Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.99.6 (NADPH oxidase)
 10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47(phox), resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKCdelta phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47(phox) phosphorylation. Using transfected COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS-phox cells), we found that a functionally active PKCdelta is required for p47(phox) phosphorylation and reconstitution of NADPH oxidase. PKCbetaII cannot replace PKCdelta for this function. Characterization of PKCdelta/PKCbetaII chimeras has led to the identification of the catalytic domain of PKCdelta as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr(505) in the activation loop of mouse PKCdelta. Mutation of Thr(505) to alanine abolishes the ability of PKCdelta to catalyze p47(phox) phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKCdelta phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKCdelta phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.
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PMID:A critical role of protein kinase C delta activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47(phox) and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase. 1802 18

We used Western blot analysis to examine the effect of dietary K intake on the expression of serine/threonine protein phosphatase in the kidney. K restriction significantly decreased the expression of catalytic subunit of protein phosphatase (PP)2B but increased the expression of PP2B regulatory subunit in both rat and mouse kidney. However, K depletion did not affect the expression of PP1 and PP2A. Treatment of M-1 cells, mouse cortical collecting duct (CCD) cells, or 293T cells with glucose oxidase (GO), which generates superoxide anions through glucose metabolism, mimicked the effect of K restriction on PP2B expression and significantly decreased expression of PP2B catalytic subunits. However, GO treatment increased expression of regulatory subunit of PP2B and had no effect on expression of PP1, PP2A, and protein tyrosine phosphatase 1D. Moreover, deletion of gp91-containing NADPH oxidase abolished the effect of K depletion on PP2B. Thus superoxide anions or related products may mediate the inhibitory effect of K restriction on the expression of PP2B catalytic subunit. We also used patch-clamp technique to study the effect of inhibiting PP2B on renal outer medullary K (ROMK) channels in the CCD. Application of cyclosporin A or FK506, inhibitors of PP2B, significantly decreased ROMK channels, and the effect of PP2B inhibitors was abolished by blocking p38 mitogen-activated protein kinase (MAPK) and ERK. Furthermore, Western blot demonstrated that inhibition of PP2B with cyclosporin A or small interfering RNA increased the phosphorylation of ERK and p38 MAPK. We conclude that K restriction suppresses the expression of PP2B catalytic subunits and that inhibition of PP2B decreases ROMK channel activity through stimulation of MAPK in the CCD.
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PMID:K restriction inhibits protein phosphatase 2B (PP2B) and suppression of PP2B decreases ROMK channel activity in the CCD. 1818 75

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is structurally similar to the neurotoxin 1-methyl-4-phenyl-4-phenylpyridium ion (MPP+), the active metabolite of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which can induce the parkinsonism property in rodents, nonhuman primates, and human. In contrast to the neurotoxic effects of paraquat, little is known about its effects on glial cells. Here, we examined the mechanisms of paraquat toxicity in glial cells in culture. Paraquat treatment also reduced the viability of C6 glial cells in primary astrocyte cultures, and cell death was mostly apoptotic in nature. PKCdelta played a central role in the paraquat-induced glial cell death: (1) the PKCdelta-specific inhibitor rottlerin blocked paraquat-induced glial cell death; (2) paraquat induced tyrosine and threonine phosphorylation of PKCdelta; and (3) transfection of the dominant-negative mutant of PKCdelta attenuated paraquat toxicity. PKCdelta was also involved in the generation of reactive oxygen species (ROS), which mediated the paraquat toxicity. The nicotinamide adenine dinucleotide phosphate (reduced form) oxidase (NADPH oxidase) inhibitor diphenyleneiodonium blocked the paraquat-induced ROS production and subsequent cell death, indicating the involvement of NADPH oxidase in the cytotoxic action of paraquat in glia. PKCdelta was also important in glial cell death induced by MPP+ but not in that induced by rotenone. Last, Rac1 appeared to antagonize paraquat toxicity in glia. These results indicate a gliotoxic effect of paraquat and an opposing role of PKCdelta and Rac1 in paraquat-induced glial cell death.
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PMID:Role of protein kinase Cdelta in paraquat-induced glial cell death. 1833 19

