Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGK␥-C1A and DGK␤-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGK␥-C1A, human-DGK␥-C1A, and human-DGK␤-C1A gave K d values of 3.6, 2.8, and 14.6 nM, respectively, suggesting that DGK␥ and DGK␤ are new targets of phorbol esters. An A12T mutation of human-DGK␤-C1A enhanced the affinity to bind PDBu, indicating that the ␤-hydroxyl group of Thr-12 significantly contributes to the binding. The K d value for PDBu of FLAG-tagged whole rat-DGK␥ (4.4 nM) was nearly equal to that of rat-DGK␥-C1A (3.6 nM). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGK␥ and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGK␥ to cause its translocation.Diacylglycerol kinase (DGK) 1 and protein kinase C (PKC) both interact with the second messenger diacylglycerol (DG) (1, 2). DGK phosphorylates DG to produce phosphatidic acid, whereas PKC is allosterically activated by DG in the presence of phosphatidylserine. Therefore, DGK may inhibit the activation of PKC by attenuating DG levels, contributing to the regulation of intracellular signal transduction.To date, nine subtypes of mammalian DGKs have been cloned (3-15). All DGK isozymes consist of a conserved catalytic domain and two or three cysteine-rich C1 domains designated as C1A, C1B, and C1C (16). These isozymes are classified into five classes according to the other functional domains (Fig. 1). The class I isozymes (DGK␣, -␤, and -␥) have calcium binding domains (EF-hands). The class II isozymes (DGK␦ and -) have a pleckstrin homology domain at the N terminus, and their catalytic region is split into two domains unlike the other DGK isozymes. DGK⑀ has a simple structure and is classified as a class III isozyme. The class IV isozymes DGK and have a myristoylated alanine-rich C kinase substrate homology domain and four ankyrin repeats. DGK , which has three C1 domains unlike other DGK and PKC isozymes, is the only isozyme in class V.The similarity between DGK and PKC isozymes in structure is in the cysteine-rich C1 domains. Recent investigations using NMR spectroscopy and x-ray crystallography have revealed the three-dimensional structure of C1B domains of PKC␣, PKC␥,. Each PKC C1 domain has six conserved cysteines and two histidines in the typical core structure HX 12 CX 2 CX 13-14 CX 2 CX 4 HX 2 CX 7 C (where X is any amino acid) that coordinates two atoms of zinc in...

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...

No abstract