Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified two major proteins in human neutrophils that are phosphorylated in vitro by protein kinase C (PKC) as lipocortins III and a fragment of a lipocortin-like 68-kDa protein. In electroporated cells, the 68-kDa protein was phosphorylated during stimulation of the cells with either FMLP or PMA. Lipocortins are of interest because of their Ca2(+)- and phospholipid-dependent actin binding properties and ability to inhibit phospholipase A2. Two crude fractions of enzymes and proteins exposed to [gamma-32]PATP in the presence of Ca2+, Mg2+, phosphatidylserine and 1,2-oleoyl-acetyl-rac-glycerol were analyzed by gel electrophoresis and autoradiography. A number of proteins in a detergent-free fraction, including proteins at 36 and 32 kDa, were phosphorylated in the presence of these cofactors. In contrast, only two major proteins (35 and 32 kDa) were phosphorylated in a detergent-extracted fraction. Phosphorylation of the 36, 35, and 32 kDa proteins required the presence of Ca2+, Mg2+, and phosphatidylserine in our soluble fraction and detergent extract, indicating PKC-dependent phosphorylation. The 32-kDa protein phosphorylated in both the soluble fraction and detergent extract was identified as lipocortin III by immunoprecipitation with a cross-reactive antibody that recognized lipocortin I and comparison of cyanogen bromide (CNBr) cleavage patterns of this protein with a lipocortin III standard. The 68-kDa protein was identified as a lipocortin VI-like protein by immunoprecipitation with anti-calelectrin. Additionally, the CNBr cleavage pattern of the 68-kDa protein was similar to that of the 36-kDa protein phosphorylated in our soluble fraction. Autoradiograms of the 68- and 36-kDa fragments immunoprecipitated from our soluble fraction with anticalelectrin and cleaved with CNBr showed that both of these proteins were phosphorylated in this sample. Because phosphorylation is known to change the functional characteristics of the lipocortins, the potential exists to link PKC and lipocortins in neutrophils to regulation of granulemembrane interactions or mediation of inflammation.
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PMID:Lipocortins are major substrates for protein kinase C in extracts of human neutrophils. 169 66

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.
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PMID:Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells. 171 Sep 33

1. Intracellular mechanisms and second messengers involved in chloride current activation by intracellular GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] in bovine chromaffin cells were studied using the whole-cell patch-clamp technique combined with measurements of intracellular calcium [Ca2+]i. 2. No correlation between the time of current activation and the appearance of [Ca2+]i transients was observed; intracellular introduction of sufficient EGTA (10 mM) to suppress the [Ca2+]i transients did not affect the current activation by GTP gamma S. 3. The cyclic nucleotides, cyclic AMP or cyclic GMP, did not activate the current when introduced intracellularly (50-250 microM). The ability of GTP gamma S to activate the current decreased when cyclic GMP (250 microM), together with MgATP (2 mM), was added to the perfusate. 4. Neomycin (0.5-1 mM), a presumed inhibitor of phospholipase C effectively prevented the current activation by GTP gamma S but it did not prevent [Ca2+]i transients. 5. Modulation of protein kinase C activity using specific inhibitors (H-7, 300 microM; polymyxin B, 400 U/ml) or activators (phorbol ester PMA, 100 nM, 20-90 min at 37 degrees C) did not affect the current activation by GTP gamma S nor did it cause current activation in the absence of GTP gamma S. 6. Activation of the current by GTP gamma S could be prevented by incubating the cells for 10-15 min with 2.5 microM p-bromophenacyl bromide (p-BPB), an inhibitor of phospholipase A2 activity. Exogenous arachidonic acid (5-10 microM), applied extracellularly or intracellularly, neither activated the current itself nor did it interfere with its activation by GTP gamma S. 7. Activation of the current by GTP gamma S could also be prevented by incubating the cells with 1 microM-nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, but not with indomethacin (2 microM), an inhibitor of cyclo-oxygenase pathway of arachidonic acid metabolism. 8. It is suggested that chloride current activation by GTP gamma S in bovine chromaffin cells involves G protein-mediated stimulation of phospholipase A2 activity and subsequent formation of lipoxygenase product(s) of arachidonic acid metabolism.
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PMID:Second messengers mediating activation of chloride current by intracellular GTP gamma S in bovine chromaffin cells. 171 42

