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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.
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PMID:Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors. 131 4

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.
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PMID:Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors. 132 58

Recently, we demonstrated that aggregation of the high affinity IgE receptor in rat basophilic leukemia (RBL-2H3) cells results in rapid tyrosine phosphorylation of a 72-kDa protein (pp72). Here we investigated the relationship of pp72 phosphorylation to guanine nucleotide-binding protein (G protein) activation and phosphatidylinositol hydrolysis. The activation of G proteins by NaF in intact cells or by guanosine 5'-O-(3-thiotriphosphate) in streptolysin O-permeabilized cells induced both phosphatidylinositol hydrolysis and histamine release without tyrosine phosphorylation of pp72. Similarly, in RBL-2H3 cells expressing the G protein-coupled muscarinic acetylcholine receptor, carbachol activated phospholipase C and induced secretion without concomitant pp72 phosphorylation. Therefore, pp72 phosphorylation was not induced by G protein activation or as a consequence of phosphatidylinositol hydrolysis. To investigate whether pp72 tyrosine phosphorylation precedes the activation of phospholipase C, we studied the effect of the tyrosine kinase inhibitor genistein. Preincubation of cells with genistein decreased, in parallel, antigen-induced tyrosine phosphorylation of pp72 (IC50 = 34 micrograms/ml) and histamine release (IC50 = 31 micrograms/ml). However, genistein at concentrations of up to 60 micrograms/ml did not inhibit phosphatidylinositol hydrolysis nor did it change the amount of the secondary messenger inositol (1,4,5)-triphosphate. Previous observations showed that there was no pp72 tyrosine phosphorylation after activation of protein kinase C or after an increase in intracellular calcium. Taken together, these results suggest that pp72 tyrosine phosphorylation represents a distinct, independent signaling pathway induced specifically by aggregation of the Fc epsilon RI.
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PMID:Fc epsilon RI-induced protein tyrosine phosphorylation of pp72 in rat basophilic leukemia cells (RBL-2H3). Evidence for a novel signal transduction pathway unrelated to G protein activation and phosphatidylinositol hydrolysis. 137 2

We investigated the possible role of tyrosine phosphorylation in the activation process of mast cells by cross-linking of cell-bound IgE antibodies. Bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE antiDNP mAb and then challenged with multivalent Ag DNP conjugates of human serum albumin. Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that cross-linking of cell-bound IgE antibodies induced a marked increase in tyrosine phosphorylation of several proteins. To obtain direct evidence for activation of protein-tyrosine kinases (PTK), phosphotyrosine-containing proteins in lysates of mast cells were affinity purified, and kinase activity of the immunoprecipitates was assessed by an in vitro kinase assay. The results clearly showed activation of PTK upon cross-linking of Fc epsilon RI. Activation of PTK was not detected by the same assay when the sensitized BMMC were challenged with monovalent DNP-lysine. Treatment of sensitized BMMC with either Ca2+ ionophore or PMA failed to induce the activation of PTK. A representative IgE-independent secretagogue, thrombin, induced histamine release from BMMC but failed to induce activation of PTK. The results excluded the possibility that PTK activation is the consequence of an increase in intracellular Ca2+ or activation of protein kinase C. Addition of genistein, a PTK inhibitor, to sensitized BMMC before Ag challenge inhibited not only Ag-induced PTK activation, but also inositol 1,4,5-trisphosphate production, and histamine release in a similar dose-response relationship. Other PTK inhibitors, such as lavendustin A and tyrphostin RG50864, also inhibited the Ag-induced activation of PTK and histamine release. The results collectively suggest that activation of PTK is an early event upstream of the activation of phospholipase C, and is involved in transduction of IgE-dependent triggering signals to mediator release.
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PMID:Tyrosine phosphorylation is required for mast cell activation by Fc epsilon RI cross-linking. 137 48

