Activation of p47 , a Cytosolic Subunit of the Leukocyte NADPH Oxidase

Johnson, Jack; Park, Jeen‐Woo; Benna, Jamel El; Faust, LaRosa P.; Inanami, Osamu; Babior, Bernard M.

doi:10.1074/jbc.273.52.35147

Cited by 131 publications

(46 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In an earlier study in which phorbol myristate acetate, an activator of PKC, was used to stimulate the neutrophils, we found that phosphorylation of one of the pairs (S303, S304) together with another of the pairs (S359, S370) was necessary for oxidase activation (19,20). PKC did not phosphorylate S328.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Modulation of p47 ^PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

Hoyal

Gutierrez²,

Young³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

169

128

View full text Add to dashboard Cite

T he antimicrobial activity of phagocytes depends in part on the cells' ability to reduce oxygen to reactive microbicidal oxidants by means of an NADPH oxidase. In resting phagocytes, the NADPH oxidase is in a dormant state, but exposure of the cell to any of a variety of stimuli can activate the enzyme, causing it to release large amounts of O 2 Ϫ by reducing oxygen at the expense of NADPH. The oxidase is activated by the phosphorylation of one of its cytosolic subunits, p47 PHOX , on particular serines (1). In whole cells, the stimulation of an appropriate receptor activates PKC and phosphatidylinositol 3-kinase (PI3-kinase) (2-4). Although NADPH oxidase activation by PKC has been well characterized (5, 6), the association between PI3-kinase and NADPH oxidase activation remains to be established. A possible connection is through the activation of Akt (7), whose role in oxidase activation is suggested by the finding that Akt is activated rapidly when neutrophils are treated with oxidase-activating agents (8) and by the finding that oxidase activation is inhibited by wortmannin (9). The experiments described here show that Akt is able to activate the oxidase by phosphorylating p47 PHOX on serines S304 and S328. Experimental ProceduresMaterials. Active Akt was obtained from Upstate Biotechnology (Lake Placid, NY) or from J.-H.L., whose recombinant material was 95% pure by SDS͞PAGE. [␥-32 P]ATP was purchased from New England Nuclear. Phosphorylated peptides were obtained from Sigma.Isolation and Fractionation of Neutrophils. Neutrophils were obtained from normal subjects by dextran sedimentation and Ficoll͞Hypaque fractionation of freshly drawn citrateanticoagulated blood (10). After treatment on ice for 10-20 min with 5 l of 0.54 M diisopropyl fluorophosphate, the neutrophils, suspended at 10 8 cells per ml in a modified relaxation buffer (0.1 M KCl͞3 mM NaCl͞3.5 mM MgCl 2 ͞10 mM Pipes buffer, pH 7.3), were subjected to nitrogen cavitation. Membranes and cytosol were separated by centrifugation through a Percoll gradient. Aliquots of membrane and cytosol were stored at Ϫ70°C until use.Recombinant Oxidase Components. Recombinant fusion proteins composed of an upstream GST linked to a downstream p47 PHOX , p67 PHOX , or Rac2 were isolated from Escherichia coli transformed with pGEX-6P3 plasmids containing cDNA inserts encoding the downstream proteins. The fusion proteins were isolated by the addition of a prewashed 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences) in PBS (133 mM NaCl͞16 mM Na 2 HPO 4 ͞2.7 mM KCl͞1.5 mM KH 2 PO 4 , pH 7.3). The tube was rotated end-over-end for 1 h at 4°C and then microcentrifuged for 3 min at 200 ϫ g to sediment the glutathione-Sepharose beads. The beads were washed four times with 10 bead volumes of PBS, and the bound fusion proteins were eluted by incubating for 30 min at 4°C with three 1-ml washes of 50 mM Tris⅐HCl, pH 8.0͞20 mM glutathione͞0.2 M NaCl. Excess glutathione was removed from the purified recombinant protein by dialysis against relaxation buffer and conce...

show abstract

Section: Discussionmentioning

confidence: 99%

“…We previously have explored the manipulation of several serines identified as targets for phosphorylation during NADPH oxidase activation (19,20), and, with the current findings, it will be important to explore the functional role of S304 and S328 further.…”

Section: Discussionmentioning

confidence: 99%

Modulation of p47 ^PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

Hoyal

Gutierrez²,

Young³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

169

128

View full text Add to dashboard Cite

show abstract

“…Upon activation, phosphorylation induces a conformational change in p47 phox , which unmasks the tandem SH3 domains, thereby allowing binding to p22 phox and translocation to the membrane. Babior et al (26) have shown that phosphorylation of Ser-359 or Ser-370 is absolutely required for translocation as well as oxidase activity. Interestingly, both of these serines are adjacent to the poly-proline motif, which is the target of the C-terminal SH3 domain of p67 phox , suggesting that phosphorylation might interfere with this interaction and may potentially induce the release of p67 phox rendering this sequence accessible for p40 phox .…”

