We examined the translocation of diacylglycerol kinase (DGK) ␣ and ␥ fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGK␣ and ␥ were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGK␥, but not DGK␣, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGK␥ was inhibited by the mutation of C1A but not C1B domain of DGK␥ and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGK␥ from the cytoplasm to the plasma membrane, whereas DGK␣ showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGK␥ and ␣ to the plasma membrane. The timing of the ATP-induced translocation of DGK␥ roughly coincided with that of PKC␥ re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKC␥ was obviously hastened by co-expression with DGK␥ and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction. Diacylglycerol (DG)1 is a second messenger regulating various cellular responses (1, 2). One of the important roles of DG is the activation of protein kinase C (PKC) (1, 3, 4). Thus, DG is very important for regulation of PKC activity and cellular response. DG is produced physiologically as a result of the signal-induced hydrolysis of phosphatidylinositol by phospholipase C and also from phosphatidylcholine by phospholipase D. Generated DG is phosphorylated to phosphatidic acid by diacylglycerol kinase (DGK) or cleavage by DG lipase (2, 5, 6). DGK is an important enzyme for inactivating PKC by attenuation of the DG level, contributing to regulation of the cellular response. In addition, phosphatidic acid itself activates PKC and PLC␥1 (7, 8) and modulates Ras GTPase-activating protein (9). DGKs have additional important functions for various cellular responses.To date at least nine subtypes of mammalian DGKs have been cloned and divided into five groups based on structure (2). Generally, all DGKs have cysteine-rich regions homologous to the C1A and C1B motifs of PKCs in the regulatory domain at the N terminus of the protein and possess a conserved catalytic domain in the C terminus of the protein. Type I DGKs, including DGK␣, -␤, and -␥, have EF-hand motifs and two cysteinerich regions in the regulatory domain (10 -12). Type II DGKs such as DGK␦ and -, have a pleckstrin homology domain instead of the EF-hand motif in addition to two cysteine-rich regions (13,14). Interestingly, the catalytic domains of DGK␦ and -are separated. Type III, consisting of DGK⑀, has only two cysteine-rich regions in the regulatory domain. Type IV, DGK and ...

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