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)

Cytosolic components of the phagocyte NADPH oxidase (p47phox, p67phox, and Rac2) translocate to the plasma membrane on cell activation where they interact with a membrane-bound cytochrome b to generate superoxide anion. Phosphorylation reactions are known to be important for activity of NADPH oxidase. Translocation of Rac2, p47phox, and p67phox were all enhanced in formyl-Met-Leu-Phe-stimulated neutrophils treated with 50 nM of the protein phosphatase 1/2A inhibitor calyculin A. Rac translocation was blocked by the tyrosine kinase inhibitors genistein (50 microM) and herbimycin (17 microM), whereas movement of p47phox and p67phox were not inhibited. Cell-free analysis of Rac translocation also demonstrated that translocation of p47phox and p67phox were not linked to the movement or availability of Rac2. Thus, Rac2 does not appear to regulate NADPH oxidase by controlling movements of the cytosolic components to the membrane-associated enzyme but may exert its effect at the level of the assembled complex. Tyrosine kinase activity is required for translocation of Rac in the chemoattractant-stimulated human neutrophil.
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PMID:Dissociation of Rac translocation from p47phox/p67phox movements in human neutrophils by tyrosine kinase inhibitors. 761 2

Protein phosphatase inhibitors, okadaic acid and calyculin A, potentiated and elongated N-formyl-methionyl-leucyl-phenylalanine-induced superoxide anion (O2-) production in guinea pig neutrophils. The activity of NADPH oxidase in the membrane fraction prepared from phorbol 12-myristate 13-acetate-stimulated neutrophils was inactivated by the addition of the cytosol from resting neutrophils, such inactivation of NADPH oxidase was also suppressed by the protein phosphatase inhibitors. We previously reported that phosphorylation of the 46-kDa protein by protein kinase C is one of the activation mechanisms of NADPH oxidase-dependent superoxide anion production. In the cytosol fraction, we found protein phosphatase activity that catalyzed dephosphorylation of 32P-labeled phosphoproteins including the 46-kDa protein. Dephosphorylation of the 46-kDa protein was inhibited by the addition of okadaic acid and calyculin A. These results indicate that dephosphorylation of the 46-kDa protein by protein phosphatase is involved in the inactivation of NADPH oxidase. NADPH oxidase activity in guinea pig neutrophil may be regulated by the phosphorylation/dephosphorylation state of the 46-kDa protein by protein kinase C and protein phosphatase.
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PMID:Cytosolic protein phosphatase may turn off activated NADPH oxidase in guinea pig neutrophils. 821 5

The non-12-O-tetadecanoylphorbol-13-acetate (TPA)-type tumor promoters, okadaic acid (OA) and calyculin-A (CAL-A), which neither interact with the phorbol ester receptor nor directly activate protein kinase C, mimic the stimulatory effects of and thapsigargin on hydroperoxide (HPx) production in mouse epidermis in vivo. The time course and dose dependency for the stimulation of HPx production by O and TPA are similar. HPx production is maximally stimulated 16 h after two applications of 2 nmol of OA at a 48-h interval. However CAL-A is a stimulator of HPx production about 4 times more potent than OA or TPA. Combinations of TPA and OA or CAL-A have subadditive effects on HPx production. The discrepancies between the abilities of various serine/threonine protein phosphatase (PP) inhibitors to stimulate HPx production suggest that PP inhibition alone is not sufficient for this response. Cycloheximide, Ca2+ antagonists, oxypurinol, diphenyliodonium, nordihydroguaiaretic acid, bromophenacyl bromide, antiinflammatory agents, and antihistamines block or decrease OA-stimulated HPx production. Although most of these inhibitors may have more than one action, their effects suggest that protein synthesis, Ca2+, xanthine oxidase and NADPH oxidase activities, the lipoxygenase pathway of arachidonic acid metabolism, and vascular permeability may be involved in the inflammatory and HPx responses that occur after tumor promoter treatment. The increased HPx-producing activity of the epidermis, therefore, may be a common event resulting from the inflammatory and tumor-promoting actions of diverse TPA- and non-TPA-type agents.
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PMID:Ability of okadaic acid and other protein phosphatase inhibitors to mimic the stimulatory effects of 12-O-tetradecanoylphorbol-13-acetate on hydroperoxide production in mouse epidermis in vivo. 855 15

We examined the effects of okadaic acid, a protein phosphatase 1 and 2A inhibitor, on superoxide generation in human neutrophils. Superoxide generation induced by fMLP was inhibited by low-dose okadaic acid (10-100 nM), but it had no effect on superoxide synthesis by PMA, and the fMLP-induced rise of the intracellular Ca2+ concentration was not affected by low-dose okadaic acid. These findings suggested that the inhibitory mechanism of okadaic acid might involve PKC-independent and Ca(2+)-independent pathways in fMLP induced NADPH oxidase activation. Both fMLP-stimulated phosphorylation of serine residues in p47phox and its translocation to the plasma membrane were suppressed by low-dose okadaic acid. On the other hand, PMA-induced phosphorylation and translocation of p47phox were not affected by such a low dose of okadaic acid. These findings suggested that fMLP induced phosphorylation of serine residues in p47phox was regulated by protein phosphatase 2A, and its phosphorylation was necessary for translocation and superoxide generation in fMLP-activated human neutrophils.
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PMID:Involvement of protein phosphatase 2A in PKC-independent pathway of neutrophil superoxide generation by fMLP. 865 38

