DGKepsilon is S-palmitoylated at the cysteine located at the cytoplasmic end of its N-terminal transmembrane fragment

Abstract

Diacylglycerol kinase-{varepsilon} (DGK{varepsilon}) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGK{varepsilon} is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity, and was also found crucial for lipid metabolism. DGK{varepsilon} dysfunction is linked with the development of atypical hemolytic uremic syndrome and possibly other human diseases. Despite the DGK{varepsilon} significance, data on its regulation by co/posttranslational modifications are scarce. Here we report that DGK{varepsilon} is S-palmitoylated at Cys38/40 (mouse/human DGK{varepsilon}) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGK{varepsilon} was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry, and with acyl-biotin and acyl-PEG exchange assays. The S-acyltransferases zDHHC7 and zDHHC17, and the zDHHC6/16 tandem were found to catalyze DGK{varepsilon} S-palmitoylation which also increased the DGK{varepsilon} abundance. Mouse DGK{varepsilon}-Myc ectopically expressed in HEK293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated while hyperpalmitoylation of wild type DGK{varepsilon} reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. Additionally, the substitution of neighboring Pro31 with Ala also diminished the activity of DGK{varepsilon}. Taken together, our data indicate that S-palmitoylation can fine-tune DGK{varepsilon} activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.