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1
Gravina, Teresa, Chiara Maria Teresa Boggio, Elisa Gorla, Luisa Racca, Silvia Polidoro, Sara Centonze, Daniela Ferrante, et al. "Role of Diacylglycerol Kinases in Acute Myeloid Leukemia." Biomedicines 11, no. 7 (July 1, 2023): 1877. http://dx.doi.org/10.3390/biomedicines11071877.
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Diacylglycerol kinases (DGKs) play dual roles in cell transformation and immunosurveillance. According to cancer expression databases, acute myeloid leukemia (AML) exhibits significant overexpression of multiple DGK isoforms, including DGKA, DGKD and DGKG, without a precise correlation with specific AML subtypes. In the TGCA database, high DGKA expression negatively correlates with survival, while high DGKG expression is associated with a more favorable prognosis. DGKA and DGKG also feature different patterns of co-expressed genes. Conversely, the BeatAML and TARGET databases show that high DGKH expression is correlated with shorter survival. To assess the suitability of DGKs as therapeutic targets, we treated HL-60 and HEL cells with DGK inhibitors and compared cell growth and survival with those of untransformed lymphocytes. We observed a specific sensitivity to R59022 and R59949, two poorly selective inhibitors, which promoted cytotoxicity and cell accumulation in the S phase in both cell lines. Conversely, the DGKA-specific inhibitors CU-3 and AMB639752 showed poor efficacy. These findings underscore the pivotal and isoform-specific involvement of DGKs in AML, offering a promising pathway for the identification of potential therapeutic targets. Notably, the DGKA and DGKH isoforms emerge as relevant players in AML pathogenesis, albeit DGKA inhibition alone seems insufficient to impair AML cell viability.
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Tabet, Ricardos, Enora Moutin, Jérôme A. J. Becker, Dimitri Heintz, Laetitia Fouillen, Eric Flatter, Wojciech Krężel, et al. "Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons." Proceedings of the National Academy of Sciences 113, no. 26 (May 27, 2016): E3619—E3628. http://dx.doi.org/10.1073/pnas.1522631113.
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Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.
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YAMADA, Keiko, Fumio SAKANE, Norio MATSUSHIMA, and Hideo KANOH. "EF-hand motifs of α, β and γ isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes." Biochemical Journal 321, no. 1 (January 1, 1997): 59–64. http://dx.doi.org/10.1042/bj3210059.
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The three diacylglycerol kinase isoenzymes (DGKα, DGKβ and DGKγ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca2+-binding activities of these motifs have not been tested except for those present in DGKα. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to α-DKE (9.9 µM) was an order of magnitude greater than those estimated for β-DKE (0.89 µM) and γ-DKE (0.40 µM). Experiments with 2-p-toluidinylnaphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to β-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of α-DKE and γ-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGKα, DGKβ and DGKγ possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca2+-induced conformational changes.
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Baldanzi, Gianluca, and Mario Malerba. "DGKα in Neutrophil Biology and Its Implications for Respiratory Diseases." International Journal of Molecular Sciences 20, no. 22 (November 13, 2019): 5673. http://dx.doi.org/10.3390/ijms20225673.
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Diacylglycerol kinases (DGKs) play a key role in phosphoinositide signaling by removing diacylglycerol and generating phosphatidic acid. Besides the well-documented role of DGKα and DGKζ as negative regulators of lymphocyte responses, a robust body of literature points to those enzymes, and specifically DGKα, as crucial regulators of leukocyte function. Upon neutrophil stimulation, DGKα activation is necessary for migration and a productive response. The role of DGKα in neutrophils is evidenced by its aberrant behavior in juvenile periodontitis patients, which express an inactive DGKα transcript. Together with in vitro experiments, this suggests that DGKs may represent potential therapeutic targets for disorders where inflammation, and neutrophils in particular, plays a major role. In this paper we focus on obstructive respiratory diseases, including asthma and chronic obstructive pulmonary disease (COPD), but also rare genetic diseases such as alpha-1-antitrypsin deficiency. Indeed, the biological role of DGKα is understudied outside the T lymphocyte field. The recent wave of research aiming to develop novel and specific inhibitors as well as KO mice will allow a better understanding of DGK’s role in neutrophilic inflammation. Better knowledge and pharmacologic tools may also allow DGK to move from the laboratory bench to clinical trials.
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Gharbi, Severine I., Esther Rincón, Antonia Avila-Flores, Pedro Torres-Ayuso, María Almena, María Angeles Cobos, Juan Pablo Albar, and Isabel Mérida. "Diacylglycerol kinase ζ controls diacylglycerol metabolism at the immunological synapse." Molecular Biology of the Cell 22, no. 22 (November 15, 2011): 4406–14. http://dx.doi.org/10.1091/mbc.e11-03-0247.
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Diacylglycerol (DAG) generation at the T cell immunological synapse (IS) determines the correct activation of antigen-specific immune responses. DAG kinases (DGKs) α and ζ act as negative regulators of DAG-mediated signals by catalyzing DAG conversion to phosphatidic acid (PA). Nonetheless, the specific input of each enzyme and their spatial regulation during IS formation remain uncharacterized. Here we report recruitment of endogenous DGKα and DGKζ to the T cell receptor (TCR) complex following TCR/CD28 engagement. Specific DGK gene silencing shows that PA production at the activated complex depends mainly on DGKζ, indicating functional differences between these proteins. DGKζ kinase activity at the TCR is enhanced by phorbol-12-myristate-13-acetate cotreatment, suggesting DAG-mediated regulation of DGKζ responsiveness. We used GFP-DGKζ and -DGKα chimeras to assess translocation dynamics during IS formation. Only GFP-DGKζ translocated rapidly to the plasma membrane at early stages of IS formation, independent of enzyme activity. Finally, use of a fluorescent DAG sensor confirmed rapid, sustained DAG accumulation at the IS and allowed us to directly correlate membrane translocation of active DGKζ with DAG consumption at the IS. This study highlights a DGKζ-specific function for local DAG metabolism at the IS and offers new clues to its mode of regulation.
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Katagiri, Yuji, Tsukasa Ito, Sachiko Saino-Saito, Yasukazu Hozumi, Akira Suwabe, Kazuhisa Otake, Makoto Sata, et al. "Expression and localization of diacylglycerol kinase isozymes and enzymatic features in rat lung." American Journal of Physiology-Lung Cellular and Molecular Physiology 288, no. 6 (June 2005): L1171—L1178. http://dx.doi.org/10.1152/ajplung.00237.2004.
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Diacylglycerol kinase (DGK) catalyzes phosphorylation of diacylglycerol to generate phosphatidic acid, and both molecules are known to serve as second messengers as well as important intermediates for the synthesis of various lipids. In this study, we investigated the spatiotemporal expression patterns of DGK isozymes together with the developmental changes of the mRNA expression and enzymatic property in rat lung. Northern blot and RT-PCR analyses showed that mRNAs for DGKα, -ε, and -ζ were detected in the lung. By immunohistochemical examination, DGKα and -ζ were shown to be coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes were localized at distinct subcellular locations, i.e., DGKα in the cytoplasm and DGKζ in the nucleus, suggesting different roles for these isozymes. In the developing lung, the expression for DGKα and -ζ was transiently elevated on embryonic day 21 (E21) to levels approximately two- to threefold higher than on postnatal day 0 (P0). On the other hand, the expression for DGKε was inversely elevated approximately twofold on P0 compared with that on E21. These unique changes in the expression pattern during the perinatal period suggest that each isozyme may play a distinct role in the adaptation of the lung to air or oxygen breathing at birth.
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Topham, Matthew K., and Stephen M. Prescott. "Diacylglycerol Kinase ζ Regulates Ras Activation by a Novel Mechanism." Journal of Cell Biology 152, no. 6 (March 12, 2001): 1135–44. http://dx.doi.org/10.1083/jcb.152.6.1135.
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Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGKζ, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGKζ also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGKζ and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGKζ in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGKζ, which controls local accumulation of DAG that would otherwise activate RasGRP.
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ROCHE, Marc A. de la, Janet L. SMITH, Maribel RICO, Silvia CARRASCO, Isabel MERIDA, Lucila LICATE, Graham P. CÔTÉ, and Thomas T. EGELHOFF. "Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian θ isoforms." Biochemical Journal 368, no. 3 (December 15, 2002): 809–15. http://dx.doi.org/10.1042/bj20021027.
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Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the θ subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system.
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DU, Xiangnan, Ying JIANG, Weijun QIAN, Xiaolan LU, and James P. WALSH. "Fatty acids inhibit growth-factor-induced diacylglycerol kinase α activation in vascular smooth-muscle cells." Biochemical Journal 357, no. 1 (June 25, 2001): 275–82. http://dx.doi.org/10.1042/bj3570275.
