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1
CLEJAN, Sanda, Conrad MALLIA, David VINSON, Robert DOTSON, and Barbara S. BECKMAN. "Erythropoietin stimulates G-protein-coupled phospholipase D in haematopoietic target cells." Biochemical Journal 314, no. 3 (March 15, 1996): 853–60. http://dx.doi.org/10.1042/bj3140853.
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A murine haematopoietic stem-cell line, B6SUt.EP, responsive to erythropoietin (EPO), has been found to exhibit both early and late changes in diacylglycerol (DAG) and phosphatidic acid (PA) as measured by HPLC and TLC. DAG levels peaked at 5 s with a 28.1% increase compared with control levels (from 17.3 to 22.2 pmol/106 cells) with a later peak at 30 min (84.2% increase from 17.3 to 31.9 pmol). These changes were concentration-dependent from 0.025 to 10 units/ml EPO (5 s, EC50 = 0.82 unit/ml; 30 min, EC50 = 0.10 unit/ml). In addition, PA levels increased 752.3% compared with control levels (from 8.6 to 64.7 μg/106 cells) with an early peak at 20 s, as measured by both HPLC and TLC (5 s, EC50 = 0.07 unit/ml). G-protein regulation was investigated by studying the effects of the non-hydrolysable GTP analogue guanosine 5´-[γ-thio]triphosphate (GTP[S]) on PA synthesis. The addition of GTP[S] (10 μM) in permeabilized cells increased PA content from 6.3 μg to 48.6 μg per 106 cells. In the presence of EPO and GTP[S], PA levels increased to 64.8 μg. An antagonist of G-proteins, guanosine 5´-[β-thio]diphosphate (GDP[S]), had no effect on control levels of PA (5.9 μg/106 cells) but blocked the effect of EPO on PA (30.6 μg/106 cells). Thus, EPO stimulated both lipid second messengers, DAG and PA. Our results demonstrate DAG kinetics to be biphasic, as observed with a high concentration of EPO, or monophasic, as observed with low concentrations of EPO. The PA accumulation preceding that of DAG in the slower and sustaining phase suggests that PA was not derived from DAG. This was confirmed by the stimulation of PA (without ATP) by GTP[S], effectively excluding phosphorylation of DAG by DAG kinase in the formation of PA. In addition, phospholipase D (PLD) activation was demonstrated with a maximal increase in phosphatidylethanol at 5 min, suggesting that EPO increases PA via a guanine nucleotide-binding protein coupled to PLD. The temporal relationship of the evolution of PA and DAG is further strengthened by experiments with ethanol and propranolol as inhibitors of the DAG/PA phosphohydrolase reaction and R59022 as an inhibitor of the DAG kinase reaction.
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Ganesan, Suriakarthiga, Brittney N. Shabits, and Vanina Zaremberg. "Tracking Diacylglycerol and Phosphatidic Acid Pools in Budding Yeast." Lipid Insights 8s1 (January 2015): LPI.S31781. http://dx.doi.org/10.4137/lpi.s31781.
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Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed.
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Song, J. G., L. M. Pfeffer, and D. A. Foster. "v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine." Molecular and Cellular Biology 11, no. 10 (October 1991): 4903–8. http://dx.doi.org/10.1128/mcb.11.10.4903.
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Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.
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Song, J. G., L. M. Pfeffer, and D. A. Foster. "v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine." Molecular and Cellular Biology 11, no. 10 (October 1991): 4903–8. http://dx.doi.org/10.1128/mcb.11.10.4903-4908.1991.
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Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.
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Farese, R. V., D. R. Cooper, T. S. Konda, G. Nair, M. L. Standaert, J. S. Davis, and R. J. Pollet. "Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes." Biochemical Journal 256, no. 1 (November 15, 1988): 175–84. http://dx.doi.org/10.1042/bj2560175.
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We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)
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BECKMAN, Barbara S., Conrad MALLIA, and Sanda CLEJAN. "Molecular species of phospholipids in a murine stem-cell line responsive to erythropoietin." Biochemical Journal 314, no. 3 (March 15, 1996): 861–67. http://dx.doi.org/10.1042/bj3140861.
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The generation of the lipid signalling molecules, diacylglycerol (DAG) and phosphatidic acid (PA), has been implicated in the transduction events essential for proliferation of murine B6SUt.EP stem cells responsive to erythropoietin (EPO). Some of the responses were rapid and transient while others were slower and sustained. In an attempt to better understand the biphasic nature of DAG and PA appearance in response to EPO, an analysis of the molecular species of DAG, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PA in control and EPO-treated B6SUt.EP cells was made by HPLC and TLC. Fifteen to eighteen species were identified, which were increased non-uniformly by 0.2 unit/ml EPO. Greater increases (×6) were observed in 16:0,20:4 and 18:0,20:4 DAGs than in other species. The molecular species profiles of the stimulated DAGs were compared with the profiles of molecular species contained in the phospholipids. Comparison of the increase in DAG species caused by EPO with the molecular species present in PC and PI showed both PI and PC as the source of the fast DAG accumulation and only PC as the source of the slow DAG accumulation. PE was involved in both phases. We found a consistent formation of ethanolamine over time, in larger amounts than choline, providing strong evidence that, in addition to PC, PE is a major substrate. In addition, changes in molecular species of PA in response to EPO suggest that PI cannot account for the mass of PA formed during the first 30 s incubation with EPO, nor for PA formed during 30 min with EPO. It is concluded that the majority of PA was formed by a direct action of phospholipase D on PC.
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Farah, Carole A., Ikue Nagakura, Daniel Weatherill, Xiaotang Fan, and Wayne S. Sossin. "Physiological Role for Phosphatidic Acid in the Translocation of the Novel Protein Kinase C Apl II in Aplysia Neurons." Molecular and Cellular Biology 28, no. 15 (May 27, 2008): 4719–33. http://dx.doi.org/10.1128/mcb.00178-08.
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ABSTRACT In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKCε, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.
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Peterson, M. W., and M. E. Walter. "Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial cells." American Journal of Physiology-Cell Physiology 263, no. 6 (December 1, 1992): C1216—C1224. http://dx.doi.org/10.1152/ajpcell.1992.263.6.c1216.
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Calcium ionophore exposure generates diglycerides (DAG) from phosphatidylcholine (PC) hydrolysis in Madin-Darby canine kidney (MDCK) epithelial cells. This study compares calcium ionophore-activated PC hydrolysis with the previously described phorbol ester-stimulated PC hydrolysis pathway using MDCK cells labeled with [14C]-linoleic acid. Lipid species were measured using thin-layer chromatography. DAG resulted in part from PC hydrolysis because DAG increased in cells labeled with [palmitoyl-2-14C]phosphatidylcholine. Neither protein kinase C (PKC) inhibitors nor PKC depletion affected the ionomycin (IONO)-induced increase in DAG. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the increased DAG after IONO but not after phorbol 12,13-dibutyrate (PDBu) exposure. The EGTA effect was reversed by adding excess calcium but was not reversed by adding excess Mg2+. IONO exposure also increased phosphatidic acid (PA) production. The PA was produced by phospholipase D (PLD) because phosphatidylethanol was produced when IONO was added to the cells in the presence of ethanol. Although increasing concentrations of ethanol resulted in progressively less PA, it had no effect on increased DAG after IONO exposure at any time point tested. These data are consistent with both increased phospholipase C (PLC) and increased PLD activity following ionomycin. In contrast to IONO exposure, ethanol completely prevented the increase in DAG after PDBu exposure, consistent with DAG produced by PLD activation. These results demonstrate that calcium activates both PC-specific PLC and PLD in MDCK cells and that the calcium-activated pathway is independent of the previously described PKC activation pathways.