Spermatozoa undergoing capacitation, a necessary prerequisite event to successful fertilization that can be induced in vitro by reactive oxygen species (ROS), generate superoxide anion (O2.-). Because, in neutrophils, the generation of O2.- is associated with tyrosine phosphorylation of several proteins, the aim of the present study was to investigate the association between protein-tyrosine phosphorylation and ROS-induced human sperm capacitation. Human spermatozoa express two major phosphotyrosine-containing proteins of 105 and 81 kDa, the phosphotyrosine content of which is increased when spermatozoa are incubated under capacitating conditions. Superoxide dismutase and catalase abolish both sperm capacitation and tyrosine phosphorylation of p105 and p81, suggesting the involvement of O2.- and hydrogen peroxide in these two processes. Inhibitors of NADPH oxidase, the enzyme responsible for the neutrophil's respiratory burst, decrease both p105 and p81 tyrosine phosphorylation and sperm capacitation while hydrogen peroxide stimulates these two processes. Tyrosine phosphorylation of p105 and p81 occurs through a herbimycin A-sensitive tyrosine kinase, and sperm incubation with phosphotyrosine-protein phosphatase inhibitors results in an increase in phosphotyrosine content of these two proteins. Indirect immunocytochemical studies reveal phosphotyrosine-containing proteins mostly in the principal piece of the flagellum, in agreement with the localization of p105 and p81 in the human sperm fibrous sheath. Although tyrosine phosphorylation of p105 and p81 and sperm capacitation are related in a time-dependent fashion, some discrepancies are observed in the regulation of these two processes according to the redox status of the spermatozoa.
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PMID:Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives. 901 27

The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3-16 motifs, which form scaffolds to mediate protein-protein interactions and often the assembly of multiprotein complexes. TPR-containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90-binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel alpha-helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel alpha-helices. The uniform angular and spatial arrangement of neighbouring alpha-helices defines a helical structure and creates an amphipathic groove. Multiple-TPR motif proteins would fold into a right-handed super-helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of alpha-helices in the PP5-TPR domain is similar to those within 14-3-3 proteins.
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PMID:The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions. 948 16

Changes in amount and activity of enzyme protein are critical factors in regulating intracellular metabolisms. However, since the metabolisms are proceeding in environment with complex architecture consisted of various membranes, spatial factors should be taken into consideration for the regulation. In this review, involvement of interaction between cytosolic and membrane proteins in metabolic regulation are discussed. It had been reported that hexokinase activity was found in mitochondrial fraction in spite of almost exclusive distribution of other glycolytic enzymes to soluble fraction, the tendency being marked in the brain and many types of tumor cells whereas mitochondrial hexokinase activity was quite low in the liver. Interested in such enzyme and tissue specificities, we investigated the significance and mechanism of the unique intracellular distribution of hexokinase. We found that mitochondria-bound hexokinase was more active than the cytosolic type in producing glucose 6-phosphate (G6P), probably due to the advantage in utilizing ATP produced in mitochondria. In addition, we also found that the binding stabilized hexokinase against G6P inhibition. As to the binding, it was reported that G6P released hexokinase from mitochondria while Mg2+ promoted the binding. In this respect, we found that polyamines promoted the binding at much lower concentration than that of Mg2+, and mitochondria-bound form had small hydrophobic domain at terminal region for the binding to porin on the outer membrane. Then, we found a protease which specifically cleaved the domain with little effect on catalytic activity and molecular size of the bindable form. Such a modifying protease was purified and identified as lysosomal cathepsin L. The protease activity was high in the liver and low in the brain, suggesting that the difference in the activity was responsible for the afore-mentioned tissue specificity. On the other hand, we examined regulatory mechanism for active oxygen production in neutrophils, since the production of superoxide anion (O2-) by NADPH oxidase was very low at the resting state while markedly increased on phagocytosis and chemical stimulation. Since the stimulants for the activation were so various in chemical nature, we postulated mechanism to converge the stimulation to the activation. Incidentally, we found increase in phosphorylation of 46-47 K protein, irrespective of the type of stimulation. Use of inhibitors and examination on the phosphorylation condition indicated protein kinase C (PKC) as the phosphorylating enzyme. In addition, we observed the 46-47 K protein existed in cytosol at resting state, while it was translocated to cell membranes in concurrence with the phosphorylation. Similar findings were obtained in many laboratories and those proteins were named cytosolic activating factors (and then p47-phox, etc.). These proteins associate with membrane proteins to constitutes the active from of NADPH oxidase. Next, we examined mechanism to shut off the O2- production, and found that the inactivation through disassembly of the constituents was attained by dephosphorylation of phosphorylated p47-phox by cytosolic protein phosphatase. Then we have also found that protein kinases other than PKC were involved in regulation of NADPH oxidase activity. Though phosphorylation of p47-phox etc. is deeply involved in the activation of NADPH oxidase, membrane perturbation, so-called priming, is required for the activation. We also reported some possible indications for the priming, and possible involvement of cytoskeletons in O2- production. Apart from protein phosphorylation, it has been reported that amphiphilic acidic compounds are potent activator for NADPH oxidase. We also have examined their effects to find that these compounds also caused the assembly of the NADPH oxidase constituents. Reversely, amphiphilic basic compounds suppressed suggesting significance of introduction of negative charge in NADPH oxidase activat
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PMID:[Cooperation of membrane proteins and cytosolic proteins in metabolic regulation--involvement of binding of hexokinase to mitochondria in regulation of glucose metabolism and association and complex formation between membrane proteins and cytosolic proteins in regulation of active oxygen production]. 992 8

