Affinage

NCF4

Neutrophil cytosol factor 4 · UniProt Q15080

Length
339 aa
Mass
39.0 kDa
Annotated
2026-06-10
75 papers in source corpus 45 papers cited in narrative 45 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NCF4 (p40phox) is a cytosolic adaptor subunit of the phagocyte NADPH oxidase that couples phosphoinositide signaling to regulated, compartmentalized reactive oxygen species (ROS) production (PMID:8280052, PMID:16990793). It is held in the resting cytosol as a tight 1:1 complex with p67phox, an association mediated by a novel PC motif in its C-terminal region binding the PB1 domain of p67phox; loss of p67phox abolishes p40phox and prevents its membrane translocation (PMID:8147882, PMID:9490029, PMID:12456638, PMID:8670049). Upon stimulation, p40phox is recruited to phagosomal membranes where its PX domain binds PtdIns(3)P generated by PI3K, an interaction that sustains oxidase retention and activity rather than initiating assembly (PMID:11433300, PMID:11433301, PMID:18711001). In the resting state PtdIns(3)P binding is held off by an intramolecular PX–PB1 contact resolved in the full-length crystal structure, an autoinhibition relieved at the phagosomal membrane (PMID:17290225, PMID:17122360). PKCδ-mediated phosphorylation of Thr154 provides an additional positive signal required for full oxidase activation and p47phox translocation (PMID:20861461, PMID:9804763). The PX domain also binds F-actin directly and anchors the complex in the cytoskeleton, while the SH3 domain engages the proline-rich regions of p47phox and p22phox (PMID:20637895, PMID:15657040, PMID:17803994). Genetic ablation of p40phox in mice produces CGD-like defects in superoxide generation and bacterial killing, confirming its physiological role (PMID:16880254, PMID:16990793). Beyond classical antimicrobial oxidase function, NCF4-dependent intracellular ROS controls antigen presentation of cysteine peptides, plasma cell differentiation, and inflammasome assembly through interaction with ASC, linking it to autoimmune and inflammatory disease (PMID:38547647, PMID:36095971, PMID:38886341, PMID:27231144).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 1993 High

    Established NCF4/p40phox as a previously unrecognized cytosolic component of the NADPH oxidase, defining the protein and its membership in the activation complex.

    Evidence Co-purification, cDNA cloning and fractionation from neutrophils, with absence in p67phox-deficient CGD patients

    PMID:8280052

    Open questions at the time
    • Domain basis of partner binding not yet mapped
    • No functional contribution to oxidase activity established
  2. 1996 High

    Resolved how p40phox associates with the complex by showing its C-terminus, not its SH3 domain, mediates p67phox binding and that p67phox is required for p40phox membrane recruitment.

    Evidence Yeast two-hybrid domain mapping, in vitro binding/SPR, and fractionation of CGD patient neutrophils lacking p67phox or p47phox

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

    Open questions at the time
    • Precise interaction module within the C-terminus not yet defined
    • Functional consequence of recruitment unresolved
  3. 1998 High

    Identified the PC motif as the specific module mediating p40phox–p67phox binding, defining a novel protein-protein interaction surface.

    Evidence Yeast two-hybrid and in vitro binding with site-directed mutagenesis of the PC motif

    PMID:9490029

    Open questions at the time
    • The reciprocal p67phox surface (PB1 domain) not yet defined
    • Role in translocation not directly tested
  4. 1998 High

    Mapped p40phox phosphorylation to Thr154 and Ser315 during oxidase activation and implicated a PKC-type kinase, establishing post-translational regulation.

    Evidence Phosphoamino acid analysis, peptide mapping, mutagenesis of PKC consensus sites, and in vitro kinase assays in HL-60/neutrophils

    PMID:10577519 PMID:9370364 PMID:9804763

    Open questions at the time
    • Specific PKC isoform not identified
    • Functional role of each phosphosite unresolved
  5. 2001 High

    Defined the lipid-sensing mechanism: the PX domain specifically binds PtdIns(3)P, which directly stimulates oxidase ROS production and targets p40phox to PtdIns(3)P-enriched membranes.

