Purification, cDNA‐cloning and expression of human diacylglycerol kinase

Schaap, Dick; Widt, John de; Wal, J van der; Vandekerckhove, Joël; Damme, Josef Van; Güssow, Detlef; Ploegh, Hidde L.; Blitterswijk, Whim J. van; Bend, R L van der

doi:10.1016/0014-5793(90)81461-v

Cited by 119 publications

(101 citation statements)

References 39 publications

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“…Class I Dgk, comprising a-, b-and g-isoforms, shares a common N-terminal autoinhibitory motif and a pair of EF-hand calcium-binding domains preceding the Zn fingers. Dgk-a is abundant in T lymphocytes, but is also expressed in endothelial and epithelial cells, fibroblasts and oligodendrocytes (Schaap et al 1990;Cutrupi et al, 2000; data not shown). The biological functions and biochemical regulation of Dgk enzymes are currently under investigation (reviewed in van Blitterswijk and Houssa, 2000).…”

Section: Introductionmentioning

confidence: 88%

Activation of diacylglycerol kinase α is required for VEGF-induced angiogenic signaling in vitro

et al. 2004

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Section: Introductionmentioning

confidence: 88%

Activation of diacylglycerol kinase α is required for VEGF-induced angiogenic signaling in vitro

et al. 2004

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“…A dominant-active form of Rac1 (Rac1-G12V) was generated by replacing Gly 12 with Val using the QuikChange site-directed mutagenesis kit. A dominant-negative mutant of Rac1 (Rac1-T17N) was generated by replacing Thr 17 with Asn. The authenticity of the cDNA constructs was confirmed by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…1, panels g-hЈ) when the cells were stimulated with platelet-derived growth factor (PDGF), which is known to induce these F-actin-driven structures (33)(34)(35). On the other hand, DGK␣, which is known to be cytosolic (17,22,36), showed cytoplasmic localization and failed to show such marked accumulation at lamellipodia/ruffles (Fig. 1, panels i-l).…”

Section: Kinase-dead Dgk␥ Induces Lamellipodium/membrane Ruffle Formamentioning

confidence: 98%

Diacylglycerol Kinase γ Serves as an Upstream Suppressor of Rac1 and Lamellipodium Formation

Tsushima

Kai

Yamada

et al. 2004

Journal of Biological Chemistry

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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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“…However, it is not known which DGK isozyme so far cloned accounts for the DGK activity variably distributed within the cells. In the present work we focussed on DGKc~, which is predominantly expressed in thymus and peripheral T-cells in addition to oligodendroglial cells [3][4][5].…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Translocation of diacylglycerol kinase α to the nuclear matrix of rat thymocytes and peripheral T‐lymphocytes

et al. 1996

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The cytosolic ~-diacylglycerol kinase (DGK) was translocated to and tightly associated with the nuclear matrix when rat thymocytes and peripheral T-lymphocytes were stimulated with concanavalin A or anti-T-cell receptor antibody. This translocation occurred rather slowly and was completed in 3-4 h after cell stimulation. We also detected significant accumulation of nuclear phosphatidic acid interpreted as being formed by the translocated enzyme. The enzyme translocation is not directly linked to pbospboinositide turnover and protein pbospborylation, since pborbol myristate acetate and calcium ionopbore did not affect the cellular DGK(x and since we detected no covalent modification of the enzyme molecule. Although the mechanisms underlying the enzyme translocation remain unknown, our results indicate that DGKcx participates in nuclear pbospbollpid metabolism occurring at the intermediate stage of lymphocyte activation.Key words." Diacylglycerol kinase, c~-isozyme; T-lymphocyte, rat; Translocation; Nuclear matrix elucidated by the finding that DGKa could account for the most of the DGK activity, which was rapidly increased after IL-2 stimulation of T-cell lines [18]. However, the role of DGKc~ in phosphoinositide turnover and in other biochemical events preceding the expression of IL-2 receptors remains unknown. For example, exogenous short-chain DG repeatedly administered to human T-lymphocytes was phosphorylated by cellular DGK only to a negligible extent [19]. Van der Bend et al. [20] proposed a topological sequestration of cellular DGK molecules based on the finding that DG artificially generated by treating Jurkat and other cells with bacterial phospholipase C could not serve as substrate for the cellular DGK. These observations indicate that the metabolic role of DGKa abundantly contained in T-lymphocytes is still largely unknown and that the cellular DGKs are operating under strict control mechanisms. Here we describe a targeting of DGKc~ to the nuclear matrix upon T-cell activation and suggest the participation of this DGK isozyme in the nuclear phospholipid metabolism occurring in stimulated lymphocytes.

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Purification, cDNA‐cloning and expression of human diacylglycerol kinase

Cited by 119 publications

References 39 publications

Activation of diacylglycerol kinase α is required for VEGF-induced angiogenic signaling in vitro

Activation of diacylglycerol kinase α is required for VEGF-induced angiogenic signaling in vitro

Diacylglycerol Kinase γ Serves as an Upstream Suppressor of Rac1 and Lamellipodium Formation

Translocation of diacylglycerol kinase α to the nuclear matrix of rat thymocytes and peripheral T‐lymphocytes

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