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Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes.

ABSTRACT: NADPH oxidase is the enzyme complex responsible for the production of oxygen radicals in phagocytes. On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox and p47phox, as well as the Ras-related G-protein rac 2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b to form a functional complex. Besides rac 2, rac 1 G-protein is also involved in the activation of the NADPH oxidase, but, to date, it has not been documented whether it is also translocated in activated neutrophils. In this paper we show that: (a) in neutrophils stimulated with formylmethionyl-leucylphenylalanine, concanavalin A or phorbol 12-myristate 13-acetate, both rac 1 and rac 2 are translocated from cytosol to the membranes; (b) in neutrophils from a patient with a form of chronic granulomatous disease in which p67phox is absent, rac 2 and p47phox were translocated as in normal neutrophils on stimulation with the above agonists, but rac 1 failed to be translocated from the cytosol to the membranes. This is the first demonstration that, in activated neutrophils, rac 1 is translocated from the cytosol to the membranes and this translocation requires p67phox. These results, coupled with those showing that rac 2 is not translocated in activated neutrophils lacking p47phox [El Benna, Ruedi and Babior (1994) J. Biol. Chem. 269, 6729-6734], may suggest that the assembly of the cytosolic components of NADPH oxidase on the plasma membrane takes place through selective coupling of activated rac 1 and rac 2 with p67phox and p47phox respectively.

SUBMITTER: Dusi S

PROVIDER: S-EPMC1136820 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox.

Project description:On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox, p47phox, p40phox, as well as the Ras-related G-proteins Rac1 and Rac2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b, forming a functional complex responsible for the production of oxygen radicals in phagocytes. In this paper we show that (a) in neutrophils from a patient with a form of chronic granulomatous disease (CGD) in which p67phox is absent, p47phox and Rac2, but not p40phox and Rac1 were translocated from the cytosol to the membrane on stimulation with formylmethionyl-leucylphenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA); (b) in neutrophils from a patient with a form of CGD in which p47phox is absent, p67phox, p40phox and Rac1 failed to associate with the membrane on stimulation with fMLP or PMA, whereas Rac2 was translocated as in normal neutrophils. We also show that in neutrophils from a patient lacking p67phox, the amount of cytosolic p40phox was decreased by about 40%. These findings indicate that, on neutrophil stimulation, p67phox mediates the translocation of p40phox and Rac1 from the cytosol to cell membranes and that Rac2 associates with the membranes independently of p47phox and p67phox.

| S-EPMC1217064 | biostudies-other

The Rac effector p67phox regulates phagocyte NADPH oxidase by stimulating Vav1 guanine nucleotide exchange activity.

Project description:The phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for microbial defense. Electron transport through the oxidase flavocytochrome is activated by the Rac effector p67(phox). Previous studies suggest that Vav1 regulates NADPH oxidase activity elicited by the chemoattractant formyl-Met-Leu-Phe (fMLP). We show that Vav1 associates with p67(phox) and Rac2, but not Rac1, in fMLP-stimulated human neutrophils, correlating with superoxide production. The interaction of p67(phox) with Vav1 is direct and activates nucleotide exchange on Rac, which enhances the interaction between p67(phox) and Vav1. This provides new molecular insights into regulation of the neutrophil NADPH oxidase, suggesting that chemoattractant-stimulated superoxide production can be amplified by a positive feedback loop in which p67(phox) targets Vav1-mediated Rac activation.

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Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox.

Project description:Activation of human neutrophil NADPH oxidase requires the interaction of cytosolic and membrane-associated components. Evidence has been accumulated that in phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils, the translocation to the plasma membrane of the cytosolic components p47phox and p67phox and the phosphorylation of p47phox are essential steps in activation of NADPH oxidase. No direct evidence has been presented to date as to whether p67phox is also phosphorylated. To address this problem we have immunoprecipitated p67phox from neutrophil cytosol and membrane fractions. The results indicate that, very soon after activation with PMA (20 s), p67phox was present in a phosphorylated form in the cytosol and in the membranes. At later times (1-3 min) the extent of p67phox phosphorylation continuously increased both in the cytosol and in the membrane fraction, while oxygen consumption reached the maximal rate within 40 s, and then remained linear. p67phox was also phosphorylated in formyl-methionyl-leucyl-phenylalanine-activated neutrophils. That the phosphorylated p67 protein we identified in immunoprecipitation experiments was p67phox was confirmed by the observation that no phosphorylated band of 67 kDa was immunoprecipitated from the cytosol and membranes of PMA-stimulated neutrophils from a p67phox-deficient chronic granulomatous disease patient. In this case, p47phox was normally phosphorylated. These data demonstrate that: (1) the phosphorylation of p67phox is correlated with activation of NADPH oxidase, and (2) continuous phosphorylation of p67phox is required in order to maintain the linearity of the respiratory burst.

