Identification of cyclin‐dependent kinase phosphorylation sites in yeast phosphatidate phosphatase

Abstract

The PAH1‐encoded phosphatidate (PA) phosphatase (PAP) plays a major role in controlling the cellular content of PA in the yeast Saccharomyces cerevisiae. The enzyme is phosphorylated in vivo on multiple residues, and one of the protein kinases involved is cyclin‐dependent kinase (CDK). In vitro, CDK phosphorylates purified recombinant PAP on both serine and threonine residues. An analysis of purified truncated forms of PAP revealed that the sites of CDK phosphorylation were at the C‐terminal end of the enzyme. A site‐directed mutagenesis study showed that Ser602 and Thr723 were the major sites of phosphorylation. In vitro, the S723A mutation caused a 50 % reduction in the CDK phosphorylation of PAP, whereas the S602A mutation caused the complete loss of the CDK phosphorylation of the enzyme. This indicated that phosphorylation of Ser602 was required for the phosphorylation of Thr723. Yeast strains that express S602A, T723A, and S602A T723A mutant enzymes have been constructed to examine the effects of CDK phosphorylation on PAP function in lipid metabolism. Supported by NIH grant GM‐50679.