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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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Harrris, W. E., and S. L. Bursten. "Lipid A stimulates phospholipase D activity in rat mesangial cells via a G-protein." Biochemical Journal 281, no. 3 (February 1, 1992): 675–82. http://dx.doi.org/10.1042/bj2810675.
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Stimulation of mesangial cells (MC) with the bacterial endotoxin Lipid A activated two enzymes involved in lipid metabolism. First, a phospholipase D hydrolyses phosphatidylethanolamine (PE) to phosphatidic acid (PA), followed by dephosphorylation of PA to 1,2-diacylglycerol (DAG) by PA phosphohydrolase. MC or microsomes from these cells were pre-labelled with [3H]glycerol. A 30-60 s stimulation with 10-100 ng of Lipid A/ml caused a decrease in [3H]glycerol in PE and increased radioactive glycerol in PA. The enzyme responsible for this hydrolysis preferred PE containing unsaturated acyl side chains. DAG was formed from PA within the first 1 min after Lipid A stimulation. Microsomes incubated with 25 mM-NaF to inhibit phospholipase C and to stimulate GTP-binding proteins also caused PE to be converted into PA. The [3H]glycerol and acyl mass of phosphatidylcholine, phosphatidylserine and phosphatidylinositol did not change with either Lipid A or NaF. Addition of guanosine 5′-[gamma-thio]triphosphate to MC microsomes caused the rapid decrease in proportion of PE and increase in PA, followed by an increase in DAG unsaturated acyl mass. These data suggest the concurrent G-protein-dependent activation by Lipid A of a PE-directed phospholipase D and a PA phosphohydrolase.
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Wen, Y., M. C. Cabot, E. Clauser, S. L. Bursten, and J. L. Nadler. "Lipid signal transduction pathways in angiotensin II type 1 receptor-transfected fibroblasts." American Journal of Physiology-Cell Physiology 269, no. 2 (August 1, 1995): C435—C442. http://dx.doi.org/10.1152/ajpcell.1995.269.2.c435.
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A stable Chinese hamster ovary fibroblast line expressing the rat vascular type 1a angiotensin II (ANG II) receptor was used to study the lipid-derived signal transduction pathways elicited by type 1a ANG II receptor activation. ANG II caused a biphasic and dose-dependent increase in diacylglycerol (DAG) accumulation with an initial peak at 15 s (181 +/- 11% of control, P < 0.02) and a second sustained peak at 5-10 min (214 +/- 10% of control, P < 0.02). The late DAG peak was derived from phosphatidylcholine (PC), and the formation was blocked by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANG II also increased phosphatidic acid (PA) production nearly fourfold by 7.5 min. In the presence of ethanol, ANG II markedly increased phosphatidylethanol (PEt) formation, indicating activation of phospholipase D (PLD). ANG II was shown to increase the mass of three separate PA species, one of which apparently originated from DAG kinase action on PC-phospholipase C (PLC)-produced DAG, providing evidence for PC-PLC activity. ANG II also formed a third PA species, which originated neither from PLD nor from DAG kinase. These results demonstrate that multiple lipid signals propagated via collateral stimulation of PLC and PLD are generated by specific activation of the vascular type 1a ANG II receptor.
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Carlsson, L., and K. Dahlberg. "Ha en bra dag!: Att vara boende pa servicehus." Nordic Journal of Nursing Research 22, no. 1 (March 1, 2002): 20–24. http://dx.doi.org/10.1177/010740830202200104.
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Lin, P., W. J. Fung, and A. M. Gilfillan. "Phosphatidylcholine-specific phospholipase D-derived 1,2-diacylglycerol does not initiate protein kinase C activation in the RBL 2H3 mast-cell line." Biochemical Journal 287, no. 1 (October 1, 1992): 325–31. http://dx.doi.org/10.1042/bj2870325.
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We examined the role of phosphatidylcholine-specific phospholipase D (PC-PLD) in the IgE-dependent activation of protein kinase C (PKC) in RBL 2H3 cells (a model for mast-cell function). Cells were sensitized with mouse monoclonal anti-trinitrophenol (TNP) IgE (0.5 micrograms/ml) and were then triggered with an optimal concentration (10 ng/ml) of TNP-ovalbumin conjugate (TNP-OVA). This resulted in an immediate biphasic increase in the production of 1,2-diacylglycerol (DAG) and activation of PKC. The initial increase in DAG production reached a peak within 30 s, and the second phase reached a plateau within 5 min after stimulation. TNP-OVA-induced PC-PLD activation followed the initial increase in DAG formation in response to IgE-receptor cross-bridging, but coincided with the second peak. Phosphatidic acid (PA), derived from the PC-PLD pathway, is metabolized to DAG by the action of PA phosphohydrolase (PAPase). Propranolol (0.3 mM), which inhibits PAPase, blocked the IgE-dependent increase in DAG, activation of PKC, and subsequently degranulation. The PKC inhibitor staurosporine (0.1 microM) inhibited the second, but not first, peak of DAG accumulation, reversed PKC translocation after 10 min and inhibited subsequent mediator release. Taken together, these results demonstrate that PC-PLD does not initiate, but may play a latent role in, IgE-dependent DAG production, PKC activation and mediator release from RBL 2H3 cells.
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Cybulsky, A. V., and M. D. Cyr. "Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9." American Journal of Physiology-Renal Physiology 265, no. 4 (October 1, 1993): F551—F560. http://dx.doi.org/10.1152/ajprenal.1993.265.4.f551.
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In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria. In cultured rat GEC, C5b-9 stimulates a phosphoinositide-directed phospholipase (PL) C and products of PLC downregulate C5b-9-mediated GEC injury. We now report that C5b-9-induced hydrolysis of phosphatidylcholine (PC) provides an additional source of 1,2-diacylglycerol (DAG). PC was labeled in intact GEC by brief incubation with 1-O-[alkyl-3H]2-lyso-PC. Assembly of C5b-9 stimulated an increase in PC-derived [3H]DAG (173 +/- 18% control), which was reduced in GEC depleted of protein kinase C (PKC) by prolonged preincubation with phorbol 12-myristate 13-acetate (PMA). Similar to C5b-9, [3H]DAG was released from PC after brief incubation of GEC with Ca2+ ionophore A23187 plus PMA. The increases in [3H]DAG induced by C5b-9 and A23187 plus PMA were paralleled by increases in DAG mass. C5b-9 also increased [3H]phosphatidic acid (PA; 182 +/- 37% control), but there was no significant interconversion of DAG and PA. Thus DAG probably originated via PLC. PC-directed PLC activity was also studied in GEC homogenates by release of [14C]DAG from exogenous 1-palmitoyl-2-[arachidonoyl-14C]PC. PLC activity was present at physiological Ca2+ concentration (200-1,200 nM), and PMA stimulated PLC activity in cell homogenates (in presence of ATP). These results demonstrate directly that PMA stimulates release of DAG from PC and are in keeping with the effect of PMA in [3H]lyso-PC-labeled GEC. Thus GEC contain a PC-directed PLC, whose activity is physiologically regulated and is present at nanomolar Ca2+ concentration. C5b-9 stimulates PC-directed PLC, leading to production of DAG. This DAG might trigger a mechanism for limiting injury during complement attack.
