Isolation and Expression of an Isoform of Human CDP-Diacylglycerol Synthase cDNA
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Phosphatidic acid (PA) is a phospholipid involved in signal transduction and in glycerolipid biosynthesis. CDP-diacylglycerol synthase (CDS) or CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41) catalyzes the conversion of PA to CDP-diacylglycerol (CDP-DAG), an important precursor for the synthesis of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We describe in this study the isolation and characterization of a human cDNA clone that encodes amino acid sequences homologous to Escherichia coli, yeast, and Drosophila CDS sequences. Expression of this human cDNA under the control of a GAL1 promoter in a null cds1 mutant yeast strain complements its growth defect and produces CDS activity when induced with galactose. Transfection of this cDNA into mammalian cells leads to increased CDS activity in cell-free extracts using an in vitro assay that measures the conversion of [alpha-32P]CTP to [32P]CDP-DAG. This increase in CDS activity also leads to increased secretion of tumor necrosis factor-alpha and interleukin-6 from endothelial ECV304 cells upon stimulation with interleukin-1beta, suggesting that CDS overexpression may amplify cellular signaling responses from cytokines.We previously described the purification of a membrane-bound diacylglycerol kinase highly selective for sn-1-acyl-2-arachidonoyl diacylglycerols (Walsh, J. P., Suen, R., Lemaitre, R. N., and Glomset, J. A. (1994) J. Biol. Chem.269, 21155‐21164). This enzyme appears to be responsible for the rapid clearance of the arachidonaterich pool of diacylglycerols generated during stimulusinduced phosphoinositide turnover. We have now shown phosphatidylinositol 4,5-bisphosphate to be a potent and specific inhibitor of arachidonoyl-diacylglycerol kinase. Kinetic analyses indicated a Ki for phosphatidylinositol 4,5-bisphosphate of 0.04 mol %. Phosphatidic acid also was an inhibitor with a Ki of 0.7 mol %. Other phospholipids had only small effects at these concentrations. A series of multiply phosphorylated lipid analogs also inhibited the enzyme, indicating that the head group phosphomonoesters are the primary determinants of the polyphosphoinositide effect. However, these compounds were not as potent as phosphatidylinositol 4,5-bisphosphate, indicating some specificity for the polyphosphoinositide additional to its total charge. Five other diacylglycerol kinases were activated to varying degrees by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, suggesting that inhibition by acidic lipids may be specific for the arachidonoyl-DAG kinase isoform. Given the presumed role of arachidonoyl-diacylglycerol kinase in the phosphoinositide cycle, this inhibition may represent a mechanism for polyphosphoinositides to regulate their own synthesis.
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PHOSPHOLIPID COMPOSITION AND [14C]GLYCEROL INCORPORATION INTO GLYCEROLIPIDS OF TOAD RETINA AND BRAIN
The phospholipid composition as well as the in vivo [14C]glycerol uptake in lipids was found to be similar in the toad brain and retina. The choroid lipid labeling was markedly different. An in vitro time-course study of [14C]glycerol incorporation in toad retina lipids disclosed that under the conditions of these experiments: (1) retina is able to rapidly synthesize phosphatidic acid from the radioactive precursor; (2) the sequence phosphatidic acid-diacylglycerol-triacylglycerol operates; (3) a high rate of phosphatidylinositol de novo biosynthesis takes place; (4) phosphoglycerides of choline and of ethanolamine are also heavily labeled after a lag period; (5) in vivo labeling profiles resembled those obtained in vitro mainly regarding phosphatidylinositol biosynthesis; and (6) the presence of glycerol kinase in the CNS is suggested.
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Phosphorylation of endogenous phosphatidylinositol was transiently increased following partial hepatectomy but was suppressed during peak DNA synthesis. Formation of inositol trisphosphate was decreased while generation of diacylglycerol and its breakdown to phosphatidic acid was increased. In response to partial hepatectomy protein kinase C was activated due to translocation from cytosol to particulate fraction, but the membrane bound activity was decreased during regeneration. Alteration of certain parameters in the signal transduction pathway apparently facilitates cell proliferation.
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In the preceding chapter, the intent was to provide the reader with a broad perspective on the chemical characteristics of cellular phospholipids. At the same time, emphasis was placed on the potential usefulness of this information in dissecting the importance of phospholipids in cellular events, such as signal transduction. There is no doubt that the large number of observations reported on the close relationship of phospholipids to the transduction process has stimulated a widespread (and gratifying) interest in these compounds. Certainly it is very clear now that stimulus-induced activation of cells leads to the turnover of specific membrane phospholipids. The following diagram reemphasizes several, but not all, possible reaction pathways that can be invoked during an agonist (stimulus)–induced activation of a cell and gives the possible sequelae: In each of the above reactions, the substrates phosphatidylcholine and phosphatidylinositol bisphosphate normally are considered biologically inactive in membranes. Then, subsequent to activation of cellular phospholipases by a stimulus, biologically active products are formed from these compounds. Thus, inositol bisphosphate triggers the release of calcium ions from intracellular stores, diacylglycerol is implicated in the translocation and activation of protein kinase C, arachidonic acid can be converted to biologically active prostaglandins, and phosphatidic acid can be an agonist in its own right. The major point to be stressed here is that phospholipid turnover is intimately associated with the signal transduction pathway in cells. Hence an understanding of the chemistry of these phospholipids is of major relevance to delineating the complicated process of signal transduction. While investigation of the behavior of phospholipids in this pathway in platelets has been a consuming interest of this author, the main thrust in this book will be simply to acquaint the reader with the chemistry of phospholipids of major importance in signal transduction and also to discuss other phospholipids found in mammalian membranes. Inasmuch as most investigations on stimulus response in cells utilize quite small numbers of cells—for example, a typical experiment on human platelets might use 1 x 109 cells, which would yield ∼50 μg of total lipid—this poses a challenge to an investigator to be able to isolate and identify these lipids.
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Addition of phytohemagglutinin to JURKAT cells, a human T-cell leukemia line, induced a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (and may also be phosphatidylinositol 4-phosphate) and an accumulation of phosphatidic acid. The accumulation and disappearance of the various molecular species of phosphatidic acid, diacylglycerol and phosphatidylinositol (PtdIns) in response to phytohemagglutinin was studied in JURKAT cells. The cells were prelabeled with [2-3H]glycerol for 2 days and 3H-labeled lipids were isolated from the cells after incubation for 2 min at 37 degrees C in the absence or in the presence of phytohemagglutinin. The isolated 3H-labeled lipids were separated into individual molecular species by reverse-phase HPLC after conversion to their 1,2-[3H]diacylglycerol acetate derivatives either by acetolysis or by acetylation. Stimulation with phytohemagglutinin induced a 2-fold increase in [3H]phosphatidic acid. The molecular species of the accumulated [3H]phosphatidic acid consisted of polyenoic species, which were almost absent in the [3H]phosphatidic acid of the unstimulated cells. Stearoylarachidonoyl species of [3H]phosphatidic acid accumulated most prominently. Although an accumulation of [3H]diacylglycerol was hardly measurable in the phytohemagglutinin-stimulated cells, the HPLC analysis of the molecular species of [3H]diacylglycerol showed a 2-fold increase in the stearoylarachidonoyl species in the stimulated cells. Stimulation with phytohemagglutinin had almost no effect on the composition of molecular species of [3H]PtdIns. The stearoylarachidonyl species is the most abundant molecular species of PtdIns in JURKAT cells. These results suggest that the [3H]diacylglycerol moiety of [3']phosphatidic acid originates from inositol lipid(s). The results also suggest a rapid and preferential phosphorylation of the diacylglycerol formed by receptor-stimulated hydrolysis of inositol lipid(s).
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