EC:2.7.11.13 - FACTA Search

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Query: EC:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stimulation of PMN with inflammatory mediators markedly augments Fc and CR1 receptor-mediated ingestion. However, CD11/CD18-deficient PMN from three patients with complete leukocyte adhesion deficiency (LAD) failed to recruit phagocytic function in response to phorbol esters, cytokine, or Arg-Gly-Asp-containing ligand stimulation. Because stimulated ingestion is protein kinase C (PKC)-dependent, our data indicate that LAD PMN exhibit only PKC-independent phagocytosis. The defect in PKC-dependent ingestion is specific for CD11b/CD18 and not secondary to the chronic or recurrent infections which occur in this disease. The LAD phenotype for phagocytic function can be reproduced in normal PMN by the anti-CD11b MAbs OKM1 and OKM10. In contrast, MAb Mo1 (anti-CD11b) and MAb IB4 (anti-CD18) inhibit both CD11b/CD18-dependent and -independent mechanisms of ingestion by normal PMN. Their ability to inhibit CD11b/CD18-independent ingestion may be mediated by cAMP, as shown by experiments with a protein kinase A inhibitor HA1004 and by direct measurement of cAMP levels in immune complex- and FMLP-stimulated PMN. These data indicate that CD11b/CD18-independent and -dependent mechanisms of phagocytosis exist and that some effects of anti-CD11b/CD18 MAbs may be mediated by alterations in cAMP levels.
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PMID:Leukocyte adhesion-deficient neutrophils fail to amplify phagocytic function in response to stimulation. Evidence for CD11b/CD18-dependent and -independent mechanisms of phagocytosis. 167 46

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

We have compared the transmembrane signals generated in human basophils by two distinct stimuli, anti-IgE antibody and FMLP (f-met peptide). Although both stimuli resulted in the activation of protein kinase C (PKC) and an increase in intracellular free calcium, there were substantial differences between the two which suggested that distinct signal transduction mechanisms were operating. We have confirmed an earlier observation that the cross-linking of IgE led to an increase in membrane PKC activity with no apparent concomitant loss of cytosolic PKC and established that in contrast, the univalent stimulus, f-met peptide, resulted in the canonical translocation of cytosolic PKC to the membrane. Furthermore, unlike anti-IgE-stimulated basophils, there was no clear relationship between the increase in PKC activity and the subsequent release of histamine. Two PKC inhibitors, staurosporine (0.1 to 1 nM) and sphingosine (25 to 50 microM), inhibited anti-IgE induced release, yet, potentiated the release of mediators after a challenge with 1 microM f-met peptide. Both stimuli led to an increase in the intracellular Ca2+ levels that correlated well with the release of histamine, however, the anti-IgE-induced responses were typically only 50% of those required to give equivalent histamine release when f-met peptide initiated release. Pharmacologic evidence suggested that the up-regulation of PKC was required for a full IgE-mediated Ca2+ response and that PKC contributed to the elevated Ca2+ levels that persist for up to 15 min after the addition of anti-IgE. In contrast, the PKC inhibitor, staurosporine, did not affect the initial increase in Ca2+ after the addition of f-met peptide but reduced the rate at which Ca2+ was removed from the cytosol. Experiments with the phorbol ester, PMA, suggested that substantial degranulation can occur in the absence of any increase in intracellular Ca2+. The addition of 10 ng/ml PMA 10 min before the addition of f-met peptide did not affect the magnitude of the initial Ca2+ transient but increased the rate at which Ca2+ levels returned to a stable baseline. Similar pretreatment with PMA almost completely abolished the anti-IgE antibody-induced Ca2+ response. These experiments, together with other previous data, suggest that the activation of PKC is a prodegranulatory component of the IgE-mediated signal transduction pathway, yet serves principally to modulate the Ca2+ signal when f-met peptide initiates release.
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PMID:Signal transduction events in human basophils. A comparative study of the role of protein kinase C in basophils activated by anti-IgE antibody and formyl-methionyl-leucyl-phenylalanine. 169 13

