DGK? regulates presynaptic release during mGluR-dependent LTD.
ABSTRACT: Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGK?. In addition, we establish that DGK? acts presynaptically, a function that contrasts with the known postsynaptic function of DGK?, a close relative of DGK?. Deficiency of DGK? in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGK?-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (?2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGK?-/- synapses. These results suggest that DGK? requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.
Project description: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. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta 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 DGK zeta 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 DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.
Project description:Diacylglycerol kinases (DGKs) inhibit diacylglycerol (DAG) signaling by phosphorylating DAG. DGK-1, the Caenorhabditis elegans ortholog of human neuronal DGK, inhibits neurotransmission to control behavior. DGK-1, like DGK, has three cysteine-rich domains (CRDs), a pleckstrin homology domain, and a kinase domain. To identify DGK domains and amino acid residues critical for terminating DAG signaling in vivo, we analyzed 20 dgk-1 mutants defective in DGK-1-controlled behaviors. We found by sequencing that the mutations included nine amino acid substitutions and seven premature stop codons that impair the physiological functions of DGK-1. All nine amino acid substitutions are in the second CRD, the third CRD, or the kinase domain. Thus, these domains are important for the termination of DAG signaling by DGK-1 in vivo. Seven of the substituted amino acid residues are present in all human DGKs and likely define key residues required for the function of all DGKs. An ATP-binding site mutation expected to inactivate the kinase domain retained very little physiological function, but we found two stop codon mutants predicted to truncate DGK-1 before its kinase domain that retained significantly more function. We detected novel splice forms of dgk-1 that can reconcile this apparent conflict, as they skip exons containing the stop codons to produce DGK-1 isoforms that contain the kinase domain. Two of these isoforms lack an intact pleckstrin homology domain and yet appear to have significant function. Additional novel isoform(s) account for all of the DGK-1 function necessary for one behavior, dopamine response.
Project description:Diacylglycerol (DAG) is a critical second messenger that mediates T cell receptor (TCR)-stimulated signaling. The abundance of DAG is reduced by the diacylglycerol kinases (DGKs), which catalyze the conversion of DAG to phosphatidic acid (PA) and thus inhibit DAG-mediated signaling. In T cells, the predominant DGK isoforms are DGK? and DGK?, and deletion of the genes encoding either isoform enhances DAG-mediated signaling. We found that DGK?, but not DGK?, suppressed the development of natural regulatory T (T(reg)) cells and predominantly mediated Ras and Akt signaling downstream of the TCR. The differential functions of DGK? and DGK? were not attributable to differences in protein abundance in T cells or in their localization to the contact sites between T cells and antigen-presenting cells. RasGRP1, a key DAG-mediated activator of Ras signaling, associated to a greater extent with DGK? than with DGK?; however, in silico modeling of TCR-stimulated Ras activation suggested that a difference in RasGRP1 binding affinity was not sufficient to cause differences in the functions of each DGK isoform. Rather, the model suggested that a greater catalytic rate for DGK? than for DGK? might lead to DGK? exhibiting increased suppression of Ras-mediated signals compared to DGK?. Consistent with this notion, experimental studies demonstrated that DGK? was more effective than DGK? at catalyzing the metabolism of DAG to PA after TCR stimulation. The enhanced effective enzymatic production of PA by DGK? is therefore one possible mechanism underlying the dominant functions of DGK? in modulating T(reg) cell development.
Project description:Diacylglycerol kinases (DGKs) transform diacylglycerol (DAG) into phosphatidic acid (PA), balancing the levels of these key metabolic and signaling lipids. We previously showed that PA derived from the DGK? isoform promotes mammalian target of rapamycin complex 1 (mTORC1) activation. This function might be crucial for the growth and survival of cancer cells, especially for those resistant to the allosteric mTOR inhibitor rapamycin. How this positive function of DGK? coordinates with DAG metabolism and signaling is unknown. In this study, we used a rapamycin-resistant colon cancer cell line as a model to address the role of DGK? in tumor cells. We found that DGK? predominated over other PA sources such as DGK? or phospholipase D to activate mTORC1, and that its activity was a component of the rapamycin-induced feedback loops. We show that the DGK? DAG-consuming function is central to cell homeostasis, as DAG negatively regulates levels of the lipogenic transcription factor SREBP-1. Our findings suggest a model in which simultaneous regulation of DAG and PA levels by DGK? is integrated with mTOR function to maintain tumor cell homeostasis; we provide new evidence of the crosstalk between mTOR and lipid metabolism that will be advantageous in the design of drug therapies.
Project description:Diacylglycerol kinases (DGKs) catalyze the phosphorylation and conversion of diacylglycerol (DAG) into phosphatidic acid. DGK isozymes have unique primary structures, expression patterns, subcellular localizations, regulatory mechanisms, and DAG preferences. DGK? has a hydrophobic segment that promotes its attachment to membranes and shows substrate specificity for DAG with an arachidonoyl acyl chain in the sn-2 position of the substrate. We determined the role of DGK? in the regulation of energy and glucose homeostasis in relation to diet-induced insulin resistance and obesity using DGK?-KO and wild-type mice. Lipidomic analysis revealed elevated unsaturated and saturated DAG species in skeletal muscle of DGK? KO mice, which was paradoxically associated with increased glucose tolerance. Although skeletal muscle insulin sensitivity was unaltered, whole-body respiratory exchange ratio was reduced, and abundance of mitochondrial markers was increased, indicating a greater reliance on fat oxidation and intracellular lipid metabolism in DGK? KO mice. Thus, the increased intracellular lipids in skeletal muscle from DGK? KO mice may undergo rapid turnover because of increased mitochondrial function and lipid oxidation, rather than storage, which in turn may preserve insulin sensitivity. In conclusion, DGK? plays a role in glucose and energy homeostasis by modulating lipid metabolism in skeletal muscle.
Project description:Diacylglycerol kinases (DGKs) are pivotal signaling enzymes that phosphorylate diacylglycerol (DAG) to yield phosphatidic acid (PA). The biosynthesis of PA from phospholipase D (PLD) and the coupled phospholipase C (PLC)/DGK route is a crucial signaling process in eukaryotic cells. Next to PLD, the PLC/DGK pathway is the second most important generator of PA in response to biotic and abiotic stresses. In eukaryotic cells, DGK, DAG, and PA are implicated in vital processes such as growth, development, and responses to environmental cues. A plethora of DGK isoforms have been identified so far, making this a rather large family of enzymes in plants. Herein we performed a comprehensive phylogenetic analysis of DGK isoforms in model and crop plants in order to gain insight into the evolution of higher plant DGKs. Furthermore, we explored the expression profiling data available in public data bases concerning the regulation of plant DGK genes in response to beneficial elements and other metal and metalloid ions, including silver (Ag), aluminum (Al), arsenic (As), cadmium (Cd), chromium (Cr), mercury (Hg), and sodium (Na). In all plant genomes explored, we were able to find DGK representatives, though in different numbers. The phylogenetic analysis revealed that these enzymes fall into three major clusters, whose distribution depends on the composition of structural domains. The catalytic domain conserves the consensus sequence GXGXXG/A where ATP binds. The expression profiling data demonstrated that DGK genes are rapidly but transiently regulated in response to certain concentrations and time exposures of beneficial elements and other ions in different plant tissues analyzed, suggesting that DGKs may mediate signals triggered by these elements. Though this evidence is conclusive, further signaling cascades that such elements may stimulate during hormesis, involving the phosphoinositide signaling pathway and DGK genes and enzymes, remain to be elucidated.
Project description:Diacylglycerol (DAG) generation at the T cell immunological synapse (IS) determines the correct activation of antigen-specific immune responses. DAG kinases (DGKs) ? and ? 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 DGK? and DGK? 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 DGK?, indicating functional differences between these proteins. DGK? kinase activity at the TCR is enhanced by phorbol-12-myristate-13-acetate cotreatment, suggesting DAG-mediated regulation of DGK? responsiveness. We used GFP-DGK? and -DGK? chimeras to assess translocation dynamics during IS formation. Only GFP-DGK? 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 DGK? with DAG consumption at the IS. This study highlights a DGK?-specific function for local DAG metabolism at the IS and offers new clues to its mode of regulation.
Project description:Mucosal-associated invariant T (MAIT) cells participate in both protective immunity and pathogenesis of diseases. Most murine MAIT cells express an invariant TCRV?19-J?33 (iV?19) TCR, which triggers signals crucial for their development. However, signal pathways downstream of the iV?19TCR and their regulation in MAIT cells are unknown. Diacylglycerol (DAG) is a critical second messenger that relays the TCR signal to multiple downstream signaling cascades. DAG is terminated by DAG kinase (DGK)-mediated phosphorylation and conversion to phosphatidic acid. We have demonstrated here that downregulation of DAG caused by enhanced DGK activity impairs late-stage MAIT cell maturation in both thymus and spleen. Moreover, deficiency of DGK? but not DGK? by itself causes modest decreases in MAIT cells, and deficiency of both DGK? and ? results in severe reductions of MAIT cells in an autonomous manner. Our studies have revealed that DAG signaling is not only critical but also must be tightly regulated by DGKs for MAIT cell development and that both DGK? and, more prominently, DGK? contribute to the overall DGK activity for MAIT cell development.
Project description:Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue-specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGK?, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGK?(-/-) mice are osteoporotic because of a marked increase in OC numbers. In vitro, DGK?(-/-) bone marrow macrophages (BMMs) form more numerous, larger, and highly resorptive OCs. Surprisingly, although increased DAG levels do not alter receptor activator of NF-?B (RANK)/RANK ligand (RANKL) osteoclastogenic pathway, DGK? deficiency increases responsiveness to the proliferative and pro-survival cytokine macrophage colony-stimulating factor (M-CSF). We find that M-CSF is responsible for increased DGK?(-/-) OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGK?(-/-) cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGK?/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLC?2(+/-) BMMs, which have reduced DAG levels and form fewer OCs because of impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGK? in PLC?2(+/-) mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGK?/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression.
Project description:Diacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ? isoform of DGK (DGK?) 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 DGK? was expressed at significant levels in both platelets and megakaryocytes and that DGK?-knockout (DGK?-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 DGK?-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, DGK? deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGK? as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo.