Phosphorylation of the leucocyte NADPH oxidase subunit p47phox by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity

2001 ◽  
Vol 358 (3) ◽  
pp. 783-790 ◽  
Author(s):  
Hee-Sae PARK ◽  
S. Min LEE ◽  
Jin Hyup LEE ◽  
Yun-Sook KIM ◽  
Young-Seuk BAE ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Conformational Changes ◽  
Oxidase Activity ◽  
Serine Threonine Kinase ◽  
Src Homology 3 ◽  
Oxidase Subunit ◽  
Putative Site ◽  
Src Homology ◽  
Membrane Bound ◽  
Eukaryotic Organisms

The leucocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyses the reduction of oxygen to O−2 at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to the appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47phox becomes phosphorylated on several serines and migrates to the plasma membrane. Protein kinase CK2 is an essential serine/threonine kinase present in all eukaryotic organisms. The leucocyte NADPH oxidase subunit p47phox has several putative CK2 phosphorylation sites. In the present study, we report that CK2 is able to catalyse the phosphorylation of p47phoxin vitro. Phosphoamino acid analysis of phosphorylated p47phox by CK2 indicated that the phosphorylation occurs on serine residues. CNBr mapping and phosphorylation of peptides containing the putative site of CK2 indicated that the main phosphorylated residues are Ser-208 and Ser-283 in the Src homology 3 (SH3) domains, and Ser-348 in the C-terminal domain of p47phox. Dependence of phosphorylation on the conformation of p47phox is supported by the finding that p47phox undergoes better phosphorylation by CK2 in the presence of arachidonic acid, a known activator of NADPH oxidase which induces conformational changes in p47phox. In addition, 5,6-dichloro-1-β-o-ribofuranosyl benzimidazole, a CK2 inhibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in DMSO-differentiated HL-60 cells. Taken together, we propose that CK2 is the p47phox kinase, and that phosphorylation of p47phox by CK2 regulates the deactivation of NADPH oxidase.

scholarly journals p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains

1993 ◽  
Vol 296 (3) ◽  
pp. 557-561 ◽  
Author(s):  
F B Wientjes ◽  
J J Hsuan ◽  
N F Totty ◽  
A W Segal
Keyword(s):  
Nadph Oxidase ◽  
Sequence Similarity ◽  
Granulomatous Disease ◽  
Phagocytic Cells ◽  
Src Homology 3 ◽  
N Terminus ◽  
Phagocyte Oxidase ◽  
Src Homology ◽  
Membrane Bound ◽  
Flavocytochrome B

The NADPH oxidase generates superoxide in phagocytic cells. It is important for immunity and its deficiency leads to chronic granulomatous disease (CGD). It consists of a membrane-bound flavocytochrome b that lies dormant until activated by the translocation to the plasma membrane of cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox and p21rac, a small GTP-binding protein. We show here that a novel component, p40phox, forms an activation complex with p47phox and p67phox with which it translocates to the membrane to associate with the flavocytochrome b. cDNA cloning and amino acid analysis revealed that p40phox has an src homology 3 (SH3) domain and a large region of sequence similarity with the N-terminus of p47phox. The primary association of p40phox appears to be with p67phox, and it is present in reduced amounts in patients with CGD lacking p67phox.

scholarly journals Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox.

1996 ◽  
Vol 184 (3) ◽  
pp. 893-902 ◽  
Author(s):  
S Tsunawaki ◽  
S Kagara ◽  
K Yoshikawa ◽  
L S Yoshida ◽  
T Kuratsuji ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Inhibitory Effect ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Amino Terminal ◽  
Src Homology 3 ◽  
Phagocyte Nadph Oxidase ◽  
A Cell ◽  
Src Homology ◽  
Tight Association