In human neutrophils, the chemotactic tripeptide FMLP and the protein kinase C activator PMA stimulate the breakdown of 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid ([3H]-EAPA) and 1-O-[3H]alkyl-2-acylglycerol ([3H]EAG) via mechanism(s) that may involve the activation of phospholipase D. We have investigated the regulation of phospholipase D by determining the effects of elevation of intracellular levels of cAMP on receptor-mediated and PMA-induced breakdown of [3H]-EAPC and formation of products. Pretreatment of neutrophils with either 10(-3) M dibutyryl-cAMP or 10(-5) M PGE2, in the presence of 4 x 10(-4) M isobutylmethylxanthine (IBMX), inhibited FMLP- and leukotriene B4-induced breakdown of [3H]EAPC and formation of [3H]EAPA and [3H]EAG. Inhibition was apparent at all time points of stimulation examined (15-120 s). In addition, the mass of diradyl-phosphatidic acid was decreased in FMLP-stimulated neutrophils pretreated with PGE2 and IBMX. In contrast, pretreatment of cells with PGE2 and IBMX did not inhibit PMA-induced breakdown of [3H]EAPC and the formation of products at 3 and 10 min after stimulation. Furthermore, formation of 1-O-[3H]alkyl-2-acyl-phosphatidylethanol, produced by phospholipase D in the presence of ethanol by a transphosphatidylation reaction, was significantly inhibited by pretreatment of cells with PGE2 and IBMX in FMLP- but not PMA-stimulated neutrophils. This differential modulation by cAMP of receptor-mediated and PMA-induced activation of phospholipase D suggests agonist-dependent activation of separate pathways and/or activation of separate phospholipase D enzymes. Thus, cAMP elevation may exert inhibitory effects directly on the phospholipase D activated by FMLP and/or on sites proximal to the enzyme that are involved in signal transmission.
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PMID:Regulation of phospholipase D-induced hydrolysis of choline-containing phosphoglycerides by cyclic AMP in human neutrophils. 170 63

Neutrophils from cystic fibrosis (CF) patients have been shown previously to be defective in their response (beta-glucuronidase exocytosis, NADPH oxidase activation) to the chemotactic peptide FMLP. In this work, we attempted to identify the defective step in this response. We showed that stimulated CF and control neutrophils do not differ in the formation of inositol phosphates. On the other hand, direct stimulation of protein kinase C with phorbol myristate acetate (PMA) revealed a subnormal stimulation of beta-glucuronidase exocytosis in CF neutrophils. Furthermore, retroinhibition exerted by PMA-activated protein kinase C on stimulated inositol phosphates or on beta-glucuronidase exocytosis was marginal or absent in CF neutrophils, whereas it was significant in the case of control neutrophils. Our observations suggest that the CFTR gene is expressed in neutrophils and is involved in protein kinase C-mediated actions.
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PMID:Defective protein kinase C-mediated actions in cystic fibrosis neutrophils. 189 35

The effect of anti-neutrophil cytoplasm autoantibodies (ANCA) on neutrophil activation was studied by pre-incubating neutrophils with IgG and F(ab')2 prepared from ANCA patients with Wegener's granulomatosis or microscopic polyarteritis. We measured the generation of inositol triphosphate (IP3) (a product of hydrolysis of membrane phospholipid which acts as a second intracellular messenger), and the translocation of protein kinase C (PKC) upon stimulation by a chemotactic peptide, fMet-Leu-Phe (fMLP). ANCA+ F(ab')2 did not induce a significant increase in IP3 generation. Nonetheless, ANCA+ F(ab')2 and ANCA+ IgG pretreatment of human neutrophils reduced the production of inositol phosphates upon subsequent fMLP stimulation compared with experiments performed when cells were pretreated with F(ab')2 and IgG prepared from ANCA- healthy subjects. A significantly reduced generation of IP3 and inositol biphosphate (IP2) was observed. ANCA+ F(ab')2 pretreatment of neutrophils inhibited fMLP-stimulated IP3 generation in a dose-dependent manner. The membrane-bound PKC activity upon stimulation by FMLP and PMA was reduced in neutrophils pretreated with ANCA+ F(ab')2 and IgG. These results indicate that ANCA affect in vitro signal transduction (IP3 generation, and translocation of PKC) in human neutrophils. Apparently, further activation of signal transduction by chemotactic peptide is significantly blunted in cells pre-incubated with ANCA+ F(ab')2 but not with F(ab')2 from healthy controls.
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PMID:The effect of anti-neutrophil cytoplasm autoantibodies on the signal transduction in human neutrophils. 189 20

Local anesthetics have been shown to affect a variety of cellular activities, such as exocytosis, membrane transport, permeability of calcium, and platelet aggregation, among others. Despite the disparate nature of the above phenomena, the calcium ion is essential for all of these cellular processes. Recently, it has been shown that local anesthetics inhibit specific calmodulin-dependent enzymes and protein kinase C-dependent enzymes. Therefore, the mode of action of local anesthetics in the activation of guinea pig neutrophils was examined in this study in order to understand the effect of local anesthetics on the intracellular calcium messenger system. In the presence of calmodulin inhibitors, FMLP-induced chemotaxis and superoxide anion production were strongly suppressed, whereas 1-oleoyl-2-acetylglycerol(OAG)(a direct activator of protein kinase C)-induced superoxide anion production was not affected. In contrast, protein kinase C inhibitors suppressed OAG-induced superoxide anion production but no inhibiting effect on FMLP-induced chemotaxis and superoxide anion production. Local anesthetics suppressed both FMLP- and OAG-induced activation of guinea pig neutrophils. These findings suggest that the inhibiting effect of local anesthetics on the activation of the intracellular calcium messenger system may be part of the pharmacological mechanism of local anesthetics in cellular activities.
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PMID:[Effect of local anesthetics on intracellular calcium messenger system]. 196 45

We have investigated the motility, superoxide anion production and tumor cell lysis of neutrophils from five patients affected by hairy cell leukemia. Random locomotion and chemotaxis towards denaturated casein and activated serum was normal as well as the superoxide production by opsonized zymosan. In contrast, chemotaxis and superoxide generation induced by FMLP and TPA were markedly reduced. The low responsiveness of neutrophils to TPA was also observed by evaluating cell lysis, against either non-immunized K562 target cells or antibody-coated tumor targets. In hairy cell leukemia neutrophils showed a selective reduced response to TPA and FMLP that, directly or indirectly, activate PKC, suggesting an impairment in the system of signal transduction.
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PMID:Neutrophil defect associated with hairy cell leukemia. 196 34

The characteristics of homotypic neutrophil aggregation, mediated by the adhesion molecule CD11b/CD18, differ according to whether activation takes place via intracellular protein kinase C(PKC) inducers or chemoattractants. In response to diacylglycerol (DAG) analogues such as PMA and 1,2-dioctanoyl-sn-glycerol, a prolonged cellular aggregation occurs that is associated with intense phosphorylation of the CD18 beta-chain. In response to the chemoattractant FMLP, a more transient aggregation event results that is associated with minimal beta-chain phosphorylation. By using the PKC inhibitor staurosporine, we now show that these differences are likely to reflect two different pathways of activation. Both aggregation and phosphorylation induced by DAG analogues are completely abolished by staurosporine in a parallel dose-dependent manner. Conversely, FMLP-induced aggregation is enhanced and prolonged by staurosporine whereas the associated minimal phosphorylation event is further diminished by staurosporine. Accordingly, activation of neutrophil aggregation by DAG analogues is associated with and presumably due to phosphorylation of the CD18 beta-chain. This intense phosphorylation occurs via a staurosporine-sensitive kinase such as PKC. FMLP, on the other hand, appears to activate CD11b/CD18 by a distinct mechanism. This latter mechanism does not seem to be dependent on what may be a minor PKC-induced phosphorylation of the beta-chain, and indeed is enhanced by inhibition of PKC. Of note, staurosporine was also found to cause selective release of specific granules with concomitant increase in surface display of CD11b/CD18. These data further support previous observations that up-regulation of this adhesive molecule is not the primary event in the induction of cellular adhesiveness.
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PMID:Two pathways of CD11b/CD18-mediated neutrophil aggregation with different involvement of protein kinase C-dependent phosphorylation. 197 98

Essentially pure preparations of normal density eosinophils obtained from patients with hypereosinophilic syndrome (HES) were stimulated with complement factor 5a (C5a), platelet-activating factor (PAF), FMLP and neutrophil-activating peptide (NAP-1/IL-8). Three responses were studied, the transient rise in cytosolic free calcium concentration ([Ca2+]i) (derived from indo-1 fluorescence), shape changes (measured by laser turbidimetry), and exocytosis of eosinophil peroxidase (EPO) (assessed by H2O2/luminol-dependent chemiluminescence). Responses were obtained with all four agonists, but C5a and PAF were by far more potent than FMLP and NAP-1/IL-8, which induced only minor effects. Pretreatment of the cells with pertussis toxin attenuated [Ca2+]i changes, EPO release and, to a lesser extent, shape changes, indicating that GTP-binding proteins of Gi-type are involved in receptor-dependent signal transduction processes leading to these responses. A clear dissociation was observed in the control of the shape change response and EPO exocytosis. The shape change was not affected by Ca2+ depletion or treatment with the protein kinase inhibitor staurosporine, but exocytosis was prevented by Ca2+ depletion and markedly enhanced by staurosporine. The activation of the contractile system, leading to shape changes and motility, thus appears to be independent of the classical signal transduction pathway involving phospholipase C, a [Ca2+]i rise and protein kinase C activation. Exocytosis is, as expected, Ca2+ dependent and appears to be under a negative control involving protein phosphorylations.
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PMID:Shape changes, exocytosis, and cytosolic free calcium changes in stimulated human eosinophils. 204 Jun 92

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