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)

The involvement of ginsenosides in the signal cascade that stimulates cellular growth was investigated. It was found that ginsenosides Rh1 and Rh2 extracted from the root of Panax ginseng inhibited cellular proliferation in NIH 3T3 fibroblasts. Both ginsenosides Rh1 and Rh2 effectively reduced phospholipase C activity resulting in a decrease in the intracellular level of diacylglycerol, an endogenous activator of protein kinase C. The treatment of cells with Rh1 or Rh2 was thus found to reduce intracellular protein kinase C activity. We also observed that the phosphorylation of myristoylated alanine-rich C kinase substrate, one of the major substrates of protein kinase C in cells, was inhibited by the ginsenosides. Data suggest that the ginsenoside Rh1 or Rh2 exerts antiproliferative effects by inhibiting phospholipase C, which produces second messengers necessary for the activation of protein kinase C.
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PMID:Modulation of protein kinase C activity in NIH 3T3 cells by plant glycosides from Panax ginseng. 934 39

Catecholamines as well as phorbol esters can induce the phosphorylation and desensitization of the alpha1B-adrenergic receptor (alpha1BAR). In this study, phosphoamino acid analysis of the phosphorylated alpha1BAR revealed that both epinephrine- and phorbol ester-induced phosphorylation predominantly occurs at serine residues of the receptor. The findings obtained with receptor mutants in which portions of the C-tail were truncated or deleted indicated that a region of 21 amino acids (393-413) of the carboxyl terminus including seven serines contains the main phosphorylation sites involved in agonist- as well as phorbol ester-induced phosphorylation and desensitization of the alpha1BAR. To identify the serines invoved in agonist- versus phorbol ester-dependent regulation of the receptor, two different strategies were adopted, the seven serines were either substituted with alanine or reintroduced into a mutant lacking all of them. Our findings indicate that Ser394 and Ser400 were phosphorylated following phorbol ester-induced activation of protein kinase C, whereas Ser404, Ser408, and Ser410 were phosphorylated upon stimulation of the alpha1BAR with epinephrine. The observation that overexpression of G protein-coupled kinase 2 (GRK2) could increase agonist-induced phosphorylation of Ser404, Ser408, and Ser410, strongly suggests that these serines are the phosphorylation sites of the alpha1BAR for kinases of the GRK family. Phorbol ester-induced phosphorylation of the Ser394 and Ser400 as well as GRK2-mediated phosphorylation of the Ser404, Ser408, and Ser410, resulted in the desensitization of alpha1BAR-mediated inositol phosphate response. This study provides generalities about the biochemical mechanisms underlying homologous and heterologous desensitization of G protein-coupled receptors linked to the activation of phospholipase C.
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PMID:Characterization of the phosphorylation sites involved in G protein-coupled receptor kinase- and protein kinase C-mediated desensitization of the alpha1B-adrenergic receptor. 935 40

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA-INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD-NORPA association in vivo, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells.
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PMID:Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo. 935 10

We recently reported that replacement of Tyr302 for Ala in the human angiotensin II type 1 receptor (hAT1) severely impaired its ability to activate phospholipase C (PLC). Another study demonstrated that the same mutation in the rat AT1 receptor only slightly impaired its ability to activate PLC. The most striking difference between the two studies was the use of LiCl in the experimental conditions. Thus, in the present report we evaluated the effect of LiCl on the rate of accumulation of inositol trisphosphate (IP3) in transfected cells stimulated with angiotension II (Ang II). In the presence of LiCl, Ang II caused a significant accumulation of IP3 in COS-7 cells transfected with the hAT1Y302A mutant receptor. In stably expressing CHO cells, stimulation of hAT1Y302A did not induce any IP3 elevation even in the presence of LiCl whereas the hAT1 wild-type receptor increased the production of IP3 exclusively in the presence of LiCl. These results show that LiCl is a convenient tool to enhance the sensitivity of PLC assays. However, in structure-activity relationship studies, it may underestimate or mask the debilitating effect of some mutations.
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PMID:Use of LiCl in phospholipase C assays masks the impaired functionality of a mutant angiotensin II receptor. 937 18

The nonspecific phospholipase C from Bacillus cereus is a zinc metalloenzyme that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. Glu-4 has been proposed as a potential candidate for the general base in the hydrolysis reaction and was shown to interact with the substrate headgroup. Site-specific mutagenesis studies suggest that Glu-4 is important for substrate binding but not for catalysis. This residue is also critical for the enzyme's preference for a phosphodiester substrate. PA, both monomeric and micellar, is shown to be a poor substrate and inhibitor of wild-type PLC. When Glu-4 was mutated to an alanine, a significant increase in PA hydrolysis and a decrease in PC hydrolysis were observed. Unlike the wild type, kinetic studies suggest that the Glu-4-->Ala mutant does not exhibit interfacial activation and processive catalysis. Glu-4 is part of a highly flexible loop flanking the entrance to the active site, suggesting that this loop might constitute an interfacial binding recognition site. This is the first evidence for the presence of an interfacial binding site distinct from the active site in the nonspecific PLC.
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PMID:Engineering of the nonspecific phospholipase C from Bacillus cereus: replacement of glutamic acid-4 by alanine results in loss of interfacial catalysis and enhanced phosphomonoesterase activity. 952 50

The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2'-deoxy-N6-methyladenosine 3', 5'-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5'-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a "cross-docking" procedure to obtain energetically refined 3D structures of the ligand-receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain.
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PMID:Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites. 955 79

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.
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PMID:Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1. 956 85

In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (Kd) of (1.5 +/- 0.3) x 10(-9) and (2.1 +/- 0.4) x 10(-9) mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125I-labelled Tyr-Ala-hexarelin. The binding of 125I-labelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release. In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.
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PMID:Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland. 961 63

The mechanism by which protein kinase A (PKA) inhibits Galphaq -stimulated phospholipase C activity of the beta subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta3 by PKA results in inhibition of Galphaq -stimulated PLCbeta3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galphaq with PLCbeta3 or PLCbeta (Ser1105--> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galphaq, PLCbeta3, or PLCbeta3 (Ser1105 --> Ala mutant alone. Whereas Galpha1-stimulated PLCbeta3, activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galphaq-stimulated PLCbeta3 (Ser1105 --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha1-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta3 inhibits Galphaq-stimulated PLCbeta3 activity. This can at least partially explain the inhibitory effect of PKA on Galphaq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.
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PMID:Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cbeta3 stimulation by Galphaq. 966 Jul 57

The role of the inflammatory cytokine interleukin 1beta (IL-1beta) as potent agonist of the PMN respiratory burst signal transduction cascade has been described. We hypothesized that this phenomenon is self-limiting and that polymorphonuclear leukocyte (PMN)-derived reactive oxygen intermediates (ROI) might provide feedback regulation on the IL-1beta surface receptor (IL-1betaR)-G-protein-effector enzyme transducing tripartite complex that ultimately leads to NADPH oxidase activation. Therefore, we separately assessed either baseline or IL-1beta-induced activation of each member of the IL-1betaR-G-protein-phospholipase D (PLD) or IL-1betaR-G-protein-phospholipase C (PLC) signaling systems in the presence or absence of one of several specific ROI scavengers/antioxidants. Purified human PMN were lipopolysaccharide primed, adhered for 2 h, and stimulated with 100 ng/mL IL-1beta with or without 1% v/v dimethyl sulfoxide, 10 mM NaN3, 30 mM L-alanine, 200 U catalase, or 300 U superoxide dismutase (SOD). To validate the use of these antioxidants, the production of O2-, H2O2, hypochlorous acid, or myeloperoxidase (MPO) in the employed experimental model was confirmed in a separate set of experiments. The expression of IL-1betaR type I or II was assessed by binding with corresponding 125I-labeled monoclonal antibodies and corrected for nonspecific binding. PLD activation was assessed by measuring phosphatidyl ethanol formation in the presence of ethanol. PLC activation was determined by quantitative measurement of diacylglycerol. The level of Galpha stimulatory and inhibitory subunits was assessed by Western blotting. IL-1betaR type I expression was significantly up-regulated in the presence of catalase and SOD. PLD activation was increased by dimethyl sulfoxide and NaN3, and PLC activation was up-regulated by NaN3, L-alanine, SOD, and catalase. After 5 min of stimulation with IL-1beta, Gialpha expression was significantly down-regulated by NaN3 and SOD, whereas SOD had an up-regulating effect on the expression of Gs alpha. Increasing concentrations of externally added authentic MPO progressively down-regulated both PLD and PLC activity. Thus, PMN-derived ROI, in addition to their role as antibacterial/fungal agents, serve as second messengers in IL-1beta signal transduction, with MPO having the most ubiquitous role as a modulator of PMN second messenger pathways.
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PMID:The role of neutrophil-derived oxidants as second messengers in interleukin 1beta-stimulated cells. 968 92


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