Affinage

NCF2

Neutrophil cytosol factor 2 · UniProt P19878

Length
526 aa
Mass
59.8 kDa
Annotated
2026-04-29
100 papers in source corpus 38 papers cited in narrative 38 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NCF2 encodes p67phox, an essential cytosolic activator of the phagocyte NADPH oxidase (NOX2) that functions as the central scaffold of the resting p40phox–p47phox–p67phox heterotrimer and as the direct catalytic activator of electron transfer from NADPH to the FAD center of flavocytochrome b558 (PMID:1512217, PMID:7896790). Its N-terminal TPR domain binds GTP-loaded Rac2 (preferentially over Rac1), inducing a conformational change in the activation domain (centered on Val204) that stimulates oxidase catalysis, while its C-terminal SH3 domain anchors the trimer via interaction with p47phox and its PB1 domain mediates interaction with p40phox and the guanine nucleotide exchange factor Vav1 (PMID:11090627, PMID:9642219, PMID:7890694, PMID:22203994). p67phox lacks intrinsic membrane-targeting capacity and depends on p47phox for plasma-membrane/phagosomal recruitment and on p40phox for endosomal recruitment; upon stimulation, it is phosphorylated by PKC, ERK2, p38 MAPK, and PKCδ on multiple serine/threonine residues including Thr233, modulating oxidase assembly and activity (PMID:17122360, PMID:9202043, PMID:12693948, PMID:15591124, PMID:9931304). Loss-of-function mutations in NCF2 cause autosomal recessive chronic granulomatous disease (CGD), and coding variants that impair Rac2 or Vav1 binding are linked to very early onset inflammatory bowel disease and systemic lupus erythematosus susceptibility (PMID:8879195, PMID:21900546, PMID:22203994).

Mechanistic history

Synthesis pass · year-by-year structured walk · 20 steps
  1. 1992 High

    Establishing that p67phox is an indispensable component for reconstituting NADPH oxidase activity resolved the question of whether all four cytosolic/membrane factors are individually required, positioning p67phox as a direct catalytic activator rather than an accessory factor.

    Evidence Cell-free reconstitution with purified recombinant p67phox, p47phox, Rac1, and cytochrome b-245

    PMID:1512217

    Open questions at the time
    • Mechanism of p67phox activation of electron transfer unknown
    • Which domain(s) of p67phox are catalytically essential not yet defined
  2. 1994 High

    Identification of p67phox as the direct effector of Rac GTPase answered which oxidase subunit reads the Rac activation signal, establishing the Rac–p67phox axis as the trigger for oxidase assembly.

    Evidence Co-immunoprecipitation with GTP-dependence and Rac effector-site mutagenesis; two-hybrid confirmation with Rac2 preference

    PMID:8036496 PMID:8550629

    Open questions at the time
    • Structural basis of Rac–p67phox recognition unknown
    • Whether Rac2 selectivity matters in vivo not tested
  3. 1994 High

    Demonstrating that p67phox translocation to membranes requires p47phox, while p67phox itself carries p40phox and Rac1 to the membrane, defined a hierarchical assembly pathway in which p67phox acts as the central scaffold bridging upstream carriers and downstream effectors.

    Evidence Subcellular fractionation of stimulated normal and CGD (p47phox-deficient and p67phox-deficient) neutrophils

    PMID:8120032 PMID:8670049

    Open questions at the time
    • Whether p40phox provides an alternative membrane-targeting route not yet explored
    • Phagosomal versus plasma membrane targeting not distinguished
  4. 1994 High

    Mapping the domain architecture of the p67phox-centered trimer — SH3 domains required for membrane association and activity, C-terminal SH3 binding p47phox, inter-SH3 region binding p40phox — established p67phox as the central bridging molecule of the resting cytosolic complex.

