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)

To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 x 10(8) cells) or Aspergillus fumigatus (1 x 10(8) cells) was prolonged 3 to 5 days by the injection of 10 microg of peptide 2 (a lactoferrin peptide) and 10 microg of alpha-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 microg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 microg of amphotericin B. When mice received a combined injection of peptide 2 (10 microg/day) with amphotericin B (0.5 microg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as alpha-defensin 1, beta-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O2-) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O2- -generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47phox as well as p67phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.
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PMID:Regulation of fungal infection by a combination of amphotericin B and peptide 2, a lactoferrin peptide that activates neutrophils. 1553 15

A complex of atypical PKC and Par6 is a common regulator for cell polarity-related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKCiota and Par6alpha PB1 domains to a resolution of 1.5 A. Both PB1 domains adopt a ubiquitin fold. PKCiota PB1 presents an OPR, PC, and AID (OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6alpha PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKCiota PB1 and the conserved lysine residue on the Par6alpha PB1. Structural comparison of the PKCiota and Par6alpha PB1 complex with the p40phox and p67phox PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.
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PMID:Structure of a cell polarity regulator, a complex between atypical PKC and Par6 PB1 domains. 1559 Jun 54

Phosphorylation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox accompanies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opsonized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphorylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phosphorylation. Although protein kinase C (PKC)alpha has previously been shown to regulate NADPH oxidase activity, we found that inhibition of PKCalpha had no effect on p67phox phosphorylation. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribonucleotides specific for PKCdelta or rottlerin, a selective inhibitor for PKCdelta, inhibited the phosphorylation of p67phox in monocytes, and Go6976, a specific inhibitor for conventional PKCs, PKCalpha and PKCbeta, had no such inhibitory effect. Additional studies indicate that ZOP stimulation of monocytes induces PKCdelta and p67phox to form a complex. We also demonstrate that lysates from activated monocytes as well as PKCdelta immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rottlerin blocked the phosphorylation in each case. We further show that recombinant PKCdelta can phosphorylate p67phox in vitro. Finally, we show that PKCdelta-deficient monocytes produce significantly less superoxide anion in response to ZOP stimulation, thus emphasizing the functional significance of the PKCdelta regulation of p67phox phosphorylation. Taken together, this is the first report to describe the requirement of PKCdelta in regulating the phosphorylation of p67phox and the related NADPH oxidase activity in primary human monocytes.
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PMID:Protein kinase Cdelta regulates p67phox phosphorylation in human monocytes. 1559 Nov 24

Amyloid beta peptides generate oxidative stress in hippocampal astrocytes through a mechanism sensitive to inhibitors of the NADPH oxidase [diphenylene iodonium (DPI) and apocynin]. Seeking evidence for the expression and function of the enzyme in primary hippocampal astrocytes, we confirmed the expression of the subunits of the phagocyte NADPH oxidase by Western blot analysis and by immunofluorescence and coexpression with the astrocyte-specific marker glial fibrillary acidic protein both in cultures and in vivo. Functional assays using lucigenin luminescence, dihydroethidine, or dicarboxyfluorescein fluorescence to measure the production of reactive oxygen species (ROS) demonstrated DPI and apocynin-sensitive ROS generation in response to the phorbol ester PMA and to raised [Ca2+]c after application of ionomycin or P2u receptor activation. Stimulation by PMA but not Ca2+ was inhibited by the protein kinase C (PKC) inhibitors staurosporine and hispidin. Responses were absent in transgenic mice lacking gp91phox. Expression of gp91phox and p67phox was increased in reactive astrocytes, which showed increased rates of both resting and stimulated ROS generation. NADPH oxidase activity was modulated by intracellular pH, suppressed by intracellular alkalinization, and enhanced by acidification. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone suppressed basal ROS generation but markedly increased PMA-stimulated ROS generation. This was independent of mitochondrial ROS production, because it was unaffected by mitochondrial depolarization with rotenone and oligomycin. Thus, the NADPH oxidase is expressed in astrocytes and is functional, activated by PKC and intracellular calcium, modulated by pHi, and upregulated by astrocyte activation. The astrocytic NADPH oxidase is likely to play important roles in CNS physiology and pathology.
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PMID:Expression and modulation of an NADPH oxidase in mammalian astrocytes. 1620 77

Rac2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, rac2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47phox and p67phox to the plasma membrane was not impaired in rac2-/- neutrophils. Combined stimulation of rac2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47phox and p67phox as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCzeta, in superoxide production by wild-type and rac2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A2 expression in rac2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated rac2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47phox and p67phox, Rac2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.
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PMID:Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid. 1627 90

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.
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PMID:Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation. 1662 5

Previously we have demonstrated that 3',4'-dihydroxyflavonol (DiOHF), a novel synthetic flavonol, protects against ischemia reperfusion injury in both heart and brain. In this study, we characterized the pharmacological effects of DiOHF on phagocytic and vascular NADPH oxidase. Superoxide release (lucigenin-enhanced chemiluminescence or cytochrome c reduction), NADPH oxidase activation (membrane translocation of p47phox), and subunit expression (real-time polymerase chain reaction and Western blot) were examined in differentiated HL-60 cells, human neutrophils, vascular endothelial and smooth muscle cells, and mouse aorta. DiOHF concentration dependently suppressed superoxide accumulation (EC(50) = 8.4 +/- 1.7 microM) in vascular smooth muscle cells, which appears to be attributable to its superoxide scavenging activity (EC(50) = 6.1 +/- 1.1 microM measured in a cell-free system). DiOHF had similar effects in HL-60 cells and isolated aortic rings. In HL-60 cells, but not endothelial or smooth muscle cells, DiOHF and quercetin (10 and 30 microM) significantly reduced the protein expression of p47phox, whereas p67phox was not altered. DiOHF did not affect phorbol ester-induced membrane translocation of either p47phox or protein kinase C in leukocytes. Our results suggest that suppression of NADPH oxidase-dependent superoxide accumulation may contribute to the cytoprotective actions of DiOHF during ischemia-reperfusion injury.
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PMID:Modulation of nicotinamide adenine dinucleotide phosphate oxidase expression and function by 3',4'-dihydroxyflavonol in phagocytic and vascular cells. 1791 58

