Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Recent evidence suggests a critical role for Syk in mast cell activation upon high affinity IgE receptor (FcepsilonRI) aggregation. A rat basophilic leukemia cell line, RBL-2H3, expresses similar levels of two Syk isoforms that differ with respect to the presence of a 23-amino acid insert within the "linker" region located between the second Src homology 2 and the catalytic domain. Although they exhibit comparable intrinsic enzymatic activity, functional differences between the two isoforms are unknown. Here we report that the deleted Syk isoform can mediate signal transduction in RBL-2H3 cells. Aggregation of chimeric kinase, consisting of either form of Syk fused to the transmembrane and extracellular domains of guinea pig type II IgG Fc receptor, on RBL transfectants resulted in degranulation, release of leukotrienes, and enhanced gene expression of tumor necrosis factor-alpha. The chimeras as well as phospholipase C-gamma1 and Vav became tyrosine-phosphorylated upon aggregation of chimeras. We also found that both Syk isoforms from transiently transfected COS-7 cells were capable of binding to phosphorylated FcepsilonRI, and their kinase activities were similarly up-regulated in the presence of tyrosine-phosphorylated synthetic peptides based on the sequence of the gamma subunit of FcepsilonRI. Thus, these results establish that both isoforms of Syk can mediate signal transduction in mast cells and suggest that the 23-amino acid insert in the linker region of Syk may not be obligatory for FcepsilonRI signaling.
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PMID:Evidence for involvement of two isoforms of Syk protein-tyrosine kinase in signal transduction through the high affinity IgE receptor on rat basophilic leukemia cells. 960 11

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcepsilonR1) leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.
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PMID:Calcium-dependent clustering of inositol 1,4,5-trisphosphate receptors. 961 87

Antibody-mediated cross-linking of Thy-1 glycoprotein on the surface of rat mast cells and rat basophilic leukemia (RBL) cells initiates biochemical events which culminate in secretion of allergy mediators. Thy-1, like some other glycosylphosphatidylinositol (GPI)-anchored proteins, forms detergent-insoluble complexes containing protein tyrosine kinases (PTK) and some other molecules which are implicated in the signaling pathway. On the surface of a rat mast cell there are more than 10(6) Thy-1 molecules; however, it is not known which fraction of them is involved in transmembrane signaling, and what exactly is the heterogeneity of Thy-1 complexes. Using sucrose density gradient ultracentrifugation of detergent-lysed RBL cells we found that the density of Thy-1 complexes depended on the detergent used and the lysis conditions employed. Sepharose 4B gel chromatography fractionation followed by density gradient ultracentrifugation revealed both size and density heterogeneity of Thy-1 and Lyn PTK complexes. Cross-linking of surface Thy-1 caused significant changes in the density of these complexes, and an increase in Lyn kinase activity in low/medium-density fractions. Thy-1 in low-density fractions was relatively resistant to cleavage with phosphatidylinositol-specific phospholipase C (PI-PLC). Interestingly, removal of only a small fraction of surface Thy-1 by PI-PLC abolished the cell activation as determined by tyrosine phosphorylation of certain proteins. When Triton X-100 lysates were fractionated at 12000 x g, about 50 % of Thy-1 remained associated with the nuclear/cytoskeleton pellet; this fraction of Thy-1 exhibited an increased sensitivity to PI-PLC. Confocal laser scanning microscopy on fixed cells revealed that the total Thy-1 was relatively homogeneously distributed over the plasma membrane, whereas the PI-PLC-resistant Thy-1 was found mostly in small clusters. The combined data suggest that specialized membrane microdomains enriched in Thy-1 with increased sensitivity to PI-PLC are directly involved in coupling Thy-1 aggregation to transmembrane signaling.
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PMID:Functional heterogeneity of Thy-1 membrane microdomains in rat basophilic leukemia cells. 964 66

The protein tyrosine kinase Syk plays a pivotal role in mediating the high-affinity IgE receptor (Fc epsilonRI)-induced degranulation of mast cells. To examine the mechanism of Syk regulation, the two tyrosine residues at 519 and 520 in the putative activation loop of rat Syk were mutated to phenylalanine either singly or in combination. The various mutants were expressed in a Syk-negative variant of the RBL-2H3 (rat basophilic leukemia 2H3) mast cell line. In these transfected cell lines, mutant Syk did show increased tyrosine phosphorylation in vivo and increased enzymatic activity in vitro after Fc epsilonRI aggregation. There were conformational changes detected by an Ab when the wild-type and mutant Syk were either tyrosine phosphorylated or bound to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these mutant Syk were incapable of transducing Fc epsilonRI signaling. In cells in which the expression level of mutant Syk was similar to that of the wild-type Syk, Fc epsilonRI cross-linking induced no increase in cellular protein tyrosine phosphorylation, no increase in tyrosine phosphorylation of phospholipase C-gamma2 and mitogen-activated protein kinase, and no histamine release. Overexpression of Y519F or Y520F Syk mutants partially reconstituted the signaling pathways. These results indicate that these tyrosines in the putative activation loop are not essential for the enzymatic activity of Syk or for the conformational changes induced by binding of tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these tyrosines are necessary for Syk-mediated propagation of Fc epsilonRI signaling.
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PMID:Mutations in the activation loop tyrosines of protein tyrosine kinase Syk abrogate intracellular signaling but not kinase activity. 978 Feb 14

