Cloning of rat p47phox and comparison with human p47phox.

A cDNA encoding rat p47phox was cloned from rat spleen cDNA library, utilizing rapid amplification of cDNA ends. The open reading frame corresponded to 389 amino acids: It contained the phagocyte oxidase homology domain, two Src homology 3 domains and a proline rich region, all of which are conserved in mammalian p47phox sequences. Rat p47phox displayed the highest degree of identity to mouse p47phox (94%). We expressed and purified rat p47phox as a glutathione S-transferase fusion protein, and found that the rat protein could replace human p47phox in a cell-free activation system for human NADPH oxidase, giving about half activity. Although rat 12-lipoxygenase interacted with human p47phox in a yeast two-hybrid system, this was not the case for rat p47phox.     

Direct interaction of actin with p47(phox) of neutrophil NADPH oxidase

The cell-free activation of human neutrophil NADPH oxidase is enhanced by actin, and actin filaments formed during activation are suggested to stabilize the oxidase. In an attempt to elucidate the mechanism, we examined the protein-protein interactions between actin and cytosolic components of the oxidase. Far-Western blotting using recombinant phox proteins showed that both alpha- and beta-actin interacted with p47(phox) and rac1, and weakly with rac2. A deletion mutant of p47(phox) proved that its C-terminal region was essential for the interaction. The dissociation constant (K(d)) for interaction between actin and p47(phox) was estimated to be 0.45 microM by surface plasmon resonance, and that between actin and rac1 or rac2 was 1.7 or 4.6 microM, respectively. Far-Western blotting using cytosol as a target showed an interaction between actin and endogenous p47(phox) and rac proteins. These results suggest that actin can directly interact with p47(phox) and possibly with rac in the cells.     

Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCdelta, -epsilon, -theta;, and -eta) and conventional PKC (including PKCalpha and -beta), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCepsilon, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCdelta, -theta;, or -eta is required. Furthermore, rottlerin (at doses that inhibit PKCdelta activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O2 production at similar doses. In addition to pharmacological inhibitors, PKCdelta-specific antisense oligodeoxyribonucleotides were used. PKCdelta antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCdelta in vitro. Furthermore, lysate-derived PKCdelta from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCdelta plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.     

Regulation of protein kinase CKII by direct interaction with the C-terminal region of p47(phox)

Protein kinase CKII is a Ser/Thr kinase which is involved in many proliferation-related processes in the cell. p47(phox) is a component of the leukocyte NADPH oxidase, which is an important element of host defense against microbial infection. In this study, we demonstrate that a truncated form of the p47(phox) lacking its N-terminal region (p47(phox)/SH3-C), but not a truncated form of the p47(phox) lacking its C-terminal region (p47(phox)/N-SH3), undergoes better phosphorylation by CKII in the presence of arachidonic acid. The yeast two-hybrid test and the glutathione S-transferase (GST) pull-down assay showed that p47(phox) interacts specifically with the regulatory beta subunit (CKIIbeta), but not with the catalytic alpha subunit (CKIIalpha) of the tetrameric CKII holoenzyme. The binding of p47(phox) to CKIIbeta requires the C-terminal region of p47(phox) and is completely abolished by addition of spermine, indicating that a highly basic region in the C-terminal region of p47(phox) contributes to binding to CKIIbeta. In addition, p47(phox) stimulates the catalytic activity of CKII holoenzyme; this stimulation also requires the C-terminal region of p47(phox). Coimmunoprecipitation experiments showed that CKII holoenzyme interacts with p47(phox) in human neutrophils. Taken together, the present data indicate that the C-terminal region of p47(phox) plays a significant role in the arachidonic acid-dependent phosphorylation of p47(phox) by CKII and that the same region of p47(phox) associates directly with CKIIbeta and can modulate the catalytic activity of CKII holoenzyme.     

Phosphorylation induces conformational changes in the leukocyte NADPH oxidase subunit p47(phox).

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(phox) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that phosphorylation of p47(phox) with protein kinase C induces conformational changes, as reflected by a fluorescence change of N, N'-di-methyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD)-labeled p47(phox). We propose that this alteration in conformation results in the appearance of a binding site through which p47(phox) interacts with cytochrome b558 during the activation process. In addition, the present study indicates that other oxidase components, such as p67(phox) and p22(phox), influence the conformation of p47(phox).     

p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox

p40(phox) activated phagocyte NADPH oxidase without p47(phox) in a cell-free system consisting of p67(phox), Rac and cytochrome b(558) relipidated with phosphatidylinositol 3-phosphate. The activation reached to 70% of that by p47(phox). Addition of p47(phox) slightly increased the activation, but not additively. p40(phox) improved the efficiency of p67(phox) in the activation. The C-terminus-truncated p67(phox), p40(phox)(D289A), p40(phox)(R58A), or p40(phox)(W207R) showed an impaired activation. A peptide corresponding to the p22(phox) Pro-rich region suppressed the activation, and far-western blotting revealed its interaction with p40(phox) SH3 domain. Thus, p40(phox) can substitute for p47(phox) in the activation, interacting with p22(phox) and p67(phox) through their specific regions.     