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We have previously shown that unsaturated fatty acids amplify platelet-derived-growth-factor (PDGF)-induced protein kinase C (PKC) activation in vascular smooth-muscle cells (VSMCs). Diacylglycerol-induced PKC activation is normally terminated by diacylglycerol kinases (DGKs). We thus hypothesized that fatty acids act by inhibiting a DGK. Fractionation of VSMC extracts demonstrated that the DGK α isoform was the major DGK activity present. PDGF markedly increased the DGK activity of cultured cells. An inhibitor selective for the DGK α isoform,R59949[3-{2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl}-2,3-dihydro-2-thioxo-4(1H)-quinazolinone], abolished the growth-factor-induced increase in DGK activity, but had little effect on basal activity. PDGF thus selectively activates DGKα. Epidermal growth factor and α-thrombin stimulated total DGK activity similarly to PDGF. Activation by epidermal growth factor was sensitive to R59949, again suggesting involvement of DGKα. However, the α-thrombin-induced activity was unaffected by this agent. Unsaturated fatty acids inhibited growth-factor-induced DGKα activation, but had no effect on basal activity. Fatty acids also amplified the PDGF-induced increase in cell diacylglycerol content. These results indicate that inhibition of DGKα contributes to fatty-acid-induced amplification of PKC activation. Increased levels of fatty acids in diabetes may thus contribute to chronic PKC activation associated with this disorder.
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Dougan, Stephanie K. "Abstract SY12-04: Lowering the TCR signaling threshold with a DGKa/z dual inhibitor potentiates anti-tumor immunity." Cancer Research 83, no. 7_Supplement (April 4, 2023): SY12–04—SY12–04. http://dx.doi.org/10.1158/1538-7445.am2023-sy12-04.
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Abstract Checkpoint blockade immunotherapies expand neoantigen- or virus-specific T cells, and poor responsiveness to immunotherapy is associated with lower mutational burden in tumors of non-viral origin. Although mouse models demonstrate that lower affinity T cells recognizing self-antigens can contribute to tumor control if sufficiently activated, therapeutic options for enhancing T cell priming are limited. Diacylglycerol kinases (DGKs) suppress DAG signaling by converting DAG to phosphatidic acid, thereby attenuating pathways downstream of TCR signaling. Using a novel dual DGKa/z inhibitor (DGKi), tumor-specific CD8 T cells with different affinities (TRP1high and TRP1low), and a series of altered peptide ligands, we demonstrate that inhibition of DGKa/z can lower the signaling threshold for T cell priming. TRP1high and TRP1low CD8 T cells produced more IL-2 and IFNγ in the presence of cognate antigen and DGKi. Effector TRP1high- and TRP1low-mediated cytolysis of tumor cells with low antigen load was MHC-restricted, mediated by IFNg and augmented by DGKi. Adoptive T cell transfer into mice bearing pancreatic or melanoma tumors synergized with single-agent DGKi or DGKi and αPD1, with increased expansion of low affinity T cells and increased cytokine production observed in tumor infiltrates of treated mice. Collectively, our findings highlight DGKa/z as therapeutic targets for augmenting tumor-specific CD8 T cell function. Citation Format: Stephanie K. Dougan. Lowering the TCR signaling threshold with a DGKa/z dual inhibitor potentiates anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY12-04.
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Takahashi, Daisuke, and Fumio Sakane. "Expression and purification of human diacylglycerol kinase α from baculovirus-infected insect cells for structural studies." PeerJ 6 (August 10, 2018): e5449. http://dx.doi.org/10.7717/peerj.5449.
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Diacylglycerol kinases (DGKs) are lipid kinases that modulate the levels of lipid second messengers, diacylglycerol and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. DGKα consists of the N-terminal regulatory domains including EF-hand motifs and C1 domains, and the C-terminal catalytic domain (DGKα-CD). To date, however, no structures of mammalian DGKs including their CDs have yet been reported, impeding our understanding on the catalytic mechanism of DGKs and the rational structure-based drug design. Here we attempted to produce DGKα-CD or a full-length DGKα using bacterial and baculovirus-insect cell expression system for structural studies. While several DGKα-CD constructs produced using both bacterial and insect cells formed insoluble or soluble aggregates, the full-length DGKα expressed in insect cells remained soluble and was purified to near homogeneity as a monomer with yields (1.3 mg/mL per one L cell culture) feasible for protein crystallization. Following enzymatic characterization showed that the purified DGKα is in fully functional state. We further demonstrated that the purified enzyme could be concentrated without any significant aggregation, and characterized its secondary structure by circular dichroism. Taken together, these results suggest that the presence of N-terminal regulatory domains suppress protein aggregation likely via their intramolecular interactions with DGKα-CD, and demonstrate that the baculovirus-insect cell expression of the full-length form of DGKα, not DGKα-CD alone, represents a promising approach to produce protein sample for structural studies of DGKα. Thus, our study will encourage future efforts to determine the crystal structure of DGK, which has not been determined since it was first identified in 1959.
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Hernandez-Lara, Miguel Angel, Santosh K. Yadav, Sushrut D. Shah, Mariko Okumura, Yuichi Yokoyama, Raymond B. Penn, Taku Kambayashi, and Deepak A. Deshpande. "Regulation of Airway Smooth Muscle Cell Proliferation by Diacylglycerol Kinase: Relevance to Airway Remodeling in Asthma." International Journal of Molecular Sciences 23, no. 19 (October 6, 2022): 11868. http://dx.doi.org/10.3390/ijms231911868.
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Airway remodeling in asthma involves the hyperproliferation of airway smooth muscle (ASM) cells. However, the molecular signals that regulate ASM growth are not completely understood. Gq-coupled G protein-coupled receptor and receptor tyrosine kinase signaling regulate ASM cell proliferation via activation of phospholipase C, generation of inositol triphosphate (IP3) and diacylglycerol (DAG). Diacylglycerol kinase (DGK) converts DAG into phosphatidic acid (PA) and terminates DAG signaling while promoting PA-mediated signaling and function. Herein, we hypothesized that PA is a pro-mitogenic second messenger in ASM, and DGK inhibition reduces the conversion of DAG into PA resulting in inhibition of ASM cell proliferation. We assessed the effect of pharmacological inhibition of DGK on pro-mitogenic signaling and proliferation in primary human ASM cells. Pretreatment with DGK inhibitor I (DGKI) significantly inhibited platelet-derived growth factor-stimulated ASM cell proliferation. Anti-mitogenic effect of DGKI was associated with decreased mTOR signaling and expression of cyclin D1. Exogenous PA promoted pro-mitogenic signaling and rescued DGKI-induced attenuation of ASM cell proliferation. Finally, house dust mite (HDM) challenge in wild type mice promoted airway remodeling features, which were attenuated in DGKζ-/- mice. We propose that DGK serves as a potential drug target for mitigating airway remodeling in asthma.
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AbdulSalam, Safnas F., Mia M. Eason, Holly A. Fowle, Anna L. Stuart, and Kurumi Y. Horiuchi. "Abstract 170: Development of diacylglycerol kinase assays to facilitate isoform specific inhibitor discovery." Cancer Research 82, no. 12_Supplement (June 15, 2022): 170. http://dx.doi.org/10.1158/1538-7445.am2022-170.
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Abstract Diacylglycerol (DAG) and phosphatidic acid (PA) are two key second messengers in signaling and metabolic pathways. Diacylglycerol kinase (DGK) phosphorylate DAG to produce PA, acting as a central switch between the various signal transduction pathways activated by these second messengers. Ten DGK isoforms (α, β, γ, δ, ε, ζ, η, θ, ι, and κ) have been identified and categorized into five classes based on their structural features. DGKα is highly expressed in several cancers including hepatoma, lymphoma and melanoma, and has been shown to promote cancer survival by positively regulating proliferative activity through several signaling pathways, attracting interest to DGKα as a potential therapeutic target. DGKα and ζ are expressed at high levels in T cells, and shown to promote T cell anergy, pointing to inhibition of these DGK isoforms as a means of enhancing T cell anti-tumor activity and restoring T cells function. Several DGK isoforms have also been implicated in disease contexts such as hypertension, autoimmunity, type II diabetes, epilepsy, and bipolar disorder. Current DGK inhibitor discovery efforts are focused on DGKα and ζ, but to our knowledge no compounds have proceeded to clinical trial-to date. To facilitate these efforts, we have developed a DGK screening panel including compound profiling assays for all DGK isoforms. This is an essential tool for identification of potent and isoform selective inhibitors towards therapeutically relevant DGK isoforms. Further, we present kinetic characterization of all isoforms and lipid substrate specificity studies of DGKs. Citation Format: Safnas F. AbdulSalam, Mia M. Eason, Holly A. Fowle, Anna L. Stuart, Kurumi Y. Horiuchi. Development of diacylglycerol kinase assays to facilitate isoform specific inhibitor discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 170.
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Hernández-Montiel, Wilber, Nubia Noemi Cob-Calan, Lilia E. Cahuich-Tzuc, José A. Rueda, Jorge Quiroz-Valiente, Víctor Meza-Villalvazo, and Roberto Zamora-Bustillos. "Runs of Homozygosity and Gene Identification in Pelibuey Sheep Using Genomic Data." Diversity 14, no. 7 (June 28, 2022): 522. http://dx.doi.org/10.3390/d14070522.
