Immunoquantitation of 80 kDa diacylglycerol kinase in pig and human lymphocytes and several other cells

Yamada, Katsushi; Sakane, Fumio; Kanoh, Hideo

doi:10.1016/0014-5793(89)80572-1

Cited by 46 publications

(32 citation statements)

References 24 publications

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“…DGK␣ isolated from rat, human, and porcine sources is a very abundant protein in tissues of hematopoietic origin (11,14). The data presented in this study confirm earlier studies describing restricted tissue distribution of DGK␣ mRNA and protein, and demonstrate tissue-specific genetic control of this isoform.…”

Section: Discussionsupporting

confidence: 87%

“…When different mouse tissues were analyzed, a 80-kDa band was recognized by the Ab in spleen and thymus (Fig. 1D), suggesting that, as previously described for rat and pig orthologs (11,14), DGK␣ is highly expressed in those tissues. Despite similar or higher mRNA expression levels, DGK␣ protein was undetectable in skeletal muscle and testis (Fig.…”

Section: Cloning and Expression Of Murine Dgk␣supporting

confidence: 75%

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T Cell Activation In Vivo Targets Diacylglycerol Kinase α to the Membrane: A Novel Mechanism for Ras Attenuation

Sanjuán

Pradet‐Balade

Jones

et al. 2003

The Journal of Immunology

110

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Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGKα, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGKα, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGKα to the membrane fraction. At early time points, DGKα translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGKα activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGKα mutants. We show that membrane localization of DGKα is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGKα, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.

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Section: Discussionsupporting

confidence: 87%

Section: Cloning and Expression Of Murine Dgk␣supporting

confidence: 75%

T Cell Activation In Vivo Targets Diacylglycerol Kinase α to the Membrane: A Novel Mechanism for Ras Attenuation

Sanjuán

Pradet‐Balade

Jones

et al. 2003

The Journal of Immunology

110

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“…It is likely that myeloid cells are the main source of our purified WBC enzyme as WBC preparations consisted for the predominant part of polymorphonuclear (myeloid) ceils. This argues against the suggestion [24] that, among the different types of peripheral blood cells, the presence of DG kinase (the structure of which we report) would be restricted to lymphocytes. In addition, the 86 kDa DG kinase could also be isolated from blast cells from a patient suffering from acute myeloid leukemia (results not shown).…”

Section: I Purification and Churacterization Of Dg Kimtse From Hucontrasting

confidence: 97%

Purification, cDNA‐cloning and expression of human diacylglycerol kinase

et al. 1990

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Diacylglycerol (DG) kinase attenuates the level of the second messenger DG in signal transduction, and therefore possibly modulates protein kinase C (PKC). DG kinase was purified to homogeneity from human white blood cells, showing an M 1 of 86 kDa as determined by SDS‐PAGE and gel filtration. Two amino acid sequences of tryptic peptides from DG kinase were determined and degenerate oligonucleotides were prepared and used in the polymerase chain reaction. An amplified DNA fragment was subsequently used to clone the full‐length human DG kinase cDNA. This sequence is the human homolog of porcine DG kinase cDNA sequence reported recently [1]. The sequence contains a double EF‐ hand structure typical for Ca2+ binding proteins. DG kinase further contains a double cysteine repeat that is present in all PKC isoforms, where it constitutes the phorbol ester (and most likely diacylglycerol) binding site. Therefore we speculate that the double cysteine repeat in DG kinase is involved in DG binding. DG kinase is transcribed as a single mRNA of 3.2 kb, that is highly expressed in T‐lymphocytes. The human DG kinase cDNA when transfected in mammalian cells (COS‐7) results in a 6–7‐fold increase of DG kinase activity.

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“…lA). We earlier demonstrated [25] that in thymocytes and peripheral T cells DGKc~ is present mostly in the cytosolic fraction. In accordance with this observation, we confirmed in separate experiments that the Triton X-100-soluble DGKtx represented the cytosolic enzyme and that the insoluble enzyme was associated with the total particulate fraction (not shown).…”

Section: Resultsmentioning

confidence: 93%

“…It thus appears that DGK translocation is caused by different mechanisms depending on the types of cells and DGK isozymes involved. Despite its presence in the cytosol of resting lymphocytes [25], DGKc~ was shown to be mainly located in the nuclei of IL-2-dependent lymphocyte cell line [18]. Although its intranuclear localization was not studied, nuclear DGKc~ was shown to be translocated to the perinuclear region upon IL-2 stimulation of these cells [18].…”

Section: Resultsmentioning

confidence: 95%

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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Immunoquantitation of 80 kDa diacylglycerol kinase in pig and human lymphocytes and several other cells

Cited by 46 publications

References 24 publications

T Cell Activation In Vivo Targets Diacylglycerol Kinase α to the Membrane: A Novel Mechanism for Ras Attenuation

T Cell Activation In Vivo Targets Diacylglycerol Kinase α to the Membrane: A Novel Mechanism for Ras Attenuation

Purification, cDNA‐cloning and expression of human diacylglycerol kinase

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

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