Phagocyte NADPH oxidase, dormant in resting cells, is activated upon cell stimulation to produce superoxide anion, a precursor of microbicidal oxidants. Active NADPH oxidase is found on the membrane as an enzyme complex, composed of membrane-integrated cytochrome b558 (gp91phox and p22phox subunits) and two cytosolic factors (p47phox and p67phox), each of the latter containing two src homology 3 (SH3) domains. Recently, we radioactively identified a third cytosolic factor, p40phox, as a molecule that associates with p67phox in human neutrophils. Although it has been found that this p40phox protein is defective in patients with chronic granulomatous disease (CGD) who lack p67phox, evidence to functionally relate it to the NADPH oxidase system has hitherto been lacking. In this study, we raised separate antibodies against both the COOH- and NH2-terminal polypeptides of p40phox as well as against the COOH-terminal polypeptide of p67phox to examine the mode of interaction between p40phox and p67phox in a complex. The antibody against the COOH terminus of p67phox was able to communoprecipitate p40phox in conjunction with p67phox itself as was expected. Very interestingly, however, the antibody against the COOH terminus of p40phox completely dissociated the p67phox molecule from the p40phox-p67phox complex unit without any detectable coimmunoprecipitation of p67phox, despite their tight association, whereas that against the NH2 terminus of p40phox had absolutely no dissociation effect. Similar results were found regarding their effects on the O2-generating ability of cytosol in a cell-free activation system, i.e., inhibition was noted with the COOH terminus antibody but not with that for the NH2 terminus of p40phox. However, this dissociation did not affect the translocation of the cytosolic components including p47phox to the membrane. Once the NADPH oxidase was activated, the antibody for the COOH terminus did not show any inhibitory effect on catalysis by the activated enzyme. The stimulators of NADPH oxidase, MA and SDS, did not dissociate the p40phox-p67phox complex. These results provide the first demonstration that p40phox is practically involved in the activation of NADPH oxidase through the association of its COOH-terminal, but not its NH2-terminal, with p67phox.

scholarly journals FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3867-3877 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Wenyu Ming ◽  
Chang-Il Suh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Resting Cells ◽  
Cellular Activation ◽  
Nadph Oxidase Activity ◽  
Phagocyte Nadph Oxidase ◽  
Before And After ◽  
Membrane Bound

AbstractThe phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.

scholarly journals Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox

1996 ◽  
Vol 317 (3) ◽  
pp. 919-924 ◽  
Author(s):  
Frans B. WIENTJES ◽  
George PANAYOTOU ◽  
Emer REEVES ◽  
Anthony W. SEGAL
Keyword(s):  
Nadph Oxidase ◽  
Binding Assays ◽  
Phagocytic Cells ◽  
Sh3 Domains ◽  
Cytosolic Proteins ◽  
Src Homology 3 ◽  
Vitro Binding ◽  
Src Homology ◽  
Flavocytochrome B

The NADPH oxidase of neutrophils and other bone-marrow-derived phagocytic cells is a multi-component system consisting of a flavocytochrome b in the plasma membrane and at least four cytosolic proteins. Three of the cytosolic proteins contain src homology 3 (SH3) domains, two each in p47phox and p67phox, and one in p40phox. All three translocate from the cytosol to the flavocytochrome in the membrane upon stimulation of the cells. A small G-protein, p21rac, is also involved in activation of the oxidase. The three cytosolic phox proteins occur as a complex in the cytosol and the strongest interaction appeared to be between p67phox and p40phox. We have investigated the interaction between p40phox and the other two cytosolic phox proteins by in vitro binding assays. An affinity-bead approach was used as well as a biosensor technique (surface plasmon resonance). We observed the strongest attachment between p40phox and p67phox where the binding was between the N-terminal half of p67phox and the C-terminal half of p40phox, and did not appear to involve SH3 domains and proline-rich sequences. p40phox also bound p47phox but more weakly than it did p67phox.

scholarly journals Phosphorylation of the leucocyte NADPH oxidase subunit p47phox by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity

2001 ◽  
Vol 358 (3) ◽  
pp. 783 ◽  
Author(s):  
Hee-Sae PARK ◽  
Su Min LEE ◽  
Jin Hyup LEE ◽  
Yun-Sook KIM ◽  
Young-Seuk BAE ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Oxidase Subunit

scholarly journals Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid

2003 ◽  
Vol 373 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Guihong PENG ◽  
Jin HUANG ◽  
Mellonie BOYD ◽  
Michael E. KLEINBERG
Keyword(s):  
Arachidonic Acid ◽  
Nadph Oxidase ◽  
Sh3 Domain ◽  
Free System ◽  
Cell Free System ◽  
Src Homology 3 ◽  
Rich Domain ◽  
Phagocyte Nadph Oxidase ◽  
Px Domain ◽  
Src Homology

In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b558. In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain. We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region. Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1–guanosine 5′-[γ-thio]triphosphate and neutrophil membranes. Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation. This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids. No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site. Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid. This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain.

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