Diacylglycerol (DAG) acts as an allosteric activator of protein kinase C (PKC) and is converted to phosphatidic acid by DAG kinase (DGK). Therefore, DGK is thought to be a negative regulator of PKC activation. Here we show molecular mechanisms of functional coupling of the two kinases. ␥PKC directly associated with DGK␥ through its accessory domain (AD), depending on Ca 2؉ as well as phosphatidylserine/diolein in vitro. Mass spectrometric analysis and mutation studies revealed that ␥PKC phosphorylated Ser-776 and Ser-779 in the AD of DGK␥. The phosphorylation by ␥PKC resulted in activation of DGK␥ because a DGK␥ mutant in which Ser-776 and Ser-779 were substituted with glutamic acid to mimic phosphorylation exhibited significantly higher activity compared with wild type DGK␥ and an unphosphorylatable DGK␥ mutant. Importantly, the interaction of the two kinases and the phosphorylation of DGK␥ by ␥PKC could be confirmed in vivo, and overexpression of the AD of DGK␥ inhibited re-translocation of ␥PKC. These results demonstrate that localization and activation of the functionally correlated kinases, ␥PKC and DGK␥, are spatio-temporally orchestrated by their direct association and phosphorylation, contributing to subtype-specific regulation of DGK␥ and DAG signaling.

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGK␥ came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGK␥ is transported from the cytoplasm to the nucleus. The nuclear transportation of DGK␥ didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGK␥ acts as a nuclear transport signal. Furthermore, to address the function of DGK␥ in the nucleus, we produced stable cell lines of wild-type DGK␥ and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGK␥ were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGK␥ regulates cell cycle. Diacylglycerol (DG)3 is a second messenger regulating various cellular responses (1, 2). One of the important roles of DG is an activating of protein kinase C (PKC) (1,3,4). DG is physiologically produced as a result of the signal-induced hydrolysis of phosphatidylinositol by phospholipase C. The generated DG is phosphorylated to phosphatidic acid by diacylglycerol kinase (DGK) or metabolized by DG lipase (2, 5, 6). Thus, DGK is an important enzyme to inactivate PKC by reducing the DG level, contributing to regulating of the cellular response. In addition, phosphatidic acid itself activates PKC (7), phosphatidylinositol 4-phosphate 5-kinase (8, 9), and mammalian target of rapamycin (10), and modulates Ras GTPase-activating protein (11).Molecular cloning studies revealed that mammalian DGK family consists of at least nine subtypes (2). Although all DGKs have cysteinerich repeats similar to the C1A and C1B domains of PKCs in the N terminus and a catalytic domain in the C terminus, they are divided into five groups on the primary structure of these DGKs. In an effort to elucidate the function of DGK, we unexpectedly found that GFP-fused DGK␥ (GFP-DGK␥) became localized in the nucleus as well as the cytoplasm a few days after transfection but was localized mainly in the cytoplasm just after expressed in CHO-K1 cells. Although nuclear transportation of DGK␥ has never been reported, expression of DGK and DGK in the nucleus has been already described (20,21). In addition, DGK is thought to be involved in the regulation of cell cycle (21). These findings, together with the facts that phosphatidylinositol turnover exists within the nucleus and DG may be involved in the regulation of cell cycle (22-27), suggest that DGK␥ has some physiological function in the nucleus. However, mechanism of the nuclear transportation and physiological functions of DGK␥ are unknown. W...

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Background: c-Abl and DGK␣ are important enzymes for many cellular processes. However, substrates of c-Abl and the regulation mechanism of DGK␣ are not fully understood. Results: c-Abl directly phosphorylates DGK␣ at Tyr-218. Conclusion:The c-Abl phosphorylation regulates the serum-induced nuclear export of DGK␣. Significance: The c-Abl phosphorylation contributes to the spatio-temporal regulation of DGK␣.

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