Ca2+ metabolism and its relationship to arachidonic acid release were studied in cultured pig aortic endothelial cells. When cells were treated with bradykinin, a rapid rise in intracellular Ca2+ concentration ([Ca2+]i) occurred. Arachidonic acid release from cells prelabelled with [3H]arachidonic acid and subjected to flow-through conditions closely followed the changes in [Ca2+]i. Attenuation of the Ca2+ response by chelating extracellular and intracellular Ca2+ or by desensitization of receptors led to comparable attenuation of arachidonate release. Activation of protein kinase C inhibited Ca2+ mobilization in response to bradykinin and stimulated arachidonic acid release. Inhibition of protein kinase C had no effect on bradykinin-stimulated arachidonic acid release, suggesting that protein kinase C does not mediate the bradykinin response. The role of GTP-binding regulatory proteins (G-proteins) in mediating the bradykinin response was also investigated. Bradykinin-stimulated arachidonic acid release was not diminished by preincubation with pertussis toxin. Treatment with the G-protein activator AlF4- resulted in the release of a large pool of arachidonic acid and the formation of lysophospholipids. Combined treatment with AlF4- and bradykinin resulted in a greater than additive effect on arachidonic acid release. In contrast with bradykinin, AlF(4-)-stimulated arachidonic acid release was not dependent on the presence of extracellular Ca2+ or the mobilization of intracellular Ca2+. These results demonstrate Ca(2+)-dependent (bradykinin) and Ca(2+)-independent (AlF4-) pathways of phospholipase A2 activation.
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PMID:Regulation of arachidonic acid release in vascular endothelium. Ca(2+)-dependent and -independent pathways. 174 1

Expression of lyso paf-acether (lyso paf):acetyl-CoA acetyltransferase and its activation above basal levels by specific agonists controls the rate of paf biosynthesis in proinflammatory cells. Acetyltransferase activation in these cells is due to the rapid postranslational modification of an inactive precursor by phosphorylation, most probably catalyzed by a cAMP-dependent kinase. However, the possibility exists that a calcium/calmodulin-dependent kinase can be implicated as well. Unlike murine cultured mast cells, human neutrophils form paf when stimulated with phorbol myristate acetate (PMA) or diacylglycerol. In both cell types, acetyltransferase is activated by PMA. Controversy exists as to whether PMA activates the remodeling pathway, i.e. the activation of phospholipase A2 and acetyltransferase, or the de novo route through CDPcholine cholinephosphotransferase action on alkylacetylglycerol. There is some indication that PKC might regulate paf biosynthesis. The implication of a GTP-regulated protein has also been postulated in signal transduction leading to paf formation in endothelial cells, neutrophils, and mast cells. The topography of paf formation is discussed in light of the subcellular distribution of acetyltransferase in neutrophils and Krebs II cells.
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PMID:Transmembrane signalling and paf-acether biosynthesis. 181 87

When rat pleural mononuclear leukocytes were stimulated with 1 microM phorbol myristate acetate (PMA), platelet-activating factor (PAF)-like activity was detected in the supernatant and the cellular fractions of the incubation mixture, as measured by rabbit platelet aggregation. C16PAF activity peaked at 30 min in both fractions. Acetyltransferase activity in the microsomal fraction of the stimulated cells also increased rapidly and showed a peak at 10 min. A protein kinase C inhibitor, staurosporine, and an inhibitor of phospholipase A2, p-bromophen-acylbromide, inhibited stimulated PAF formation in both fractions. Staurosporine also inhibited PMA induced acetyltransferase activity. The data suggest that PMA stimulates PAF synthesis by the remodeling pathway in rat pleural cells through activation of both phospholipase A2 and acetyltransferase, and that the acetyltransferase, in turn, may be activated through activation of protein kinase C.
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PMID:Phorbol ester stimulates PAF synthesis via the activation of protein kinase C in rat leukocytes. 181 89

