Phosphorylation of the Nem1‐Spo7/Pah1 Phosphatase Cascade by Pkc1 Protein Kinase C

In the yeast Saccharomyces cerevisiae , the Pah1 phosphatidate phosphatase is responsible for catalyzing the penultimate step in the synthesis of triacylglycerol. Phosphatidate phosphatase dephosphorylates phosphatidate to form diacylglycerol, the precursor of triacylglycerol. Pah1 exists in the cytosol as a phosphoprotein, but it must be dephosphorylated by the Nem1‐Spo7 protein phosphatase for its interaction with the ER membrane to associate with the substrate phosphatidate. In this work, we examined the regulation of the Nem1‐Spo7/Pah1 phosphatase cascade by its phosphorylation by Pkc1 protein kinase C (PKC). Pah1, Nem1, and Spo7 were expressed and purified from Escherichia coli to utilize non‐phosphorylated pristine substrates for PKC, which was purified from yeast. PKC phosphorylated each component of the phosphatase cascade on a serine residue, and the reactions were dependent on phosphatidylserine (0.5 mM) and diacylglycerol (0.15 mM) for the maximum phosphorylations. This was a surprising result given the fact that previous data indicated that yeast PKC does not require lipids for its activity, at least in the absence of GTP‐bound Rho1. Owing that prior work on yeast PKC utilized a peptide substrate, we examined the PKC activity using peptide substrates with sequences derived from Nem1, Spo7, or Pah1. With these substrates, PKC did not require phosphatidylserine and/or diacylglycerol for its activity. We found that mammalian PKC, using Pah1 or the Pah1‐derived peptide as substrates, were dependent on phosphatidylserine and diacylglycerol for activity. These results indicated a fundamental difference in the mechanisms of the reactions catalyzed by the yeast and mammalian PKC enzymes, and that yeast PKC is likely to require lipids for activity in vivo . Support or Funding Information Supported by NIH grant GM050679