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The runs of homozygosity (ROHs), the inbreeding coefficient, and the effective population size (Ne) in Pelibuey sheep were analyzed in 24 Pelibuey ewes from two lambs at parturition and 24 ewes that gave birth to a single lamb using the Ilumina OvineSNP50 BeadChip. The Ne decreased from 535 to 192 in the first ten generations. A total of 2194 ROHs were identified on the basis of single nucleotide polymorphisms (SNPs), were identified in the prolific group and 2185 SNPs in ROH in the non-prolific group. The distribution of the lengths of the ROH, considering both groups, were found to be: 4065 less than 6 Mb, 213 between 6 and 12 Mb, 72 between 12 and 24 Mb, twenty between 24 and 48 Mb and 8 greater than 48 Mb. In prolific sheep, the ROH associated with prolificacy were identified near the LINGO2, FLRT2, ADGRB3 genes, related to “positive regulation of synapse assembly”; and the DGKG, DGKE, DGKB and DGKI genes, related to “protein kinase C-activating G-protein coupled receptor signaling pathway”. The present work present genes that can function as signal mediators or have activity in embryonic development, which is relevant to the economic activity of this species.
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Kakehi, Tomoko, Keiko Yagi, Naoaki Saito, and Yasuhito Shirai. "Effects of vitamin E and its derivatives on diabetic nephropathy in Rats and identification of diacylglycerol kinase subtype involved in the improvement of diabetic nephropathy." Functional Foods in Health and Disease 7, no. 10 (October 31, 2017): 816. http://dx.doi.org/10.31989/ffhd.v7i10.386.
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Background: Diabetes is a significant social issue. Controlling diabetic complications such as nephropathy is very important for QOL of diabetic patients. One of the mechanisms which causes diabetic complications is the abnormal activation of protein kinase C (PKC) by increased diacylglycerol (DG) from hyperglycemia. Diacylglycerol kinase (DGK) can attenuate PKC activity by converting DG to phosphatidic acid. Thus far, d-a-tocopherol (VtE) treatment has been shown to prevent early changes of diabetic renal dysfunctions by activating DGK. However, it is still unknown whether VtE derivatives improve diabetic nephropathy similarly to VtE, and which DGK subtype is activated by VtE and/or the derivatives. Objective: The purpose of the study was to investigate effects of VtE and its derivatives on diabetic nephropathy in rats, in addition to identifying the DGK subtype involved in the improvement of nephropathy in vivo. Methods: To induce diabetes in rats, six weeks old male Sprague-Dawley rats were intraperitonealy administrated 65 mg/kg streptozocin (STZ) in 20 mM citrate buffer. For two or eight weeks, 40 mg/kg VtE, 44 mg/kg acetate VtE (aVtE) or 49.3 mg/kg succinate VtE (sVtE) was intraperitonealy administrated every other day after STZ administration. The blood glucose level, body weight, and kidney weight, in addition to urinary volume, albumin, and BUN were measured every week after STZ administration. Additionally, in order to identify the DGK subtype activated by VtE and aVtE, the DGK subtype expressed in the rat glomerulus was checked by RT-PCR and western blotting, and the activity in the glomerulus from the rats before and after the VtE and aVtE treatments were measured in the presence or absence of EGTA.Results: Averages of kidney weight and BUN of rats treated with VtE, aVtE and sVtE for 8 weeks were reduced, compared to the control. However, the intraperitoneal administration of sVtE was toxic. Additionally, the amount of urine volume and urinary albumin significantly improved by the two-weeks treatment of VtE and aVtE. mRNA of DGKa, g, d, e, and z were detected in the glomerulus, but only the protein of DGKa and DGKd were detected as calcium-dependent and independent subtype respectively. Both VtE and aVtE activated DGK in the glomerulus. However, the calcium dependent DGK subtype was mainly activated by aVtE, with not only the calcium-dependent subtype but also the calcium-independent subtype being activated in the case of VtE. In other words, DGKa was activated by aVtE and DGKd was additionally enhanced in the case of VtE.Conclusion: These results clearly indicated that aVtE as well as VtE improved diabetic nephropathy, with the activation of DGKa and/or d being potentially involved with this improvement. Key words: diabetic nephropathy, diacylglycerol kinase, vitamin E
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Imai, Shin-ichi, Masahiro Kai, Satoshi Yasuda, Hideo Kanoh, and Fumio Sakane. "Identification and Characterization of a Novel Human Type II Diacylglycerol Kinase, DGKκ." Journal of Biological Chemistry 280, no. 48 (October 6, 2005): 39870–81. http://dx.doi.org/10.1074/jbc.m500669200.
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Liu, Cheng-hu, Fabiana S. Machado, Rishu Guo, Kim E. Nichols, A. Wesley Burks, Julio C. Aliberti, and Xiao-Ping Zhong. "Diacylglycerol kinase zeta regulates microbial recognition and host resistance to Toxoplasma gondii (51.16)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S99. http://dx.doi.org/10.4049/jimmunol.178.supp.51.16.
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Abstract Mammalian Toll-like receptors (TLRs) recognize microbial pathogen-associated molecular patterns and are critical for innate immunity against microbial infection. Diacylglycerol kinases (DGKs) regulate the intracellular levels of two important second messengers involved in signaling from many surface receptors by converting diacylglycerol (DAG) to phosphatidic acid (PA). Here, we demonstrate that the ζ isoform of the DGK family (DGKζ) is expressed in macrophages (Mϕ) and dendritic cells (DC). DGKζ deficiency results in impaired IL-12 and TNFα production following TLR stimulation in vitro and in vivo, increased resistance to endotoxin shock, and enhanced susceptibility to Toxoplasma gondii infection. We further show that DGKζ negatively controls the PI3K-Akt pathway and that inhibition of PI3K activity can restore LPS-induced IL-12 production by DGKζ deficient macrophages. Collectively, our data provide the first genetic evidence that an enzyme involved in DAG/PA metabolism plays an important role in innate immunity and indicate that DGKζ promotes TLR responses via a pathway involving inhibition of PI3K.
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Fazio, Antonietta, Eric Owusu Obeng, Isabella Rusciano, Maria Vittoria Marvi, Matteo Zoli, Sara Mongiorgi, Giulia Ramazzotti, et al. "Subcellular Localization Relevance and Cancer-Associated Mechanisms of Diacylglycerol Kinases." International Journal of Molecular Sciences 21, no. 15 (July 26, 2020): 5297. http://dx.doi.org/10.3390/ijms21155297.
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An increasing number of reports suggests a significant involvement of the phosphoinositide (PI) cycle in cancer development and progression. Diacylglycerol kinases (DGKs) are very active in the PI cycle. They are a family of ten members that convert diacylglycerol (DAG) into phosphatidic acid (PA), two-second messengers with versatile cellular functions. Notably, some DGK isoforms, such as DGKα, have been reported to possess promising therapeutic potential in cancer therapy. However, further studies are needed in order to better comprehend their involvement in cancer. In this review, we highlight that DGKs are an essential component of the PI cycle that localize within several subcellular compartments, including the nucleus and plasma membrane, together with their PI substrates and that they are involved in mediating major cancer cell mechanisms such as growth and metastasis. DGKs control cancer cell survival, proliferation, and angiogenesis by regulating Akt/mTOR and MAPK/ERK pathways. In addition, some DGKs control cancer cell migration by regulating the activities of the Rho GTPases Rac1 and RhoA.
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Moroi, Alyssa J., Nicole M. Zwifelhofer, Matthew J. Riese, Debra K. Newman, and Peter J. Newman. "Diacylglycerol kinase ζ is a negative regulator of GPVI-mediated platelet activation." Blood Advances 3, no. 7 (April 9, 2019): 1154–66. http://dx.doi.org/10.1182/bloodadvances.2018026328.
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Abstract Diacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ζ isoform of DGK (DGKζ) has been reported to inhibit T-cell responsiveness by downregulating intracellular levels of DAG. However, its role in platelet function remains undefined. In this study, we show that DGKζ was expressed at significant levels in both platelets and megakaryocytes and that DGKζ-knockout (DGKζ-KO) mouse platelets were hyperreactive to glycoprotein VI (GPVI) agonists, as assessed by aggregation, spreading, granule secretion, and activation of relevant signal transduction molecules. In contrast, they were less responsive to thrombin. Platelets from DGKζ-KO mice accumulated faster on collagen-coated microfluidic surfaces under conditions of arterial shear and stopped blood flow faster after ferric chloride–induced carotid artery injury. Other measures of hemostasis, as measured by tail bleeding time and rotational thromboelastometry analysis, were normal. Interestingly, DGKζ deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGKζ as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo.
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Liu, Cheng-Hu, Fabiana S. Machado, Rishu Guo, Kim E. Nichols, A. Wesley Burks, Julio C. Aliberti, and Xiao-Ping Zhong. "Diacylglycerol kinase ζ regulates microbial recognition and host resistance to Toxoplasma gondii." Journal of Experimental Medicine 204, no. 4 (March 19, 2007): 781–92. http://dx.doi.org/10.1084/jem.20061856.