Macrophages produce a large volume of ROS (reactive oxygen species) through respiratory burst. However, the influence of iNOS [inducible NOS (nitric oxide synthase)] activation on ROS production remains unclear. In the present study, the kinetic generation of ROS in RAW264.7 murine macrophages was monitored by chemiluminescence. PMA induces a robust chemiluminescence in RAW264.7 cells, suggesting PKC (protein kinase C)-related assembly and activation of NOX (NADPH oxidase). The effects of iNOS induction on ROS production were examined. Induction of iNOS expression in RAW264.7 cells with LPS (lipopolysaccharide; 1 microg/ml) causes a significant increase in PMA-induced chemiluminescence, which could be enhanced by the NOS substrate, L-arginine, and could be abolished by the NOS inhibitor, L-NNA (NG-nitro-L-arginine). Further experiments reveal that induction of iNOS expression enhances the PMA-stimulated phosphorylation of the p47phox subunit of NOX, and promotes the relocalization of cytosolic p47phox and p67phox subunits to the membrane. Inhibition of PKCzeta by its myristoylated pseudosubstrate significantly decreased the PMA-stimulated phosphorylation of the p47phox in LPS-pretreated cells, suggesting that PKCzeta is involved in the iNOS-dependent assembly and activation of NOX. Taken together, the present study suggests that the induction of iNOS upregulates the PMA-induced assembly of NOX and leads to the enhanced production of ROS via a PKCzeta-dependent mechanism.
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PMID:Induction of inducible nitric oxide synthase increases the production of reactive oxygen species in RAW264.7 macrophages. 1967 2

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91phox and p22phox, and the cytosolic proteins, p40phox, p47phox and p67phox. In the present study, we clone and sequence the full-length cDNAs coding for the Atlantic salmon (Salmo salar) phagocyte NADPH oxidase components, p47phox, p67phox and gp91phox, using a homology cloning approach. The sequences of these cDNAs showed that the S. salar p47phox, p67phox and gp91phox genes contained single open reading frames, which encoded predicted proteins of 413, 504 and 565 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the S. salar p47phox, p67phox and gp91phox sequences shared 51, 45 and 68% identity with those of human components, respectively. Despite this relatively low homology between salmon and mammalian NADPH oxidase subunits, their functional domains are highly conserved. We also found that the mRNA levels of p47phox, p67phox and gp91phox expression were higher in immune-related tissues, such as kidney, spleen and gill. In addition, infection of the salmon macrophage cell line SHK-1 with Piscirickettsia salmonis induced the expression of p47phox, but had no effect on p67phox and gp91phox expression. Finally, we show for the first time in fish that activation of macrophages with lipopolysaccharide promotes the activation of protein kinase C, which in turn phosphorylates p47phox, leading to NADPH oxidase activation and reactive oxygen species generation. Collectively, these results suggest that the mechanisms of activation of phagocyte NADPH oxidase are well conserved from fish to mammals.
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PMID:Lipopolysaccharide primes the respiratory burst of Atlantic salmon SHK-1 cells through protein kinase C-mediated phosphorylation of p47phox. 2062 Nov 16

Neutrophils play a key role in host defense and inflammation through the production of superoxide anion and other reactive oxygen species (ROS) by the enzyme complex NADPH oxidase. The cytosolic NADPH oxidase component, p67phox, has been shown to be phosphorylated in human neutrophils but the pathways involved in this process are largely unknown. In this study, we show that p67phox is constitutively phosphorylated in resting human neutrophils and that neutrophil stimulation with PMA further enhanced this phosphorylation. Inhibition of the constitutively active serine/threonine phosphatases type 1 and type 2A (PP1/2A) by calyculin A resulted in the enhancement of p67phox phosphorylation. Constitutive and calyculin A-induced phosphorylation of p67phox was completely inhibited by the protein tyrosine kinase inhibitor genistein and partially inhibited by the MEK1/2 inhibitor PD98059, but was unaffected by GF109203X, wortmannin and SB203580, inhibitors of PKC, PI3K and p38MAP kinase, respectively. Two-dimensional phosphopeptide mapping revealed that constitutive and calyculin A-induced p67phox phosphorylation occurred on the same major sites. Interestingly, calyculin A enhanced formyl-Met-Leu-Phe (fMLP)-induced superoxide production, while genistein inhibited this process. Taken together, these results suggest that (i) p67phox undergoes a continual cycle of phosphorylation/dephosphorylation in resting cells; (ii) p67phox phosphorylation is controlled by MEK1/2 and an upstream tyrosine kinase; (iii) PP1/2A directly or indirectly antagonize this process. Thus, these pathways could play a role in regulating ROS production by human neutrophils at inflammatory sites.
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PMID:The NADPH oxidase cytosolic component p67phox is constitutively phosphorylated in human neutrophils: Regulation by a protein tyrosine kinase, MEK1/2 and phosphatases 1/2A. 2178 60


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