Affinage

NCF4

Neutrophil cytosol factor 4 · UniProt Q15080

Length
339 aa
Mass
39.0 kDa
Annotated
2026-04-29
75 papers in source corpus 50 papers cited in narrative 51 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NCF4 (p40phox) is a cytosolic adaptor and regulatory subunit of the phagocyte NADPH oxidase (NOX2) complex that couples PI3K signaling to phagosomal superoxide production and additionally links ROS sensing to inflammasome activation and antigen presentation. NCF4 constitutively associates with p67phox via its C-terminal PB1 (PC motif) domain and with p47phox via its SH3 domain, forming a cytosolic ternary complex; in resting cells, an intramolecular PB1–PX domain interaction maintains an autoinhibited conformation that is relieved when PtdIns(3)P on phagosomal membranes engages the PX domain, as demonstrated by crystal structures, knock-in R58A mice with impaired PtdIns(3)P binding, and reconstitution systems (PMID:17290225, PMID:16990793, PMID:16880255). Thr154 phosphorylation by PKCδ is required for full oxidase activation and p47phox phagosomal translocation, while phosphorylation-driven redistribution of NCF4 to the perinuclear region promotes ASC oligomerization and NLRP3/AIM2 inflammasome activation (PMID:20861461, PMID:38886341). Biallelic loss-of-function mutations in NCF4 cause an atypical form of chronic granulomatous disease with severely impaired particle-induced but partially preserved PMA-induced oxidase activity, and endosomal ROS controlled by NCF4 regulates cysteine-peptide antigen presentation and plasma cell differentiation, protecting against autoimmunity (PMID:29969437, PMID:38547647, PMID:36095971).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 1993 High

    Discovery that p40phox exists as a third cytosolic oxidase component resolved the question of whether additional factors beyond p47phox and p67phox participate in NADPH oxidase assembly, establishing p40phox as an SH3-domain-containing protein that primarily associates with p67phox and translocates to membranes upon activation.

    Evidence cDNA cloning, co-immunoprecipitation, and cell fractionation of neutrophils

    PMID:8280052

    Open questions at the time
    • Functional contribution of p40phox to oxidase activity was unclear
    • Domain requirements for partner interactions were unmapped
  2. 1996 High

    Mapping the domain architecture of p40phox interactions established that its C-terminal region (later identified as the PC/PB1 motif) mediates binding to p67phox while the SH3 domain binds the proline-rich region of p47phox, and that p67phox is essential for p40phox membrane translocation—defining the modular logic of the cytosolic oxidase complex.

    Evidence Yeast two-hybrid, surface plasmon resonance, CGD patient neutrophil fractionation, domain-specific antibody perturbation, cell-free oxidase assay

    PMID:7890694 PMID:8670049 PMID:8679714 PMID:8760383 PMID:9064349

    Open questions at the time
    • Lipid ligand of the PX domain was unknown
    • Positive vs. negative regulatory role of p40phox was debated
  3. 1997 High

    The finding that the p40phox SH3 domain competes with p67phox for binding to the p47phox proline-rich region, thereby inhibiting oxidase activity in cell-free assays, provided the first evidence that p40phox could function as a negative modulator of the oxidase—raising the question of how context determines its regulatory sign.

    Evidence Cell-free oxidase assay and K562 cell co-transfection with recombinant SH3 domains and mutants

    PMID:9083043

    Open questions at the time
    • Cellular conditions favoring positive vs. negative regulation were undefined
    • Phosphorylation consequences for SH3 competition were unknown
  4. 1998 High

    Identification of Thr154 and Ser315 as stimulus-dependent phosphorylation sites targeted by PKC-type kinases established that p40phox is post-translationally regulated during oxidase activation, and the PC motif was identified as a novel conserved interaction module essential for p67phox binding.

    Evidence Phosphoamino acid analysis, tryptic peptide mapping, site-directed mutagenesis, PKC inhibitors; yeast two-hybrid and recombinant protein binding for PC motif

    PMID:9490029 PMID:9804763

    Open questions at the time
    • Which specific PKC isoform phosphorylates p40phox in vivo was unknown
    • Functional consequences of Thr154 phosphorylation on oxidase activity were untested
  5. 2001 High

    The convergent demonstration that the p40phox PX domain specifically binds PtdIns(3)P, targets p40phox to early endosomes, and stimulates ROS production in reconstituted systems identified the missing link between PI3K signaling and oxidase activation; the crystal structure at 1.7 Å revealed how a CGD-associated Arg mutation disrupts lipid recognition.

    Evidence Lipid overlay and liposome sedimentation assays, GFP-fusion imaging, site-directed mutagenesis, X-ray crystallography of PX domain–PtdIns(3)P complex, cell-free NADPH oxidase assay with defined lipids

    PMID:11433300 PMID:11433301 PMID:11684018

    Open questions at the time
    • Whether PtdIns(3)P binding was essential in vivo (whole animal) was unproven
    • How autoinhibition of the PX domain was achieved structurally was unknown
  6. 2002 High

    Demonstration that p40phox enhances translocation of p67phox and p47phox to membranes through its PC motif–PB1 domain interaction with p67phox established p40phox as a positive regulator of oxidase assembly in intact cells, resolving the ambiguity about its regulatory role.

    Evidence COS7-phox cell reconstitution with D289A (PC) and K355A (p67phox PB1) point mutations, superoxide and translocation assays

    PMID:12456638

    Open questions at the time
    • Relative contribution of p40phox in phagocytic vs. soluble stimuli was unclear
    • In vivo validation in primary phagocytes was lacking
  7. 2006 High

    Genetic validation in mice—p40phox knockout causing CGD-like bacterial killing defects and the R58A PtdIns(3)P-binding knock-in reducing phagosomal oxidase activity—proved that PtdIns(3)P binding by p40phox is a physiologically essential signal for oxidase activation during phagocytosis, and that p40phox stabilizes p67phox protein levels.

    Evidence p40phox−/− mice and R58A knock-in mice with ROS assays, bacterial killing, Fc𝛾R-mediated phagocytosis reconstitution

    PMID:16880254 PMID:16880255 PMID:16990793

    Open questions at the time
    • Structural basis for PX domain autoinhibition in the full-length protein was unknown
    • Whether p40phox contributes to non-phagosomal oxidase activation was untested
  8. 2007 High

    The full-length crystal structure of p40phox revealed that the PB1 domain intramolecularly masks the PX domain's PtdIns(3)P-binding site on a face opposite to the p67phox-binding interface, explaining how autoinhibition is maintained in the cytosol even while complexed with p67phox; disruption of this closed conformation enhances oxidase activity ~2.5-fold.

    Evidence X-ray crystallography of full-length p40phox, in vivo/in vitro PtdIns(3)P binding assays, open-conformation mutant analysis in permeabilized cells

    PMID:17290225 PMID:18029359

    Open questions at the time
    • Mechanism triggering opening of the PX–PB1 clamp in cells was not defined
    • Whether p40phox contributes to oxidase regulation independently of p47phox was debated
  9. 2010 High

    In vivo rescue experiments with defined p40phox mutants established that PKCδ-mediated Thr154 phosphorylation is required for full oxidase activation and p47phox phagosomal recruitment, while deletion of the SH3 domain enhances activity—reconciling prior conflicting data about positive and negative regulatory functions by assigning them to distinct domains.

    Evidence Retroviral transduction of mutant p40phox into p40phox−/− bone marrow, radiation chimeras, multiple stimulation conditions

    PMID:20861461

    Open questions at the time
    • Precise structural mechanism by which Thr154 phosphorylation relieves inhibition was undefined
    • Whether SH3 domain deletion effects hold in all stimulus contexts was untested
  10. 2016 High

    Comparison of Ncf4−/− and Ncf4R58A mice separated total oxidase assembly (dependent on p40phox scaffolding of p67phox) from endosomal-specific oxidase activation (dependent on PtdIns(3)P binding), revealing that selective loss of intracellular ROS promotes autoimmune arthritis while total p40phox loss delays neutrophil apoptosis and broadly enhances innate immunity.

    Evidence Ncf4−/− and R58A knock-in mice with collagen-induced arthritis, ROS compartment assays, neutrophil apoptosis

    PMID:27231144

    Open questions at the time
    • Whether endosomal vs. phagosomal ROS compartments have distinct immunoregulatory targets was unclear
    • B cell-intrinsic vs. myeloid contributions to autoimmunity were unresolved
  11. 2018 High

    Identification of biallelic NCF4 loss-of-function mutations in patients with granulomatous disease established NCF4 deficiency as a human immunodeficiency, with a distinctive phenotype of impaired particle-induced but partially preserved PMA-induced oxidase activity and intact antifungal killing—distinguishing it from classic CGD.

    Evidence 24-patient cohort from 12 families, LOF mutation overexpression in NB4/EBV-B cells, NADPH oxidase and fungal killing assays

    PMID:29969437

    Open questions at the time
    • Range of infectious susceptibility in NCF4-deficient patients was incompletely defined
    • Whether residual oxidase activity is p47phox-dependent was not established
  12. 2024 High

    Two studies expanded p40phox function beyond the oxidase: NCF4 phosphorylation-driven redistribution to the perinuclear region promotes ASC oligomerization and NLRP3/AIM2 inflammasome activation, while NCF4-dependent endosomal ROS maintains cysteine peptides in an oxidized state that prevents their presentation to autoreactive T cells—providing mechanistic explanations for how intracellular ROS compartmentalization controls both innate and adaptive immunity.

    Evidence IP-MS of ASC, NCF4 KO inflammasome assays, confocal imaging; R58A knock-in mice with clodronate depletion, T cell activation, and peptide presentation assays

    PMID:38547647 PMID:38886341

    Open questions at the time
    • Signal that triggers NCF4 phosphorylation-dependent release from the oxidase complex to ASC is unknown
    • Whether cysteine-peptide oxidation mechanism applies beyond the GPI325-339 model antigen is untested
    • Structural basis for NCF4–ASC interaction is undetermined

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key open questions include the molecular trigger that switches p40phox from oxidase-bound to inflammasome-associated states, the structural basis for the PX–PB1 autoinhibition release in physiological membranes, and whether NCF4-regulated endosomal ROS controls antigen presentation broadly or only for cysteine-containing peptides.
  • No high-resolution structure of the full activated oxidase complex including p40phox
  • Relative contributions of p40phox in different tissue macrophage and DC subsets are poorly characterized
  • Role of NCF4 in non-phagocytic cells remains largely based on overexpression studies

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0008289 lipid binding 5 GO:0060090 molecular adaptor activity 4 GO:0098772 molecular function regulator activity 4
Localization
GO:0005768 endosome 4 GO:0005829 cytosol 4 GO:0005856 cytoskeleton 3 GO:0005886 plasma membrane 2 GO:0031410 cytoplasmic vesicle 2
Pathway
R-HSA-168256 Immune System 6 R-HSA-162582 Signal Transduction 3 R-HSA-5357801 Programmed Cell Death 1
Partners
ASCCYBACYBBMSNNCF1NCF2PRKCДRAC1
Complex memberships
NADPH oxidase (NOX2) cytosolic complex (p40phox–p67phox–p47phox)

Evidence

Reading pass · 51 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1993 p40phox (NCF4) was identified as a novel cytosolic component of the NADPH oxidase that forms an activation complex with p47phox and p67phox, translocates to the membrane upon cell stimulation, contains an SH3 domain, and shows primary association with p67phox. cDNA cloning, co-immunoprecipitation, cell fractionation The Biochemical journal High 8280052
1994 p40phox forms a tight 1:1 complex with p67phox in cytosol, co-immunoprecipitates consistently with p67phox, and is absent in CGD patients lacking p67phox, indicating p67phox-dependent stabilization. Immunoprecipitation, column chromatography, Western blot of CGD patient cells Biochemical and biophysical research communications High 8147882
1995 The SH3 domain of p40phox is sufficient for interaction with p47phox, whereas the C-terminus of p40phox (but not its SH3 domain) mediates interaction with p67phox. Yeast two-hybrid system with truncation mapping The Journal of biological chemistry High 7890694
1996 p67phox mediates the translocation of p40phox and Rac1 from cytosol to cell membranes upon neutrophil stimulation; in cells lacking p67phox, p40phox fails to translocate, whereas in cells lacking p47phox, both p67phox and p40phox fail to translocate. Cell fractionation of CGD patient neutrophils lacking p67phox or p47phox The Biochemical journal High 8670049
1996 The interaction between p40phox and p67phox is mediated by the C-terminal half of p40phox binding to the N-terminal half of p67phox, and does not involve SH3 domains or proline-rich sequences; p40phox also binds p47phox more weakly. In vitro binding assays (affinity beads, surface plasmon resonance biosensor) with truncation constructs The Biochemical journal High 8760383
1996 The C-terminal domain of p40phox is required for its interaction with p67phox and for activation of NADPH oxidase; antibodies against the C-terminus of p40phox dissociate the p40phox-p67phox complex and inhibit superoxide production, whereas N-terminal antibodies have no effect. Immunoprecipitation with domain-specific antibodies, cell-free oxidase activation assay The Journal of experimental medicine High 9064349
1996 Yeast two-hybrid analysis mapped p40phox interactions: its SH3 domain interacts with the polyproline motif of p47phox, its C-terminal tail interacts with the region between the two SH3 domains of p67phox (a novel non-SH3/polyproline interaction), and the p40phox C-terminal interaction with p67phox is maintained under activating conditions. Yeast two-hybrid system with domain fragments Biochimica et biophysica acta High 8679714
1997 p40phox down-regulates NADPH oxidase activity through its SH3 domain by competing with the p67phox SH3 domain for binding to the proline-rich region of p47phox (residues 358–390); the isolated SH3 domain of p40phox was even more inhibitory than full-length p40phox in cell-free and cell-based assays. Cell-free oxidase assay with recombinant proteins, co-transfection in K562 cells The Journal of biological chemistry High 9083043
1997 p40phox is in a basal phosphorylated state in resting cells and undergoes further phosphorylation on multiple sites upon NADPH oxidase stimulation (PMA or fMLP); the extent of phosphorylation correlates with superoxide production, and the kinase(s) responsible differ from those phosphorylating p47phox. 2D electrophoresis, immunoprecipitation of 32P-labeled HL60 cells European journal of biochemistry High 9370364
1998 p40phox is phosphorylated on Thr154 and Ser315 during NADPH oxidase activation, mediated by a PKC-type kinase; these sites were identified by phosphoamino acid analysis, tryptic peptide mapping, and directed mutagenesis of all PKC consensus sites. Phosphoamino acid analysis, tryptic peptide maps of in vivo/in vitro phosphorylated p40phox, site-directed mutagenesis, PKC inhibitors The Journal of biological chemistry High 9804763
1998 The PC motif in the C-terminal region of p40phox is a novel evolutionarily conserved protein–protein interaction module essential for binding to p67phox; intensive site-directed mutagenesis of the PC motif abolished interaction with p67phox. Yeast two-hybrid system, in vitro binding with recombinant proteins, site-directed mutagenesis European journal of biochemistry High 9490029
1999 Rac1-GTP displaces p67phox from its binding site on p40phox; this displacement is blocked by a synthetic peptide corresponding to p67phox amino acids 170–199 (the Rac binding domain), suggesting that Rac-GTP binding to p67phox induces a conformational change causing p40phox dissociation. In vitro pull-down/binding competition assays with recombinant Rac1-GTP, Cdc42, and peptides Biochemical and biophysical research communications Medium 10486263
1999 Thioredoxin interacts with p40phox in a yeast two-hybrid screen; this interaction requires the reducing-active form of thioredoxin (abolished by C32S/C35S mutation), suggesting redox-dependent regulation of p40phox. Yeast two-hybrid screen and confirmation assays Immunology letters Low 10397171
1999 p40phox is phosphorylated by protein kinase C during neutrophil activation (PMA stimulation), and PKC inhibitor H-7 blocks both p40phox phosphorylation and its translocation to membranes; purified PKC directly phosphorylates p40phox in the p40/p47/p67 complex. 32P labeling of guinea pig neutrophils, kinase inhibitors, in vitro kinase assay with purified PKC Journal of leukocyte biology High 10577519
1999 p40phox associates with the Triton X-100-insoluble cytoskeletal fraction in resting neutrophils via its association with p67phox; PMA stimulation does not alter this cytoskeletal association; p40phox co-localizes with filamentous actin by immunofluorescence. Cell fractionation, immunofluorescence confocal microscopy Journal of leukocyte biology Medium 10614785
1999 Ku70 (a DNA-dependent protein kinase regulatory component) interacts with p40phox; the 186 C-terminal amino acids of Ku70 bind two distinct regions of p40phox (the central core aa 50–260 and C-terminal aa 260–339); Ku70 and p40phox co-localize in B lymphocytes. Yeast two-hybrid screen, affinity chromatography, co-localization in B lymphocytes and COS-7 cells Journal of cell science Medium 9914162
2000 p40phox promotes NADPH oxidase activation by increasing the affinity of p47phox for flavocytochrome b558 approximately 3-fold in a cell-free system. Cell-free NADPH oxidase activation assay with recombinant proteins The Biochemical journal Medium 10861218
2001 The PX domain of p40phox specifically binds phosphatidylinositol 3-phosphate (PtdIns(3)P) and localizes to PtdIns(3)P-enriched early endosomes; an Arg-to-Gln CGD-associated mutation in the PX domain eliminates phosphoinositide binding and disrupts endosomal localization; PI(3)K inhibition also disrupts this localization. Lipid-binding assays (protein-lipid overlay, liposome sedimentation), GFP-fusion imaging in cells, site-directed mutagenesis Nature cell biology High 11433300 11433301
2001 PtdIns(3)P stimulates ROS formation specifically through binding to the PX domain of p40phox when p40phox is added to the minimal NADPH oxidase core complex; this defines PI(3)K signaling to the oxidase and PtdIns(3)P as the cellular ligand of the p40phox PX domain. In vitro reconstituted NADPH oxidase assay with lipid supplementation, PX domain mutants Nature cell biology High 11433301
2001 Crystal structure of the p40phox PX domain bound to PtdIns(3)P at 1.7 Å resolution reveals that the domain embraces the 3-phosphate on one side of a water-filled positively charged pocket; a CGD-associated mutation maps to a conserved Arg that stabilizes a critical lipid-binding loop; the SH3 domain does not affect PtdIns(3)P binding to the isolated PX domain. X-ray crystallography (1.7 Å), lipid-binding assays, mutagenesis Molecular cell High 11684018
2001 The p40phox PX domain localizes to early endosomes via direct PtdIns(3)P binding; the p47phox PX domain localizes to the plasma membrane via an indirect PI3K-dependent mechanism; in p47phox, an intramolecular SH3–PX interaction prevents nonspecific membrane penetration, whereas such interaction is absent in p40phox. EGFP-fusion imaging in HEK293 cells, surface plasmon resonance, monolayer penetration assays, mutagenesis The Journal of biological chemistry High 12556460
2001 Both p47phox and p40phox bind moesin (an ERM family F-actin-binding protein) through their PX domains in a phosphoinositide-dependent manner; the N-terminal part of moesin mediates this interaction. Co-immunoprecipitation, pulldown from neutrophil lysates Biochemical and biophysical research communications Medium 11716484
2001 Small angle neutron scattering confirmed that p40phox binds full-length p47phox in solution (without phosphorylation) through its SH3 domain interacting with the C-terminal proline-rich region of p47phox; the C-terminal end of p47phox is essential for this interaction. Small angle neutron scattering, gel filtration with truncation constructs Biochemistry High 11258927
2001 p40phox phosphorylation in B lymphocytes is mediated by PKC-delta in a redox-dependent manner; oxidant treatment (H2O2/vanadate) activates PKC-delta via tyrosine phosphorylation, and PKC-delta then phosphorylates p40phox on serine/threonine residues. Phosphoamino acid analysis, PKC inhibitors (GFX, rottlerin), co-stimulation of B cells Biochemical and biophysical research communications Medium 11573965
2002 p40phox enhances membrane translocation of p67phox and p47phox in stimulated cells, thereby facilitating superoxide production; this enhancement requires the PC motif of p40phox binding the PB1 domain of p67phox (D289A mutation in PC or K355A in p67phox PB1 abolishes the effect). Transfection in COS7-phox cells, superoxide assay, membrane translocation by fractionation, site-directed mutagenesis The EMBO journal High 12456638
2003 Membrane binding of the p40phox PX domain is initiated by nonspecific electrostatic interactions followed by membrane penetration of hydrophobic residues; this penetration is induced specifically by PtdIns(3)P; specific membrane penetration is important for subcellular localization of the PX domain in cells. Surface plasmon resonance, monolayer techniques, electrostatic potential calculations, GFP-PX imaging in HEK293 cells The Journal of biological chemistry High 12556460
2004 Phosphorylated p40phox (specifically Thr154 phosphorylation) acts as a negative regulator of NADPH oxidase in a cell-free system; phosphorylated p40phox inhibits oxidase activity even when added after full activation; phosphorylation induces a conformational change but does not disrupt interactions with p47phox or p67phox. Cell-free NADPH oxidase assay, site-directed mutagenesis of PKC consensus sites, pulldown assays, thrombin proteolysis conformation assay Biochemistry High 15035643
2004 TRAF3 associates with p40phox and p85 (PI3K subunit) in B cells; this association is required for CD40-induced ROS production via NADPH oxidase (dominant-negative TRAF3 blocks ROS generation from NADPH oxidase). Co-immunoprecipitation, dominant-negative overexpression, PI3K inhibitor treatment in WEHI 231 B cells Journal of immunology Medium 14688330
2005 Crystal structure of the p40phox SH3 domain alone and in complex with a 12-residue proline-rich region of p47phox at 1.46 Å resolution; SH3p40 binds both a canonical polyproline and a noncanonical motif of the p47phox C-terminus; phosphorylation of Ser379 of p47phox destabilizes binding to both SH3p40 and C-SH3p67. X-ray crystallography (1.46 Å), intrinsic tryptophan fluorescence measurements, phosphomimetic peptides The Journal of biological chemistry High 15657040
2006 Knockout of p40phox in mice severely impairs NADPH oxidase responses (up to 97% reduction for TNFα/fibrinogen stimulus) and causes a CGD-like defect in killing of Staphylococcus aureus both in vitro and in vivo; p67phox expression is reduced ~55% in the absence of p40phox. p40phox-/- mouse model, ROS measurement, bacterial killing assays The Journal of experimental medicine High 16880254
2006 p40phox is required to activate NADPH oxidase during FcγIIA-mediated phagocytosis; activation requires both PI(3)P binding by the p40phox PX domain and intact SH3/PB1 domains; PI(3)P generated in phagosomal membranes is the critical signal linking FcγR phagocytosis to oxidase activation. Stable reconstitution in COS7 cells, point mutations in PX/SH3/PB1 domains, PI3K inhibitors, superoxide assay The Journal of experimental medicine High 16880255
2006 The R58A mutation in the p40phox PX domain (which selectively prevents PtdIns(3)P binding) causes significantly reduced oxidase responses in neutrophils (e.g., 60% reduction in response to S. aureus phagocytosis) and compromises bacterial killing in vivo, establishing PtdIns(3)P binding as a physiological signal for oxidase activation. Knock-in mouse model (R58A mutation), ROS measurement, in vivo bacterial killing assay, wortmannin inhibition The EMBO journal High 16990793
2006 GFP-p40phox translocates to early endosomes (via PX domain–PtdIns(3)P interaction), while GFP-p47phox translocates to plasma membrane (via arachidonic acid); p67phox requires either p40phox or p47phox as a carrier for membrane targeting; p40phox has an autoinhibitory head-to-tail (PX–PB1 domain) intramolecular interaction in its resting state. Live cell GFP imaging, domain deletion and point mutations, binding assays Molecular biology of the cell High 17122360
2006 Vav proteins (specifically Vav3 and Vav1) are required for FcγR-mediated PI3K-dependent phosphorylation of p40phox; this pathway operates independently of Rac activation and actin polymerization. Vav1/Vav3 knockout neutrophils, Rac1/2 knockout, actin inhibitors, p40phox phosphorylation assay Journal of immunology Medium 17056570
2007 Full-length crystal structure of p40phox reveals that the PB1 domain inhibits PtdIns(3)P-binding activity of the PX domain through intramolecular PB1–PX interaction; the PB1 domain interface for the PX domain is on the opposite side from the p67phox PB1 binding interface, allowing simultaneous PB1-PB1 association with p67phox while the PX domain remains autoinhibited. X-ray crystallography of full-length p40phox, in vivo and in vitro PtdIns(3)P-binding assays The EMBO journal High 17290225
2007 Mutation of Arg-57 in the p40phox PX domain (abrogating PtdIns(3)P binding) produces a dominant inhibitory effect on superoxide production and membrane translocation; the mutant p40phox displays increased association with actin and moesin in the Triton X-100-insoluble cytoskeletal fraction, sequestering p67phox and p47phox; cytochalasin B treatment or introduction of a second D289A mutation (disrupting p67phox binding) abolishes the dominant inhibitory effect. Site-directed mutagenesis, COS-phox cell reconstitution, cytoskeletal fractionation, superoxide assay The Journal of biological chemistry High 17698849
2007 p40phox can substitute for p47phox as an organizer in Nox2 activation in a cell-free system containing PtdIns(3)P-relipidated cytochrome b558; p40phox interacts with the proline-rich region of p22phox through its SH3 domain (shown by far-western blotting), reaching ~70% of p47phox-induced activity. Cell-free NADPH oxidase assay with relipidated cytochrome b558, peptide competition, far-western blotting FEBS letters Medium 17803994
2007 Depletion of p40phox reduces ROS production and bacterial killing in differentiated PLB-985 neutrophils; the p40phox PX domain has both PtdIns(3)P-dependent and -independent functions: PX domain (but not PtdIns(3)P binding) is required for p67phox translocation, while oxidase activation requires both PtdIns(3)P binding and a functional SH3 domain; mutations disrupting the closed autoinhibited form of p40phox increase oxidase activity ~2.5-fold. Lentiviral shRNA knockdown, permeabilized cell reconstitution, chimeric PX domains, mutagenesis, ROS assay The Journal of biological chemistry High 18029359
2008 p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly; p67phox and p47phox accumulate on nascent phagosomes independently of p40phox or PI3K; p40phox translocates to phagosomes via p67phox binding (not PI3P), but PI3P binding is required for p40phox retention after phagosome internalization and for stimulating superoxide production. Real-time live-cell imaging of YFP-tagged phox proteins, immunofluorescence, point mutations in p40phox, wortmannin inhibition Blood High 18711001
2008 PAF-induced endosome formation leads to membrane translocation of the p40phox-p67phox complex localizing to gp91phox; this requires PI3K and Akt1 phosphorylation of p40phox and p67phox, and is p47phox-independent; EEA-1 provides a scaffold for recruitment of this complex. Subcellular fractionation, electron microscopy, PI3K inhibitors, Akt1 identification, immunofluorescence in human neutrophils Journal of immunology Medium 18523285
2010 Phosphorylation of p40phox on Thr154 (by PKCδ) is required for full NADPH oxidase activation in response to fMLP, opsonized S. aureus, and IgG-coated erythrocytes; Thr154 phosphorylation is required for full translocation of p47phox to phagosomes; the SH3 domain is not required and its deletion enhances oxidase activity. Retroviral transduction of wild-type and mutant p40phox into p40phox-/- bone marrow, radiation chimeras, ROS assay, phagosome fractionation Blood High 20861461
2010 The PX domain of p40phox directly interacts with F-actin; this interaction is lipid-independent (lipid-binding mutant p40PX still co-localizes with F-actin); p40phox co-localizes with F-actin in COS cells, and disruption of actin with cytochalasin D alters p40phox localization. Affinity chromatography from neutrophil extracts, pure actin binding assay, GFP-fusion imaging, cytochalasin D treatment The international journal of biochemistry & cell biology Medium 20637895
2011 p40phox acquires PI(3)P-binding capability through conformational changes induced by H2O2 in the cytoplasm or through direct/indirect membrane targeting; p40phox is essential when p47phox is only partially phosphorylated during FcγR-mediated oxidase activation; PI binding to p47phox is less critical when the autoinhibitory PX-PB1 interaction in p40phox is disrupted. Phosphorylation-mimicking mutants, HEK293-Nox2/FcγR cells, RAW264.7 p40/p47 knockdown, GFP-imaging, H2O2 treatment The Journal of biological chemistry Medium 21956105
2012 p40phox deficiency in mice enhances intestinal inflammation (colitis), with increased proinflammatory cytokines, neutrophil recruitment, and colonic tissue injury; p40phox expression in neutrophils is necessary for restitution during the recovery phase; p40phox deficiency leads to upregulation of CXCR1 and downregulation of fucosyltransferases and sialyltransferases. p40phox-/- mice, DSS and innate immune colitis models, cytokine assays, neutrophil depletion, transcriptomics Journal of immunology Medium 22914050
2013 p40phox (NCF4) overexpression activates the NOX2 complex and produces ROS, which drives epithelial-mesenchymal transition (EMT) in HeLa cells via upregulation of NOX2/NOX5, matrix metalloproteinases, Snail, Slug, vimentin, and YB-1, with downregulation of E-cadherin; ROS scavenging reverses these changes. NCF4 overexpression in HeLa cells, ROS measurement, qRT-PCR, antioxidant rescue experiments Cellular signalling Medium 24378533
2013 p40phox gene transcription in myeloid cells is driven by the AhR (aryl hydrocarbon receptor/dioxin receptor); 3-methylcholanthrene increases p40phox expression in an AhR-dependent manner; promoter analysis confirmed p40phox as an AhR transcriptional target; silencing p40phox abolishes AhR-dependent NADPH oxidase activity and ROS production. AhR knockout mice, siRNA knockdown, promoter-reporter assay, qRT-PCR, ROS assay Molecular pharmacology Medium 23478803
2016 Ncf4-/- mice show severe reduction in overall ROS due to concomitant reduction of NCF1 and NCF2, delayed neutrophil apoptosis, and enhanced innate immune responses, with aggravated collagen-induced arthritis (CIA) and psoriatic arthritis-like disease; the R58A PtdIns(3)P-binding mutation (Ncf4*/*) selectively reduces intracellular NOX2 activity without affecting overall ROS, showing milder effects on innate immunity but clearly promoting CIA susceptibility. Ncf4-/- and Ncf4R58A knock-in mouse models, ROS assays, neutrophil apoptosis, CIA and MIP models Antioxidants & redox signaling High 27231144
2018 Biallelic loss-of-function mutations in NCF4 cause severely impaired particle-induced NADPH oxidase activity in neutrophils and EBV-B cells, but normal or mildly impaired PMA-induced DHR oxidation; neutrophil killing of Candida albicans and Aspergillus fumigatus is conserved unlike in classic CGD; mononuclear phagocyte oxidase activity is normal. Patient cohort (24 patients, 12 families), LOF mutation overexpression in NB4/EBV-B cells, NADPH oxidase activity assays, fungal killing assays The Journal of clinical investigation High 29969437
2022 The NCF4 R58A mutation (disrupting endosomal PtdIns(3)P binding) reduces intracellular but not extracellular ROS in B cells, and leads to enhanced plasma cell differentiation with altered CXCR3/CXCR4 expression; this effect is B cell-intrinsic as shown by chimeric B cell transfer experiments. Ncf4R58A knock-in mice, B cell transfer experiments, in vitro LPS/CD40L/anti-IgM stimulation, flow cytometry, ELISA Redox biology High 36095971
2024 NCF4 interacts with ASC (apoptosis-associated speck-like protein containing CARD) and cooperates with NCF1 and NCF2 to promote NLRP3 and AIM2 inflammasome activation; NCF4 phosphorylation causes redistribution from the NADPH complex to the perinuclear region, mediating ASC oligomerization and speck formation; NCF4 acts as a ROS sensor to balance ROS production and inflammasome activation. Immunoprecipitation-mass spectrometry of ASC, co-IP, NCF4 KO mouse models, inflammasome assays, phosphorylation studies, confocal imaging Nature communications High 38886341
2024 NCF4-dependent intracellular ROS maintains cysteine peptides in an oxidized crosslinked state in phagocytic cells, preventing their presentation to autoreactive T cells; NCF4 R58A mutation (reducing intracellular ROS) leads to efficient presentation of cysteine-containing GPI325-339 peptides to arthritogenic T cells, promoting autoimmune arthritis. NCF4R58A knock-in mice, clodronate macrophage depletion, FYT720 T cell trapping, T cell activation assays, peptide presentation experiments Redox biology High 38547647

Source papers

Stage 0 corpus · 75 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2001 The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nature cell biology 524 11433300
2001 PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox). Nature cell biology 337 11433301
1993 p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. The Biochemical journal 249 8280052
2001 The crystal structure of the PX domain from p40(phox) bound to phosphatidylinositol 3-phosphate. Molecular cell 234 11684018
2006 Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing. The Journal of experimental medicine 141 16880254
2003 Membrane binding mechanisms of the PX domains of NADPH oxidase p40phox and p47phox. The Journal of biological chemistry 124 12556460
2002 The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase. The EMBO journal 117 12456638
2006 The phosphoinositide-binding protein p40phox activates the NADPH oxidase during FcgammaIIA receptor-induced phagocytosis. The Journal of experimental medicine 111 16880255
2001 The p40phox and p47phox PX domains of NADPH oxidase target cell membranes via direct and indirect recruitment by phosphoinositides. The Journal of biological chemistry 111 11729195
1996 Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox. The Biochemical journal 107 8670049
1995 Mapping the domains of interaction of p40phox with both p47phox and p67phox of the neutrophil oxidase complex using the two-hybrid system. The Journal of biological chemistry 101 7890694
2006 PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation. The EMBO journal 100 16990793
2018 Inherited p40phox deficiency differs from classic chronic granulomatous disease. The Journal of clinical investigation 98 29969437
1994 A novel cytosolic component, p40phox, of respiratory burst oxidase associates with p67phox and is absent in patients with chronic granulomatous disease who lack p67phox. Biochemical and biophysical research communications 95 8147882
1997 p40(phox) down-regulates NADPH oxidase activity through interactions with its SH3 domain. The Journal of biological chemistry 93 9083043
1998 p40(phox) is phosphorylated on threonine 154 and serine 315 during activation of the phagocyte NADPH oxidase. Implication of a protein kinase c-type kinase in the phosphorylation process. The Journal of biological chemistry 89 9804763
2007 A case-control study of rheumatoid arthritis identifies an associated single nucleotide polymorphism in the NCF4 gene, supporting a role for the NADPH-oxidase complex in autoimmunity. Arthritis research & therapy 79 17897462
2008 Fc gamma R-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome. Blood 77 18711001
2006 A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox. Molecular biology of the cell 74 17122360
1996 Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox. The Biochemical journal 73 8760383
1996 Involvement of p40phox in activation of phagocyte NADPH oxidase through association of its carboxyl-terminal, but not its amino-terminal, with p67phox. The Journal of experimental medicine 71 9064349
1998 The PC motif: a novel and evolutionarily conserved sequence involved in interaction between p40phox and p67phox, SH3 domain-containing cytosolic factors of the phagocyte NADPH oxidase. European journal of biochemistry 70 9490029
2006 Vav proteins in neutrophils are required for FcgammaR-mediated signaling to Rac GTPases and nicotinamide adenine dinucleotide phosphate oxidase component p40(phox). Journal of immunology (Baltimore, Md. : 1950) 68 17056570
2001 The NADPH oxidase components p47(phox) and p40(phox) bind to moesin through their PX domain. Biochemical and biophysical research communications 68 11716484
1998 Functional modules and expression of mouse p40(phox) and p67(phox), SH3-domain-containing proteins involved in the phagocyte NADPH oxidase complex. European journal of biochemistry 67 9490028
2005 Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains. The Journal of biological chemistry 64 15657040
1996 Genomic structure, chromosomal localization, start of transcription, and tissue expression of the human p40-phox, a new component of the nicotinamide adenine dinucleotide phosphate-oxidase complex. Blood 63 8839867
2004 Phosphorylated p40PHOX as a negative regulator of NADPH oxidase. Biochemistry 61 15035643
2013 Germline variation in NCF4, an innate immunity gene, is associated with an increased risk of colorectal cancer. International journal of cancer 58 23982929
2000 p40(phox) Participates in the activation of NADPH oxidase by increasing the affinity of p47(phox) for flavocytochrome b(558). The Biochemical journal 58 10861218
2007 Full-length p40phox structure suggests a basis for regulation mechanism of its membrane binding. The EMBO journal 51 17290225
2004 Role of TNF receptor-associated factor 3 in the CD40 signaling by production of reactive oxygen species through association with p40phox, a cytosolic subunit of nicotinamide adenine dinucleotide phosphate oxidase. Journal of immunology (Baltimore, Md. : 1950) 48 14688330
1996 Topological organization of the cytosolic activating complex of the superoxide-generating NADPH-oxidase. Pinpointing the sites of interaction between p47phoz, p67phox and p40phox using the two-hybrid system. Biochimica et biophysica acta 47 8679714
1999 P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox. Journal of leukocyte biology 46 10614785
2013 Aryl hydrocarbon receptor modulates NADPH oxidase activity via direct transcriptional regulation of p40phox expression. Molecular pharmacology 44 23478803
2012 p40phox expression regulates neutrophil recruitment and function during the resolution phase of intestinal inflammation. Journal of immunology (Baltimore, Md. : 1950) 44 22914050
2007 Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase. The Journal of biological chemistry 42 18029359
2010 Phosphorylation of threonine 154 in p40phox is an important physiological signal for activation of the neutrophil NADPH oxidase. Blood 41 20861461
2013 Activation of NADPH oxidase subunit NCF4 induces ROS-mediated EMT signaling in HeLa cells. Cellular signalling 39 24378533
2011 Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding. The Journal of biological chemistry 37 21956105
1999 Demonstration of the interaction of thioredoxin with p40phox, a phagocyte oxidase component, using a yeast two-hybrid system. Immunology letters 36 10397171
1999 Rac1 disrupts p67phox/p40phox binding: a novel role for Rac in NADPH oxidase activation. Biochemical and biophysical research communications 36 10486263
1997 The 40-kDa component of the phagocyte NADPH oxidase (p40phox) is phosphorylated during activation in differentiated HL60 cells. European journal of biochemistry 34 9370364
2008 Platelet-activating factor-mediated endosome formation causes membrane translocation of p67phox and p40phox that requires recruitment and activation of p38 MAPK, Rab5a, and phosphatidylinositol 3-kinase in human neutrophils. Journal of immunology (Baltimore, Md. : 1950) 32 18523285
2002 Multiple PU.1 sites cooperate in the regulation of p40(phox) transcription during granulocytic differentiation of myeloid cells. Blood 30 12036891
2016 A Reduction in Intracellular Reactive Oxygen Species Due to a Mutation in NCF4 Promotes Autoimmune Arthritis in Mice. Antioxidants & redox signaling 29 27231144
2013 Evolutionary dynamics of the human NADPH oxidase genes CYBB, CYBA, NCF2, and NCF4: functional implications. Molecular biology and evolution 26 23821607
1999 Phosphorylation of p40-phox during activation of neutrophil NADPH oxidase. Journal of leukocyte biology 26 10577519
2007 Characterization of a mutation in the Phox homology domain of the NADPH oxidase component p40phox identifies a mechanism for negative regulation of superoxide production. The Journal of biological chemistry 25 17698849
2012 Regulation of alveolar macrophage p40phox: hierarchy of activating kinases and their inhibition by PGE2. Journal of leukocyte biology 24 22544939
2001 Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox. Biochemistry 24 11258927
2000 Relationships of p40(phox) with p67(phox) in the activation and expression of the human respiratory burst NADPH oxidase. Journal of biochemistry 23 11056390
2009 rs224136 on chromosome 10q21.1 and variants in PHOX2B, NCF4, and FAM92B are not major genetic risk factors for susceptibility to Crohn's disease in the German population. The American journal of gastroenterology 20 19262523
2006 Orientation and penetration depth of monolayer-bound p40phox-PX. Biochemistry 20 17087510
2015 Role of an SNP in Alternative Splicing of Bovine NCF4 and Mastitis Susceptibility. PloS one 19 26600390
1996 Translocation of guinea pig p40-phox during activation of NADPH oxidase. Biochimica et biophysica acta 18 8982388
2024 NCF4 attenuates colorectal cancer progression by modulating inflammasome activation and immune surveillance. Nature communications 15 38886341
2001 Involvement of cytosolic prolyl endopeptidase in degradation of p40-phox splice variant protein in myeloid cells. Journal of leukocyte biology 15 11404383
1999 The Ku70 autoantigen interacts with p40phox in B lymphocytes. Journal of cell science 15 9914162
2010 Subcellular localisation of the p40phox component of NADPH oxidase involves direct interactions between the Phox homology domain and F-actin. The international journal of biochemistry & cell biology 14 20637895
2001 Oxidant-dependent phosphorylation of p40phox in B lymphocytes. Biochemical and biophysical research communications 14 11573965
2018 The effect of the SNP g.18475 A>G in the 3'UTR of NCF4 on mastitis susceptibility in dairy cattle. Cell stress & chaperones 12 29476341
2022 NCF4 dependent intracellular reactive oxygen species regulate plasma cell formation. Redox biology 11 36095971
2019 Early Onset Granulomatous Colitis Associated with a Mutation in NCF4 Resolved with Hematopoietic Stem Cell Transplantation. The Journal of pediatrics 11 31027832
2017 ABCG2 and NCF4 polymorphisms are associated with clinical outcomes in diffuse large B-cell lymphoma patients treated with R-CHOP. Oncotarget 11 28938556
2007 p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox. FEBS letters 11 17803994
1999 Identification of a splice variant mRNA of p40phox, an NADPH oxidase component of phagocytes. FEBS letters 11 10437784
2020 Association of NCF2, NCF4, and CYBA Gene Polymorphisms with Rheumatoid Arthritis in a Chinese Population. Journal of immunology research 10 33145364
2001 Molecular cloning and identification of bottle-nosed dolphin p40(phox), p47(phox) and p67(phox). Veterinary immunology and immunopathology 10 11182145
2024 NCF4 regulates antigen presentation of cysteine peptides by intracellular oxidative response and restricts activation of autoreactive and arthritogenic T cells. Redox biology 6 38547647
2021 Genotype Analysis of ABCC1, NCF4 and CBR3 Polymorphism and the Association With Childhood Acute Lymphoblastic Leukemia in Mexican Childhood Population. Frontiers in pharmacology 3 33613283
2006 Crystallization and preliminary crystallographic analysis of p40phox, a regulatory subunit of NADPH oxidase. Acta crystallographica. Section F, Structural biology and crystallization communications 3 17012801
2023 Polymorphisms of the NCF4 Gene Increase the Risk of Chronic Heart Failure in Patients with Type 2 Diabetes Mellitus. Bulletin of experimental biology and medicine 1 38085396
2023 Anti-TNF Therapy Regulates Phagosome Pathway by Inhibiting NCF4 Expression to Treat Ankylosing Spondylitis. Journal of musculoskeletal & neuronal interactions 0 37654221
2021 Role of p40phox in host defense against Citrobacter rodentium infection. FEBS open bio 0 33780601