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Mammalian Toll-like receptors (TLRs) recognize microbial pathogen-associated molecular patterns and are critical for innate immunity against microbial infection. Diacylglycerol (DAG) kinases (DGKs) regulate the intracellular levels of two important second messengers involved in signaling from many surface receptors by converting DAG to phosphatidic acid (PA). We demonstrate that the ζ isoform of the DGK family (DGKζ) is expressed in macrophages (Mφ) and dendritic cells. DGKζ deficiency results in impaired interleukin (IL) 12 and tumor necrosis factor α production following TLR stimulation in vitro and in vivo, increased resistance to endotoxin shock, and enhanced susceptibility to Toxoplasma gondii infection. We further show that DGKζ negatively controls the phosphatidylinositol 3–kinase (PI3K)–Akt pathway and that inhibition of PI3K activity or treatment with PA can restore lipopolysaccharide-induced IL-12 production by DGKζ-deficient Mφ. Collectively, our data provide the first genetic evidence that an enzyme involved in DAG/PA metabolism plays an important role in innate immunity and indicate that DGKζ promotes TLR responses via a pathway involving inhibition of PI3K.
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Gu, Wangxian, Guoqing Wan, Yanjun Zheng, Xintong Yang, Peng Zhang, Changlian Lu, and Xuefeng Gu. "BIOM-55. DGKζ-TARGETED REGULATION OF MIR-34A IN THE PROLIFERATION AND TUMORIGENICITY OF HUMAN GLIOBLASTOMA." Neuro-Oncology 22, Supplement_2 (November 2020): ii13. http://dx.doi.org/10.1093/neuonc/noaa215.052.
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Abstract Diacylglycerol kinase (DGK) is a lipid kinase that catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), which uses ATP as a phosphate donor. Diacylglycerol kinases ζ(DGKζ) is characterized as specific type IV due to its myristoylated alanine-rich C-kinase substrate (MARCKS), ankyrin, and PDZ binding domain. Similar to other DGKs, DGKζ is also reported to be abnormally expressed in human colorectal cancer cells, and it is indispensable for the proliferation of cancer cells. However, its implications in human glioblastoma (GBM) is largely unknown. Both the mRNA and protein levels of DGKζ were significantly higher in GBM tissues than in precancerous lesions. Knockdown of DGKζ inhibited GBM cell proliferation, cell cycle and promoted apoptosis of GBM cells. Moreover, down-regulation of DGKζ markedly reduced in vitro colony formation and in vivo tumorigenic capability. Furthermore, we confirmed that DGKζ was the downstream target of miR-34a. The expression level of DGKζ was negatively correlated with miR-34a in GBM tissues. Overexpression of DGKζ reversed the tumor suppressive roles of miR-34a in GBM cells. Taken together, DGKζ can act as a potential prognostic biomarker for GBM patients and promote the growth of GBM cells was regulated by miR-34a, and it may represent a promising therapeutic target for patients with GBM.
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SAKANE, Fumio, Masahiro KAI, Ikuo WADA, Shin-ichi IMAI, and Hideo KANOH. "The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain." Biochemical Journal 318, no. 2 (September 1, 1996): 583–90. http://dx.doi.org/10.1042/bj3180583.
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All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGKα and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGKα. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs α, β and γ expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.
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Sun, Deheng, Hongfu Lu, Huaxing Yu, Feng Wang, Mike Korzinkin, Xin Cai, Xiao Ding, Sujata Rao, Feng Ren, and Alex Zhavoronkov. "Abstract 1855: Targeting DGKA for immuno-oncology therapy: ISM4312A, a novel DGKA inhibitor with robust anti-tumor activity." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1855. http://dx.doi.org/10.1158/1538-7445.am2023-1855.
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Abstract Diacylglycerol kinases (DGKs), a family of isozymes that phosphorylate the membrane lipid, diacylglycerol (DAG), to phosphatidic acid (PA), are important players in signal transduction cascades. DAG and PA act as vital second messengers that regulate multiple cellular signal transduction pathways including PKC and MAPK. DGKα (DGKA), one of the ten human DGK isoforms, has been reported to play a role in mediating numerous aspects of cancer progression including survival, migration, and invasion of cancer cells. Emerging data indicate that DGKA mediates T-cell dysfunction during anti-PD-1 therapy, playing a role in the development of resistance to PD-1 blockade. This suggests that DGKA inhibition offers a promising strategy to improve the efficacy of immunotherapy in the treatment of cancer. Although serotonin (5-HT) antagonists ritanserin, R59022 and R59949 have been previously repurposed as DGKA inhibitors, their potency and selectivity profiles are largely unsatisfactory. Therefore, there is a significant unmet clinical need to develop potent and selective DGKA inhibitors. Here, we used our proprietary PandaOmics platform to explore targets that can overcome the resistance to anti-PD1 therapy. In order to do so, we created a project that included bulk RNAseq data from 4 datasets alongside with 2 single-cell RNAseq datasets. Based on the calculation results, DGKA inhibitors may overcome anti-PD-1 resistance and expand the responder patient population in cancer immunotherapy. We then designed and evaluated a series of compounds in biochemical and cellular assays, and report that ISM4312A is a novel DGKA inhibitor with excellent potency (IC50 of 120 pM) and high selectivity (>25000 fold) for DGKA versus 5-HT1A, 5-HT1B, 5-HT2A, and 5-HT2B. Further, ISM4312A showed high selectivity against 468 other kinases. In the cellular T-cell activation assay, ISM4312A induced IL-2 production in a dose-dependent manner and enhanced T-cell activity at concentrations as low as 20 nM. In the MC38 in vivo syngeneic model, ISM4312A at 3 mg/kg BID showed robust anti-tumor activities with (Tumor Growth Inhibition, TGI, 93.4%, 37.5% mice achieved CR) or without (TGI, 25.2%) anti-PD-1 therapy. Data from this combination study indicate synergistic activity as compared with the single anti-PD-1 treatment (TGI, 50.8% at 5 mg/kg BID, no mice achieved CR). ISM4312A also exhibited favorable ADME, excellent oral bioavailability, and tolerance. Together, the data support the ongoing clinical development of ISM4312A as a potential first-in-class DGKA inhibitor in cancer immunotherapy. Citation Format: Deheng Sun, Hongfu Lu, Huaxing Yu, Feng Wang, Mike Korzinkin, Xin Cai, Xiao Ding, Sujata Rao, Feng Ren, Alex Zhavoronkov. Targeting DGKA for immuno-oncology therapy: ISM4312A, a novel DGKA inhibitor with robust anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1855.
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Baldanzi, Gianluca, Beatrice Ragnoli, and Mario Malerba. "Potential role of diacylglycerol kinases in immune-mediated diseases." Clinical Science 134, no. 13 (July 1, 2020): 1637–58. http://dx.doi.org/10.1042/cs20200389.
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Abstract The mechanism promoting exacerbated immune responses in allergy and autoimmunity as well as those blunting the immune control of cancer cells are of primary interest in medicine. Diacylglycerol kinases (DGKs) are key modulators of signal transduction, which blunt diacylglycerol (DAG) signals and produce phosphatidic acid (PA). By modulating lipid second messengers, DGK modulate the activity of downstream signaling proteins, vesicle trafficking and membrane shape. The biological role of the DGK α and ζ isoforms in immune cells differentiation and effector function was subjected to in deep investigations. DGK α and ζ resulted in negatively regulating synergistic way basal and receptor induced DAG signals in T cells as well as leukocytes. In this way, they contributed to keep under control the immune response but also downmodulate immune response against tumors. Alteration in DGKα activity is also implicated in the pathogenesis of genetic perturbations of the immune function such as the X-linked lymphoproliferative disease 1 and localized juvenile periodontitis. These findings suggested a participation of DGK to the pathogenetic mechanisms underlying several immune-mediated diseases and prompted several researches aiming to target DGK with pharmacologic and molecular strategies. Those findings are discussed inhere together with experimental applications in tumors as well as in other immune-mediated diseases such as asthma.
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Sakane, Fumio, Fumi Hoshino, and Chiaki Murakami. "New Era of Diacylglycerol Kinase, Phosphatidic Acid and Phosphatidic Acid-Binding Protein." International Journal of Molecular Sciences 21, no. 18 (September 16, 2020): 6794. http://dx.doi.org/10.3390/ijms21186794.
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Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Mammalian DGK consists of ten isozymes (α–κ) and governs a wide range of physiological and pathological events, including immune responses, neuronal networking, bipolar disorder, obsessive-compulsive disorder, fragile X syndrome, cancer, and type 2 diabetes. DG and PA comprise diverse molecular species that have different acyl chains at the sn-1 and sn-2 positions. Because the DGK activity is essential for phosphatidylinositol turnover, which exclusively produces 1-stearoyl-2-arachidonoyl-DG, it has been generally thought that all DGK isozymes utilize the DG species derived from the turnover. However, it was recently revealed that DGK isozymes, except for DGKε, phosphorylate diverse DG species, which are not derived from phosphatidylinositol turnover. In addition, various PA-binding proteins (PABPs), which have different selectivities for PA species, were recently found. These results suggest that DGK–PA–PABP axes can potentially construct a large and complex signaling network and play physiologically and pathologically important roles in addition to DGK-dependent attenuation of DG–DG-binding protein axes. For example, 1-stearoyl-2-docosahexaenoyl-PA produced by DGKδ interacts with and activates Praja-1, the E3 ubiquitin ligase acting on the serotonin transporter, which is a target of drugs for obsessive-compulsive and major depressive disorders, in the brain. This article reviews recent research progress on PA species produced by DGK isozymes, the selective binding of PABPs to PA species and a phosphatidylinositol turnover-independent DG supply pathway.