    Evidence SH3 deletion mutagenesis in reconstituted CGD B-cells; two-hybrid and in vitro binding with domain constructs; co-IP of native p40phox–p67phox complex

    PMID:7890694 PMID:8147882 PMID:8206939

    Open questions at the time
    • Stoichiometry and shape of native complex not determined
    • Whether SH3 domains contact membrane targets directly unknown
  5. 1995 High

    Showing that p67phox is specifically required for NADPH→FAD electron transfer while p47phox is additionally needed for FAD→heme transfer dissected the catalytic roles of individual cytosolic subunits for the first time.

    Evidence Cell-free oxidase reconstitution with CGD patient cytosol selectively depleted of each subunit; spectrophotometric electron-flow assays

    PMID:7896790

    Open questions at the time
    • Whether p67phox directly contacts the FAD-binding site of gp91phox unknown
    • Structural basis for activation domain function not defined
  6. 1996 High

    Discovery of an NADPH-binding site on p67phox itself suggested a substrate-channeling or regulatory role beyond simple protein–protein scaffolding, adding catalytic complexity to the activation mechanism.

    Evidence NADPH dialdehyde affinity labeling of purified recombinant p67phox; competition with NADPH; cell-free rescue assay

    PMID:8770870

    Open questions at the time
    • Physiological significance of p67phox NADPH binding versus gp91phox NADPH binding not resolved
    • No structural data for NADPH-binding site
  7. 1996 High

    A CGD patient mutation (ΔLys58) that disrupts p67phox–Rac1 interaction and abolishes superoxide production provided the first human genetic proof that the Rac–p67phox interaction is essential for oxidase function in vivo.

    Evidence Patient genetic analysis; co-IP of mutant p67phox with Rac1; membrane translocation and oxidase activity in patient neutrophils

    PMID:8879195

    Open questions at the time
    • Full spectrum of CGD-causing NCF2 mutations not catalogued
    • Whether partial loss of Rac binding causes intermediate phenotypes unknown
  8. 1997 High

    Identification of PKC-dependent and PKC-independent phosphorylation of p67phox on serine residues upon neutrophil activation opened the question of how post-translational modifications regulate oxidase assembly dynamics.

    Evidence 32P-labeling of intact neutrophils; 2D tryptic peptide mapping; in vitro PKC kinase assay

    PMID:9202043

    Open questions at the time
    • Specific phosphorylation sites not yet mapped
    • Functional consequences of phosphorylation on oxidase activity not tested
  9. 1998 High

    Identification of an activation domain (residues ~199–210, Val204 critical) that is essential for catalysis independently of Rac or p47phox binding defined the minimal catalytic surface on p67phox and provided a dominant-negative tool.

    Evidence Systematic truncation and single amino acid mutagenesis; cell-free oxidase reconstitution; dominant-negative competition assay

    PMID:9642219

    Open questions at the time
    • How Val204 region contacts flavocytochrome b558 structurally unknown
    • Whether activation domain undergoes conformational change upon Rac binding not shown
  10. 1999 High

    Mapping Thr233 as the major phosphorylation site and identifying MAP kinase as the responsible kinase provided a specific molecular handle for studying phospho-regulation of p67phox.

    Evidence Phosphopeptide mapping by CNBr digestion/HPLC-MS; Thr233Ala mutagenesis; in vitro kinase assay

    PMID:9931304

    Open questions at the time
    • Functional consequence of Thr233 phosphorylation on oxidase activation not determined
    • Additional phosphorylation sites not yet mapped
  11. 2000 High

    The crystal structure of the p67phox TPR domain–Rac·GTP complex at 2.7 Å revealed a novel Rho effector recognition mode mediated by an insertion between TPR motifs, explaining GTPase specificity and providing a structural basis for CGD mutations in this region.

    Evidence X-ray crystallography of Rac·GTP–p67phox(1–203) complex

    PMID:11090627

    Open questions at the time
    • Full-length p67phox structure not available
    • How Rac binding transmits conformational change to the activation domain structurally unresolved
  12. 2001 High

    The 1.8 Å structure of the active N-terminal fragment revealed the four-TPR fold, mapped CGD mutations G78E and A128V to the hydrophobic core, and identified a C-terminal helix (187–193) as part of the activation domain, connecting structure to disease-causing loss of function.