    Evidence Protein-lipid overlay, GFP live-cell imaging with PI3K inhibition, cell-free reconstitution with lipid vesicles, and high-resolution PX-domain crystal structure

    PMID:11258927 PMID:11433300 PMID:11433301 PMID:11684018 PMID:11729195

    Open questions at the time
    • How PtdIns(3)P access is gated in full-length protein not yet resolved
    • Physiological in vivo requirement not yet tested
  6. 2002 High

    Showed the PC–PB1 interaction enhances translocation of p67phox and p47phox and superoxide production, linking the binding module to functional oxidase activation.

    Evidence Cell-based translocation/oxidase assays with reciprocal D289A (PC) and K355A (PB1) mutagenesis

    PMID:12456638

    Open questions at the time
    • Quantitative contribution relative to other organizers unresolved
    • Phosphorylation crosstalk with this interaction not addressed
  7. 2003 High

    Clarified the biophysics of membrane engagement, showing PtdIns(3)P triggers hydrophobic membrane penetration and that p40phox lacks the intramolecular SH3–PX autoinhibition found in p47phox.

    Evidence SPR, lipid monolayer assays, electrostatic modeling, and GFP cell imaging with full-length protein

    PMID:12556460

    Open questions at the time
    • Mechanism restraining PX access in resting cells not yet defined
    • Did not yet identify the PX–PB1 autoinhibition later described
  8. 2004 Medium

    Established that Thr154-phosphorylated p40phox induces a conformational change that inhibits the oxidase in vitro without disrupting partner binding, indicating phosphorylation-dependent regulation.

    Evidence Cell-free reconstitution with site mutagenesis and limited proteolysis conformational probing

    PMID:15035643

    Open questions at the time
    • In vitro inhibitory effect contrasts with later in vivo positive role of Thr154
    • Cellular context of the conformational switch unresolved
  9. 2006 High

    Demonstrated the physiological requirement for p40phox and its PtdIns(3)P-binding in phagocytosis-driven oxidase activation and bacterial killing in vivo, validating the PI3K–PtdIns(3)P–p40phox axis.

    Evidence p40phox knockout and R58A knock-in mice, FcγR phagocytosis reconstitution in COS cells with domain mutants, and PI3K inhibition in primary macrophages

    PMID:16880254 PMID:16880255 PMID:16990793

    Open questions at the time
    • Concomitant ~55% p67phox reduction complicates clean attribution in full KO
    • Extracellular vs intracellular oxidase roles not yet separated
  10. 2007 High

    Resolved the autoinhibition mechanism with the full-length structure, showing an intramolecular PB1–PX contact blocks PtdIns(3)P binding while leaving the p67phox-binding face free, and confirmed cytoskeletal anchoring as a co-regulatory layer.

    Evidence Full-length crystal structure, in vitro lipid binding with truncations, cell localization, and Triton-fractionation/imaging of PX mutants

    PMID:17122360 PMID:17290225 PMID:17698849 PMID:17803994

    Open questions at the time
    • Trigger that relieves PX–PB1 autoinhibition at the membrane not yet defined
    • Quantitative interplay between actin anchoring and lipid binding unresolved
  11. 2008 High

    Defined the temporal logic of recruitment, showing p67phox-driven translocation precedes PI(3)P accumulation, while PI(3)P binding sustains retention and activity after phagosome sealing, positioning p40phox as a regulator of activity rather than assembly.

    Evidence Real-time imaging of YFP-phox proteins and PI(3)P probe with wortmannin and lipid-binding mutants

    PMID:18029359 PMID:18711001

    Open questions at the time
    • Signals coupling phagosome sealing to PI(3)P generation not addressed
    • Generalizability beyond phagocytic stimuli unresolved
  12. 2010 High

    Identified PKCδ-mediated Thr154 phosphorylation as required for full oxidase activation and p47phox phagosomal translocation in primary neutrophils, and showed the SH3 domain is dispensable for activation.

    Evidence In vivo reconstitution of p40phox-/- bone marrow with phosphosite mutants, radiation chimaeras, PKCδ inhibitor/siRNA, and phagosomal imaging

    PMID:20861461

    Open questions at the time
    • Reconciliation with earlier in vitro inhibitory role of Thr154 phosphorylation incomplete
    • Structural effect of phosphorylation on autoinhibition not resolved
  13. 2013 Medium

    Showed NCF4 expression is transcriptionally controlled by the aryl hydrocarbon receptor, extending regulation beyond protein-level mechanisms to gene expression in non-phagocytic tissue.

    Evidence AhR-knockout mice, promoter-reporter analysis, ChIP, and RNAi knockdown with oxidase activity readout

    PMID:23478803

    Open questions at the time
    • Physiological context of AhR-driven NCF4 expression unresolved
    • Single lab; not independently corroborated
  14. 2024 High

    Established NCF4-dependent intracellular ROS as a regulator of adaptive and innate immunity beyond microbial killing, controlling cysteine-peptide antigen presentation, plasma cell differentiation, and ASC-dependent inflammasome assembly, with consequences for autoimmunity.

    Evidence NCF4-/- and R58A knock-in mice in arthritis models, chimeric B-cell transfer, antigen presentation and T-cell assays, IP-MS identifying ASC, and inflammasome activation assays

    PMID:27231144 PMID:36095971 PMID:38547647 PMID:38886341

    Open questions at the time
    • Direct molecular mechanism coupling ROS levels to ASC oligomerization not fully defined
    • ASC interaction validated largely within single study

Open questions

Synthesis pass · forward-looking unresolved questions
  • The precise signal that releases the PX–PB1 autoinhibition at the phagosomal membrane, and how phosphorylation, lipid binding, and cytoskeletal anchoring are integrated into a single activation switch, remain to be defined.
  • No unified structural model of the activated, membrane-bound full-length complex
  • Mechanistic link between intracellular ROS sensing and ASC oligomerization unresolved

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0008289 lipid binding 4 GO:0060090 molecular adaptor activity 3 GO:0098772 molecular function regulator activity 3 GO:0008092 cytoskeletal protein binding 2 GO:0140299 molecular sensor activity 1
Localization
GO:0005768 endosome 3 GO:0005829 cytosol 3 GO:0005856 cytoskeleton 2 GO:0005886 plasma membrane 2
Pathway
R-HSA-168256 Immune System 4 R-HSA-1643685 Disease 3 R-HSA-8953897 Cellular responses to stimuli 3
Partners
ACTBASCCYBANCF1NCF2PRKCDRAC1
Complex memberships
NADPH oxidase (NOX2) complex

Evidence

Reading pass · 45 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 activation complex; it contains an SH3 domain and forms a complex with p47phox and p67phox, translocating with them to the membrane upon stimulation. Its primary association is with p67phox, and it is absent in CGD patients lacking p67phox. Co-purification, cDNA cloning, immunoprecipitation, subcellular fractionation The Biochemical journal High 8280052
1994 p40phox forms a tight 1:1 molar complex with p67phox in the cytosol; free p40phox is absent from neutrophil cytosol, and it is missing in CGD patients who lack p67phox. Immunoprecipitation, column chromatography, SDS-PAGE analysis Biochemical and biophysical research communications Medium 8147882
1995 The SH3 domain of p40phox is sufficient for interaction with p47phox, whereas the C-terminus of p40phox (not the SH3 domain) mediates interaction with p67phox, as mapped by yeast two-hybrid screening. Yeast two-hybrid assay with truncation constructs The Journal of biological chemistry Medium 7890694
1996 p40phox translocation to the membrane requires p67phox: in p67phox-deficient neutrophils p40phox fails to translocate upon stimulation, whereas in p47phox-deficient neutrophils p67phox and p40phox both fail to translocate, establishing that p67phox mediates p40phox membrane recruitment. Subcellular fractionation of CGD patient neutrophils lacking p67phox or p47phox, Western blotting The Biochemical journal High 8670049
1996 p40phox interacts with both p67phox (via the N-terminal half of p67phox and the C-terminal half of p40phox, independent of SH3/proline-rich interactions) and with p47phox (more weakly), as shown by in vitro affinity-bead binding and surface plasmon resonance. Affinity bead pulldown, surface plasmon resonance (Biacore) The Biochemical journal Medium 8760383
1996 Two-hybrid analysis mapped the p67phox–p40phox interaction to the 150-aa stretch between the two SH3 domains of p67phox and the C-terminal tail of p40phox (which contains neither SH3 nor polyproline motif, indicating a novel type of interaction). The p47phox polyproline motif also mediates interaction with the SH3 domain of p40phox. Yeast two-hybrid with systematic deletion constructs Biochimica et biophysica acta Medium 8679714
1996 The C-terminal region of p40phox (containing the PC motif) interacts with p67phox, while the SH3 domain of p40phox does not. Antibody against the C-terminus of p40phox dissociates the p40phox–p67phox complex and inhibits NADPH oxidase activity in a cell-free system, establishing a functional role for this interaction in oxidase activation. Co-immunoprecipitation with epitope-specific antibodies, cell-free NADPH oxidase activity assay, membrane translocation assay The Journal of experimental medicine Medium 9064349
1997 p40phox is phosphorylated on multiple sites during NADPH oxidase activation in HL-60 cells and neutrophils; the extent of phosphorylation correlates strongly with superoxide production; dephosphorylation follows the end of the respiratory burst; the kinases responsible differ from those phosphorylating p47phox. 2D electrophoresis, immunoprecipitation of 32P-labeled cells, kinase inhibitor studies European journal of biochemistry Medium 9370364
1997 p40phox inhibits NADPH oxidase activity via its SH3 domain, which competes with the p67phox SH3 domain for binding to the proline-rich region of p47phox (residues 358–390). The isolated SH3 domain of p40phox inhibits whole-cell oxidase activity; deletion of the C-terminal p67phox-binding domain does not relieve inhibition. In vitro cell-free NADPH oxidase assay with recombinant proteins, K562 cell co-transfection with oxidase activity measurement, domain deletion constructs The Journal of biological chemistry Medium 9083043
1998 The PC motif in the C-terminal region of p40phox is critical for its interaction with p67phox (specifically the region between the two SH3 domains of p67phox). Site-directed mutagenesis of the PC motif abolished binding, identifying it as a novel protein-protein interaction module. Yeast two-hybrid, in vitro binding assays with recombinant proteins, site-directed mutagenesis European journal of biochemistry High 9490029
1998 p40phox is phosphorylated on Thr154 and Ser315 during NADPH oxidase activation. In vitro phosphorylation studies and kinase inhibitor experiments implicate a PKC-type kinase in this phosphorylation. Phosphoamino acid analysis, tryptic peptide mapping, directed mutagenesis of PKC consensus sites, in vitro kinase assays with PKC and CK2, inhibitor studies in HL-60 cells and neutrophils The Journal of biological chemistry High 9804763
1999 p40phox is phosphorylated by protein kinase C during neutrophil activation; PKC inhibitor H-7 blocked p40phox phosphorylation and its membrane translocation, and purified PKC directly phosphorylated p40phox within the p40phox/p47phox/p67phox complex in vitro. 32P labeling of guinea pig neutrophils, PKC inhibitors, in vitro kinase assay with purified PKC Journal of leukocyte biology Medium 10577519
1999 Rac1-GTP (but not Cdc42) displaces p67phox from p40phox in vitro; a peptide corresponding to p67phox residues 170–199 (Rac-binding region) inhibits this displacement, suggesting Rac-GTP binding causes a conformational change in p67phox that disrupts its association with p40phox. In vitro binding/displacement assay with recombinant proteins and synthetic peptides Biochemical and biophysical research communications Medium 10486263
1999 Thioredoxin (TRX) interacts with p40phox, as identified by yeast two-hybrid screening; this interaction requires the reducing activity of TRX (a redox-inactive C32S/C35S mutant does not interact), suggesting redox-dependent regulation of p40phox by TRX. Yeast two-hybrid screening of B-cell library, histidine prototrophy and β-galactosidase assays with TRX mutants Immunology letters Low 10397171
1999 Ku70, a DNA-dependent protein kinase regulatory component, interacts with p40phox via two distinct regions (central core aa 50–260 and C-terminal aa 260–339 of p40phox); they co-localize in B lymphocytes and co-migrate in successive chromatography steps. Additionally, p47phox and p67phox are substrates for DNA-PK in vitro and are present in the nucleus of B cells. Yeast two-hybrid, affinity pulldown of recombinant proteins, co-elution in multi-step column chromatography, immunofluorescence co-localization Journal of cell science Medium 9914162
2000 p40phox promotes NADPH oxidase activation in a cell-free system by increasing the affinity of p47phox for flavocytochrome b558 approximately 3-fold. Cell-free NADPH oxidase reconstitution assay with recombinant components The Biochemical journal Medium 10861218
2001 The PX domain of p40phox specifically binds PtdIns(3)P and localizes to PtdIns(3)P-enriched early endosomes in cells; an Arg-to-Gln mutation in the PX domain eliminates phosphoinositide binding and disrupts endosomal localization. PI3K inhibition also disrupts this localization. Lipid-binding assays (protein-lipid overlay), GFP-fusion live-cell imaging, PI3K inhibitor treatment, site-directed mutagenesis Nature cell biology High 11433300
2001 PtdIns(3)P, generated by PI3K, stimulates NADPH oxidase-dependent ROS formation specifically when p40phox is added to the minimal core complex; this effect is mediated by binding of PtdIns(3)P to the PX domain of p40phox. Cell-free NADPH oxidase reconstitution assay with lipid vesicles and recombinant p40phox; PX domain mutant analysis Nature cell biology High 11433301
2001 Crystal structure of the p40phox PX domain bound to PtdIns(3)P at 1.7 Å resolution shows the PX domain embraces the 3-phosphate in a water-filled, positively charged pocket; 3-phosphoinositide specificity is achieved by the binding pocket geometry. An Arg residue mutated in CGD-associated p47phox stabilizes a critical lipid-binding loop but does not directly contact the phosphoinositide. The SH3 domain in full-length p40phox does not affect soluble PtdIns(3)P binding. X-ray crystallography (1.7 Å), site-directed mutagenesis, lipid binding assays Molecular cell High 11684018
2001 The p40phox PX domain specifically binds PtdIns(3)P in vitro and localizes to endosomes in intact cells; the Y59A/L65Q PX domain mutant with reduced PtdIns(3)P affinity fails to target EGFP-p40-PX to endosomes. In vitro phosphoinositide binding assay, GFP-fusion confocal microscopy in intact cells, site-directed mutagenesis The Journal of biological chemistry Medium 11729195
2001 p40phox (and p47phox) bind to moesin via their PX domains in a phosphoinositide-dependent manner; the N-terminal part of moesin mediates this interaction. Co-immunoprecipitation from neutrophil lysates, affinity pulldown with recombinant proteins Biochemical and biophysical research communications Low 11716484
2001 Small-angle neutron scattering showed that p40phox binds full-length p47phox in solution; the C-terminal end of p47phox is essential for this interaction, and the SH3 domain of p40phox interacts with the C-terminal proline-rich region of p47phox. Small-angle neutron scattering (SANS), gel filtration, domain truncation analysis Biochemistry Medium 11258927
2002 p40phox enhances membrane translocation of p67phox and p47phox in stimulated cells via its PC motif binding to the PB1 domain of p67phox, facilitating superoxide production. Mutations D289A in the p40phox PC motif or K355A in the p67phox PB1 domain each abolish this enhancement, defining the PB1-PC interaction as the mechanism. Transfection in cell lines, oxidase activity assay, membrane fractionation, site-directed mutagenesis of PC and PB1 domains The EMBO journal High 12456638
2003 The p40phox PX domain membrane binding is initiated by nonspecific electrostatic interactions followed by hydrophobic membrane penetration triggered by PtdIns(3)P. In contrast to p47phox, p40phox lacks an intramolecular SH3–PX interaction that would prevent nonspecific membrane penetration. PtdIns(3)P-induced membrane penetration is required for correct subcellular localization of p40phox in cells. Surface plasmon resonance, lipid monolayer technique, electrostatic potential calculations, GFP-fusion cell imaging, full-length protein studies The Journal of biological chemistry High 12556460
2004 Phosphorylated p40phox (but not unphosphorylated) inhibits NADPH oxidase in a cell-free system; phosphorylation of Thr154 specifically is required for the inhibitory effect. Phosphorylation induces a conformational change in p40phox (altered thrombin proteolysis pattern) without disrupting binding to p47phox or p67phox. Cell-free NADPH oxidase reconstitution assay, directed mutagenesis of PKC sites, pulldown assays, limited proteolysis Biochemistry Medium 15035643
2004 TRAF3 associates with p40phox and with p85 (PI3K subunit) in B cells, and this association mediates CD40-stimulated ROS production via NADPH oxidase; dominant-negative TRAF3 or N17Rac1 prevented full CD40-induced ROS production. Co-immunoprecipitation, dominant-negative overexpression, ROS measurement with antioxidants and kinase inhibitors Journal of immunology Low 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 shows that SH3p40 binds both a consensus polyproline motif and a noncanonical motif of the p47phox C-terminus. Phosphorylation of p47phox Ser-379 (but not Ser-359/370) destabilizes the interaction with both SH3p40 and the C-terminal SH3 of p67phox. X-ray crystallography (1.46 Å), intrinsic tryptophan fluorescence binding measurements, systematic phosphomimetic peptide binding studies The Journal of biological chemistry High 15657040
2006 p40phox-deficient mice show severe defects in NADPH oxidase responses (up to 97% reduction depending on stimulus) and CGD-like defects in S. aureus killing in vitro and in vivo; p67phox expression is also reduced ~55% in the absence of p40phox. p40phox knockout mice (p40phox-/-), ROS measurement by luminometry and flow cytometry, bacterial killing assays in vitro and in vivo The Journal of experimental medicine High 16880254
2006 p40phox is required for FcγR-mediated phagocytosis-induced NADPH oxidase activation in COS(phox)FcγR cells; this requires both PI(3)P binding by the p40phox PX domain and intact SH3/PB1 domains. PI3K inhibitors blocked phagosome NADPH oxidase activation in primary macrophages, establishing PI(3)P as the coupling signal. Stable transgene expression in COS7 cells, point mutants in PX/SH3/PB1 domains, superoxide assay, PI3K inhibitor studies in primary macrophages The Journal of experimental medicine High 16880255
2006 PtdIns(3)P binding to the p40phox PX domain (R58A mutation abrogates this) is a physiological signal for oxidase activation: p40phox(R58A/-) mice show ~60% reduced oxidase response to S. aureus phagocytosis and are significantly compromised in S. aureus killing in vivo. Wortmannin inhibition correlates with R58A-dependent reduction, showing the PI3K–PtdIns(3)P–p40phox axis mediates the response. Knock-in mice (R58A point mutation), primary neutrophil oxidase assays, phagosomal PtdIns(3)P accumulation measurement, in vivo S. aureus killing The EMBO journal High 16990793
2006 GFP-p40phox translocates to early endosomes via its PX domain (PtdIns(3)P binding required), whereas GFP-p47phox translocates to plasma membrane; GFP-p67phox depends on p40phox for endosomal targeting and on p47phox for plasma membrane targeting. A head-to-tail (PX–PB1 domain) intramolecular interaction in p40phox keeps the PX domain inaccessible in the resting state. GFP-fusion live-cell imaging, domain mutants disrupting phospholipid binding or protein-protein interactions, deletion mutagenesis, in vitro binding assays Molecular biology of the cell High 17122360
2006 Vav3-dependent Rac2 activation and a separate Vav1/Vav3-dependent, Rac-independent, PI3K-dependent pathway both contribute to FcγR-mediated NADPH oxidase activation; the latter pathway is proximal to oxidase activation and involves PI3K-dependent phosphorylation of p40phox. Vav1/Vav3 and Rac1/Rac2 knockout neutrophils, superoxide measurement, PI3K inhibitor treatment, p40phox phosphorylation analysis Journal of immunology Medium 17056570
2007 Crystal structure of full-length p40phox reveals that the PB1 domain inhibits PtdIns(3)P binding by the PX domain through an intramolecular PB1–PX interaction; the PB1–PX interface is on the opposite side of the PB1 domain from the p67phox-binding surface, so PB1-mediated PX regulation occurs without preventing PB1–PB1 association with p67phox. X-ray crystallography of full-length p40phox, in vitro PtdIns(3)P binding assays with truncation/deletion constructs, cell-based localization assays The EMBO journal High 17290225
2007 p40phox can substitute for p47phox as an organizer of NADPH oxidase activation in a cell-free system reconstituted with PtdIns(3)P-containing membranes, reaching ~70% of p47phox-dependent activity; p40phox SH3 domain interacts with the p22phox proline-rich region, as shown by far-western blotting, providing an alternative docking mechanism. Cell-free NADPH oxidase reconstitution with relipidated cytochrome b558 and PtdIns(3)P vesicles, domain mutants (D289A, R58A, W207R), far-western blotting with p22phox peptide FEBS letters Medium 17803994
2007 p40phox bearing the R57Q PX domain mutation (abolishing PtdIns(3)P binding) acts as a dominant inhibitor of oxidase activation and shows increased association with actin and moesin in the Triton-insoluble fraction, sequestering p67phox and p47phox in the cytoskeleton. A second mutation at Asp-289 (disrupting p67phox interaction) reverses these effects, indicating that cytoskeletal anchoring via the PX domain and PtdIns(3)P-binding together constitute a dual regulatory mechanism. Transgenic COS(phox) cell expression, superoxide assay, Triton X-100 fractionation, immunofluorescence, site-directed mutagenesis The Journal of biological chemistry Medium 17698849
2007 p40phox depletion in PLB-985 cells reduces ROS production and impairs bacterial killing even when p67phox levels are unchanged; PtdIns(3)P-independent PX domain function is required for p67phox translocation, while both PtdIns(3)P binding and SH3 domain function are required for full oxidase activation. An autoinhibited PX–PB1 conformation was identified; disrupting it increases oxidase activity ~2.5-fold. Lentiviral shRNA knockdown, cytosol-reconstituted permeabilized neutrophil core system, PX chimeric and deletion constructs, p47PX–p40 chimeras, bacterial killing assay The Journal of biological chemistry High 18029359
2008 p40phox translocates to nascent phagosomes before phagosome internalization and before PI(3)P accumulation, driven by p67phox binding (not PI(3)P); after phagosome internalization, PI(3)P accumulates and p40phox PI(3)P binding sustains p40phox retention and stimulates oxidase activity. p40phox functions primarily to regulate oxidase activity rather than assembly, via a PI(3)P signal that increases after phagosome sealing. Real-time fluorescence imaging of YFP-tagged phox proteins, PI(3)P probe imaging, wortmannin inhibition, PI(3)P-binding domain mutants, immunofluorescence Blood High 18711001
2010 Phosphorylation of p40phox on Thr154, but not Ser315, is required for full NADPH oxidase activation in response to fMLP, serum-opsonized S. aureus, and IgG-opsonized red blood cells in primary mouse neutrophils; this phosphorylation is mediated by PKCδ and is required for full translocation of p47phox to phagosomes. An intact SH3 domain is not required for oxidase activation; deletion of the entire SH3 domain enhances oxidase responses. Retroviral transduction of p40phox-/- bone marrow with wild-type and phosphorylation-site mutants, radiation chimaera reconstitution, primary neutrophil oxidase assays, PKCδ inhibitor/siRNA, phagosomal p47phox translocation imaging Blood High 20861461
2010 The PX domain of p40phox directly binds F-actin in a phosphoinositide-independent manner; both full-length p40phox and the isolated PX domain co-localize with F-actin in a peripheral lamellar compartment in COS cells; disruption of actin with cytochalasin D relocates p40phox. Actin was identified as a direct binding partner of p40PX by affinity chromatography from neutrophil extracts. GFP-fusion cell imaging, PX domain deletion and lipid-binding mutants, affinity chromatography with neutrophil extracts, pure actin binding assay, cytochalasin D treatment The international journal of biochemistry & cell biology Medium 20637895
2011 p40phox acquires PI(3)P-binding capability through: (1) H2O2-induced conformational changes in the cytoplasm, and (2) direct or indirect membrane targeting dependent on p47phox. p40phox is essential when p47phox is only partially phosphorylated during FcγR-mediated oxidase activation, but dispensable when p47phox is fully phosphorylated. The autoinhibitory PX–PB1 interaction in p40phox prevents PI(3)P binding in the resting state but is released on the phagosomal membrane. HEK293(Nox2/FcγRIIa) cells, RAW264.7 with p40/p47 knockdown, phosphorylation-mimicking p47phox mutants, p40phox PX-PB1 disruption mutants, membrane targeting constructs The Journal of biological chemistry Medium 21956105
2013 p40phox expression is transcriptionally regulated by the aryl hydrocarbon receptor (AhR); AhR directly binds the p40phox promoter (identified by promoter analysis), and AhR activation increases p40phox expression and NADPH oxidase activity in liver/hepatoma cells; RNAi knockdown of p40phox abolishes AhR-induced ROS elevation. AhR-knockout mice, promoter-reporter deletion analysis, RNAi knockdown, NADPH oxidase activity assay, ChIP Molecular pharmacology Medium 23478803
2016 Ncf4-/- mice show severe defects in overall ROS production (due to concomitant NCF1/NCF2 reduction) and develop aggravated autoimmune arthritis with delayed neutrophil apoptosis. Mice with a selective R58A mutation in NCF4 (disrupting PtdIns(3)P binding) show selectively impaired intracellular NOX2 activity with milder effects on innate immunity but clearly promote susceptibility to collagen-induced arthritis, establishing a specific role for NCF4-mediated intracellular ROS in autoimmunity. Ncf4-/- and Ncf4(R58A) knock-in mice, collagen-induced arthritis model, mannan-induced psoriatic arthritis model, ROS assays, neutrophil apoptosis assays Antioxidants & redox signaling High 27231144
2022 NCF4 with the R58A mutation (impaired endosomal PtdIns(3)P binding) reduces intracellular but not extracellular ROS in B cells; this selectively enhances plasma cell differentiation and autoantibody production in collagen-immunized mice, an effect B-cell intrinsic as confirmed by chimeric B cell transfer and in vitro stimulation. NCF4(R58A) knock-in mice, chimeric B cell transfer, in vitro LPS/CD40L/anti-IgM stimulation, flow cytometry for plasma cells, CXCR3/CXCR4 expression analysis Redox biology High 36095971
2024 NCF4 interacts with ASC (apoptosis-associated speck-like protein containing a CARD) and cooperates with NCF1 and NCF2 to promote NLRP3 and AIM2 inflammasome activation. NCF4 phosphorylation causes redistribution from the NADPH oxidase complex to the perinuclear region, mediating ASC oligomerization, speck formation and inflammasome activation. NCF4 functions as a ROS sensor that couples ROS levels to inflammasome assembly. Immunoprecipitation–mass spectrometry (ASC interactome), co-IP validation, fluorescence imaging of NCF4 puncta/redistribution, inflammasome activation assays (IL-1β, caspase-1), genetic deficiency models in mice, colorectal cancer model Nature communications Medium 38886341
2024 NCF4-dependent intracellular ROS (via endosomal PtdIns(3)P binding) maintains cysteine peptides in an oxidized, cross-linked state in macrophages, preventing their presentation to non-tolerized autoreactive T cells; NCF4(R58A) macrophages efficiently present uncross-linked cysteine peptides (GPI325–339), leading to strong arthritogenic T cell responses and arthritis, which is abolished by clodronate depletion of antigen-presenting cells. NCF4(R58A) knock-in mice, antigen presentation assays with cysteine-containing GPI peptides, T cell activation readouts, clodronate depletion, FTY720 lymphocyte trapping, arthritis scoring Redox biology Medium 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 525 11433300
2001 PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox). Nature cell biology 338 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 235 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 108 8670049
2018 Inherited p40phox deficiency differs from classic chronic granulomatous disease. The Journal of clinical investigation 102 29969437
2006 PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation. The EMBO journal 101 16990793
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
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 78 18711001
2006 A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox. Molecular biology of the cell 75 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 45 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 38 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
2016 A Reduction in Intracellular Reactive Oxygen Species Due to a Mutation in NCF4 Promotes Autoimmune Arthritis in Mice. Antioxidants & redox signaling 30 27231144
2002 Multiple PU.1 sites cooperate in the regulation of p40(phox) transcription during granulocytic differentiation of myeloid cells. Blood 30 12036891
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
2015 Role of an SNP in Alternative Splicing of Bovine NCF4 and Mastitis Susceptibility. PloS one 20 26600390
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
2024 NCF4 attenuates colorectal cancer progression by modulating inflammasome activation and immune surveillance. Nature communications 18 38886341
1996 Translocation of guinea pig p40-phox during activation of NADPH oxidase. Biochimica et biophysica acta 18 8982388
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
2007 p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox. FEBS letters 12 17803994
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
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

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