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You, Jae-Sung, Hannah C. Lincoln, Chan-Ran Kim, John W. Frey, Craig A. Goodman, Xiao-Ping Zhong, and Troy A. Hornberger. "The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy." Journal of Biological Chemistry 289, no. 3 (December 3, 2013): 1551–63. http://dx.doi.org/10.1074/jbc.m113.531392.
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The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.
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Masai, I., A. Okazaki, T. Hosoya, and Y. Hotta. "Drosophila retinal degeneration A gene encodes an eye-specific diacylglycerol kinase with cysteine-rich zinc-finger motifs and ankyrin repeats." Proceedings of the National Academy of Sciences 90, no. 23 (December 1, 1993): 11157–61. http://dx.doi.org/10.1073/pnas.90.23.11157.
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The Drosophila visual mutant, carrying the retinal degeneration A gene (rdgA), has photoreceptor cells that degenerate within a week after eclosion. Morphological studies suggested that this mutant harbors abnormalities in membrane turnover of the photoreceptor cells. Biochemically, the rdgA mutant lacks an eye-specific and membrane-associated diacylglycerol kinase (DGK; EC 2.7.1.107) activity in a gene-dosage-dependent manner, suggesting that rdgA gene encodes a DGK. We report the molecular cloning and characterization of a DGK gene, which maps to the rdgA locus. This gene, designated as DGK2, has a single open reading frame that encodes 1454 amino acids. Like porcine DGK, DGK2 has two cysteine-rich zinc-finger motifs as well as a DGK catalytic domain. The DGK2 protein contains four ankyrin-like repeats at the C-terminal region, suggesting that DGK2 is likely anchored to the membrane or cytoskeleton. Northern blot analysis and tissue in situ hybridization to adult sections revealed that DGK2 is expressed exclusively in the adult retina and that the amount of its mRNA is reduced in some of the rdgA mutant alleles. Furthermore, in two rdgA alleles, rdgA1 and rdgA2, nonsense and missense mutations occur within their DGK2 gene, respectively. Thus, we conclude that rdgA encodes an eye-specific DGK, the absence of which leads to rhabdomere degeneration due to defective phospholipid turnover.
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WALKER, Anthony J., Annette DRAEGER, Brahim HOUSSA, Wim J. VAN BLITTERSWIJK, Vasken OHANIAN, and Jacqueline OHANIAN. "Diacylglycerol kinase θ is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries." Biochemical Journal 353, no. 1 (December 18, 2000): 129–37. http://dx.doi.org/10.1042/bj3530129.
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Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51–56]. Here, we have identified this DGK activity as DGKθ, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKθ was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKθ translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30s, followed by a return to near-basal levels at 5min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKθ activity in immunoprecipitates (126% at 60s). Membrane translocation and DGKθ activation were regulated differently: NA-induced DGKθ activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKθ suggests that this pathway may have a physiological role in vascular smooth-muscle responses.
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Luo, Bai, Stephen M. Prescott, and Matthew K. Topham. "Association of diacylglycerol kinase ζ with protein kinase C α." Journal of Cell Biology 160, no. 6 (March 10, 2003): 929–37. http://dx.doi.org/10.1083/jcb.200208120.
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Activation of PKC depends on the availability of DAG, a signaling lipid that is tightly and dynamically regulated. DAG kinase (DGK) terminates DAG signaling by converting it to phosphatidic acid. Here, we demonstrate that DGKζ inhibits PKCα activity and that DGK activity is required for this inhibition. We also show that DGKζ directly interacts with PKCα in a signaling complex and that the binding site in DGKζ is located within the catalytic domain. Because PKCα can phosphorylate the myristoylated alanine-rich C-kinase substrate (MARCKS) motif of DGKζ, we tested whether this modification could affect their interaction. Phosphorylation of this motif significantly attenuated coimmunoprecipitation of DGKζ and PKCα and abolished their colocalization in cells, indicating that it negatively regulates binding. Expression of a phosphorylation-mimicking DGKζ mutant that was unable to bind PKCα did not inhibit PKCα activity. Together, our results suggest that DGKζ spatially regulates PKCα activity by attenuating local accumulation of signaling DAG. This regulation is impaired by PKCα-mediated DGKζ phosphorylation.
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Hsu, Ku-Lung, Adam L. Borne, Jeffrey W. Brulet, Sean T. Campbell, Caroline E. Franks, Tao Huang, Rebecca L. McCloud, Myungsun Shin, and Timothy B. Ware. "Towards identification of a lipid metabolic checkpoint for immuno-oncology." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 71.7. http://dx.doi.org/10.4049/jimmunol.202.supp.71.7.
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Abstract Lipid phosphorylation is an understudied regulatory mechanism for T cell metabolism and signaling. Specifically, diacylglycerol kinases (DGKs) modulate intracellular levels of the secondary messengers diacylglycerol and phosphatidic acid, which are implicated in regulation of T cell activation and anergy. Development of isoform-selective DGK inhibitors is challenging but needed to understand specificity of DGK biology in vivo. Towards this goal, we use ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, the DGK ATP-binding site (DAGKc/DAGKa subdomains) and propose a potential role for C1 domains in DGK catalytic function. We discovered fragments from old drugs (ritanserin) that reveal pharmacophores required for selective binding to DGK active sites that are not in the canonical ATP binding pocket. We provide evidence of a single contiguous inhibitor binding site composed of DAGKc, DAGKa, and C1 sites by demonstrating equipotent inhibition using a fragment inhibitor. Our current working model is that C1 domains of DGKs are involved in substrate and inhibitor recognition of type 1, 4, and 5 DGKs and is a site for pursuing development of isoform-selective inhibitors. We describe new lead compounds that will be optimized into the first selective and in vivoactive DGK-alpha inhibitor with potential applications for immuno-oncology. We also describe our efforts to use mass spectrometry-metabolomics to annotate substrate specificity of all 10 mammalian DGKs in live cells to identify biomarkers for future applications in studying T cell biology.
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Mérida, Isabel, Javier Arranz-Nicolás, Cristina Rodríguez-Rodríguez, and Antonia Ávila-Flores. "Diacylglycerol kinase control of protein kinase C." Biochemical Journal 476, no. 8 (April 18, 2019): 1205–19. http://dx.doi.org/10.1042/bcj20180620.
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Abstract The diacylglycerol kinases (DGK) are lipid kinases that transform diacylglycerol (DAG) into phosphatidic acid (PA) in a reaction that terminates DAG-based signals. DGK provide negative regulation to conventional and novel protein kinase C (PKC) enzymes, limiting local DAG availability in a tissue- and subcellular-restricted manner. Defects in the expression/activity of certain DGK isoforms contribute substantially to cognitive impairment and mental disorders. Abnormal DGK overexpression in tumors facilitates invasion and resistance to chemotherapy preventing tumor immune destruction by tumor-infiltrating lymphocytes. Effective translation of these findings into therapeutic approaches demands a better knowledge of the physical and functional interactions between the DGK and PKC families. DGKζ is abundantly expressed in the nervous and immune system, where physically and functionally interacts with PKCα. The latest discoveries suggest that PDZ-mediated interaction facilitates spatial restriction of PKCα by DGKζ at the cell–cell contact sites in a mechanism where the two enzymes regulate each other. In T lymphocytes, DGKζ interaction with Sorting Nexin 27 (SNX27) guarantees the basal control of PKCα activation. SNX27 is a trafficking component required for normal brain function whose deficit has been linked to Alzheimer's disease (AD) pathogenesis. The enhanced PKCα activation as the result of SNX27 silencing in T lymphocytes aligns with the recent correlation found between gain-of-function PKCα mutations and AD and suggests that disruption of the mechanisms that provides a correct spatial organization of DGKζ and PKCα may lie at the basis of immune and neuronal synapse impairment.
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Niizeki, Takeshi, Yasuchika Takeishi, Takanori Arimoto, Hiroki Takahashi, Tetsuro Shishido, Yo Koyama, Kaoru Goto, Richard A. Walsh, and Isao Kubota. "Cardiac-specific overexpression of diacylglycerol kinase ζ attenuates left ventricular remodeling and improves survival after myocardial infarction." American Journal of Physiology-Heart and Circulatory Physiology 292, no. 2 (February 2007): H1105—H1112. http://dx.doi.org/10.1152/ajpheart.00927.2006.
