There are ten isozymes of diacylglycerol kinase (DGK), and they regulate diverse patho-physiological functions. Here, we investigated the lipid-binding properties of DGK isozymes using protein-lipid overlay and liposome-binding assays. DGK gamma showed a strong binding activity compared with other DGK isozymes for phosphatidic acid (PA) among the various glycerophospholipids tested. However, DGK gamma failed to interact with DG and lyso-PA. Moreover, the isozyme was capable of binding to ceramide-l-phosphate but not to ceramide or sphingosine-1-phosphate. The isozyme bound more strongly to PA containing unsaturated fatty acid than to PA having only saturated fatty acid. An analysis using a series of deletion mutants of DGK gamma revealed that the N-terminal region, which contains a recoverin homology domain and EF-hand motifs, is responsible for the PA binding activity of DGK gamma. Taken together, these results indicate that DGK gamma is an anionic phospholipid binding protein that preferably interacts with a small highly charged head group that is very close to the glycerol or sphingosine backbone. (C) 2014 Elsevier Inc. All rights reserved.