Regulation of the Yeast DAG Kinase by Phosphorylation

Abstract

In the yeast Saccharomyces cerevisiae, DGK1‐encoded diacylglycerol (DAG) kinase is a membrane‐associated enzyme that catalyzes the CTP‐dependent phosphorylation of DAG to form phosphatidic acid (PA). This enzyme, in conjunction with PAH1‐encoded PA phosphatase, controls the levels of PA and DAG for phospholipid synthesis, membrane growth, and lipid droplet formation. When cells resume vegetative growth from stasis in the absence of de novo fatty acid synthesis, DAG kinase is required. In addition, the activity of DAG kinase is induced when cells resume growth and it is not due to the increased protein expression. Therefore, we speculated that the increased activity was due to a biochemical mechanism such as phosphorylation. Several phosphorylation sites are located in the hydrophilic N‐terminal region of Dgk1p. We expressed and purified the N‐terminal His6‐Dgk1p1‐77 (residues 1‐77) for in vitro phosphorylation studies. His6‐Dgk1p1‐77 was phosphorylated on serine residues by casein kinase II (CKII) and cAMP‐dependent protein kinase A (PKA). Site‐directed mutagenesis coupled with two‐dimensional phosphopeptide mapping analyses identified Ser‐44, Ser‐45, and Ser‐46 as target sites of CKII and Ser‐25 and Ser‐26 as target sites of PKA. Phosphorylation‐deficient and phosphorylation‐mimic mutations will be introduced into full‐length Dgk1p to examine the effects of phosphorylation by CKII and PKA on DAG kinase function in vivo. (Supported by NIH grant GM028140).