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Friedlander, G., C. Le Grimellec, J. Sraer, and C. Amiel. "12-HETE modulates Na-coupled uptakes in proximal tubular cells: role of diacylglycerol kinase inhibition." American Journal of Physiology-Renal Physiology 259, no. 5 (November 1, 1990): F816—F822. http://dx.doi.org/10.1152/ajprenal.1990.259.5.f816.
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Inhibition of diacylglycerol (DAG) kinase, an alternative way to increase the cellular DAG level, was shown to reproduce, in renal proximal tubular cells, the inhibitory effect of protein kinase C (PKC) activators on Na-Pi and Na-alpha-methyl-D-glucopyranoside (MGP) cotransport. To evaluate whether 12S-hydroxyeicosatetraenoic acid (12S-HETE) or 12R-HETE, a DAG kinase inhibitor in endothelial cells, has a similar effect in proximal tubular cells, we studied the influence of this lipoxygenase product on Na-dependent uptake of Pi, MGP, and alanine, as well as on [14C]arachidonate-DAG content and [32P]phosphatidic acid (PA) content in rabbit proximal tubular cells grown as a primary culture. 12-HETE (1-10 microM) decreased [32P]PA content and stimulated [14C]DAG accumulation in a concentration-dependent manner. The labeled phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin contents were not modified. 12-HETE also decreased DAG kinase activity of cell membranes. 12-HETE (10 microM) decreased the maximum velocity of Pi uptake by 36% and that of MGP uptake by 44% but did not affect alanine uptake. The effect of 12-HETE on transport was potentiated by calcium ionophore A23187 and was blunted by PKC downregulation. The effects of 12-HETE on lipid composition and transport were mimicked by R 59022, a pharmacological DAG kinase inhibitor. Neither arachidonic acid nor prostaglandin E2 reproduced the effects of 12-HETE. We conclude that in the proximal tubule, 12-HETE affected Na-dependent Pi and MGP cotransport through stimulation of PKC and that 12-HETE-induced activation of PKC is mediated by the inhibition of DAG kinase.
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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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Qin, Haixia, Michael A. Frohman, and Wendy B. Bollag. "Phospholipase D2 Mediates Acute Aldosterone Secretion in Response to Angiotensin II in Adrenal Glomerulosa Cells." Endocrinology 151, no. 5 (March 10, 2010): 2162–70. http://dx.doi.org/10.1210/en.2009-1159.
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In primary bovine adrenal glomerulosa cells, the signaling enzyme phospholipase D (PLD) is suggested to mediate priming, the enhancement of aldosterone secretion after pretreatment with and removal of angiotensin II (AngII), via the formation of persistently elevated diacylglycerol (DAG). To further explore PLD’s role in priming, glomerulosa cells were pretreated with an exogenous bacterial PLD. Using this approach, phosphatidic acid (PA) is generated on the outer, rather than the inner, leaflet of the plasma membrane. Although PA is not readily internalized, the PA is nonetheless rapidly hydrolyzed by cell-surface PA phosphatases to DAG, which efficiently flips to the inner leaflet and accesses the cell interior. Pretreatment with bacterial PLD resulted in priming upon subsequent AngII exposure, supporting a role of DAG in this process, because the increase in DAG persisted after exogenous PLD removal. To determine the PLD isoform mediating aldosterone secretion, and presumably priming, primary glomerulosa cells were infected with adenoviruses expressing GFP, PLD1, PLD2, or lipase-inactive mutants. Overexpressed PLD2 increased aldosterone secretion by approximately 3-fold over the GFP-infected control under basal conditions, with a significant enhancement to about 16-fold over the basal value upon AngII stimulation. PLD activity was also increased basally and upon stimulation with AngII. In contrast, PLD1 overexpression had little effect on aldosterone secretion, despite the fact that PLD activity was enhanced. In both cases, the lipase-inactive PLD mutants showed essentially no effect on PLD activity or aldosterone secretion. Our results suggest that PLD2 is the isoform that mediates aldosterone secretion and likely priming.
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Friedlaender, M. M., D. Jain, Z. Ahmed, D. Hart, R. L. Barnett, and E. P. Nord. "Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+." American Journal of Physiology-Renal Physiology 264, no. 5 (May 1, 1993): F845—F853. http://dx.doi.org/10.1152/ajprenal.1993.264.5.f845.
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Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.
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Noh, Ji-Yoon, Kyung-Min Lim, Ok-Nam Bae, Seung-Min Chung, Sang-Wook Lee, Kyung-Mi Joo, Sin-Doo Lee, and Jin-Ho Chung. "Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid." American Journal of Physiology-Heart and Circulatory Physiology 299, no. 2 (August 2010): H347—H355. http://dx.doi.org/10.1152/ajpheart.01144.2009.
Full textAbstract:
Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca2+-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A2or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca2+increase and the resultant activation of Ca2+-dependent PKC-α appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.
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Dey, Prabuddha, Gil-Soo Han, and George M. Carman. "A review of phosphatidate phosphatase assays." Journal of Lipid Research 61, no. 12 (September 22, 2020): 1556–64. http://dx.doi.org/10.1194/jlr.r120001092.
Full textAbstract:
Phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate, phosphatidate (PA), and product, DAG; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of PAP activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product, DAG, or Pi. However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive PA, to analyze radioactive and nonradioactive products, DAG and Pi, and discusses the advantages and disadvantages of each PAP assay.
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Noh, Ji-Yoon, Kyung-Min Lim, and Jin-Ho Chung. "Phosphatidic Acid Enhances Procoagulant Activity and Thrombosis through Phosphatidylserine Exposure in Human Erythrocytes." Blood 112, no. 11 (November 16, 2008): 3846. http://dx.doi.org/10.1182/blood.v112.11.3846.3846.
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Abstract The increased phospholipase D (PLD) activity and phosphatidic acid (PA) level are frequently observed in various disease states including cancers, diabetes, inflammation, sepsis, and thrombosis. While PA has been previously regarded as a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG), increasing evidence suggests the biological activities of PA, itself. Here we demonstrated that the PA can enhance procoagulant activities in human erythrocytes and thrombus formation mediated through phosphatidylserine (PS) exposure. Conspicuously, the PS exposure by PA was substantially greater than that of LPA and we examined its mechanism of action in an effort to elucidate the biological significance of PA. In human erythrocytes, PA treatment resulted in PS exposure without microvesicle generation or hemolysis as determined by flow cytometry. These effects were not attenuated by inhibitors of phospholipase A2 and phosphatidate phosphatase, that convert PA to LPA and DAG, respectively, suggesting that PA directly induced PS exposure. PA exposed erythrocytes showed significantly high intracellular calcium level and resultant protein kinase C (PKC) a activation. Consistent with these findings, the activity of scramblase was enhanced by PA treatment, while that of flippase was inhibited. Furthermore, PA-exposed erythrocytes were aggregated, accelerated thrombin generation, and increased adherence to endothelial cells, implying PA treatment enhanced the thrombogenic activities of erythrocytes indeed. Of note, these procoagulant activations by PA were confirmed in rat in vivo venous thrombosis model. These results suggest that PA can contribute to enhanced thrombosis, mediated through PS exposure on erythrocytes. With this study, we believe a novel insight was given into the role of PA in cardiovascular diseases.
