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)

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O-2) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67(phox). In this study, we found that activation of human neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67(phox). Using an anti-p67(phox) antibody or an anti-p47(phox) antibody, we showed that phosphorylated p67(phox) and p47(phox) form a complex. Phosphoamino acid analysis of the phosphorylated p67(phox) revealed only 32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67(phox) is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67(phox) in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67(phox) was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67(phox) is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.
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PMID:Phosphorylation of the respiratory burst oxidase subunit p67(phox) during human neutrophil activation. Regulation by protein kinase C-dependent and independent pathways. 920 43

Previous studies have shown that platelet stimulation with cathepsin G rapidly results in cytoplasmic calcium ([Ca2+]i) increase and activation of protein kinase C (PKC). To elucidate the relationship between these two biochemical events and their relative contribution to the regulation of platelet response to cathepsin G, arachidonic acid (AA) release and serotonin (5HT) secretion were studied. Platelets made Ca2+-depleted and -permeable by treatment with A23187 were compared to intact platelets to better dissociate calcium changes from other receptor-stimulated events. AA release elicited by cathepsin G in intact platelets was prevented by the Ca2+ chelator BAPTA; in Ca2+-depleted, -permeable platelets AA was released in direct response to added Ca2+ and was not increased by simultaneous stimulation with cathepsin G. In intact platelets, PKC inhibition by Ro 31-8220 or PKC induction with PMA either enhanced or reduced, respectively, cathepsin G-induced AA release. Both BAPTA and Ro 31-8220 prevented 5HT secretion from intact platelets; however, in Ca2+-depleted, -permeable platelets, cathepsin G was able to evoke 5HT secretion and p47 phosphorylation independently of [Ca2+]i increase, both effects being hampered by Ro 31-8220. Ca2+ and PKC therefore regulate PLA2 activity and 5HT secretion in cathepsin G-stimulated platelets in a different manner: the former is mainly triggered by [Ca2+]i increase, while PKC represents the major factor in determining dense granule secretion.
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PMID:Different requirement of intracellular calcium and protein kinase C for arachidonic acid release and serotonin secretion in cathepsin G-activated platelets. 926 95

Monochloramine derivatives are long lived physiological oxidants produced by neutrophils during the respiratory burst. The effects of chemically prepared monochloramine (NH2Cl) on protein kinase C (PKC) and PKC-mediated cellular responses were studied in elicited rat peritoneal neutrophils and human Jurkat T cells. Neutrophils pretreated with NH2Cl (30-50 microM) showed a marked decrease in the respiratory burst activity induced by phorbol 12-myristate 13-acetate (PMA), which is a potent PKC activator. These cells, however, were viable and showed a complete respiratory burst upon arachidonic acid stimulation, which induces the respiratory burst by a PKC-independent mechanism. The NH2Cl-treated neutrophils showed a decrease in both PKC activity and PMA-induced phosphorylation of a 47-kDa protein, which corresponds to the cytosolic factor of NADPH oxidase, p47(phox). Jurkat T cells pretreated with NH2Cl (20-70 microM) showed a decrease in the expression of the interleukin-2 receptor alpha chain following PMA stimulation. This was also accompanied by a decrease in both PKC activity and nuclear transcription factor-kappaB activation, also without loss of cell viability. These results show that NH2Cl inhibits PKC-mediated cellular responses through inhibition of the inducible PKC activity.
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PMID:Monochloramine inhibits phorbol ester-inducible neutrophil respiratory burst activation and T cell interleukin-2 receptor expression by inhibiting inducible protein kinase C activity. 933 93

A role for protein kinase C (PKC) isotypes is implicated in the activation of phagocytic cell functions. An antisense approach was used to selectively deplete beta-PKC, both betaI- and betaII-PKC, but not alpha-PKC, delta-PKC, or zeta-PKC in HL60 cells differentiated to a neutrophil-like phenotype (dHL60 cells). Depletion of beta-PKC in dHL60 cells elicited selective inhibition of O-2 generation triggered by fMet-Leu-Phe, immune complexes, or phorbol myristate acetate, an activator of PKC. In contrast, neither ligand-elicited beta-glucuronidase (azurophil granule) release nor adherence to fibronectin was inhibited by beta-PKC depletion. Ligand-induced phosphorylation of a subset of proteins was reduced in beta-PKC-depleted dHL60 cells. Phosphorylation of p47(phox) and translocation of p47(phox) to the membrane are essential for activation of the NADPH oxidase and generation of O-2. beta-PKC depletion had no effect on the level of p47(phox) in dHL60 cells but did significantly decrease ligand-induced phosphorylation of this protein. Furthermore, translocation of p47(phox) to the membrane in response to phorbol myristate acetate or fMet-Leu-Phe was reduced in beta-PKC-depleted cells. These results indicate that beta-PKC is essential for signaling for O-2 generation but not cell adherence or azurophil degranulation. Depletion of beta-PKC inhibited ligand-induced phosphorylation of p47(phox), translocation of p47(phox) to the membrane, and activation of O-2 generation.
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PMID:Selective role for beta-protein kinase C in signaling for O-2 generation but not degranulation or adherence in differentiated HL60 cells. 976 54

Using a model of the enzyme structure and the results from a series of free and myristylated peptides, we provide evidence that peptides corresponding to the pseudosubstrate sequence of protein kinase C bind to the enzyme substrate binding site in an essentially extended conformation. This and the nearly symmetrical location of positive charges around the substrate phosphoritable site allow the peptide to bind to the enzyme in either an N-to-C orientation or its C-to-N opposite orientation. The latter is favoured by a change in residue chirality or when the peptide bears a myristoyl chain at its N-terminus. A myristyl binding site was also identified in the enzyme structure and its location in a region proximal to the C-terminal residue of pseudosubstrate bound in the N-to-C direction suggested that C-myristylation of peptide substrates should be more effective than N-myristoylation in antagonizing the enzyme. A peptide (H-RFARKGALRQKN-CONH-Myr) which contains the myristyl chain covalently linked to the C-terminal residue of the pseudosubstrate was thus made and shown to be a potent inhibitor of the histone kinase reaction of protein kinase C and the phosphorylation of p47 in intact cells.
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PMID:The interaction of myristylated peptides with the catalytic domain of protein kinase C revealed by their sequence palindromy and the identification of a myristyl binding site. 979 30

The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67(phox) (phagocyte oxidase), p47(phox), and p40(phox), which translocate to the membrane upon activation. In a previous paper, we reported that p40(phox) undergoes phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol 12-myristate 13-acetate or by formyl peptide with a time course that is strongly correlated with that of superoxide production (Fuchs, A., Bouin, A. P., Rabilloud, T., and Vignais, P. V. (1997) Eur. J. Biochem. 249, 531-539). In this study, through phosphoamino acid and tryptic peptide maps of in vivo and in vitro phosphorylated p40(phox), we show that p40(phox) is phosphorylated on serine and threonine residues during activation of the NADPH oxidase in dimethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolated human neutrophils. In vitro phosphorylation studies using casein kinase II and protein kinase C (PKC) as well as the effect of various protein kinase inhibitors on the isoelectric focusing pattern of p40(phox) in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40(phox). Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40(phox) are phosphorylated during activation of the NADPH oxidase.
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PMID:p40(phox) is phosphorylated on threonine 154 and serine 315 during activation of the phagocyte NADPH oxidase. Implication of a protein kinase c-type kinase in the phosphorylation process. 980 63

Vitamin E (alpha-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. alpha-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O-2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of alpha-tocopherol on O-2 production by human monocytes. In activated monocytes O-2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, alpha-tocopherol (but not beta-tocopherol) inhibited O-2 production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that alpha-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47(phox) were reduced in monocytes preincubated with alpha-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47(phox) was similar to that of neutrophil p47(phox), indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47(phox) was immunoprecipitated from alpha-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by alpha-tocopherol in a specific manner compared with that of beta-tocopherol or Trolox(R). Membrane translocation of PKC was not affected. These results show that alpha-tocopherol inhibits O-2 production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) results from a decrease in PKC activity.
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PMID:alpha-tocopherol inhibits the respiratory burst in human monocytes. Attenuation of p47(phox) membrane translocation and phosphorylation. 983 25

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(phox) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that phosphorylation of p47(phox) with protein kinase C induces conformational changes, as reflected by a fluorescence change of N, N'-di-methyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD)-labeled p47(phox). We propose that this alteration in conformation results in the appearance of a binding site through which p47(phox) interacts with cytochrome b558 during the activation process. In addition, the present study indicates that other oxidase components, such as p67(phox) and p22(phox), influence the conformation of p47(phox).
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PMID:Phosphorylation induces conformational changes in the leukocyte NADPH oxidase subunit p47(phox). 1033 12

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.
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PMID:Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system. 1033 47

GTPgammaS activates the NADPH oxidase and this activity declines rapidly with time after preexposure to streptolysin O. This was not due to loss of p47(phox), p67(phox), or Rac. To identify the component(s) leaking out of the permeabilized cell responsible for loss of activity, a GTPgammaS-dependent reconstitution assay was established. Neutrophil cytosol was subjected to chromatographic fractionation steps for purification of the minimum fraction required to restore activity. The reconstitution of the GTPgammaS-stimulated activity was dependent on ATP. The inhibitors staurosporine and calphostin C greatly reduced the activity in the reconstitution assay, implicating the involvement of a protein kinase C (PKC) pathway. PKC isoforms beta and delta were eliminated as the active factors in the most pure reconstitution fraction. With this novel cell-based reconstitution assay, we have identified the requirement for a protein kinase, or its substrate, for the restoration of GTPgammaS activation of the NADPH oxidase.
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PMID:Reconstitution of GTPgammaS-induced NADPH oxidase activity in streptolysin-O-permeabilized neutrophils by specific cytosol fractions. 1054 86


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