Dami human leukemia cells express G protein-coupled thrombin receptors that operate through the phospholipase C pathway. When these receptors are activated by alpha-thrombin or by thrombin receptor-activating peptide, an elevation in cytosolic Ca2+ concentration develops that is accompanied by hyperpolarization of the plasma membrane. This transitory phase of hyperpolarization is primarily mediated by inwardly rectifying, Ca2+-activated K+ channels that have an inward conductance of approximately 24 pS. In cell-attached patches the channels open within seconds after superfusion of the cell with thrombin receptor-activating peptide. In inside-out patches, perfusion of submicromolar Ca2+ onto the cytosolic surface of the membrane is sufficient to activate the channels. In outside-out patches, channel opening can be blocked by nanomolar concentrations of charybdotoxin. The function of these intermediate-sized inwardly rectifying, Ca2+-activated K+ channels has not been established; however, by analogy with other cell systems, they may serve to regulate cell volume during cellular activation or to increase the electromotive drive that sustains Na+ and/or Ca2+ influx through ligand-gated cation channels.
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PMID:Analysis of a Ca2+-activated K+ channel that mediates hyperpolarization via the thrombin receptor pathway. 981 83

We examined the mechanism of action of lysophosphatidylcholine (LPC), which is suggested to be involved in the pathogenesis of atherosclerosis and inflammatory disorders, in HL-60 leukaemia cells. Extracellular 1-palmitoyl LPC increased the intracellular Ca2+ concentration in association with production of inositol phosphate. These actions of LPC were markedly inhibited by treatment of the cells with pertussis toxin and U73122, a phospholipase C inhibitor. The lipid-induced stimulation of the phospholipase C/Ca2+ system was also attenuated in the dibutyryl cAMP-induced differentiated (neutrophil-like) cells, in which phospholipase C activation induced by NaF or formyl-Met-Leu-Phe was enhanced. In contrast with the stimulatory action of 1-palmitoyl LPC, 1-stearoyl LPC was inhibitory for the phospholipase C/Ca2+ system stimulated by NaF as well as by 1-palmitoyl LPC or other Ca2+-mobilizing agonists. In a cell-free system, only an inhibitory effect on phospholipase C activity was observed even by 1-palmitoyl LPC; 1-stearoyl LPC was more inhibitive than 1-palmitoyl LPC. Taken together, these results suggest that atherogenic and inflammatory LPC exerts both stimulatory and inhibitory actions on the phospholipase C/Ca2+ system depending on the species of fatty acid residue of the lipid; the stimulatory effect is possibly mediated through G-protein-coupled receptors; the inhibitory effect might be caused by dysfunction of the components involved in the enzyme system owing to the amphiphilic nature of the lipid. 1-Palmitoyl LPC prefers the former receptor stimulation at least in intact cells, but 1-stearoyl LPC preferentially exerts the latter inhibitory action.
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PMID:Stimulatory and inhibitory actions of lysophosphatidylcholine, depending on its fatty acid residue, on the phospholipase C/Ca2+ system in HL-60 leukaemia cells. 982 Aug 28

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.
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PMID:Formyl peptide receptor signaling in HL-60 cells through sphingosine kinase. 993 90

Continuous stimulation of anaphylatoxin receptors C3aR and C5aR with their cognate ligands engenders, within minutes, diminished responsiveness of these receptors. We tested the hypothesis that agonist-induced desensitization involves C3aR and C5aR phosphorylation by G protein-coupled receptor kinases (GRK). When expressed in rat basophilic leukemia cells and exposed to C3a, the C3aR underwent rapid (t(1/2) approximately 15 s), dose-dependent (EC50 approximately 10 nM) and reversible phosphorylation by a kinase refractory to the effects of PKC inhibitors. Phosphoamino acid analysis revealed that the C3aR is phosphorylated on serine and threonine, but not on tyrosine residues. Overexpression of GRK2, GRK3, GRK5 or GRK6 together with C3aR in COS-7 cells enhanced the C3a-induced C3aR phosphorylation 1.5 - 1.9-fold (p < 0.05), but each kinase reduced ligand-stimulated phospholipase C activity differently. Conversely, antibody-mediated inhibition of endogenous GRK2 and GRK3 significantly inhibited C3aR phosphorylation in permeabilized cells. GRK overexpression in cells which co-expressed C5aR and were exposed to C5a resulted in the hyperphosphorylation of the C5aR. These findings are of physiological relevance, since we observed anaphylatoxin-induced phosphorylation of C3aR and C5aR endogenously expressed in human mast cells (HMC-1) which contain significant intracellular levels of GRK2 and GRK3.
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PMID:Ligand-induced phosphorylation of anaphylatoxin receptors C3aR and C5aR is mediated by "G protein-coupled receptor kinases. 1050 78

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.
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PMID:Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2. 1073 56

The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.
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PMID:Differential roles of the NPXXY motif in formyl peptide receptor signaling. 1123 59


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