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Schütze, S., D. Berkovic, O. Tomsing, C. Unger, and M. Krönke. "Tumor necrosis factor induces rapid production of 1'2'diacylglycerol by a phosphatidylcholine-specific phospholipase C." Journal of Experimental Medicine 174, no. 5 (November 1, 1991): 975–88. http://dx.doi.org/10.1084/jem.174.5.975.
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Tumor necrosis factor (TNF) is a proinflammatory polypeptide that is able to induce a great diversity of cellular responses via modulating the expression of a number of different genes. One major pathway by which TNF receptors communicate signals from the membrane to the cell nucleus involves protein kinase C (PKC). In the present study, we have addressed the molecular mechanism of TNF-induced PKC activation. To this, membrane lipids of the human histiocytic cell line U937 were labeled by incubation with various radioactive precursors, and TNF-induced changes in phospholipid, neutral lipid, and water-soluble metabolites were analyzed by thin layer chromatography. TNF treatment of U937 cells resulted in a rapid and transient increase of 1'2'diacylglycerol (DAG), a well-known activator of PKC. The increase in DAG was detectable as early as 15 s after TNF treatment and peaked at 60 s. DAG increments were most pronounced (approximately 360% of basal levels) when cells were preincubated with [14C]lysophosphatidylcholine, which was predominantly incorporated into the phosphatidylcholine (PC) pool of the plasma-membranes. Further extensive examination of changes in metabolically labeled phospholipids indicated that TNF-stimulated hydrolysis of PC is accompanied by the generation of phosphorylcholine and DAG. These results suggest the operation of a PC-specific phospholipase C. Since no changes in phosphatidic acid (PA) and choline were observed and the production of DAG by TNF could not be blocked by either propranolol or ethanol, a combined activation of phospholipase D and PA-phosphohydrolase in DAG production appears unlikely. TNF-stimulated DAG production as well as PKC activation could be blocked by the phospholipase inhibitor p-bromophenacylbromide (BPB). Since BPB did not inactivate PKC directly, these findings underscore that TNF activates PKC via formation of DAG. TNF stimulation of DAG production could be inhibited by preincubation of cells with a monoclonal anti-TNF receptor (p55-60) antibody, indicating that activation of a PC-specific phospholipase C is a TNF receptor-mediated event.
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NIGOU, Jérôme, and Gurdyal S. BESRA. "Cytidine diphosphate-diacylglycerol synthesis in Mycobacterium smegmatis." Biochemical Journal 367, no. 1 (October 1, 2002): 157–62. http://dx.doi.org/10.1042/bj20020370.
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Recent studies have demonstrated that, during infection of macrophages by mycobacteria, phospholipids (PLs) are released from the mycobacterial cell wall within infected macrophages and transported out of this compartment into intracellular vesicles. The release of these PLs may have functions that influence the outcome of mycobacterial infections. Despite their important role, little is known about the biosynthesis of PLs in mycobacteria. In all organisms, PL biosynthesis begins with acylation of sn-glycerol 3-phosphate to form phosphatidic acid (PA), which is then converted to the central liponucleotide intermediate, cytidine diphosphate-diacylglycerol (CDP-DAG) via the CDP-DAG synthase (CDS). The present work examines CDS activity in Mycobacterium smegmatis extracts, with regard to subcellular localization, pH dependence, bivalent and univalent cation requirement, substrate specificity and regulation by nucleotides. We show that CDS activity, which is mainly found within the cytoplasmic membrane, is Mg2+-dependent and activated by K+ ions. Among PAs containing saturated fatty acids, dipalmitoyl-PA is the preferred substrate [Km = 0.23±0.03mM for Triton X-100 (v/v)/PA in the ratio 5:1]. Moreover, CDS activity is inhibited by the reaction products PPi (IC50 = 1.5mM), CDP-DAG (IC50 = 0.3mM) and the nucleotides ATP, UTP and GTP. This study contributes to the delineation of PL biosynthesis in mycobacteria.
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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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Latijnhouwers, Maita, Teun Munnik, and Francine Govers. "Phospholipase D in Phytophthora infestans and Its Role in Zoospore Encystment." Molecular Plant-Microbe Interactions® 15, no. 9 (September 2002): 939–46. http://dx.doi.org/10.1094/mpmi.2002.15.9.939.
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We show that differentiation of zoospores of the late blight pathogen Phytophthora infestans into cysts, a process called encystment, was triggered by both phosphatidic acid (PA) and the G-protein activator mastoparan. Mastoparan induced the accumulation of PA, indicating that encystment by mastoparan most likely acts through PA. Likewise, mechanical agitation of zoospores, which often is used to induce synchronized encystment, resulted in increased levels of PA. The levels of diacylglycerolpyrophosphate (DGPP), the phosphorylation product of PA, increased simultaneously. Also in cysts, sporangiospores, and mycelium, mastoparan induced increases in the levels of PA and DGPP. Using an in vivo assay for phospholipase D (PLD) activity, it was shown that the mastoparan-induced increase in PA was due to a stimulation of the activity of this enzyme. Phospholipase C in combination with diacylglycerol (DAG) kinase activity also can generate PA, but activation of these enzymes by mastoparan was not detected under conditions selected to highlight 32P-PA production via DAG kinase. Primary and secondary butanol, which, like mastoparan, have been reported to activate G-proteins, also stimulated PLD activity, whereas the inactive tertiary isomer did not. Similarly, encystment was induced by n- and sec-butanol but not by tert-butanol. Together, these results show that Phytophthora infestans contains a mastoparan- and bu-tanol-inducible PLD pathway and strongly indicate that PLD is involved in zoospore encystment. The role of G-proteins in this process is discussed.
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Ha, K. S., E. J. Yeo, and J. H. Exton. "Lysophosphatidic acid activation of phosphatidylcholine-hydrolysing phospholipase D and actin polymerization by a pertussis toxin-sensitive mechanism." Biochemical Journal 303, no. 1 (October 1, 1994): 55–59. http://dx.doi.org/10.1042/bj3030055.
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Incubation of IIC9 fibroblasts with lysophosphatidic acid (LPA) induced an increase in the amount of filamentous actin (F-actin), which was concentration-dependent with a maximal effect at 100 ng/ml. Phosphatidic acid (PA) also produced a concentration-dependent increase of F-actin, but it was less potent than LPA. The LPA-induced increase in F-actin was rapid and sustained for at least 60 min. LPA rapidly increased the levels of PA and choline, with maximal increases at 5 min and 30 s respectively. LPA also caused a monophasic increase in diacylglycerol (DAG) which lagged behind the increases in PA and choline. LPA stimulated phosphatidylbutanol formation in the presence of butanol and produced a small increase in inositol phosphates that was much less than that induced by alpha-thrombin. Pretreatment of cells with pertussis toxin (PTX) caused greater than 50% inhibition of the LPA-stimulated increases in PA, DAG and choline. PTX increased the LPA concentration required to induce half-maximal actin polymerization by about 10-fold. PTX caused a similar shift in the dose-response curve for LPA-induced PA formation. These results suggest that LPA induces an increase in PA by activating a phosphatidylcholine-hydrolysing phospholipase D via a PTX-sensitive G-protein and that the increase in PA is involved in the activation of actin polymerization.