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Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca(2+)-depleted human neutrophils.

Project description:Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.

| S-EPMC1132398 | biostudies-other

PtdIns3P and Rac direct the assembly of the NADPH oxidase on a novel, pre-phagosomal compartment during FcR-mediated phagocytosis in primary mouse neutrophils.

Project description:The generation of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate oxidase is an important mechanism by which neutrophils kill pathogens. The oxidase is composed of a membrane-bound cytochrome and 4 soluble proteins (p67(phox), p40(phox), p47(phox), and GTP-Rac). These components form an active complex at the correct time and subcellular location through a series of incompletely understood mutual interactions, regulated, in part, by GTP/GDP exchange on Rac, protein phosphorylation, and binding to lipid messengers. We have used a variety of assays to follow the spatiotemporal assembly of the oxidase in genetically engineered primary mouse neutrophils, during phagocytosis of both serum- and immunoglobulin G-opsonized targets. The oxidase assembles directly on serum-Staphylococcus aureus-containing phagosomes within seconds of phagosome formation; this process is only partially dependent (∼ 30%) on PtdIns3P binding to p40(phox), but totally dependent on Rac1/2 binding to p67(phox). In contrast, in response to immunoglobulin G-targets, the oxidase first assembles on a tubulovesicular compartment that develops at sites of granule fusion to the base of the emerging phagosome; oxidase assembly and activation is highly dependent on both PtdIns3P-p40(phox) and Rac2-p67(phox) interactions and delivery to the phagosome is regulated by Rab27a. These results define a novel pathway for oxidase assembly downstream of FcR-activation.

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The major phosphorylation site of the NADPH oxidase component p67phox is Thr233.

Project description:Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC-MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.

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Anthracyclines Suppress Both NADPH Oxidase- Dependent and -Independent NETosis in Human Neutrophils.

Project description:Neutrophil extracellular traps (NETs) are cytotoxic DNA-protein complexes that play positive and negative roles in combating infection, inflammation, organ damage, autoimmunity, sepsis and cancer. However, NETosis regulatory effects of most of the clinically used drugs are not clearly established. Several recent studies highlight the relevance of NETs in promoting both cancer cell death and metastasis. Here, we screened the NETosis regulatory ability of 126 compounds belonging to 39 classes of drugs commonly used for treating cancer, blood cell disorders and other diseases. Our studies show that anthracyclines (e.g., epirubicin, daunorubicin, doxorubicin, and idarubicin) consistently suppress both NADPH oxidase-dependent and -independent types of NETosis in human neutrophils, ex vivo. The intercalating property of anthracycline may be enough to alter the transcription initiation and lead NETosis inhibition. Notably, the inhibitory doses of anthracyclines neither suppress the production of reactive oxygen species that are necessary for antimicrobial functions nor induce apoptotic cell death in neutrophils. Therefore, anthracyclines are a major class of drug that suppresses NETosis. The dexrazoxane, a cardioprotective agent, used for limiting the side effects of anthracyclines, neither affect NETosis nor alter the ability of anthracyclines to suppress NETosis. Hence, at correct doses, anthracyclines together with dexrazoxane could be considered as a therapeutic candidate drug for suppressing unwanted NETosis in NET-related diseases.

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Project description:Nitric oxide (NO), generated by NO synthases (NOSs), has multifarious roles in signal transduction. Reactive oxygen species (ROS), generated by ubiquitous NADPH oxidases (NOXs), also participate in cellular signaling. However, the coordination of signals conveyed by NO and ROS is poorly understood. We show that the small GTPase Rac, a component of some NOXs, also interacts with and regulates the constitutively-expressed NOSs. Cellular NO and O(2)(-) production increase or decrease together following activation or inhibition of Rac, and Rac inhibition reveals transduction mechanisms that depend upon NO (vasodilation), ROS (actin polymerization) or both (cytoskeletal organization). Thus, signaling by NO and ROS may be coordinated through a common control element.

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