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Left ventricular (LV) remodeling, including cardiomyocyte necrosis, scar formation, LV geometric changes, and cardiomyocyte hypertrophy, contributes to cardiac dysfunction and mortality after myocardial infarction (MI). Although precise cellular signaling mechanisms for LV remodeling are not fully elucidated, Gq protein-coupled receptor signaling pathway, including diacylglycerol (DAG) and PKC, are involved in this process. DAG kinase (DGK) phosphorylates DAG and controls cellular DAG levels, thus acting as a negative regulator of PKC and subsequent cellular signaling. We previously reported that DGK inhibited angiotensin II and phenylephrine-induced activation of the DAG-PKC signaling and subsequent cardiac hypertrophy. The purpose of this study was to examine whether DGK modifies LV remodeling after MI. Left anterior descending coronary artery was ligated in transgenic mice with cardiac-specific overexpression of DGKζ (DGKζ-TG) and wild-type (WT) mice. LV chamber dilatation (4.12 ± 0.10 vs. 4.53 ± 0.32 mm, P < 0.01), reduction of LV systolic function (34.8 ± 8.3% vs. 28.3 ± 4.8%, P < 0.01), and increases in LV weight (95 ± 3.6 vs. 111 ± 4.1 mg, P < 0.05) and lung weight (160 ± 15 vs. 221 ± 25 mg, P < 0.05) at 4 wk after MI were attenuated in DGKζ-TG mice compared with WT mice. In the noninfarct area, fibrosis fraction (0.51 ± 0.04, P < 0.01) and upregulation of profibrotic genes, such as transforming growth factor-β1 ( P < 0.01), collagen type I ( P < 0.05), and collagen type III ( P < 0.01), were blocked in DGKζ-TG mice. The survival rate at 4 wk after MI was higher in DGKζ-TG mice than in WT mice (61% vs. 37%, P < 0.01). In conclusion, these results demonstrate the first evidence that DGKζ suppresses LV structural remodeling and fibrosis and improves survival after MI. DGKζ may be a potential novel therapeutic target to prevent LV remodeling after MI.
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Bruneau, Sarah, Mélanie Néel, Lubka T. Roumenina, Marie Frimat, Lætitia Laurent, Véronique Frémeaux-Bacchi, and Fadi Fakhouri. "Loss of DGKε induces endothelial cell activation and death independently of complement activation." Blood 125, no. 6 (February 5, 2015): 1038–46. http://dx.doi.org/10.1182/blood-2014-06-579953.
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Key Points Loss of DGKε in endothelial cells induces cell death, impairs angiogenic responses, and leads to an activated and prothrombotic phenotype. DGKE silencing in resting endothelial cells does not affect complement activation at their surface.
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Arranz-Nicolas, Javier, Cristina Rodríguez-Rodríguez, Rosa Liébana, Judith Leitner, Antonia Ávila-Flores, Peter Steinberger, and Isabel Mérida. "519 Diacylglycerol kinase ζ limits IL-2-dependent control of PD-1 expression in tumor-infiltrating T lymphocytes." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A555. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0519.
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BackgroundTumors evade T cell responses targeting them through the upregulation of tolerance-inducing mechanisms. One of the best characterized is that of PD-1/PD-1L engagement, that in healthy CD8+ T cells limits cytotoxic responses against self-antigens and that tumors employ to neutralize T cell attack. Antibody-based therapies aimed to block the PD-1/PD-1L axis have rendered notable results, but most patients eventually develop resistance. This failure is attributed to CD8+ T cells achieving an exhausted phenotype where recovery is hardly feasible. The dysfunctional phenotype of tumor-infiltrating T cells is largely triggered by the unbalance of diacylglycerol (DAG)- and Ca2+-regulated signals that results in alteration of the transcriptional T cell program. DAG kinase (DGK) ζ-dependent DAG consumption contributes to hypofunctional T cell states while DGKζ deficiency facilitates tumor rejection in mice without apparent adverse autoimmune effects. In spite of its therapeutic potential, little is known about DGKζ function in human T cells and there are not isoform-specific inhibitors targeting this DGK isoform.MethodsHere we used of a human triple parameter reporter (TPR) cell line to examine the consequences of DGKζ depletion in the transcriptional restriction imposed by PD-1 ligation. We also investigated the effect of DGKζ deficiency in the expression dynamics of PD-1, as well as the impact of the absence of this DGK isoform in the in vivo growth of a MC38 adenocarcinoma cell line.ResultsWe demonstrate that DGKζ depletion enhances DAG-regulated transcriptional programs, favoring IL-2 production and limiting PD-1 expression. Diminished PD-1 expression and enhanced expansion of cytotoxic CD8+ T cell populations is also observed even in the context of immunosuppressive milieus and correlates with the failure of MC38 adenocarcinoma cells to form tumors in DGKζ-deficient mice.ConclusionsOur results suggest the relevance of DGKζ as a therapeutic target on its own as well as a biomarker of CD8+ T cell dysfunctional states.
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Zhong, Xiaoping, Chi-Keung Wan, and Rishu Guo. "Synergistic role of diacylglycerol kinases α and ζ in T cell development and self-tolerance (137.37)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 137.37. http://dx.doi.org/10.4049/jimmunol.182.supp.137.37.
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Abstract Signal for the T cell receptor (TCR) plays critical roles in T cell development and function. TCR signaling can induce positive or negative selection of thymocytes, induces activation or anergy of mature T cells, and induce conventional T cell activation or regulatory T cells to suppress immune function. The mechanisms that modulate TCR signaling to direct these distinct outcomes have been poorly understood. Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA) through phosphorylation. In mammals, ten DGK isoforms have been identified. Using both a gain of function and a loss of function approaches, we have found that DGK ( and ( are important regulators for T cells. Enhanced DGK activity inhibits TCR signaling and T cell maturation. Deficiency of either DGK( or ( causes T cells hyperresponsive to TCR stimulation and resistant to anergy induction. Loss of both DGK( and ( in mice results in impairment of positive selection, spontaneous T cell activation, impairment of regulatory T cell function, and autoimmunity. We further demonstrate that DGKs function both as signal terminator by inhibiting DAG-mediated signaling and as signaling initiator by generating PA.
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Okada, Naoki, Ko Sugiyama, Hidemitsu Kitamura, and Akinobu Taketomi. "Inhibition of diacylglycerol kinase alpha to augment antitumor effector T cells in tumor-bearing host." Journal of Clinical Oncology 37, no. 4_suppl (February 1, 2019): 293. http://dx.doi.org/10.1200/jco.2019.37.4_suppl.293.
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293 Background: Diacylglycerol kinases (DGKs), lipid kinases transforming diacylglycerol to phosphatidic acid, play important roles in intracellular signal transduction. Diacylglycerol kinase alpha (DGKa), an isozyme of DGKs, is well-known to promote proliferation of cancer cells by suppression of the apoptosis. Additionally, a previous report demonstrated that activation of DGKa induced anergy state of T lymphocytes in vivo. In this study, we investigated whether inhibition of DGKa not only suppress the tumorigenesis of cancer cells but also activate anti-tumor immunity. Methods: We first investigated the effect of DGKa inhibitor on in vitro proliferation of murine hepatoma cell lines (Hepa1-6) by cell proliferation assay. Cytokine and Granzyme B productions by CD8+ T cells from OT-1 mice after the OVA antigen stimulation were evaluated by ELISA and flowcytometry, respectively. Next, we established a tumor-bearing mice model by injection of mCherry-transfected Hepa1-6 cells into spleen. Tumorigenesis and tumor-infiltrating T cells in the liver were evaluated by in vivo imaging system, HE staining, and immunohistochemistry. CD8+ T cells were collected from the liver and stimulated with PMA and Ca2+ ionophore and the IFN-g production levels were evaluated by flowcytometry. Results: Proliferation of Hepa1-6 cells were suppressed in the presence of DGKa inhibitor in vitro. IL-2 production levels of OT-1 CD8 T cells in control group was augmented by the addition of DGKa inhibitor (246 vs 579 pg/ml, p < 0.05). Granzyme B-positive cells in OT-1 CD8+ T cells were increased by the treatment with DGKa inhibitor compared to the control group (4.4 vs 8.9 %, P < 0.05) after the antigen stimulation. In vivo administration of DGKa inhibitor significantly suppressed the tumor size (fluorescence (AU) 2.0x1010 vs 6.3x109, area (μm2) 1.5x107 vs 0.9x107, p < 0.05) in the liver of tumor bearing mice. Then, the number of tumor-infiltrating T cells (582 vs 1506, 5 HPF, p < 0.05) and the IFN-g-producing cells (9.2 vs 16.0 %) in CD8+ T cells were elevated by the DGKa treatment. Conclusions: Inhibition of DGKa not only suppressed the proliferation of hepatoma but also activated anti-tumor effector T cells in vivo.
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Cai, Kai, and Marion B. Sewer. "Diacylglycerol kinase θ couples farnesoid X receptor-dependent bile acid signalling to Akt activation and glucose homoeostasis in hepatocytes." Biochemical Journal 454, no. 2 (August 9, 2013): 267–74. http://dx.doi.org/10.1042/bj20130609.
