80p suppressed the subsequent phosphorylation by PKC but did not affect PKA. The analysis of the S10A and S10D mutations (mimicking dephosphorylation and phosphorylation, respectively), alone or in combination with the seven alanine (7A) mutations of the sites phosphorylated by Pho85p-Pho80p and Cdc28p-cyclin B, indicated that phosphorylation at Ser-10 stabilized Pah1p abundance and inhibited its association with membranes, PAP activity, and triacylglycerol synthesis. The S10A mutation enhanced the physiological effects imparted by the 7A mutations, whereas the S10D mutations attenuated the effects of the 7A mutations. The analyses of PKC target site mutations revealed that neither 3A nor 3D Pah1p mutant affected the protein abundance, localization of pah1p and TAG synthesis. Collectively, these data indicated that the protein kinase A-mediated phosphorylation of Ser-10 functions in conjunction with the phosphorylations mediated by Pho85p-Pho80p and Cdc28p-cyclin B, and that phospho-Ser-10 should be dephosphorylated for proper PAP function. |