Activation of receptor-linked and cytoplasmic protein tyrosine kinases is crucial in the control of normal and abnormal cell growth and differentiation. Some substrates of protein tyrosine kinases such as phospholipase C gamma and ras GTPase-activating protein (GAP) contain sequences homologous to the src protein domains SH2 and SH3 (refs 3-9). The proto-oncogene vav is expressed in haematopoietic cells and its product Vav contains sequence motifs commonly found in transcription factors, such as helix-loop-helix, leucine-zipper and zinc-finger motifs and nuclear localization signals, as well as a single SH2 and two SH3 domains. Here we show that stimulation of T-cell antigen receptor on normal human peripheral blood lymphocytes or on human leukaemic T cells, and the crosslinking of IgE receptors on rat basophilic leukaemia cells, both promote the phosphorylation of tyrosine residues in Vav. Moreover, activation of the receptor for epidermal growth factor leads to marked tyrosine phosphorylation of Vav in cells transiently expressing vav, and Vav associates with the receptor through its SH2 domain. We propose that vav encodes a new class of substrates whose tyrosine phosphorylation may provide a mechanism for direct signal transduction linking receptors at the cell surface to transcriptional control.
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PMID:Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs. 161 45

Stimulation of rat basophilic leukemia (RBL-2H3) cells with oligomeric IgE elicited a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1 on tyrosine residues. Prior incubation of RBL-2H3 cells with a protein tyrosine kinase inhibitor, herbimycin A, prevented the tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of phosphatidylinositol 4,5-bisphosphate induced by oligomeric IgE. However, 5'-(N-ethyl)carboxamidoadenosine, which is known to activate PLC through a G protein, did not elicit tyrosine phosphorylation of PLC-gamma 1. These results, together with previous findings showing that tyrosine phosphorylation of PLC-gamma 1 enhances its catalytic activity, indicate that phosphorylation of PLC-gamma 1 by a nonreceptor tyrosine kinase is the mechanism by which IgE receptor aggregation triggers PLC activation.
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PMID:IgE-induced tyrosine phosphorylation of phospholipase C-gamma 1 in rat basophilic leukemia cells. 166 4

Rat basophilic leukemia (RBL 2H3) cells were passively sensitized by exposure to monoclonal anti-trinitrophenol mouse immunoglobulin E (anti-trinitrophenol IgE) (0.5 microgram/ml) and triggered by exposure to a sub-optimal concentration of trinitrophenol ovalbumin conjugate (5 ng/ml). At this concentration, trinitrophenol-ovalbumin increased histamine release from a basal rate of 4.8 +/- 0.5 to 28.5 +/- 4.6% and peptidoleukotrienes from less than 0.1 to 4.2 +/- 1.3 ng/10(6) cells in the activated cells. Ro 19-3704 and Ro 19-1400, platelet activating factor (PAF) antagonists which are structural analogs of PAF, potently inhibited both the IgE-dependent release of histamine (IC50 values of 3.0 and 3.6 microM, respectively) and LT release (IC50 values of 5.0 microM for both compounds) from the cells. These effects appeared to be independent to the ability of the compounds to act as PAF antagonists since PAF on its own had no effect on mediator release, and WEB 2086 and BN 52021, structurally distinct PAF antagonists, were relatively ineffective as inhibitors of mediator release. Ro 19-3704 and Ro 19-1400 were observed to be potent inhibitors of the soluble phospholipase A2 activity in synovial fluid from rheumatoid arthritic patients (IC50 values of 6.5 and 8.4 microM, respectively). In contrast, WEB 2086 and BN 52021 had no effect on this phospholipase A2. Ro 19-3704 significantly inhibited the IgE-dependent formation of inositol phosphates in RBL 2H3 cells (IC50 value of 7.0 microM). These data suggest that the mediator release inhibitory action of these compounds may be related to the ability of these compounds to inhibit phospholipase A2 and/or phospholipase C.
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PMID:Ro 19-3704 directly inhibits immunoglobulin E-dependent mediator release by a mechanism independent of its platelet-activating factor antagonist properties. 169 11

Crosslinking of the IgE receptor on the surface of rat basophilic leukemia (RBL) cells by multivalent antigen induces an association of these receptors with the detergent-insoluble membrane skeleton. Detergent insolubility of the receptor can also be induced on purified plasma membranes isolated from RBL cells by the use of either IgE oligomers or IgE monomers plus multivalent antigen. The critical event in initiating this interaction between the receptor and the membrane skeleton is cross-linking of the receptor. This association is rapid, and, when triggered by multivalent antigen, it is quickly reversed by the addition of excess monovalent antigen. The fact that this association occurs with the use of purified plasma membranes indicates that all of the components necessary for this interaction are present in the plasma membrane and that intracellular components are not required. Although crosslinking of the receptor activates phospholipase C and phospholipase A2 leading to the generation of several second messengers, none of these signaling mechanisms appears to be involved in IgE receptor interaction with the membrane skeleton. This interaction cannot be induced by phorbol 12-myristate 13-acetate (PMA), ionomycin, or a combination of these two reagents, although this will result in degranulation. Furthermore, receptor detergent insolubility is temperature independent when triggered by multivalent antigen, thus indicating that enzyme-catalyzed reactions are not important. This was verified by the fact that a variety of inhibitors that block phosphatidylinositol metabolism, arachidonic acid metabolism, Ca2+ influx, and protein kinase C (PKC) activation had no effect on antigen-induced association of the receptor with the membrane skeleton. These results indicate that the signaling mechanisms leading to the degranulation response are not involved in the association of the crosslinked receptor with the membrane skeleton.
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PMID:Association of the crosslinked IgE receptor with the membrane skeleton is independent of the known signaling mechanisms in rat basophilic leukemia cells. 183 Apr 93

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on mast cells by a polyvalent Ag leads to hydrolysis of phosphoinositides (PI) catalyzed by phospholipase C (PI-PLC). To understand this phenomenon in molecular terms, it is important to obtain active, cell-free preparations. In extensive preliminary studies, we could not demonstrate Fc epsilon RI-mediated activation of PI-PLC in plasma membranes prepared by conventional methods from rat basophilic leukemia cells. We now report a stepwise approach involving preparation of cytoplasts from such cells and then hypotonic lysis of the cytoplasts to obtain active membrane vesicles. These membranes, best described as "ghosts," appear to reseal after losing greater than 90% of their soluble, cytoplasmic components and contain receptors that when aggregated, activate PI-PLC to hydrolyze endogenous phospholipids. Per unit of plasma membrane, the ghosts retain approximately 25% of Fc epsilon RI-mediated stimulation of PI-PLC relative to the cells. This activity requires ATP, magnesium, phosphoenolpyruvate, and, to a limited degree, calcium. Although an adequate amount of phosphatidylinositol biphosphate is present, the predicted spike of (1,4,5)-inositol trisphosphate is not seen, and the predominant inositol phosphate isomer is (1,4)-inositol bisphosphate. This is the first report of Fc epsilon RI-mediated activation of PI-PLC in a cytoplasm-depleted system that demonstrates activation of endogenous enzyme acting on endogenous substrate. In addition, it is the first such report for any receptor of the Ig superfamily.
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PMID:Fc epsilon RI-mediated hydrolysis of phosphoinositides in ghosts derived from rat basophilic leukemia cells. 184 42

The current studies explore the role of phospholipase D (PLD) in mast cell activation. Although most investigators believe that receptor-mediated accumulation of 1,2-diacylglycerol (DAG) occurs by phospholipase C hydrolysis of phosphoinositides, our previous work indicated a modest role for these substrates and suggested that phosphatidylcholine (PC) is the more likely substrate. PLD cleaves the terminal phosphodiester bond of phospholipids to yield phosphatidic acid (PA), but in the presence of ethanol, it transfers the phosphatidyl moiety of the phospholipid substrate to ethanol producing phosphatidylethanol (PEt); a reaction termed transphosphatidylation. In purified rat mast cells prelabeled with [3H]arachidonic acid, [3H]palmitic acid, or 1-O-[3H]alkyl-lysoPC, a receptor-associated increase in PLD activity was initially suggested by the rapid accumulation of labeled PA, although other mechanisms might be involved. PLD activity was assessed more directly by the production of labeled PEt by PLD-mediated transphosphatidylation in the presence of ethanol. IgE receptor cross-linking resulted in a 3- to 10-fold increase in PLD activity during the 10 min after stimulation, approximately 50% of which occurred during the first two min. PEt formation was dependent on the concentration of ethanol and was maximal at 0.5%. At concentrations of ethanol greater than or equal to 0.2%, receptor-dependent formation of PA was reduced suggesting that the ethanol promoted transphosphatidylation at the expense of hydrolysis. The dose-related decline in PA accumulation seen in the presence of ethanol was similar to ethanol-mediated inhibition of exocytosis suggesting that receptor-mediated PA formation may be of regulatory importance. These observations indicate that PLD-mediated formation of PA occurs in stimulated mast cells and, in conjunction with separate findings of PA phosphohydrolase conversion of PA to DAG in mast cells, suggest that a major mechanism of DAG formation during mast cell activation is PC----PA----DAG.
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PMID:An indirect pathway of receptor-mediated 1,2-diacylglycerol formation in mast cells. I. IgE receptor-mediated activation of phospholipase D. 213 97


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