The production of reactive oxygen species (ROS) in activated neutrophils is catalyzed by NADPH oxidase, a multiproteins complex. Various protein kinases including protein kinase C (PKC) and mitogen activated protein kinases (MAPKs) are involved in NADPH oxidase phosphorylation and activation. The main step in the NADPH oxidase activation is phosphorylation of its cytosolic protein p47(phox). We found previously that nitric oxide (NO) donors such as metabolite of molsidomine-SIN-1 and diethylamine/NO significantly impaired the ROS production in activated human neutrophils. In this study, we investigated the effects of both NO donors on NADPH oxidase-linked signaling proteins in activated neutrophils. We found that NO donors decreased the phosphorylation of p47(phox) on tyrosine and serine/threonine residues and PKC on serine residues in neutrophils. Both NO donors did not affect the phosphorylation of MAPKs. NO donors partially but significantly lost their ability to reduce ROS production in the presence of PKC but not MAPKs inhibitors. We show that whereas NO donors have no effect on MAPKs activity, they do decrease PMA- and/or fMLP-induced phosphorylation of p47(phox) and PKC as well as PMA- and fMLP-induced ROS production.
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PMID:Effect of nitric oxide donors on NADPH oxidase signaling pathway in human neutrophils in vitro. 1924 25

Cyclooxygenase-2 (Cox-2) metabolites produced by endothelial cells, particularly prostacyclin and prostaglandin E(2), profoundly affect vascular tone, regional blood flow, and angiogenesis. We have previously shown that reactive oxygen species induce Cox-2 expression in human endothelial cells (HUVEC), either on their own or as components of the signaling pathway triggered by TNFalpha, the prototypical inflammatory cytokine. Here we investigated the role of Cox-2 induced by hydrogen peroxide (H(2)O(2)), either exogenous or endogenously generated by TNFalpha, in the repair of a mechanically wounded HUVEC monolayer and probed the sources of H(2)O(2) that are involved in TNFalpha signaling and the pathways through which H(2)O(2) modulates Cox-2 expression. Results indicate that H(2)O(2)-induced Cox-2 activity participates in the repair of wounded monolayers. Both NADPH oxidase and the mitochondrial electron transport chain are involved in H(2)O(2) generation. Signaling triggered by H(2)O(2) for Cox-2 induction acts by increasing the protein tyrosine kinase phosphorylation that follows inhibition of protein phosphatase activity. The activation of p38 MAPK and its interaction in the inhibition of serine/threonine phosphatase activity are both critical steps in this event. We conclude that Cox-2 induced by H(2)O(2) plays an important role in promoting endothelial wound repair after injury, so that the cardioprotective effect of Cox-2 is due at least in part to its power of healing damaged endothelium.
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PMID:Cyclooxygenase-2 mediates hydrogen peroxide-induced wound repair in human endothelial cells. 1926 18

Bcr-Abl causes chronic myelogenous leukemia, a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells. In this study, inducible expression of Bcr-Abl in TonB.210 cells is associated with increased production of intracellular reactive oxygen species (ROS), which is thought to play a role in survival signaling when generated at specific levels. Elevated ROS in Bcr-Abl-expressing cells were found to activate PI3k/Akt pathway members such as Akt and GSK3beta as well as downstream targets beta-catenin and Mcl-1. The activation of these proteins was inhibited by the flavoprotein inhibitor diphenyleneiodonium, which is commonly used to inhibit NADPH oxidase (Nox). This indicated that increased ROS might be related to increased activity of one member of the Nox family. Knock-down experiments using siRNA suggest that Nox-4 is the main source of increased ROS following Bcr-Abl expression. We showed that Bcr-Abl-induced ROS could also increase survival pathway signaling through redox inhibition of PP1alpha, a serine threonine phosphatase that negatively regulates the PI3k/Akt pathway. Overall our results demonstrate that Bcr-Abl expression increases Nox-4-generated ROS, which in turn increases survival signaling through PI3k/Akt pathway by inhibition of PP1alpha, thus contributing to the high level of resistance to apoptosis seen in these Bcr-Abl-expressing cells.
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PMID:Bcr-Abl-mediated redox regulation of the PI3K/AKT pathway. 1929 48

Activation of the NADPH oxidase homolog dual oxidase 1 (DUOX1) within the airway epithelium represents a key mechanism of innate airway host defense, through enhanced production of H2O2, which mediates cellular signaling pathways that regulate the production of various inflammatory mediators. Production of the CXC chemokine interleukin (IL)-8/CXCL8 forms a common epithelial response to many diverse stimuli, including bacterial and viral triggers, environmental oxidants, and other biological mediators, suggesting the potential involvement of a common signaling pathway that may involve DUOX1-dependent H2O2 production. Following previous reports showing that DUOX1 is activated by extracellular ATP and purinergic receptor stimulation, this study demonstrates that airway epithelial IL-8 production in response to several bacterial stimuli involves ATP release and DUOX1 activation. ATP-mediated DUOX1 activation resulted in the activation of ERK1/2 and NF-kappaB pathways, which was associated with epidermal growth factor receptor (EGFR) ligand shedding by ADAM17 (a disintegrin and metalloproteinase-17). Although ATP-mediated ADAM17 activation and IL-8 release were not prevented by extracellular H2O2 scavenging by catalase, these responses were attenuated by intracellular scavengers of H2O2 or related oxidants, suggesting an intracellular redox signaling mechanism. Both ADAM17 activation and IL-8 release were suppressed by inhibitors of EGFR/ERK1/2 signaling, which can regulate ADAM17 activity by serine/threonine phosphorylation. Collectively, our results indicate that ATP-mediated DUOX1 activation represents a common response mechanism to several environmental stimuli, involving H2O2-dependent EGFR/ERK activation, ADAM17 activation, and EGFR ligand shedding, leading to amplified epithelial EGFR activation and IL-8 production.
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PMID:ATP-mediated activation of the NADPH oxidase DUOX1 mediates airway epithelial responses to bacterial stimuli. 1938 3