Section: Discussionmentioning

confidence: 99%

Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Lapouge¹,

Smith²,

Groemping³

et al. 2002

Journal of Biological Chemistry

137

144

View full text Add to dashboard Cite

The phagocyte NADPH oxidase is a multiprotein enzyme whose subunits are partitioned between the cytosol and plasma membrane in resting cells. Upon exposure to appropriate stimuli multiple phosphorylation events in the cytosolic components take place, which induce rearrangements in a number of protein-protein interactions, ultimately leading to translocation of the cytoplasmic complex to the membrane. To understand the molecular mechanisms that underlie the assembly and activation process we have carried out a detailed study of the protein-protein interactions that occur in the p40-p47-p67 phox complex of the resting oxidase. Here we show that this complex contains one copy of each protein, which assembles to form a heterotrimeric complex. The apparent high molecular weight of this complex, as observed by gel filtration studies, is due to an extended, non-globular shape rather than to the presence of multiple copies of any of the proteins. Isothermal titration calorimetry measurements of the interactions between the individual components of this complex demonstrate that p67 phox is the primary binding partner of p47 phox in the resting state. These findings, in combination with earlier reports, allow us to propose a model for the architecture of the resting complex in which p67 phox acts as the bridging molecule that connects p40 phox and p47 phox .

show abstract

“…2A). Interestingly, the homologue was missing the proline-proline-argininearginine-containing region of p47 phox , which is involved in autoinhibition (17) through binding to the N-terminal SH3 domain, as well as the adjacent serine phosphorylation sites (18), which relieve autoinhibition when phosphorylated (19). This suggested a stimulus-independent activity of the homologue.…”

Section: Cloning Of Mouse P47mentioning

confidence: 99%

Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1

Bánfi¹,

Clark²,

Steger³

et al. 2003

Journal of Biological Chemistry

441

369

View full text Add to dashboard Cite

NOX1, an NADPH oxidase expressed predominantly in colon epithelium, shows a high degree of similarity to the phagocyte NADPH oxidase. However, superoxide generation by NOX1 has been difficult to demonstrate. Here we show that NOX1 generates superoxide when co-expressed with the p47 phox and p67 phox subunits of the phagocyte NADPH oxidase but not when expressed by itself. Since p47 phox and p67 phox are restricted mainly to myeloid cells, we searched for their homologues and identified two novel cDNAs. The mRNAs of both homologues were found predominantly in colon epithelium. Differences between the homologues and the phagocyte NADPH oxidase subunits included the lack of the autoinhibitory domain and the protein kinase C phosphorylation sites in the p47 phox homologue as well as the absence of the first Src homology 3 domain and the presence of a hydrophobic stretch in the p67 phox homologue. Co-expression of NOX1 with the two novel proteins led to stimulus-independent high level superoxide generation. Stimulus dependence of NOX1 was restored when p47 phox was used to replace its homologue. In conclusion, NOX1 is a superoxide-generating enzyme that is activated by two novel proteins, which we propose to name NOXO1 (NOX organizer 1) and NOXA1 (NOX activator 1).Superoxide generation by phagocytes plays a crucial role in the elimination of invading microorganisms. It is catalyzed by the phagocyte NADPH oxidase, an enzyme consisting of two transmembrane subunits, p22 phox and gp91 phox , and at least three cytosolic subunits, p47 phox , p67 phox , and Rac2 (1). Upon activation, the NADPH oxidase subunits assemble, and electrons are transported from intracellular NADPH to extracellular oxygen by the flavo-heme gp91 phox subunit (2). Recently six gp91 phox homologues have been described in mammals: NOX1 1 (3, 4), NOX3 (5, 6), and NOX4 (7, 8) with an overall structure similar to gp91 phox (alias NOX2), NOX5 with an N-terminal EF hand-containing extension (9), and DUOX1 and DUOX2 with an additional peroxidase homology domain (10 -12). NOX1 is found mainly in colon epithelium (3, 4); NOX3 in embryonic kidney (5, 6), NOX4 in the kidney cortex (7, 8), NOX5 in lymphoid organs and testis (9), DUOX1 in thyroid and lung, and DUOX2 in thyroid and colon (10 -12).Based on their primary structure all members of the NOX/ DUOX family should be flavo-heme electron transporters. However, it is not established whether all NOX enzymes transfer electrons to oxygen or whether some of them may use other electron acceptors as has been shown for a yeast homologue of gp91phox that functions as a ferric reductase (13). Among NOX enzymes, only gp91 phox and NOX5 have appeared capable of generating large amounts of superoxide, both of them in a stimulus-dependent manner (1, 9).Based on data gained with NOX1-transfected NIH 3T3 cell clones NOX1 has been suggested to be a subunit-independent, low capacity superoxide-generating enzyme involved in the regulation of mitogenesis (4, 14). However, we have not been able to measure any superoxide generat...

show abstract

Activation of p47 , a Cytosolic Subunit of the Leukocyte NADPH Oxidase

Abstract: The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O 2 . ) at the expense of PHOX to the membrane at some point after the first phosphorylation takes place.

Cited by 131 publications

References 34 publications

Modulation of p47 ^PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

Modulation of p47 ^PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1

Contact Info

Product

Resources

About

Activation of p47 , a Cytosolic Subunit of the Leukocyte NADPH Oxidase

Abstract: The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O 2 . ) at the expense of PHOX to the membrane at some point after the first phosphorylation takes place.

Cited by 131 publications

References 34 publications

Modulation of p47 PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

Modulation of p47 PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

Architecture of the p40-p47-p67 Complex in the Resting State of the NADPH Oxidase

Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1

Contact Info

Product

Resources

About

Modulation of p47 ^PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

Modulation of p47 ^PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase