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 binding of natural killer (NK) cells to either susceptible tumor cells or antibody-coated targets results in rapid activation of phospholipase C (PLC) in NK cells. PLC activation generates inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers, which, in turn, increase intracellular free calcium concentrations ([Ca2+]i) and protein kinase C (PKC) activity, respectively. These proximal signals initiate a cascade of as yet undefined biochemical events, leading eventually to the exocytosis of preformed cytotoxic granules. To investigate the signal transduction pathways involved in granule exocytosis, we utilized streptolysin-O-permeabilized human NK cells as our experimental model. Our initial studies indicated that the separate activation of either PKC (using the phorbol ester, PMA) or G protein-dependent pathways (using guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)) stimulated granule exocytosis in a time-, concentration-, and Ca(2+)-dependent manner. PMA-stimulated exocytosis was inhibited by staurosporine or a PKC pseudosubstrate antagonist peptide, but was not affected by GDP. In contrast, GTP gamma S-stimulated exocytosis was effectively inhibited by GDP, but not by staurosporine or the PKC pseudosubstrate antagonist. These observations suggest that NK cell exocytosis can be stimulated by at least two separate pathways; one involving PKC and the other involving a G protein. However, co-stimulation with PMA and GTP gamma S synergistically enhanced exocytosis, suggesting that even though the two exocytotic pathways were biochemically distinct, cross-talk between the two pathways may potently influence the exocytotic process. These results define a regulatory role for PKC- and G protein-dependent pathways during granule exocytosis from NK cells.
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PMID:Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis. 142 33

Epidermal growth factor (EGF) can stimulate inositol lipid hydrolysis in rat hepatocytes and can accelerate GTP/GDP exchange in hepatic membranes. Both of these responses can be abolished by pretreatment with pertussis toxin, suggesting that EGF may regulate phospholipase C (PLC) activity via a guanine nucleotide-binding regulatory protein (G protein) in liver cells. In contrast, in A431 human epidermoid carcinoma cells EGF can induce a rapid phosphorylation of PLC-gamma on tyrosine residues that increases the activity of immunoprecipitated PLC-gamma, suggesting that tyrosine phosphorylation of PLC-gamma may be the mechanism for EGF-stimulated inositol trisphosphate production in these cells. To determine the importance of the phosphorylation of PLC-gamma on tyrosine residues in a system where the EGF receptor apparently couples to a G protein, the effect of EGF on tyrosine phosphorylation of PLC-gamma was examined in rat hepatocytes. PLC-gamma was immunoprecipitated from cell lysates with a PLC-gamma antiserum and its tyrosine phosphorylation state was determined using both Western blot analysis with phosphotyrosine antibodies and direct measurement of phosphorylated amino acids. The results were compared with analogous experiments performed with A431 cells and another cultured cell line expressing high levels of human EGF receptors, Rat1hER fibroblasts. Although the amount of PLC-gamma in rat hepatocytes is similar to that in A431 cells and slightly higher than that in Rat1hER cells, EGF causes a barely detectable increase in the phosphorylation of PLC-gamma on tyrosine in hepatocytes, whereas it stimulates a significant degree of phosphorylation of PLC-gamma on tyrosine in Rat1hER or A431 cells. Pretreatment of hepatocytes with pertussis toxin abolishes the ability of EGF to activate PLC, as determined by an increase in intracellular Ca2+, but has no effect on the small amount of phosphate incorporated into tyrosine residues on the PLC-gamma protein, demonstrating that this low level of PLC-gamma phosphorylation does not correlate with changes in PLC activity. The data suggest that phosphorylation of PLC-gamma on tyrosine is not important for EGF-enhanced PLC activity in hepatocytes. This conclusion implies that EGF may use a mechanism to regulate PLC activity in hepatocytes that is different from that used in cultured cells expressing high levels of EGF receptors.
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PMID:Epidermal growth factor activates phospholipase C in rat hepatocytes via a different mechanism from that in A431 or rat1hER cells. 143 49

The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).
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PMID:Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C. 146 34

In this study the effect of different times of exposure to ethanol (1-7 days, 100 mM) on bradykinin and GTP(S)-stimulated activation of phospholipase C in NG 108-15 cells and on the binding of [3H]bradykinin to its receptors was investigated. Ethanol attenuated both agonist and GTP-analogue-induced hydrolysis of phosphoinositides for a period of up to 4 days of treatment, while exerting no effect on binding to bradykinin receptors. However, after 7 days of exposure to ethanol, the agonist-induced activation of phospholipase C was completely resistant to the inhibitory effects of alcohol. This finding correlated to a change in the affinity of the bradykinin receptor population after 7 days of treatment. The results indicate that bradykinin-induced breakdown of phosphatidylinositol 4,5-bisphosphate adapts to the effects of ethanol, after long-term treatment. Possible adaptative changes taking place at the level of the G protein(s), may induce a shift in the affinity of the receptor population and, consequently, serve as a compensatory mechanism to counteract the inhibitory effect of ethanol.
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PMID:Mechanisms of adaptation to the effects of ethanol on activation of phospholipase C in NG 108-15 cells. 147 23

The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In the present study, polyclonal antibodies raised against a PLC isoenzyme, PLC-alpha, reacted with a 60-kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-alpha with cell surface AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. Anti-PLC-alpha antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent-promoted protein-protein interaction. On the other hand, monoclonal antibodies against another PLC isozyme, PLC-gamma, did not precipitate AngII-Rs in solubilized N1E-115 membranes. Finally, the formation of the immunoprecipitated AngII-R-PLC-alpha complex was disrupted by the nonhydrolyzable guanine nucleotide analog GTP gamma S, suggesting that the interaction between AngII-Rs and PLC-alpha is likely to involve a heterotrimeric guanine nucleotide-binding protein in neuron-like cells.
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PMID:Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells. 151 21

The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets.
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PMID:Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide). 153 May 76

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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PMID:Lipid A stimulates phospholipase D activity in rat mesangial cells via a G-protein. 153 47

In response to concanavalin A, cytoplasmic calcium movement was observed in human platelets, both in the presence of 1 mM Ca2+ or 1 mM EGTA in the medium. Concanavalin A also caused the activation of inositide turnover and the production of inositol phosphates, suggesting that activation of phospholipase C occurs. The mechanism by which concanavalin A stimulates phospholipase C does not depend on GTP-binding transducers, because it was not inhibited by GDP beta S, while experiments performed in the presence of cytochalasin B suggested a role for membrane glycoprotein IIb-IIIa-cytoskeleton interaction in this process. Ca(2+)-proteases and Na+/H+ antiport also seemed to be related to concanavalin A-induced phospholipase C activation, as suggested by experiments performed in the presence of leupeptin and amiloride.
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PMID:Stimulation of human platelets with concanavalin A involves phospholipase C activation. 157 30

Recently, we have reported that the isolated guanine nucleotide-binding regulatory protein, Gh, couples to the alpha 1-adrenergic receptor (Im, M.-J., and Graham, R. M. (1990) J. Biol. Chem. 265, 18944-18951 and Im, M.-J., Riek, R.P., and Graham, R. M. (1990) J. Biol. Chem. 265, 18952-18960) and has a molecular mass of approximately 74 kDa, and the approximately 50-kDa protein which is copurified probably regulates guanine nucleotide binding of the 74-kDa GTP-binding protein. In this paper, we describe the role of purified Gh in the regulation of phospholipase C in the reconstitution system. The stimulation of phospholipase C activity by Gh effectively occurred at a low calcium concentration (less than or equal to 2 microM), but the phospholipase C (PLC) itself required at least 50-100 times more calcium to become fully activated. The characteristic nature of phospholipase C stimulation by Gh is its response to the calcium concentration. Thus, the enzyme activity changes in narrow submicromolar ranges and reaches maximal stimulation, but it does not extend to the levels above those stimulated by calcium alone. The calcium concentrations for the maximal stimulation of phospholipase C activity were 10-20 microM with phospholipid vesicles and 100-200 microM with detergent solution. These calcium concentrations were further decreased when Gh and phospholipase C were co-reconstituted into the phospholipid vesicles or in the detergent solution. The maximal stimulations of the PLC activity were reached at less than 5 microM calcium in both the vesicles and the detergent solution. The changes of calcium concentration for the activation of PLC are quite different from those obtained by reconstituting PLC-beta 1 with Gq-like G-proteins (Smarcka, A. V., Hepler, J. R., Brown, K. O., and Sternweis, P. C. (1991) Science 251, 804-807 and Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton, J. H. (1991) Nature 350, 516-518). The phospholipase C activity was stimulated in a Gh concentration-dependent manner in the presence of GTP gamma S. The phospholipase C activity was activated by Gh alpha in the presence of aluminum fluoride, but not by Gh beta. Furthermore, a Gh.PLC complex can be induced by incubation with aluminum fluoride in a detergent solution and partially purified without the dissociation of related proteins. Thus, our reconstitution studies show that the pattern of stimulation of PLC by AIF-4-activated Gh in the ternary complex is similar to the stimulation of PLC activated by Gh in both detergent solution and phospholipid vesicles.
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PMID:Characterization of a phospholipase C activity regulated by the purified Gh in reconstitution systems. 157 27

In isolated rat aorta, 72.7 mM KCI, 10 microM prostaglandin F2 alpha, 30 nM endothelin-1 and 1 microM norepinephrine increased muscle tension, cytosolic Ca++ concentration ([Ca++]i) and 20 kDa myosin light chain (MLC) phosphorylation. The levels of contractile tension and MLC phosphorylation at a given [Ca++]i were greatest in the presence of endothelin-1 followed by prostaglandin F2 alpha greater than norepinephrine greater than high K+. Verapamil inhibited the high K(+)-induced increments to their respective resting levels. Verapamil also almost completely inhibited the receptor agonist-induced increments in [Ca++]i and MLC phosphorylation, although a part of the contraction was not inhibited. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid further decreased [Ca++]i and muscle tension, suggesting that a part of the contraction is regulated by [Ca++]i below a resting level. Receptor agonists induced sustained contraction in the absence of external Ca++ which was not followed by the increase in [Ca++]i or MLC phosphorylation. This contraction was followed by the increments in shortening velocity and stiffness. In the rabbit mesenteric artery permeabilized with Staphylococcus aureus, alpha-toxin, norepinephrine and endothelin-1 shifted the Ca(++)-tension curve to the left in the presence of GTP. From these results, it is suggested that high K(+)-induced sustained contraction of vascular smooth muscle is attributable to an increase in [Ca++]i followed by an increase in MLC phosphorylation. In addition to this fundamental mechanism, receptor agonists increase Ca+ sensitivity of MLC phosphorylation when [Ca++]i is higher than resting level resulting in a greater contraction than that induced by high K+ for a given increase in [Ca++]i.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle. 157 67


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