In human neutrophils, TNF-elicited O(2)(-) production requires adherence and integrin activation. How this cooperative signaling between TNFRs and integrins regulates O(2)(-) generation has yet to be fully elucidated. Previously, we identified delta-PKC as a critical early regulator of TNF signaling in adherent neutrophils. In this study, we demonstrate that inhibition of delta-PKC with a dominant-negative delta-PKC TAT peptide resulted in a significant delay in the onset time of TNF-elicited O(2)(-) generation but had no effect on Vmax, indicating an involvement of delta-PKC in the initiation of O(2)(-) production. In contrast, fMLP-elicited O(2)(-) production in adherent and nonadherent neutrophils was delta-PKC-independent, suggesting differential regulation of O(2)(-) production. An important step in activation of the NADPH oxidase is phosphorylation of the cytosolic p47phox component. In adherent neutrophils, TNF triggered a time-dependent association of delta-PKC with p47phox, which was associated with p47phox phosphorylation, indicating a role for delta-PKC in regulating O(2)(-) production at the level of p47phox. Activation of ERK and p38 MAPK is also required for TNF-elicited O(2)(-) generation. TNF-mediated ERK but not p38 MAPK recruitment to p47phox was delta-PKC-dependent. delta-PKC activity is controlled through serine/threonine phosphorylation, and phosphorylation of delta-PKC (Ser643) and delta-PKC (Thr505) was increased significantly by TNF in adherent cells via a PI3K-dependent process. Thus, signaling for TNF-elicited O(2)(-) generation is regulated by delta-PKC. Adherence-dependent cooperative signaling activates PI3K signaling, delta-PKC phosphorylation, and delta-PKC recruitment to p47phox. delta-PKC activates p47phox by serine phosphorylation or indirectly through control of ERK recruitment to p47phox.
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PMID:Regulation of TNF-induced oxygen radical production in human neutrophils: role of delta-PKC. 1980

NADPH oxidase comprises both cytosolic and membrane-bound subunits, which, when assembled and activated, initiate the transfer of electrons from NADPH to molecular oxygen to form superoxide. This activity, known as the respiratory burst, is extremely important in the innate immune response as indicated by the disorder chronic granulomatous disease. The regulation of this enzyme complex involves protein-protein and protein-lipid interactions as well as phosphorylation events. Previously, our laboratory demonstrated that the small membrane subunit of the oxidase complex, p22(phox), is phosphorylated in neutrophils and that its phosphorylation correlates with NADPH oxidase activity. In this study, we utilized site-directed mutagenesis in a Chinese hamster ovarian cell system to determine the phosphorylation sites within p22(phox). We also explored the mechanism by which p22(phox) phosphorylation affects NADPH oxidase activity. We found that mutation of threonine 147 to alanine inhibited superoxide production in vivo by more than 70%. This mutation also blocked phosphorylation of p22(phox) in vitro by both protein kinase C-alpha and -delta. Moreover, this mutation blocked the p22(phox)-p47(phox) interaction in intact cells. When phosphorylation was mimicked in vivo through mutation of Thr-147 to an aspartyl residue, NADPH oxidase activity was recovered, and the p22(phox)-p47(phox) interaction in the membrane was restored. Maturation of gp91(phox) was not affected by the alanine mutation, and phosphorylation of the cytosolic component p47(phox) still occurred. This study directly implicates threonine 147 of p22(phox) as a critical residue for efficient NADPH oxidase complex formation and resultant enzyme activity.
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PMID:Phosphorylation of p22phox on threonine 147 enhances NADPH oxidase activity by promoting p47phox binding. 1994 36

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH(i) regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-delta phosphorylation sites in the human voltage-gated proton channel, H(V)1 (Hvcn1), Thr(29) and Ser(97), both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-delta. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H(V)1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H(V)1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr(29) is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.
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PMID:Identification of Thr29 as a critical phosphorylation site that activates the human proton channel Hvcn1 in leukocytes. 2003 53


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