    Evidence X-ray crystallography; temperature-shift cell-free oxidase assay with CGD mutants

    PMID:11262407

    Open questions at the time
    • Structure of activation domain in contact with flavocytochrome b558 not determined
    • No structure of full-length p67phox
  13. 2002 High

    Biophysical characterization of the 1:1:1 p40phox–p67phox–p47phox heterotrimer with p67phox as the central bridge, combined with chimeric Rac–p67phox demonstrating Rac's dual role as membrane anchor and conformational activator, provided an integrated model of oxidase assembly.

    Evidence ITC, gel filtration, analytical ultracentrifugation for trimer; cell-free reconstitution with prenylated p67phox–Rac1 chimeras

    PMID:11796733 PMID:11896062

    Open questions at the time
    • Structure of complete ternary cytosolic complex not solved
    • Kinetics of assembly in intact cells not measured
  14. 2003 High

    Demonstration that ERK2 phosphorylates the N-terminal region and p38MAPK the C-terminal region of p67phox, with a cryptic C-terminal site unmasked by domain rearrangement, revealed compartmentalized phospho-regulation linked to intramolecular conformational control.

    Evidence In vitro kinase assays with truncation constructs; MEK1/2 and p38MAPK inhibitors in intact neutrophils

    PMID:12693948

    Open questions at the time
    • Identity of C-terminal phosphorylation sites not mapped
    • Whether phosphorylation at these sites regulates oxidase activity directly not tested
  15. 2003 High

    Showing that p47phox is required for stable phagosomal recruitment of p67phox and that flavocytochrome b558 is needed for retention of both subunits on nascent phagosomes extended the assembly hierarchy to the phagosome compartment.

    Evidence Synchronized phagocytosis with immunofluorescence in CGD patient neutrophils

    PMID:14623873

    Open questions at the time
    • Role of p40phox in phagosomal targeting of p67phox not assessed here
    • Whether retention mechanism involves direct p67phox–cytochrome b558 interaction unknown
  16. 2004 High

    Identification of PKCδ as a direct kinase for p67phox in monocytes, with functional consequences for superoxide production, expanded the kinase network regulating p67phox beyond conventional PKC and MAPKs.

    Evidence Co-IP of PKCδ–p67phox; in vitro kinase assay; PKCδ antisense knockdown; rottlerin inhibitor; superoxide assay

    PMID:15591124

    Open questions at the time
    • PKCδ phosphorylation sites on p67phox not identified
    • Whether PKCδ regulates p67phox translocation or catalytic activation not distinguished
  17. 2006 High

    Live-cell imaging established that p67phox has no intrinsic membrane-targeting capacity and reaches the plasma membrane via p47phox and endosomes via p40phox, resolving the question of how p67phox accesses distinct membrane compartments.

    Evidence GFP-p67phox live imaging with PX and PB1 domain mutations in COS-7 cells

    PMID:17122360

    Open questions at the time
    • Whether p40phox-mediated endosomal route is essential for antimicrobial ROS not tested in phagocytes
    • Contribution of Rac-mediated membrane tethering in intact cells not assessed
  18. 2011 High

    Linking the NCF2 PB1-domain variant H389Q to reduced Vav1 binding and impaired FcγR-stimulated ROS production connected a lupus-risk allele to a specific defect in the Vav1-dependent arm of oxidase activation.

    Evidence Site-directed mutagenesis at position 389; computational modeling; FcγR-stimulated ROS assay

    PMID:22203994

    Open questions at the time
    • In vivo relevance in patient cells not demonstrated
    • Whether other PB1-domain partners are affected not tested
  19. 2012 High

    Demonstrating that p53 directly activates NCF2 transcription revealed a link between tumor-suppressor signaling and oxidase-derived ROS, with NCF2 knockdown reducing ROS and enhancing apoptosis.

    Evidence ChIP of p53 on NCF2 promoter; luciferase reporter; siRNA knockdown with ROS and apoptosis readouts

    PMID:23187810

    Open questions at the time
    • Whether p53-driven NCF2 expression is relevant in phagocytes or primarily in tumor cells not clarified
    • Downstream ROS targets mediating survival not identified
  20. 2019 High

    Identification of M. tuberculosis PPE2 as a direct p67phox-binding virulence factor that blocks oxidase assembly exemplified pathogen subversion of the Rac–p67phox activation axis.

    Evidence Co-IP of PPE2–p67phox; Trp236Ala mutagenesis abolishing binding; membrane translocation and ROS assays in macrophages

    PMID:31375544

    Open questions at the time
    • Which domain of p67phox is targeted by PPE2 not mapped
    • In vivo significance during TB infection not established

Open questions

Synthesis pass · forward-looking unresolved questions
  • A full-length structure of p67phox in complex with flavocytochrome b558 is lacking, leaving the precise contact surface through which the activation domain stimulates FAD reduction unresolved; the functional consequences of individual phosphorylation events and the integrated regulatory logic of multi-site phosphorylation remain to be determined.
  • No full-length p67phox structure or p67phox–cytochrome b558 complex structure available
  • Functional roles of individual phosphorylation sites (beyond Thr233) on oxidase kinetics not characterized
  • How intramolecular conformational changes couple Rac binding to activation domain rearrangement structurally unresolved

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 4 GO:0098772 molecular function regulator activity 3
Localization
GO:0005829 cytosol 3 GO:0005886 plasma membrane 3 GO:0031410 cytoplasmic vesicle 1
Pathway
R-HSA-168256 Immune System 6 R-HSA-162582 Signal Transduction 4
Partners
CYBBNCF1NCF4PRKCQRAC1RAC2S100A8VAV1
Complex memberships
NADPH oxidase (NOX2 complex)p40phox–p67phox–p47phox cytosolic heterotrimer