The phosphorylation of p47phox is widely viewed as an important step in the activation of the neutrophil respiratory burst oxidase system. The exact nature of the kinase(s) responsible remains to be elucidated. We show here that such a kinase was detected on neutrophil membranes activated by either PMA or formyl-methionyl-leucyl-phenylalanine. This enzyme is not intrinsic to the neutrophil membrane and could be eluted with 0.5 M NaCl. The kinase activity was partially purified and was found not to be due to the presence of previously suggested kinases, including protein kinase C isotypes, mitogen-activated protein kinase and protein kinase B. Gel filtration and renaturation in substrate gels suggest a molecular mass of between 45 and 51 kDa. The kinase activity was independent of calcium and lipids but was potently inhibited by staurosporine. Treatment with protein phosphatase 2Ac suggested that the kinase was activated by serine/threonine phosphorylation. Phosphopeptide maps indicated that the kinase phosphorylated p47phox on similar sites to those found in vivo. These results indicate that activation of neutrophils by PMA results in the activation of a membrane-associated kinase that may play a part in the regulation of neutrophil NADPH oxidase through its ability to phosphorylate p47phox.
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PMID:Characterization and partial purification of a novel neutrophil membrane-associated kinase capable of phosphorylating the respiratory burst component p47phox. 1002 11

We screened 93 lesion mimic mutants of rice for resistance to the blast fungus, Magnaporthe grisea, and found eight mutants that exhibited significant resistance to the fungus. We called these mutants cdr (cell death and resistance) and further analyzed three of them. Two mutations, cdr1 and cdr2, were recessive and one, Cdr3, was dominant. Many small brownish lesions developed over the entire leaf of the mutants 20-50 days after sowing. TUNEL staining revealed that DNA fragmentation occurred in leaf blade cells of the homozygous Cdr3 mutants. Autofluorescence and callose deposition were visible in leaf cells of these three mutants. Activation of two defense-related genes, PBZ1 and PR1, was observed in the leaves of the mutants; high expression of PBZ1 was correlated with the lesion formation in the three mutants, whereas PR1 was constitutively expressed in the cdr2 and Cdr3 mutants irrespective of the lesion formation. Levels of momilactone A, a major phytoalexin of rice, in these mutants were increased approximately 100-400-fold relative to the wild-type levels. Suspension-cultured cells of the cdr1 and cdr2 but not Cdr3 produced higher levels of H2O2 than the wild type when treated with calyculin A, an inhibitor of protein phosphatase 1. These results suggest that biochemical lesions of cdr1 and cdr2 lie in the early signaling steps leading to activation of the NADPH oxidase and that type-1 protein phosphatase is operative in protein dephosphorylation involved in NADPH oxidase activation.
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PMID:Lesion mimic mutants of rice with alterations in early signaling events of defense. 1020 6

Calyculin A, a protein phosphatase inhibitor, enhanced phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2-) production and translocation of the cytosolic NADPH oxidase factor, p47phox, to the plasma membrane in guinea pig polymorphonuclear leukocytes (PMNs). When PMNs were treated with t-(5-isoquino-line-sulfonyl)-3-methyl-piperazine (H-7), a protein kinase C (PKC) inhibitor, after exposure to PMA, inhibition of O2- production and of translocation of p47phox to the membrane fraction in PMA-stimulated PMNs were observed. When calyculin A was added to the PMA-stimulated PMNs after the addition of H-7, O2- production was again observed, and translocation of p47phox to the membrane fraction also occurred. The activity of NADPH oxidase, the amount of p47phox and the level of phosphorylation of p47phox in the membrane fraction prepared from PMA-stimulated PMNs, were reduced by the addition of the cytosol fraction from unstimulated PMNs. These reductions were attenuated by calyculin A. These results indicate that the active form of NADPH oxidase in PMNs can be reconstituted after the active complex of the enzyme has disappeared once, and that one of the mechanisms of regulation of this enzyme activity involves the phosphorylation of p47phox in the cyotosol and dephosphorylation of phosphorylated p47phox in the NADPH oxidase complex by protein kinase and protein phosphatase, respectively.
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PMID:Participation of cytosolic protein phosphatase in regulation of NADPH oxidase in polymorphonuclear leukocytes. 1040 25


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