Activation of phospholipase A2 (PLA2) in response to external stimuli may play a pivotal role in signal-transduction pathways via the generation of important cellular intermediates, including prostaglandins. Epidermal growth factor (EGF) has been shown to modulate prostaglandin production, possibly via direct activation of PLA2 or indirectly via interaction with a PLA2-modifying protein such as lipocortin I. We have investigated these pathways with two CHO cell-lines, one (CHOwt) transfected with the full-length human EGF receptor and the second (CHO 11) with a deletion mutant, delta 990, that has lost the autophosphorylation sites and part of the internalization domain. CHOwt cells responded to EGF with a rapid rise in lysophosphatidylcholine and arachidonic acid release concomitant with an increase in prostaglandin production. However, in the non-internalizing CHO 11 cells no such activation of PLA2 was observed. This was not due to an intrinsic lack of PLA2 in these cells, as PLA2 activation was shown on melittin addition, nor was this difference due to a defect in intracellular pathways, as arachidonic acid was released from both cell types by Ca2+ and protein kinase C modulators. However, only in CHOwt cells were these responses potentiated by concomitant addition of EGF. Thus the cytoplasmic subdomain of the EGF receptor, containing the major sites of autophosphorylation and the internalization domain, seems to be involved in the activation of PLA2 by EGF. In addition, we have shown that phosphorylation of lipocortin I is unlikely to play a role in PLA2 activation. In CHOwt cells and a positive control cell line, A431, activation of PLA2 was complete by 10 min, at which time there was no evidence of lipocortin I phosphorylation.
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PMID:Modulation of phospholipase A2 activity by epidermal growth factor (EGF) in CHO cells transfected with human EGF receptor. Role of receptor cytoplasmic subdomain. 182 22

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

Previous studies from our laboratory have determined that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to prostaglandin E2 (PGE2) production. In the present study, we have further characterized the dopamine-stimulated PGE2 response. Dopamine stimulated PGE2 production in cultured IMCD cells dose dependently (concentration for half-maximal stimulation, 11.1 microM; maximal stimulation, 235.1% of basal), an effect that was blocked by the DA2 antagonists domperidone and (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)-methyl] benzamine. Inhibition of intracellular calcium release with 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (100 microM) blocked the dopamine response, whereas voltage-dependent calcium-channel blockers had no effect. Inhibition of phospholipase A2 (PLA2) activity with quinacrine (100 microM) completely blocked the dopamine-stimulated PGE2 production, whereas inhibition of polyphosphoinositol hydrolysis with neomycin (100 microM) or inhibition of protein kinase C with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10 microM) did not. Pertussis toxin (PT) treatment completely blocked the dopamine-stimulated PGE2 production but not the arachidonic acid-stimulated PGE2 production. These results suggest that dopamine, acting through the DA2K receptor, may be an important regulator of PGE2 production in IMCD cells. Furthermore, our results are most consistent with either a direct interaction of the DA2K receptor with PLA2 through a PT-sensitive G protein or an indirect interaction with PLA2 through mobilization of intracellular calcium.
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PMID:Prostaglandin E2 production in rat IMCD cells. I. Stimulation by dopamine. 183 85

Lysophosphatidylcholine (lyso-PC), a natural product of phospholipase A2 activity, induced the secretion of both granule-associated beta-hexosaminidase and newly generated leukotriene C4 from mouse bone marrow-derived mast cells. Micromolar concentrations of lyso-PC potentiated the release of beta-hexosaminidase induced by specific antigen but not the calcium ionophore, A23187. Exogenous adenosine was relatively ineffective in enhancing beta-hexosaminidase release from cells challenged with lyso PC. Lyso-PC caused a marked increase in intracellular free-calcium levels and induced the activation of protein kinase C (PKC). These effects could not be abrogated by a prolonged preincubation with pertussis toxin. Staurosporine, an inhibitor of PKC, partially inhibited the abilities of antigen and A23187 to induce beta-hexosaminidase release but was ineffective when lyso-PC was the secretagogue. Lyso-PC appears to activate mast cell PKC, but its ability to stimulate mast cell mediator release appears to be related to its ability to elevate intracellular free calcium concentrations.
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PMID:Lysophosphatidylcholine induces mast cell secretion and protein kinase C activation. 183 66


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