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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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DeJong, Kitty, Shahrzad A. Rahbar, and Frans A. Kuypers. "Phospholipase D Activity Is Essential for PKC-Mediated Phosphatidylserine Exposure." Blood 110, no. 11 (November 16, 2007): 1715. http://dx.doi.org/10.1182/blood.v110.11.1715.1715.
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Abstract The adverse effects of red blood cells (RBC) that expose phosphatidylserine (PS) in the sickle cell circulation are well recognized. However, the mechanism that underlies the formation of these cells is not well understood. We have shown previously that activation of protein kinase C (PKC) using phorbol myristate acetate (PMA) stimulates RBC to expose PS on a subset of cells. This process was dependent on Ca2+-influx induced by this stimulation. PKC inhibitors were shown to counteract this process and also reduce PS exposure induced by high levels of intracellular Ca2+. Furthermore, PIP2 administered to the RBC similarly induces PS exposure. Because both PKC and PIP2 can stimulate phospholipase D (PLD), we investigated the role of PLD in signal transduction initiated phospholipid scrambling. PLD cleaves phospholipids to produce phosphatidic acid (PA), which can be further metabolized to lysophosphatidic acid, an important lipid mediator, or to diacylglycerol (DAG), itself a PKC activator. In presence of primary alcohols such as ethanol or 1-butanol, PLD generates phosphatidylethanol or phosphatidylbutanol, respectively. Using annexin V, we measured the fraction of PS-exposing cells after treatment of RBC with PMA in presence of these primary alcohols. Treatment with 1% ethanol as well as 0.2% 1-butanol (at 0.4% hematocrit) completely abolished the generation of PS exposure in PMA-treated cells, indicating that the formation of PA by PLD was essential for scrambling. Anandamides are structural analogues of arachidonoyl ethanolamide, an endocannabinoid. Since these structures provide feedback inhibition of the PLD-isoform that produces these compounds in the brain and were found effective on plant PLD isoforms, we evaluated their effect on PLD-mediated PS scrambling in our assay. Short-chain lauroyl ethanolamide as well as arachidonoyl ethanolamide itself induced concentration-dependent inhibition of PMA-induced PS exposure. This suggests that these compounds indeed inhibit the isoforms of PLD present in the RBC, and confirms the importance of PLD activity for PKC-mediated PS exposure. Furthermore we evaluated the effect of propranolol, which inhibits phosphatidate phosphatase, the enzyme that converts PA to DAG. PMA-induced PS exposure was more than 80% inhibited by propranolol, indicating that the conversion of DAG from PLD-generated PA is essential for PKC-mediated PS exposure. PLD inhibitors did not inhibit Ca2+-induced scrambling initiated with Ca-ionophore, indicating that loading RBC with high levels of intracellular Ca2+ can override the requirement for PLD activation, possibly because DAG is formed through direct activation of phospholipase C. These results show that PLD activation is essential for PMA-induced Ca-influx and PS exposure, and that the subsequent formation of DAG from PA is a critical step in this process. Altered Ca2+ homeostasis, increased PA generation and increased PKC activation are observed in sickle RBC. Our data indicate that activation of signal transduction pathways plays an important role in generating PS exposure in sickle cells.
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Ha, K. S., and J. H. Exton. "Activation of actin polymerization by phosphatidic acid derived from phosphatidylcholine in IIC9 fibroblasts." Journal of Cell Biology 123, no. 6 (December 15, 1993): 1789–96. http://dx.doi.org/10.1083/jcb.123.6.1789.
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alpha-Thrombin induced a change in the cell morphology of IIC9 fibroblasts from a semiround to an elongated form, accompanied by an increase in stress fibers. Incubation of the cells with phospholipase D (PLD) from Streptomyces chromofuscus and exogenous phosphatidic acid (PA) caused similar morphological changes, whereas platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) induced different changes, e.g., disruption of stress fibers and cell rounding. alpha-Thrombin, PDGF, and exogenous PLD increased PA by 20-40%, and PMA produced a smaller increase. alpha-Thrombin and exogenous PLD produced rapid increases in the amount of filamentous actin (F-actin) that were sustained for at least 60 min. However, PDGF produced a transient increase of F-actin at 1 min and PMA caused no significant change. Dioctanoylglycerol was ineffective except at 50 micrograms/ml. Phospholipase C from Bacillus cereus, which increased diacylglycerol (DAG) but not PA, did not change F-actin content. Down-regulation of protein kinase C (PKC) did not block actin polymerization induced by alpha-thrombin. H-7 was also ineffective. Exogenous PA activated actin polymerization with a significant effect at 0.01 microgram/ml and a maximal increase at 1 microgram/ml. No other phospholipids tested, including polyphosphoinositides, significantly activated actin polymerization. PDGF partially inhibited PA-induced actin polymerization after an initial increase at 1 min. PMA completely or largely blocked actin polymerization induced by PA or PLD. These results show that PC-derived PA, but not DAG or PKC, activates actin polymerization in IIC9 fibroblasts, and indicate that PDGF and PMA have inhibitory effects on PA-induced actin polymerization.
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Ogawa, Ichijo. "On yan-dag kun-rdzob (tathya-samvrti) according to Tson-kha-pa." JOURNAL OF INDIAN AND BUDDHIST STUDIES (INDOGAKU BUKKYOGAKU KENKYU) 36, no. 1 (1987): 1–9. http://dx.doi.org/10.4259/ibk.36.1.
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el Bawab, S., O. Macovschi, M. Lagarde, and A. F. Prigent. "Time-course changes in content and fatty acid composition of phosphatidic acid from rat thymocytes during concanavalin A stimulation." Biochemical Journal 308, no. 1 (May 15, 1995): 113–18. http://dx.doi.org/10.1042/bj3080113.
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Several studies have shown the potential role of phosphatidic acid (PA) as a second messenger in different cell types. Thus, PA has been shown to mimic physiological agonists leading to various cellular responses, such as neurotransmitter and hormone release, cell proliferation by modulating DNA or RNA synthesis, the expression of several proto-oncogenes and growth factors, and the stimulation of enzyme activities such as phospholipase C (PLC), protein kinases and cyclic AMP (cAMP) phosphodiesterase. Stimulation of [3H]arachidonate-labelled rat thymocytes with the mitogen lectin concanavalin A (con A) resulted in enhanced production of radiolabelled PA after only 5 min of activation. The radiolabelled PA increase corresponded to a real increase in PA mass as determined by GLC quantification of its fatty acid content. In the presence of ethanol (0.5%), formation of phosphatidylethanol was not observed after 5 min of con A activation. Pretreatment of cells with R 59022 (10 microM), a diacylglycerol (DAG) kinase inhibitor, showed an inhibition in the formation of radiolabelled PA and in PA mass. These results suggest that the PLC-DAG kinase may be the pathway for PA synthesis in the first minutes of mitogenic thymocyte activation. A detailed analysis of the fatty acid composition showed that the relative amount of unsaturated fatty acids was increased in PA from stimulated cells concomitantly with a decrease in saturated ones; in particular, arachidonic acid was increased approximately 2-fold only 2 min after con A addition whereas palmitic acid was decreased for the whole period investigated (20 min). These changes favour the hydolysis of phosphoinositides rather than phosphatidylcholines by PLC. As PA remains a minor phospholipid, these changes are unlikely to affect cell membrane fluidity; but PA being now well recognized as a potential second messenger, its increased content as well as its increased unsaturation in the fatty acyl moiety might modulate several signalling pathways or the activity of enzymes such as cyclic nucleotide phosphodiesterase, controlling in this way the cellular level of cAMP, a negative regulator of blastic transformation.
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Ho, J. L., B. Zhu, S. He, B. Du, and R. Rothman. "Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase." Journal of Experimental Medicine 180, no. 4 (October 1, 1994): 1457–69. http://dx.doi.org/10.1084/jem.180.4.1457.
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Interleukin 4 (IL-4) diminishes cytokine activation of human macrophage. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacyglycerol (DAG), an activator of PKC, from membrane phospholipids was investigated to define the proximal events of IL-4R signaling. IL-4 induced a statistically significant time-and dose-dependent generation of DAG. The IL-4-triggered production of DAG was not derived from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in response to IL-4. Experiments were performed using [14C-methyl]choline-labeled U937 cells and monocytes to determine whether IL-4R activated phospholipase C (PLC), PLD, or PLA2 to use membrane phosphatidylcholine (PC) to form DAG. IL-4 induced a time- and dose-dependent increase of phosphocholine (pchol) with concomitant degradation of membrane PC (p < 0.05 compared with control). The finding that the peak reduction of PC was equivalent to peak production of pchol suggested that IL-4R signaling involved the activation of a PC-specific PLC. Changes in choline (chol) or lyso-PC and glycerolphosphocholine, the respective products of PC cleavage by PLD or PLA2, were not detected in IL-4-treated cells. In contrast, exogenous PLD induced an increase in chol and concomitant loss of membrane PC. Additional investigation suggested that IL-4R signaling does not involve PLD. In cells labeled with L-lyso-3-PC 1-[1-14C]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with ethanol. Propranolol, an inhibitor of phosphatidate phosphohydrolase, and calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG production in response to FMLP but not to IL-4. In propranolol pretreated cells, PMA but not IL-4 triggered the production of PA and lowered the amount of DAG. Evidence that PLA2 is not coupled to IL-4R is the detection of arachidonate production in response to FMLP but not to IL-4. Furthermore, IL-4R is not coupled to sphingomyelinase (SMase) since IL-4, unlike exogenous SMase, did not generate ceramide but induced the hydrolysis of PC to pchol that was comparable to exogenous PLC. In summary, IL-4R signaling in monocytes and U937 cells involves PLC and not PLD, PLA2, or SMase, and it uses PC and not PIP2 to form DAG.
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Farese, Robert V. "Insulin-Sensitive Phospholipid Signaling Systems and Glucose Transport. Update II." Experimental Biology and Medicine 226, no. 4 (April 2001): 283–95. http://dx.doi.org/10.1177/153537020122600404.
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Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidyicholine with subsequent increases in phosphatidic acid (PA) and diacyiglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.
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PLO, Isabelle, Dominique LAUTIER, Thierry LEVADE, Hadef SEKOURI, JeanPierre JAFFRÉZOU, Guy LAURENT, and Ali BETTAÏEB. "Phosphatidylcholine-specific phospholipase C and phospholipase D are respectively implicated in mitogen-activated protein kinase and nuclear factor κB activation in tumour-necrosis-factor-α-treated immature acute-myeloid-leukaemia cells." Biochemical Journal 351, no. 2 (October 10, 2000): 459–67. http://dx.doi.org/10.1042/bj3510459.
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Tumour necrosis factor-α (TNFα) has been reported to induce potent growth inhibition of committed myeloid progenitor cells, whereas it is a potential growth stimulator of human CD34+CD38- multipotent haematopoietic cells. The present study was aimed at evaluating the respective role of two phospholipases, phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the response of the CD34+ CD38- KG1a cells to TNFα. In these cells TNFα triggered phosphoinositide 3-kinase (PI3K)-dependent PC hydrolysis within 4–8min with concomitant production of both diacylglycerol (DAG) and phosphocholine (P-chol). DAG and P-chol production was accompanied by extracellular-signal-related protein kinase-1 (‘ERK-1’) activation and DNA-synthesis stimulation. PC-PLC stimulation was followed by PI3K-independent PLD activation with concomitant phosphatidic acid (PA) production followed by PA-derived DAG accumulation and sustained nuclear factor κB (NF-κB) activation. PLD/NF-κB signalling activation played no role in the TNFα proliferative effect and conferred no consistent protection of KG1a cells towards antileukaemic agents. Altogether these results suggest that, in KG1a cells, TNFα can stimulate in parallel PC-PLC and PLD, whose lipid products activate in turn mitogen-activated protein kinase (MAP kinase) and NF-κB signalling respectively. Finally, our study suggests that PC-PLC, but not PLD, plays a role in the TNFα proliferative effect in immature myeloid cells.
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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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Brown, L. A., and M. Chen. "Vasopressin signal transduction in rat type II pneumocytes." American Journal of Physiology-Lung Cellular and Molecular Physiology 258, no. 6 (June 1, 1990): L301—L307. http://dx.doi.org/10.1152/ajplung.1990.258.6.l301.
Full textAbstract:
Previous studies demonstrate that in cultured type II pneumocytes, [Arg8]-vasopressin (AVP) stimulates surfactant secretion independent of adenosine 3',5'-cyclic monophosphate (cAMP). In the current study AVP stimulated a 50% loss of radioactive phosphatidylinositol 4,5-bisphosphate (PIP2) within 15 s. Consistent with AVP-induced PIP2 hydrolysis was an increased appearance of the two breakdown products 1,2-diacylglycerol (1,2-DAG) and inositol 1,4,5-trisphosphate (IP3). Also, AVP stimulated the appearance of radiolabel in phosphatidic acid (PA) suggesting that the conversion of 1,2-DAG to PA could be used for PIP2 resynthesis. The effects of AVP on PIP2 and IP3 were mimicked by the bioactive AVP fragment and inhibited by the specific AVP1 antagonist. The EC50 for AVP on IP3 production was 6 nM. AVP stimulated protein kinase C (PK-C) activity twofold over the basal activity of 0.74 +/- 0.07 nmol P.min-1.mg protein-1 but did not activate cAMP-dependent protein kinase activity. The AVP1 antagonist inhibited AVP activation of PK-C. Therefore, activation of the AVP1 receptor resulted in PIP2 hydrolysis for signal transduction, PK-C activation, and surfactant secretion.
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Pai, J. K., E. A. Dobek, and W. R. Bishop. "Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1." Cell Regulation 2, no. 11 (November 1991): 897–903. http://dx.doi.org/10.1091/mbc.2.11.897.
Full textAbstract:
Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.
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Farese, R. V., J. S. Davis, D. E. Barnes, M. L. Standaert, J. S. Babischkin, R. Hock, N. K. Rosic, and R. J. Pollet. "The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+." Biochemical Journal 231, no. 2 (October 15, 1985): 269–78. http://dx.doi.org/10.1042/bj2310269.
Full textAbstract:
We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated ‘second messenger’ which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular ‘messenger’ for controlling metabolic processes during insulin action.
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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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Cockcroft, Shamshad, and Sima Lev. "Mammalian PITPs at the Golgi and ER-Golgi Membrane Contact Sites." Contact 3 (January 2020): 251525642096417. http://dx.doi.org/10.1177/2515256420964170.
Full textAbstract:
Phosphatidylinositol (PI)-transfer proteins (PITPs) have been long recognized as proteins that modulate phosphoinositide levels in membranes through their intrinsic PI/PC-exchange activity. Recent studies from flies and mammals suggest that certain PITPs bind phosphatidic acid (PA) and possess PI/PA-exchange activity. Phosphoinositides and PA play critical roles in cell signaling and membrane trafficking, and numerous biochemical, genetic and functional studies have shown that PITPs regulate cellular lipid metabolism, various signaling pathways and intracellular membrane transport events. In this mini-review, we discuss the function of mammalian PITPs at the Golgi and ER-Golgi membrane contact sites (MCS) and highlight DAG (Diacylglycerol) as a central hub of PITPs functions. We describe PITPs-associated phospho-signaling network at the ER-Golgi interface, and share our perspective on future studies related to PITPs at MCSs.
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Kwiatek, Joanna M., and George M. Carman. "Yeast phosphatidic acid phosphatase Pah1 hops and scoots along the membrane phospholipid bilayer." Journal of Lipid Research 61, no. 8 (June 15, 2020): 1232–43. http://dx.doi.org/10.1194/jlr.ra120000937.
Full textAbstract:
PA phosphatase, encoded by PAH1 in the yeast Saccharomyces cerevisiae, catalyzes the Mg2+-dependent dephosphorylation of PA, producing DAG at the nuclear/ER membrane. This enzyme plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. As an interfacial enzyme, PA phosphatase interacts with the membrane surface, binds its substrate, and catalyzes its reaction. The Triton X-100/PA-mixed micellar system has been utilized to examine the activity and regulation of yeast PA phosphatase. This system, however, does not resemble the in vivo environment of the membrane phospholipid bilayer. We developed an assay system that mimics the nuclear/ER membrane to assess PA phosphatase activity. PA was incorporated into unilamellar phospholipid vesicles (liposomes) composed of the major nuclear/ER membrane phospholipids, PC, PE, PI, and PS. We optimized this system to support enzyme-liposome interactions and to afford activity that is greater than that obtained with the aforementioned detergent system. Activity was regulated by phospholipid composition, whereas the enzyme’s interaction with liposomes was insensitive to composition. Greater activity was attained with large (≥100 nm) versus small (50 nm) vesicles. The fatty-acyl moiety of PA had no effect on this activity. PA phosphatase activity was dependent on the bulk (hopping mode) and surface (scooting mode) concentrations of PA, suggesting a mechanism by which the enzyme operates along the nuclear/ER membrane in vivo.
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Lipschitz, DA, KB Udupa, SR Indelicato, and M. Das. "Effect of age on second messenger generation in neutrophils." Blood 78, no. 5 (September 1, 1991): 1347–54. http://dx.doi.org/10.1182/blood.v78.5.1347.1347.
Full textAbstract:
Abstract Neutrophils from healthy elderly donors generate significantly less diacylglycerol (DAG) and inositol triphosphate (IP3) than neutrophils from young donors, following stimulation by the chemotactic peptide, formyl-methionyl-leucylphenylalanine (FMLP). The defect in signal transduction occurred at a point proximal to the generation of IP3 and DAG, since the reduction in FMLP-induced superoxide generation was corrected if the intervening signal transduction steps were bypassed, either by priming with a substimulatory dose (1.62 nmol/L) of phorbol myristate acetate (PMA), by ionophore elevation of cytosolic calcium, or by using a stimulatory dose of PMA (1.62 mumol/L). FMLP receptor number and affinity were unaffected by aging. On FMLP activation, neutrophils from old, as compared with young, volunteers showed significantly greater and more long-lasting decreases in the concentrations of phosphatidylinositol (PI), phosphatidylinositol 4- monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). This indicates a reduction with age in the metabolically active precursor pools responsible for the generation of IP3 and DAG. In contrast, aging had little effect on the production of phosphatidic acid (PA), which has recently been suggested to serve as a major activator of the NADPH oxidase. This may explain why the decrease in IP3 and DAG production was not accompanied by a comparable decrement in superoxide generation, which was only 17% lower in the old than in young donor neutrophils. Thus, aging is associated with reductions in the concentration of critically important phosphoinositides, resulting in diminution in the ability to produce key second messengers. Although the aged neutrophil is largely able to compensate for the decrements in signal transduction, its reserve capacity is compromised, making it particularly vulnerable to external insults that also impair function.
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Lipschitz, DA, KB Udupa, SR Indelicato, and M. Das. "Effect of age on second messenger generation in neutrophils." Blood 78, no. 5 (September 1, 1991): 1347–54. http://dx.doi.org/10.1182/blood.v78.5.1347.bloodjournal7851347.
Full textAbstract:
Neutrophils from healthy elderly donors generate significantly less diacylglycerol (DAG) and inositol triphosphate (IP3) than neutrophils from young donors, following stimulation by the chemotactic peptide, formyl-methionyl-leucylphenylalanine (FMLP). The defect in signal transduction occurred at a point proximal to the generation of IP3 and DAG, since the reduction in FMLP-induced superoxide generation was corrected if the intervening signal transduction steps were bypassed, either by priming with a substimulatory dose (1.62 nmol/L) of phorbol myristate acetate (PMA), by ionophore elevation of cytosolic calcium, or by using a stimulatory dose of PMA (1.62 mumol/L). FMLP receptor number and affinity were unaffected by aging. On FMLP activation, neutrophils from old, as compared with young, volunteers showed significantly greater and more long-lasting decreases in the concentrations of phosphatidylinositol (PI), phosphatidylinositol 4- monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). This indicates a reduction with age in the metabolically active precursor pools responsible for the generation of IP3 and DAG. In contrast, aging had little effect on the production of phosphatidic acid (PA), which has recently been suggested to serve as a major activator of the NADPH oxidase. This may explain why the decrease in IP3 and DAG production was not accompanied by a comparable decrement in superoxide generation, which was only 17% lower in the old than in young donor neutrophils. Thus, aging is associated with reductions in the concentration of critically important phosphoinositides, resulting in diminution in the ability to produce key second messengers. Although the aged neutrophil is largely able to compensate for the decrements in signal transduction, its reserve capacity is compromised, making it particularly vulnerable to external insults that also impair function.
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Holinstat, Michael, and Heidi E. Hamm. "Protease Activated Receptors Differentially Regulate Human Platelet Activation through Phosphatidic Acid-Dependent DAG Formation." Blood 108, no. 11 (November 16, 2006): 3906. http://dx.doi.org/10.1182/blood.v108.11.3906.3906.
Full textAbstract:
Abstract Pathological conditions such as coronary artery disease are clinically controlled via therapeutic regulation of platelet activity. Thrombin, through PAR1 and PAR4, plays a central role in regulation of human platelet function as it is known to be the most potent activator of human platelets. Currently, direct thrombin inhibitors used to block platelet activation result in unwanted side effects of excessive bleeding. An alternative therapeutic strategy would be to inhibit PAR-mediated intra-cellular platelet signaling pathways. To elucidate the best target, we are studying differences between the two platelet thrombin receptors, PAR1 and PAR4, in mediating thrombin’s action. In this study we show that platelet activation by PAR1 requires a signaling pathway that generates diacylglycerol (DAG) formation independent of its PLC-mediated production. Specifically, inhibition of lipid phosphate phosphatase-1 (LPP-1) by propranolol or inhibition of the phosphatidylcholine (PC)-mediated phosphatidic acid (PA) formation with a primary alcohol significantly attenuated platelet activation by PAR1. Platelet activation by thrombin or PAR4 was insensitive to these inhibitors. Further, activation of Rap1 was significantly attenuated by these inhibitors following stimulation by PAR1, but not thrombin or PAR4. In the mouse, evidence indicates that Rap1 can be activated through the DAG-sensitive guanine nucleotide exchange factor CalDAG-GEF1, and our data indicate that the differential signaling of PARs may, in part, occur through formation of distinct DAG species following thrombin stimulation.
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Nanjundan, Meera, and Fred Possmayer. "Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase." American Journal of Physiology-Lung Cellular and Molecular Physiology 284, no. 1 (January 1, 2003): L1—L23. http://dx.doi.org/10.1152/ajplung.00029.2002.
Full textAbstract:
The lung contains two distinct forms of phosphatidic acid phosphatase (PAP). PAP1 is a cytosolic enzyme that is activated through fatty acid-induced translocation to the endoplasmic reticulum, where it converts phosphatidic acid (PA) to diacylglycerol (DAG) for the biosynthesis of phospholipids and neutral lipids. PAP1 is Mg2+ dependent and sulfhydryl reagent sensitive. PAP2 is a six-transmembrane-domain integral protein localized to the plasma membrane. Because PAP2 degrades sphingosine-1-phosphate (S1P) and ceramide-1-phosphate in addition to PA and lyso-PA, it has been renamed lipid phosphate phosphohydrolase (LPP). LPP is Mg2+independent and sulfhydryl reagent insensitive. This review describes LPP isoforms found in the lung and their location in signaling platforms (rafts/caveolae). Pulmonary LPPs likely function in the phospholipase D pathway, thereby controlling surfactant secretion. Through lowering the levels of lyso-PA and S1P, which serve as agonists for endothelial differentiation gene receptors, LPPs regulate cell division, differentiation, apoptosis, and mobility. LPP activity could also influence transdifferentiation of alveolar type II to type I cells. It is considered likely that these lipid phosphohydrolases have critical roles in lung morphogenesis and in acute lung injury and repair.
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Cybulsky, A. V., D. J. Salant, R. J. Quigg, J. Badalamenti, and J. V. Bonventre. "Complement C5b-9 complex activates phospholipases in glomerular epithelial cells." American Journal of Physiology-Renal Physiology 257, no. 5 (November 1, 1989): F826—F836. http://dx.doi.org/10.1152/ajprenal.1989.257.5.f826.
Full textAbstract:
In rat membranous nephropathy, formation of the C5b-9 membrane attack complex (MAC) leads to proteinuria in association with glomerular visceral epithelial cell (GEC) injury. These alterations in GEC function and morphology might result from changes in intracellular free Ca2+ concentration [( Ca2+]i) and activation of phospholipases. We demonstrate that in cultured rat GEC, antibody-directed formation of noncytolytic amounts of the MAC induced a rapid and sustained increase in [Ca2+]i that was partly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The MAC elevated levels of inositol bis- (IP2) and trisphosphate (IP3), as well as 1,2-diacylglycerol (DAG) and phosphatidic acid (PA). In permeabilized GEC, IP3 released Ca2+ from intracellular stores. Cellular 45Ca2+ uptake was also increased by the MAC. Thus, in GEC, the MAC induced Ca2+ mobilization from intracellular stores secondary to activation of phospholipase C and production of IP3, as well as enhanced Ca2+ influx. In addition, C5b-9 stimulated release of arachidonic acid (AA), prostaglandin F2 alpha, and thromboxane A2. Indomethacin partially inhibited the increase in DAG levels observed with the MAC, whereas the prostaglandin H2/thromboxane A2 analogue U46619 elevated DAG, suggesting that an eicosanoid product of MAC-induced AA release may enhance the activation of phospholipase C. Activation of phospholipases by the MAC may lead to altered GEC function and thereby contribute to the pathophysiological changes that characterize complement-dependent rat membranous nephropathy.
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JUNG, EunMi, Soraya BETANCOURT-CALLE, RaShawn MANN-BLAKENEY, Tasha FOUSHEE, M. Carlos ISALES, and B. Wendy BOLLAG. "Sustained phospholipase D activation in response to angiotensin II but not carbachol in bovine adrenal glomerulosa cells." Biochemical Journal 330, no. 1 (February 15, 1998): 445–51. http://dx.doi.org/10.1042/bj3300445.
Full textAbstract:
We have demonstrated previously that in bovine adrenal glomerulosa cells, phospholipase D (PLD) activity can indirectly result in the generation of sn-1,2-diacylglycerol (DAG) through its production of phosphatidic acid (PA) and the subsequent action of PA phosphohydrolase. Furthermore, the PLD-generated DAG can trigger aldosterone secretion. Therefore, we characterized PLD activation by two agonists, angiotensin II (Ang II) and carbachol, to determine if the activity of the enzyme might underlie sustained aldosterone secretion. We determined that Ang II-induced PLD activation occurred via the angiotensin-1 receptor (AT1), and that a specific AT1 antagonist, losartan, inhibited this activation, whereas the same concentration of the AT2-specific antagonist, PD 123319, had no effect. Ang II activated PLD with a dose dependence similar to that observed for aldosterone secretion, with slight increases in activity induced by 0.1 nM Ang II and maximal activation at 10 nM. We also found that Ang II induced a sustained activation of PLD, but that the effect of carbachol, a stable analogue of acetylcholine, was transient; PLD activity increased within 5 min of exposure to carbachol but then ceased by 15 min. Higher carbachol concentrations were also unable to sustain PLD activation. These results suggest that the Ang II-elicited activation of PLD is associated with a sustained increase in aldosterone secretion from glomerulosa cells and further provide the first evidence, to our knowledge, of differences in the kinetics of PLD activation in response to two physiologically relevant agonists. Finally, we speculate that this disparity correlates with different functional responses induced by the two agents.
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Liu, Guang-Hui, Jing Qu, Anne E. Carmack, Hyun Bae Kim, Chang Chen, Hongmei Ren, Andrew J. Morris, Brian N. Finck, and Thurl E. Harris. "Lipin proteins form homo- and hetero-oligomers." Biochemical Journal 432, no. 1 (October 25, 2010): 65–76. http://dx.doi.org/10.1042/bj20100584.
Full textAbstract:
Lipin family members (lipin 1, 2 and 3) are bi-functional proteins that dephosphorylate PA (phosphatidic acid) to produce DAG (diacylglycerol) and act in the nucleus to regulate gene expression. Although other components of the triacylglycerol synthesis pathway can form oligomeric complexes, it is unknown whether lipin proteins also exist as oligomers. In the present study, using various approaches, we revealed that lipin 1 formed stable homo-oligomers with itself and hetero-oligomers with lipin 2/3. Both the N- and C-terminal regions of lipin 1 mediate its oligomerization in a head-to-head/tail-to-tail manner. We also show that lipin 1 subcellular localization can be influenced through oligomerization, and the individual lipin 1 monomers in the oligomer function independently in catalysing dephosphorylation of PA. The present study provides evidence that lipin proteins function as oligomeric complexes and that the three mammalian lipin isoforms can form combinatorial units.
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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.
Full textAbstract:
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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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.
Full textAbstract:
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.