Evidence

Reading pass · 38 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1992 p67phox (NCF2 gene product) is one of four components required to fully reconstitute NADPH oxidase activity in a cell-free system; no activity is observed in its absence, establishing it as an essential cytosolic activator of flavocytochrome b-245. Cell-free reconstitution with purified recombinant p67phox, p47phox, p21rac1, and cytochrome b-245 The Journal of biological chemistry High 1512217
1994 Rac1 (GTP-bound form) interacts directly with p67phox in a GTP-dependent manner; effector-site mutations in Rac that abolish NADPH oxidase activation also abolish binding to p67phox, identifying p67phox as the Rac effector in the NADPH oxidase complex. Co-immunoprecipitation, GTP-dependence assay, Rac effector-site mutagenesis, cell-free oxidase activity Science High 8036496
1994 p67phox and p47phox translocate from cytosol to the plasma membrane/submembranous actin cytoskeleton upon neutrophil activation; translocation of p67phox and Rac2 to the cytoskeleton requires p47phox, but Rac2 translocation to the membrane can occur independently of p67phox. Subcellular fractionation of activated normal and CGD (p47phox-deficient) neutrophils, immunoblotting The Journal of biological chemistry High 8120032
1994 Rac2 translocates to membranes independently of p47phox and p67phox but depends on flavocytochrome b558 for maximal membrane stability; Rac2 membrane binding is reduced ~75% in CGD neutrophils lacking gp91phox. Cell-free translocation assay with normal and CGD neutrophil membranes/cytosols; quantitative immunoblotting of Rac1 and Rac2 in intact stimulated CGD neutrophils The Journal of biological chemistry High 7982999
1994 Deletion of either SH3 domain of p67phox dramatically reduces NADPH oxidase activity and membrane binding in intact B-cells; deletion of both SH3 domains completely abolishes membrane binding and oxidase activity, demonstrating that the SH3 domains are required for translocation and activity in whole cells but not in the cell-free system. Stable transfection of p67phox-deficient CGD B-cells with SH3 deletion mutants; whole-cell and cell-free oxidase assays The Journal of biological chemistry High 8206939
1994 p40phox forms a tight 1:1 complex with p67phox in neutrophil cytosol; p40phox is absent in CGD patients lacking p67phox, indicating that its stability depends on p67phox. Co-immunoprecipitation and column chromatography of neutrophil cytosol; gel analysis of CGD patient samples Biochemical and biophysical research communications High 8147882
1995 p67phox (not p47phox) is required for electron flow from NADPH to the flavin (FAD) center of the oxidase; p47phox is additionally required for electron transfer beyond the flavin to the heme in cytochrome b-245 and thence to oxygen. Cell-free oxidase reconstitution with individually depleted cytosol fractions from CGD patients; spectrophotometric electron-flow assays The Journal of biological chemistry High 7896790
1995 p40phox interacts with p67phox via the C-terminal SH3 domain of p40phox binding to p47phox, and via the C-terminal portion of p40phox binding to the region between the two SH3 domains of p67phox; p40phox also binds p47phox but more weakly. Affinity-bead pulldown, surface plasmon resonance (Biacore) with truncation constructs The Biochemical journal High 8760383
1995 Two-hybrid and in vitro binding studies show p67phox interacts with p47phox via the C-terminal SH3 domain of p67phox binding to the polyproline motif of p47phox; p40phox interacts with p67phox via the region between p67phox's two SH3 domains, not via SH3-polyproline interactions. Yeast two-hybrid system; in vitro binding with domain constructs The Journal of biological chemistry High 7890694
1996 p67phox interacts with Rac2 approximately 6-fold more strongly than with Rac1 in yeast two-hybrid assays; Rac effector-site mutants inactive in NADPH oxidase lose interaction with p67phox, and an activating Rac2 mutant shows increased affinity for p67phox. Yeast two-hybrid system; cell-free oxidase activity The Journal of biological chemistry High 8550629
1996 p67phox contains an NADPH-binding site (identified by NADPH dialdehyde crosslinking and competition with NADPH); this site participates in catalysis by the leukocyte NADPH oxidase. NADPH dialdehyde affinity labeling of cytosol and purified recombinant p67phox; cell-free oxidase reconstitution rescue assay The Journal of clinical investigation High 8770870
1996 p67phox mediates translocation of p40phox and Rac1 from cytosol to membrane upon neutrophil stimulation; in p67phox-deficient CGD neutrophils, p40phox and Rac1 fail to translocate, whereas Rac2 and p47phox translocate normally. Subcellular fractionation and immunoblotting of stimulated normal and CGD (p67phox-deficient and p47phox-deficient) patient neutrophils The Biochemical journal High 8670049
