Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. DGK␦ (type II isozyme) contains a pleckstrin homology domain at the N terminus and a sterile ␣ motif domain at the C terminus. We identified another DGK␦ isoform (DGK␦2, 135 kDa) that shared the same sequence with DGK␦ previously cloned (DGK␦1, 130 kDa) except for the 52 residues N-terminally extended. Analysis of panels of human normal and tumor tissue cDNAs revealed that DGK␦2 was ubiquitously expressed in all normal and tumor tissues examined, whereas the transcript of DGK␦1 was detected only in ovary and spleen, and in a limited set of tumor-derived cells. The expression of DGK␦2 was induced by treating cells with epidermal growth factor and tumor-promoting phorbol ester. In contrast, the levels of mRNA and protein of DGK␦1 were suppressed by phorbol ester treatment. It thus becomes clear that the two DGK␦ isoforms are expressed under distinct regulatory mechanisms. DGK␦1 was translocated through its pleckstrin homology domain from the cytoplasm to the plasma membrane in response to phorbol ester stimulation, whereas DGK␦2 remained in the cytoplasm even after stimulation. Further experiments showed that the ␦2-specific N-terminal sequence blocks the phorbol ester-dependent translocation of this isoform. Co-immunoprecipitation analysis of differently tagged DGK␦1 and DGK␦2 proteins showed that they were able to form homo-as well as heterooligomers. Taken together, alternative splicing of the human DGK␦ gene generates at least two isoforms, differing in their expressions and regulatory functions.

Nine diacylglycerol kinase (DGK) isozymes have been identified. However, our knowledge of their individual functions is still limited. Here, we demonstrate the role of DGK␥ in regulating Rac1-governed cell morphology. We found that the expression of kinase-dead DGK␥, which acts as a dominant-negative mutant, and inhibition of endogenous DGK␥ activity with R59949 induced lamellipodium and membrane ruffle formation in NIH3T3 fibroblasts in the absence of growth factor stimulation. Reciprocally, lamellipodium formation induced by plateletderived growth factor was significantly inhibited upon expression of constitutively active DGK␥. Moreover, the constitutively active DGK␥ mutant suppressed integrinmediated cell spreading. These effects are isoform-specific because, in the same experiments, none of the corresponding mutants of DGK␣ and DGK␤, closely related isoforms, affected cell morphology. These results suggest that DGK␥ specifically participates in the Rac1-mediated signaling pathway leading to cytoskeletal reorganization. In support of this, DGK␥ co-localized with dominant-active Rac1 especially in lamellipodia. Moreover, we found that endogenous DGK␥ was physically associated with cellular Rac1. Dominant-negative Rac1 expression blocked the lamellipodium formation induced by kinase-dead DGK␥, indicating that DGK␥ acts upstream of Rac1. This model is supported by studies demonstrating that kinase-dead DGK␥ selectively activated Rac1, but not Cdc42. Taken together, these results strongly suggest that DGK␥ functions through its catalytic action as an upstream suppressor of Rac1 and, consequently, lamellipodium/ruffle formation.It is well recognized that a variety of lipid second messengers in low abundance carry out specific tasks for a wide range of biological processes in eukaryotic cells. The cellular concentrations of such signaling lipids must be strictly regulated by the action of metabolic enzymes. Diacylglycerol kinase (DGK) 1 phosphorylates diacylglycerol (DAG) to yield phosphatidic acid (PA) (1). DAG is an established activator of conventional and novel protein kinases C (PKCs), Unc-13, and Ras guanyl nucleotide-releasing protein (2-6). PA has also been reported to regulate a number of signaling proteins such as phosphatidylinositol-4-phosphate 5-kinase, Ras GTPase-activating protein, Raf-1 kinase, atypical PKC, and chimaerins (6 -8). Moreover, specific PA-binding sites have recently been identified in several important proteins such as Raf-1 (9), mTOR (mammalian target of rapamycin) (10), and p47 phox (11). Thus, DGK can potentially participate in a diverse range of cellular events through modulating the balance between two bioactive lipids, DAG and PA.Mammalian DGK is known to exist as a large protein family consisting of nine isozymes classified into five subtypes according to their structural features (12-15). These subfamilies can be characterized by the presence of a variety of regulatory domains of known and/or predicted functions, clearly indicating their distinct functions and regulatory mecha...

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