Full textAbstract:
DGKs (diacylglycerol kinases) catalyse the conversion of diacylglycerol into PA (phosphatidic acid), a positive modulator of mTOR (mammalian target of rapamycin). We have found that chenodeoxycholic acid and the synthetic FXR (farnesoid X receptor) ligand GW4064 induce the mRNA and protein expression of DGKθ in the HepG2 cell line and in primary human hepatocytes. Reporter gene studies using 1.5 kB of the DGKθ promoter fused to the luciferase gene revealed that bile acids increase DGKθ transcriptional activity. Mutation of putative FXR-binding sites attenuated the ability of GW4046 to increase DGKθ luciferase activity. Consistent with this finding, ChIP (chromatin immunoprecipitation) assays demonstrated that bile acid signalling increased the recruitment of FXR to the DGKθ promoter. Furthermore, GW4064 evoked a time-dependent increase in the cellular concentration of PA. We also found that GW4064 and PA promote the phosphorylation of mTOR, Akt and FoxO1 (forkhead box O1), and that silencing DGKθ expression significantly abrogated the ability of GW4046 to promote the phosphorylation of these PA-regulated targets. DGKθ was also required for bile-acid-dependent decreased glucose production. Taken together, our results establish DGKθ as a key mediator of bile-acid-stimulated modulation of mTORC2 (mTOR complex 2), the Akt pathway and glucose homoeostasis.
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Singh, Brenal K., Wen Lu, Amanda M. Schmidt Paustian, Moyar Q. Ge, Cynthia J. Koziol-White, Cameron H. Flayer, Sara S. Killingbeck, et al. "Diacylglycerol kinase ζ promotes allergic airway inflammation and airway hyperresponsiveness through distinct mechanisms." Science Signaling 12, no. 597 (September 3, 2019): eaax3332. http://dx.doi.org/10.1126/scisignal.aax3332.
Full textAbstract:
Asthma is a chronic allergic inflammatory airway disease caused by aberrant immune responses to inhaled allergens, which leads to airway hyperresponsiveness (AHR) to contractile stimuli and airway obstruction. Blocking T helper 2 (TH2) differentiation represents a viable therapeutic strategy for allergic asthma, and strong TCR-mediated ERK activation blocks TH2 differentiation. Here, we report that targeting diacylglycerol (DAG) kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protected against allergic asthma by simultaneously reducing airway inflammation and AHR though independent mechanisms. Targeted deletion of DGKζ in T cells decreased type 2 inflammation without reducing AHR. In contrast, loss of DGKζ in airway smooth muscle cells decreased AHR but not airway inflammation. T cell–specific enhancement of ERK signaling was only sufficient to limit type 2 airway inflammation, not AHR. Pharmacological inhibition of DGK diminished both airway inflammation and AHR in mice and also reduced bronchoconstriction of human airway samples in vitro. These data suggest that DGK is a previously unrecognized therapeutic target for asthma and reveal that the inflammatory and AHR components of asthma are not as interdependent as generally believed.
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Abramovici, Hanan, Parmiss Mojtabaie, Robin J. Parks, Xiao-Ping Zhong, Gary A. Koretzky, Matthew K. Topham, and Stephen H. Gee. "Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI." Molecular Biology of the Cell 20, no. 7 (April 2009): 2049–59. http://dx.doi.org/10.1091/mbc.e07-12-1248.
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Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGKζ initiates RhoGDI release and Rac1 activation. In DGKζ-deficient fibroblasts PAK1 phosphorylation and Rac1–RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGKζ, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGKζ stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGKζ, and PA to the activation of Rac1: the PA generated by DGKζ activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility.
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Merino-Cortés, Sara V., Sofia R. Gardeta, Sara Roman-Garcia, Ana Martínez-Riaño, Judith Pineau, Rosa Liebana, Isabel Merida, et al. "Diacylglycerol kinase ζ promotes actin cytoskeleton remodeling and mechanical forces at the B cell immune synapse." Science Signaling 13, no. 627 (April 14, 2020): eaaw8214. http://dx.doi.org/10.1126/scisignal.aaw8214.
Full textAbstract:
Diacylglycerol kinases (DGKs) limit antigen receptor signaling in immune cells by consuming the second messenger diacylglycerol (DAG) to generate phosphatidic acid (PA). Here, we showed that DGKζ promotes lymphocyte function–associated antigen 1 (LFA-1)–mediated adhesion and F-actin generation at the immune synapse of B cells with antigen-presenting cells (APCs), mostly in a PA-dependent manner. Measurement of single-cell mechanical force generation indicated that DGKζ-deficient B cells exerted lower forces at the immune synapse than did wild-type B cells. Nonmuscle myosin activation and translocation of the microtubule-organizing center (MTOC) to the immune synapse were also impaired in DGKζ-deficient B cells. These functional defects correlated with the decreased ability of B cells to present antigen and activate T cells in vitro. The in vivo germinal center response of DGKζ-deficient B cells was also reduced compared with that of wild-type B cells, indicating that loss of DGKζ in B cells impaired T cell help. Together, our data suggest that DGKζ shapes B cell responses by regulating actin remodeling, force generation, and antigen uptake–related events at the immune synapse. Hence, an appropriate balance in the amounts of DAG and PA is required for optimal B cell function.
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Velnati, Suresh, Sara Centonze, Federico Girivetto, and Gianluca Baldanzi. "Diacylglycerol Kinase alpha in X Linked Lymphoproliferative Disease Type 1." International Journal of Molecular Sciences 22, no. 11 (May 29, 2021): 5816. http://dx.doi.org/10.3390/ijms22115816.
Full textAbstract:
Diacylglycerol kinases are intracellular enzymes that control the balance between the secondary messengers diacylglycerol and phosphatidic acid. DGKα and DGKζ are the prominent isoforms that restrain the intensity of T cell receptor signalling by metabolizing PLCγ generated diacylglycerol. Thus, their activity must be tightly controlled to grant cellular homeostasis and refine immune responses. DGKα is specifically inhibited by strong T cell activating signals to allow for full diacylglycerol signalling which mediates T cell response. In X-linked lymphoproliferative disease 1, deficiency of the adaptor protein SAP results in altered T cell receptor signalling, due in part to persistent DGKα activity. This activity constrains diacylglycerol levels, attenuating downstream pathways such as PKCθ and Ras/MAPK and decreasing T cell restimulation induced cell death. This is a form of apoptosis triggered by prolonged T cell activation that is indeed defective in CD8+ cells of X-linked lymphoproliferative disease type 1 patients. Accordingly, inhibition or downregulation of DGKα activity restores in vitro a correct diacylglycerol dependent signal transduction, cytokines production and restimulation induced apoptosis. In animal disease models, DGKα inhibitors limit CD8+ expansion and immune-mediated tissue damage, suggesting the possibility of using inhibitors of diacylglycerol kinase as a new therapeutic approach.
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Mérida, Isabel, Antonia Ávila-Flores, and Ernesto Merino. "Diacylglycerol kinases: at the hub of cell signalling." Biochemical Journal 409, no. 1 (December 11, 2007): 1–18. http://dx.doi.org/10.1042/bj20071040.
Full textAbstract:
DGKs (diacylglycerol kinases) are members of a unique and conserved family of intracellular lipid kinases that phosphorylate DAG (diacylglycerol), catalysing its conversion into PA (phosphatidic acid). This reaction leads to attenuation of DAG levels in the cell membrane, regulating a host of intracellular signalling proteins that have evolved the ability to bind this lipid. The product of the DGK reaction, PA, is also linked to the regulation of diverse functions, including cell growth, membrane trafficking, differentiation and migration. In multicellular eukaryotes, DGKs provide a link between lipid metabolism and signalling. Genetic experiments in Caenorhabditis elegans, Drosophila melanogaster and mice have started to unveil the role of members of this protein family as modulators of receptor-dependent responses in processes such as synaptic transmission and photoreceptor transduction, as well as acquired and innate immune responses. Recent discoveries provide new insights into the complex mechanisms controlling DGK activation and their participation in receptor-regulated processes. After more than 50 years of intense research, the DGK pathway emerges as a key player in the regulation of cell responses, offering new possibilities of therapeutic intervention in human pathologies, including cancer, heart disease, diabetes, brain afflictions and immune dysfunctions.
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Nagaya, Hisao, Ikuo Wada, Yan-Jun Jia, and Hideo Kanoh. "Diacylglycerol Kinase δ Suppresses ER-to-Golgi Traffic via Its SAM and PH Domains." Molecular Biology of the Cell 13, no. 1 (January 2002): 302–16. http://dx.doi.org/10.1091/mbc.01-05-0255.
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We report here that the anterograde transport from the endoplasmic reticulum (ER) to the Golgi was markedly suppressed by diacylglycerol kinase δ (DGKδ) that uniquely possesses a pleckstrin homology (PH) and a sterile α motif (SAM) domain. A low-level expression of DGKδ in NIH3T3 cells caused redistribution into the ER of the marker proteins of the Golgi membranes and the vesicular-tubular clusters (VTCs). In this case DGKδ delayed the ER-to-Golgi traffic of vesicular stomatitis virus glycoprotein (VSV G) and also the reassembly of the Golgi apparatus after brefeldin A (BFA) treatment and washout. DGKδ was demonstrated to associate with the ER through its C-terminal SAM domain acting as an ER-targeting motif. Both of the SAM domain and the N-terminal PH domain of DGKδ were needed to exert its effects on ER-to-Golgi traffic. Kinase-dead mutants of DGKδ were also effective as the wild-type enzyme, suggesting that the catalytic activity of DGK was not involved in the present observation. Remarkably, the expression of DGKδ abrogated formation of COPII-coated structures labeled with Sec13p without affecting COPI structures. These findings indicate that DGKδ negatively regulates ER-to-Golgi traffic by selectively inhibiting the formation of ER export sites without significantly affecting retrograde transport.
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Olenchock, Benjamin A., Rishu Guo, Michael A. Silverman, Jennifer N. Wu, Jeffery H. Carpenter, Gary A. Koretzky, and Xiao-Ping Zhong. "Impaired degranulation but enhanced cytokine production after FcεRI stimulation of diacylglycerol kinase ζ–deficient mast cells." Journal of Experimental Medicine 203, no. 6 (May 22, 2006): 1471–80. http://dx.doi.org/10.1084/jem.20052424.
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Calcium and diacylglycerol are critical second messengers that together effect mast cell degranulation after allergen cross-linking of immunoglobulin (Ig)E-bound FcεRI. Diacylglycerol kinase (DGK)ζ is a negative regulator of diacylglycerol-dependent signaling that acts by converting diacylglycerol to phosphatidic acid. We reported previously that DGKζ−/− mice have enhanced in vivo T cell function. Here, we demonstrate that these mice have diminished in vivo mast cell function, as revealed by impaired local anaphylactic responses. Concordantly, DGKζ−/− bone marrow–derived mast cells (BMMCs) demonstrate impaired degranulation after FcεRI cross-linking, associated with diminished phospholipase Cγ activity, calcium flux, and protein kinase C–βII membrane recruitment. In contrast, Ras-Erk signals and interleukin-6 production are enhanced, both during IgE sensitization and after antigen cross-linking of FcεRI. Our data demonstrate dissociation between cytokine production and degranulation in mast cells and reveal the importance of DGK activity during IgE sensitization for proper attenuation of FcεRI signals.
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Chianale, Federica, Santina Cutrupi, Elena Rainero, Gianluca Baldanzi, Paolo E. Porporato, Sara Traini, Nicoletta Filigheddu, et al. "Diacylglycerol Kinase-α Mediates Hepatocyte Growth Factor-induced Epithelial Cell Scatter by Regulating Rac Activation and Membrane Ruffling." Molecular Biology of the Cell 18, no. 12 (December 2007): 4859–71. http://dx.doi.org/10.1091/mbc.e07-02-0177.
Full textAbstract:
Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkα in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkα is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkα, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkα, obtained either pharmacologically by R59949 treatment, or by expression of Dgkα dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkα, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkα, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.
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Niizeki, Takeshi, Yasuchika Takeishi, Tatsuro Kitahara, Takanori Arimoto, Mitsunori Ishino, Olga Bilim, Satoshi Suzuki, et al. "Diacylglycerol kinase-ε restores cardiac dysfunction under chronic pressure overload: a new specific regulator of Gαq signaling cascade." American Journal of Physiology-Heart and Circulatory Physiology 295, no. 1 (July 2008): H245—H255. http://dx.doi.org/10.1152/ajpheart.00066.2008.
Full textAbstract:
Gαq protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in cardiac hypertrophy. DAG kinase (DGK) catalyzes DAG phosphorylation and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been reported that DGKε acts specifically on DAG produced by inositol cycling. In this study, we examined whether DGKε prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We generated transgenic mice with cardiac-specific overexpression of DGKε (DGKε-TG) using an α-myosin heavy chain promoter. There were no differences in cardiac morphology and function between wild-type (WT) and DGKε-TG mice at the basal condition. Either continuous phenylephrine infusion or thoracic transverse aortic constriction (TAC) was performed in WT and DGKε-TG mice. Increases in heart weight after phenylephrine infusion and TAC were abolished in DGKε-TG mice compared with WT mice. Cardiac dysfunction after TAC was prevented in DGKε-TG mice, and the survival rate after TAC was higher in DGKε-TG mice than in WT mice. Phenylephrine- and TAC-induced DAG accumulation, the translocation of PKC isoforms, and the induction of fetal genes were blocked in DGKε-TG mouse hearts. The upregulation of transient receptor potential channel (TRPC)-6 expression after TAC was attenuated in DGKε-TG mice. In conclusion, these results demonstrate the first evidence that DGKε restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and TRPC-6 expression. DGKε may be a novel therapeutic target to prevent cardiac hypertrophy and progression to heart failure.
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Zhu, Jili, Moumita Chaki, Dongmei Lu, Chongyu Ren, Shan-Shan Wang, Alysha Rauhauser, Binghua Li, et al. "Loss of diacylglycerol kinase epsilon in mice causes endothelial distress and impairs glomerular Cox-2 and PGE2 production." American Journal of Physiology-Renal Physiology 310, no. 9 (May 1, 2016): F895—F908. http://dx.doi.org/10.1152/ajprenal.00431.2015.
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Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε ( DGKE) that encodes the lipid kinase DGKε (Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531–536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377–384, 2013). DGKε is unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2 are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2 supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.
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Sakane, Fumio, Fumi Hoshino, Masayuki Ebina, Hiromichi Sakai, and Daisuke Takahashi. "The Roles of Diacylglycerol Kinase α in Cancer Cell Proliferation and Apoptosis." Cancers 13, no. 20 (October 16, 2021): 5190. http://dx.doi.org/10.3390/cancers13205190.
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Diacylglycerol (DG) kinase (DGK) phosphorylates DG to generate phosphatidic acid (PA). The α isozyme is activated by Ca2+ through its EF-hand motifs and tyrosine phosphorylation. DGKα is highly expressed in several refractory cancer cells including melanoma, hepatocellular carcinoma, and glioblastoma cells. In melanoma cells, DGKα is an antiapoptotic factor that activates nuclear factor-κB (NF-κB) through the atypical protein kinase C (PKC) ζ-mediated phosphorylation of NF-κB. DGKα acts as an enhancer of proliferative activity through the Raf–MEK–ERK pathway and consequently exacerbates hepatocellular carcinoma progression. In glioblastoma and melanoma cells, DGKα attenuates apoptosis by enhancing the phosphodiesterase (PDE)-4A1–mammalian target of the rapamycin pathway. As PA activates PKCζ, Raf, and PDE, it is likely that PA generated by DGKα plays an important role in the proliferation/antiapoptosis of cancer cells. In addition to cancer cells, DGKα is highly abundant in T cells and induces a nonresponsive state (anergy), which represents the main mechanism by which advanced cancers escape immune action. In T cells, DGKα attenuates the activity of Ras-guanyl nucleotide-releasing protein, which is activated by DG and avoids anergy through DG consumption. Therefore, a DGKα-specific inhibitor is expected to be a dual effective anticancer treatment that inhibits cancer cell proliferation and simultaneously enhances T cell functions. Moreover, the inhibition of DGKα synergistically enhances the anticancer effects of programmed cell death-1/programmed cell death ligand 1 blockade. Taken together, DGKα inhibition provides a promising new treatment strategy for refractory cancers.
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Murakami, Chiaki, Fumi Hoshino, Hiromichi Sakai, Yasuhiro Hayashi, Atsushi Yamashita, and Fumio Sakane. "Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains." Journal of Biological Chemistry 295, no. 10 (January 24, 2020): 2932–47. http://dx.doi.org/10.1074/jbc.ra119.012369.
Full textAbstract:
The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr–SAMD, but not SMS1–SAMD, co-immunoprecipitates with DGKδ–SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro. Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.
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Singh, Brenal, Wen Lu, Amanda Schmidt-Paustian, and Taku Kambayashi. "The loss of DGK protects against allergic airway inflammation and airway hyperresponsiveness." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 44.32. http://dx.doi.org/10.4049/jimmunol.200.supp.44.32.
Full textAbstract:
Abstract Asthma is a chronic allergic inflammatory airway disease that is caused by aberrant immune responses to inhaled allergens, which leads to airflow obstruction driven in part by increased sensitivity of airway smooth muscle to contractile agonists, a process known as airway hyperresponsiveness (AHR). The inflammation of allergic asthma is driven by type 2 cytokines released by Th2 CD4+ T cells and group 2 innate lymphoid cells (ILC2) in the lung. Here, we report that targeting DAG kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protects against allergic asthma. DGKζ knockout (KO) mice exhibited reduced type 2 airway inflammation and were completely resistant to AHR induction in a mouse model of allergic asthma. Surprisingly, the mechanism by which DGKζ protected against airway inflammation and AHR were separable. Targeted deletion of DGKζ in T cells led to decreased type 2 inflammation with no attenuation of AHR. In contrast, conditional deletion of DGKζ in airway smooth muscle cells led to decreased AHR despite no changes in airway inflammation. Importantly, the pharmacological inhibition of DGK provided protection against airway inflammation and AHR in mice, and also reduced bronchoconstriction of human airways. Together, our data suggest that DGKζ is potentially a novel therapeutic target for allergic asthma. Moreover, the targeted deletion of DGKζ reveals that the inflammatory and AHR components of allergic asthma are not as interdependent, as generally believed.