1996 A deletion of Lys58 in p67phox found in a CGD patient disrupts the interaction between p67phox and p21-Rac1, prevents translocation of p47phox and p67phox to the plasma membrane upon stimulation, and abolishes superoxide production — demonstrating that p67phox–Rac1 interaction is essential for oxidase complex assembly. CGD patient genetic analysis; co-immunoprecipitation of p67phox-Rac1; membrane translocation assay in patient neutrophils The Journal of experimental medicine High 8879195
1997 p67phox is phosphorylated on serine residues upon neutrophil activation by fMLP and PMA; PKC phosphorylates p67phox at the same tryptic peptide site in vitro and in intact cells via a PKC-dependent pathway; an additional PKC-independent pathway also phosphorylates p67phox. 32P-labeling of intact neutrophils; phosphoamino acid analysis; 2D tryptic peptide mapping; in vitro kinase assay with PKC and GST-p67phox The Journal of biological chemistry High 9202043
1997 Rac1 effector region (residues 26–45) mutations increase Kd for p67phox binding, showing this region mediates direct Rac1–p67phox interaction; insert region mutations (residues 124–135) elevate EC50 for oxidase activity without affecting p67phox binding, suggesting the insert region interacts with another component (possibly cytochrome b558). Fluorescence (MANT-GTP) direct binding assay; steady-state kinetic NADPH oxidase reconstitution; site-directed Rac1 mutagenesis The Journal of biological chemistry High 9228059
1998 An activation domain in p67phox (residues ~199–210, especially Val204) is essential for NADPH oxidase activity independently of p47phox or Rac binding; V204A mutation completely abolishes activity and acts as a dominant-negative by competing with wild-type p67phox for membrane translocation. C-terminal truncation series; single amino acid mutagenesis; cell-free oxidase reconstitution; dominant-negative competition assay; membrane translocation assay The Journal of biological chemistry High 9642219
1998 C-terminal truncations of both p47phox and p67phox relieve autoinhibition, allowing amphiphile-independent NADPH oxidase activation in vitro; the C-terminal regions of both proteins function as negative regulatory elements, and the N-terminal SH3 domain of p47phox is normally masked by intramolecular interaction with its own C-terminus. Cell-free oxidase activation with truncated recombinant proteins; p22phox binding assay The Journal of biological chemistry High 9461621
1998 The C-terminal SH3 domain of p67phox and the polyproline motif of p40phox constitute a PC motif-based interaction interface; mutagenesis of the PC motif in p40phox abolishes binding to p67phox. Yeast two-hybrid screening; in vitro binding with recombinant proteins; site-directed mutagenesis of PC motif European journal of biochemistry High 9490029
1998 Rac1 binds to p67phox at residues 170–199 (N-terminal region); deletion of the C-terminal SH3 domain or polyproline motif of p67phox increases Rac1 binding ~8-fold, revealing a cryptic Rac-binding site; PAK kinase phosphorylates p67phox adjacent to the Rac-binding site, and this phosphorylation is enhanced by C-terminal SH3 or polyproline deletion. Pulldown binding assays with p67phox truncations/deletions; in vitro PAK kinase assay; NADPH oxidase inhibition assay The Journal of biological chemistry Medium 9624165
1999 The major phosphorylation site of p67phox is Thr233; mutagenesis of Thr233 to Ala abolishes in vitro phosphorylation; this site is phosphorylated by MAP kinase and cytosol in vitro and in stimulated neutrophils. Phosphopeptide mapping by cyanogen bromide digestion and HPLC-MS; Thr233Ala mutagenesis; in vitro kinase assays The Biochemical journal High 9931304
2000 Crystal structure of the N-terminal TPR domain of p67phox bound to Rac·GTP at 2.7 Å reveals a novel Rho effector interaction mode in which an insertion between TPR motifs mediates complex formation and GTPase specificity. X-ray crystallography of Rac·GTP–p67phox TPR domain complex Molecular cell High 11090627
2001 Crystal structure of the active N-terminal fragment of p67phox (residues 1–213) at 1.8 Å resolution reveals four TPR motifs with the C-terminus folding back into a hydrophobic groove; an additional short C-terminal helix (residues 187–193) likely constitutes part of the activation domain; CGD mutations G78E and A128V map to this region, with A128V exhibiting temperature-sensitive activity. X-ray crystallography; temperature-shift cell-free oxidase reconstitution with CGD mutants The Journal of biological chemistry High 11262407
2002 In the resting cytosolic complex, p67phox acts as the central bridging molecule that connects p40phox and p47phox; the complex contains one copy of each protein forming a 1:1:1 heterotrimer with an extended non-globular shape; isothermal titration calorimetry shows p67phox is the primary binding partner of p47phox in the resting state. Gel filtration; isothermal titration calorimetry (ITC); analytical ultracentrifugation The Journal of biological chemistry High 11796733
2002 A prenylated chimera of p67phox(1–212) fused to full-length Rac1, when loaded with GTP, activates NADPH oxidase in the absence of p47phox and amphiphile by spontaneously associating with membranes, demonstrating that Rac serves as a membrane-targeting carrier for p67phox and that intrachimeric p67phox–Rac interaction induces a conformational change in the p67phox activation domain. Cell-free NADPH oxidase reconstitution with prenylated chimeric proteins; membrane association assay The Journal of biological chemistry High 11896062
2003 ERK2 phosphorylates p67phox primarily in its N-terminal fragment (residues 1–243), while p38MAPK phosphorylates sites in the C-terminal fragment (residues 244–526); a C-terminal phosphorylation site is cryptic in the intact protein due to an intramolecular interaction with the N-terminal TPR-rich region; both kinases phosphorylate p67phox in fMLP- and PMA-stimulated neutrophils. In vitro kinase assays with ERK2 and p38MAPK; phosphopeptide mapping; truncation constructs; MEK1/2 and p38MAPK inhibitors in intact neutrophils Biochemistry High 12693948
2004 Rac has a dual role in NADPH oxidase assembly: (1) tethering p67phox to the membrane via Rac's C-terminal basic residues, and (2) inducing an activating conformational change in p67phox (via intrachimeric p67phox–Rac interaction); inactive Rac moiety chimeras can be rescued by exogenous Rac1-GTP but not by exogenous p67phox, demonstrating the two functions are separable. Cell-free NADPH oxidase reconstitution with p67phox(1–212)–Rac1 chimeras bearing site-directed mutations in activation domain, Rac effector region, and C-terminal basic residues The Journal of biological chemistry High 14761978
2004 PKCdelta forms a complex with p67phox in activated monocytes and directly phosphorylates p67phox in vitro; rottlerin (PKCdelta inhibitor) and PKCdelta antisense both block p67phox phosphorylation and reduce superoxide production, identifying PKCdelta as a regulator of p67phox in human monocytes. Co-immunoprecipitation of PKCdelta–p67phox; in vitro kinase assay with recombinant PKCdelta; PKCdelta antisense knockdown; selective inhibitor studies; superoxide assay Journal of leukocyte biology High 15591124
2005 S100A8 (but not S100A9) interacts with p67phox and Rac in vitro; S100A8/A9 promotes NADPH oxidase activation by transferring arachidonic acid to the complex; S100A9 knockdown in NB4 cells and S100A9-/- mouse neutrophils show impaired oxidase activity, confirming functional relevance. Protein-protein interaction studies (pulldown); cell-free oxidase assay with S100A8/A9 mutant unable to bind arachidonic acid; S100A9 antibody neutralization; S100A9-/- mouse neutrophils; S100A9 siRNA in NB4 cells FASEB journal High 15642721
2006 p67phox depends on p40phox (via PX domain-phosphatidylinositol 3-phosphate interaction) or p47phox (via PX domain-plasma membrane interaction) for membrane targeting; GFP-p67phox expressed alone does not translocate to membranes; mutations disrupting p67phox binding to p40phox or p47phox abolish p67phox translocation to their respective membrane compartments. Live-cell GFP imaging of translocation; mutations in PX and PB1 domains; co-expression in COS-7 cells Molecular biology of the cell High 17122360
2008 PKC phosphorylates gp91phox/NOX2 in stimulated neutrophils; this phosphorylation enhances diaphorase activity of the NOX2 flavoprotein domain and increases its binding to Rac2, p67phox, and p47phox, indicating a novel mechanism of NADPH oxidase regulation through phosphorylation-enhanced assembly. 32P-labeling of intact neutrophils; in vitro PKC kinase assay on recombinant NOX2 domain; 2D tryptic peptide mapping; co-IP pulldown after phosphorylation; diaphorase activity assay FASEB journal High 19028840
2009 SAXS analysis shows p67phox in solution is a very elongated multidomain protein with semi-flexible linkers, adopting a beads-on-a-string arrangement with evidence of intramolecular interactions that constrain the overall conformation; this architecture underlies its ability to interact simultaneously with all other NADPH oxidase components. Small-angle X-ray scattering (SAXS) of full-length p67phox Journal of structural biology High 19723583
2011 A naturally occurring NCF2 missense mutation H389Q in the PB1 domain reduces binding efficiency to the Vav1 guanine nucleotide exchange factor and causes a ~2-fold decrease in ROS production stimulated via FcγR-elicited (Vav-dependent) pathway, linking this SLE-risk variant to a specific loss of Vav1-dependent oxidase activation. Computational modeling of NCF2–Vav1 interaction; site-directed mutagenesis at position 389; FcγR-stimulated ROS production assay Proceedings of the National Academy of Sciences of the United States of America High 22203994
2011 A rare NCF2 missense variant found in patients with very early onset IBD reduces binding of p67phox to RAC2, providing a direct mechanistic link between partial oxidase impairment and intestinal inflammation. Direct sequencing; functional binding assay of variant p67phox–RAC2 interaction Gut Medium 21900546
2012 NCF2/p67phox is a direct transcriptional target of p53; p53 binds to the NCF2 promoter in vivo; NCF2 knockdown by siRNA reduces ROS production and stimulates cell death, indicating p53-driven NCF2 expression generates ROS that promote cell survival against apoptosis. Microarray identification; luciferase reporter assay; chromatin immunoprecipitation (ChIP); siRNA knockdown with ROS and apoptosis readouts Cell cycle High 23187810
1999 IFN-γ-induced transcription of NCF2 (p67phox) requires cooperative recruitment of PU.1, IRF1, and ICSBP to a specific NCF2 cis element; this transcription factor complex then recruits the coactivator CBP (CREB-binding protein) to activate NCF2 transcription. Promoter deletion and mutation analysis; gel shift assays; reporter gene assays; co-IP of transcription factor complex with CBP Journal of immunology High 10570299
2015 TLR4/TLR2 signaling activates an IRAK–ERK pathway that drives ERK–p67phox interaction, p67phox translocation to membrane, and p67phox–Nox2 interaction, resulting in ROS generation that mediates IL-1β transcription and processing; disruption at any node (IRAK, ERK, p67phox, or Nox2 siRNA) blocks IL-1β production. Co-IP of ERK–p67phox and p67phox–Nox2 complexes; siRNA knockdown of Nox2; pharmacological inhibitors; membrane translocation assay; IL-1β reporter and ELISA Cellular & molecular immunology Medium 26320741
2019 M. tuberculosis PPE2 protein directly interacts with p67phox via its Trp236 residue; this interaction prevents p67phox and p47phox translocation to the membrane and reduces NADPH oxidase activity; Trp236Ala PPE2 mutant loses both p67phox binding and inhibition of ROS production. Co-IP of PPE2–p67phox; site-directed mutagenesis (Trp236Ala); membrane translocation assay; ROS quantification in macrophages; bacterial survival assay Journal of immunology High 31375544
2003 p67phox translocation to forming phagosomes is deficient in neutrophils lacking p47phox, demonstrating that p47phox is required for stable phagosomal recruitment of p67phox; in X-linked CGD neutrophils lacking flavocytochrome b558, both p47phox and p67phox accumulate transiently but are shed from nascent phagosomes once particles are internalized, showing flavocytochrome b558 is required for stable retention of p67phox at the phagosome. Synchronized phagocytosis assay with immunofluorescence microscopy; immunoblotting of phagosome fractions from CGD patient neutrophils The Journal of biological chemistry High 14623873

Source papers

Stage 0 corpus · 100 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1994 Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity. Science (New York, N.Y.) 340 8036496
2003 Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases. The Journal of biological chemistry 315 12716910
2000 Structure of the TPR domain of p67phox in complex with Rac.GTP. Molecular cell 260 11090627
1992 Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245. The Journal of biological chemistry 256 1512217
2003 Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells. The Journal of biological chemistry 241 12657628
2009 NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers. Journal of structural biology 239 19723583
1994 Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. Evidence for its interaction with flavocytochrome b558. The Journal of biological chemistry 209 7982999
1998 Angiotensin II induces p67phox mRNA expression and NADPH oxidase superoxide generation in rabbit aortic adventitial fibroblasts. Hypertension (Dallas, Tex. : 1979) 192 9719063
1998 Regulation of the neutrophil respiratory burst oxidase. Identification of an activation domain in p67(phox). The Journal of biological chemistry 188 9642219
2003 Translocation of glomerular p47phox and p67phox by protein kinase C-beta activation is required for oxidative stress in diabetic nephropathy. Diabetes 182 14514646
1994 Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation. The Journal of biological chemistry 167 8120032
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