Phosphorylation of yeast Pah1 phosphatidate phosphatase by casein kinase I

Abstract

Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate) in the synthesis of triacylglycerol. The enzyme is subject to phosphorylation (e.g. cyclin‐dependent protein kinases Pho85‐Pho80 and Cdc28‐cyclin B, protein kinases A and C, and casein kinase II) and dephosphorylation (e.g. by the Nem1‐Spo7 phosphatase complex) for regulation of its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is also phosphorylated by casein kinase I. By mass spectrometry, phosphopeptide mapping, and phosphoamino acid analysis, we showed that the phosphorylation occurs on multiple serine and threonine residues. Using Pah1 as substrate, casein kinase I activity was dependent on the time of the reaction and the amount of protein kinase, as well as the concentrations of Pah1 and ATP. The prephosphorylation of Pah1 by casein kinase I reduced its subsequent phosphorylation by Pho85‐Pho80, and the prephosphorylation by Pho85‐Pho80 reduced its subsequent phosphorylation by casein kinase I. This was attributed to both protein kinases having phosphorylation sites on Pah1 in common. The prephosphorylation by casein kinase I stimulated subsequent phosphorylation by casein kinase II, but the prephosphorylation with casein kinase II did not affect subsequent phosphorylation by casein kinase I. This indicated that the sites phosphorylated by casein kinases I and II were not common. Prephosphorylation of Pah1 by casein kinase I had no effect on the phosphorylation by protein kinase A, and vice versa. Hierarchal phosphorylation experiments with protein kinase C, along with experiments to identify sites on Pah1 phosphorylated by casein kinase II are currently in progress.Support or Funding InformationGM050679This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.