Kinase-dependent change in the conformation of the leukocyte NADPH oxidase subunit p47phox

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. In whole cells and under certain circumstances in the cell-free system, the phosphorylation of p47phox mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47phox may be an important part of the activation mechanism. The total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47phox was substantially decreased, reflecting on the conformational change that occurs when p47phox was phosphorylated with protein kinase C. We show here that the phosphorylation of p47phox by protein kinase A or mitogen-activated protein kinase, however, had little effect on the intrinsic fluorescence of p47phox. In addition, the present experiments indicate that in the mutant p47phoxS379A, only the single S-->A mutation appears to be a major importance for the function of p47phox, which is able to undergo the change in conformation that takes place when p47phox is phosphorylated by protein kinase C.     

C-terminal region of the cytosolic subunit p47(phox) is a primary target of conformational change during the activation of leukocyte NADPH oxidase

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O(2(-)) from oxygen using NADPH as the electron donor. During activation, the cytosolic oxidase components p47(phox) and p67(phox), each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane, where they associate with cytochrome b(558), a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid and the phosphorylation of p47(phox )with protein kinase C. Activators of the oxidase in vitro cause exposure of p47(phox)-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the total protein steady-state intrinsic fluorescence exhibited by the tryptophan residues of p47(phox) substantially decreased when N-terminal truncated p47(phox)-SH3-C was treated with anionic amphiphiles or phosphorylated with protein kinase C. This finding was similar to the results obtained with full-length p47(phox). However, the fluorescence of C-terminal truncated p47(phox)-N-SH3 and both C-terminal and N-terminal truncated p47(phox)-SH3 were not altered by the activators. These results indicate that the C-terminal region of p47(phox) is a primary target of the conformational change during the activation of NADPH oxidase.     

The NADPH oxidase components p47(phox) and p40(phox) bind to moesin through their PX domain.

The NADPH oxidase of phagocytes is a membrane-bound heterodimeric flavocytochrome which catalyses the transfer of electrons from NADPH in the cytoplasm to oxygen in the phagosome. A number of cytosolic proteins are involved in its activation/deactivation: p47phox, p67phox, p40phox and the small GTP-binding protein, rac. The cytosolic phox proteins interact with the cytoskeleton in human neutrophils and, in particular, an interaction with coronin has been reported (Grogan A., Reeves, E., Keep, N. H., Wientjes, F., Totty, N., Burlingame, N. L., Hsuan, J., and Segal, A. W. (1997) J. Cell Sci. 110, 3071-3081). Here, we report on the interaction of another cytoskeletal protein, moesin, with the phox proteins. Moesin belongs to the ezrin-radixin-moesin family of F-actin-binding proteins and we show that it binds to p47phox and p40phox in a phosphoinositide-dependent manner. Furthermore, we show that its N-terminal part binds to the PX domain of p47phox and p40phox.     

Binding of the PX domain of p47(phox) to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction

p47(phox) is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47(phox) PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40(phox), while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipid-binding pockets radically increases membrane affinity. Strikingly, measurements for full-length p47(phox) show that membrane interaction by the PX domain is masked by an intramolecular association with the C-terminal SH3 domain (C-SH3). Either a site-specific mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47(phox) [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX domain.     

The C-terminus of Dpb2 is required for interaction with Pol2 and for cell viability

DNA polymerase ε (Pol ε) participates in the synthesis of the leading strand during DNA replication in Saccharomyces cerevisiae. Pol ε comprises four subunits: the catalytic subunit, Pol2, and three accessory subunits, Dpb2, Dpb3 and Dpb4. DPB2 is an essential gene with unclear function. A genetic screen was performed in S. cerevisiae to isolate lethal mutations in DPB2. The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation. This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits. This resulted in the purification of a Pol2/Dpb3/Dpb4 complex that possessed high specific activity and high processivity and holoenzyme assays with PCNA, RFC and RPA on a single-primed circular template did not reveal any defects in replication efficiency. In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity. Thus, the C-terminal motif of Dpb2 that we have identified may instead be required for Dpb2 to fulfill an essential structural role at the replication origin or at the replication fork.     

Phosphorylation of p67phox in the neutrophil occurs in the cytosol and is independent of p47phox.

p67phox and p47phox are phosphorylated in the course of stimulation of the NADPH oxidase in neutrophils. Isolated neutrophil cytosol can phosphorylate both of these proteins in vitro. Phosphoamino acid analysis showed that isolated membranes can tyrosine-phosphorylate p67phox in vitro. Further experiments with anti-phosphotyrosine antibodies did not support a role for tyrosine phosphorylation of p67phox in the cell. A phosphopeptide analysis showed that the phosphorylation of p67phox is unchanged in the absence of p47phox. These results further characterise the phosphorylation of p67phox and provide evidence that this is a cytosolic event independent of interaction with p47phox and the membrane.     

Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.     

TNF-alpha induces phosphorylation of p47(phox) in human neutrophils: partial phosphorylation of p47phox is a common event of priming of human neutrophils by TNF-alpha and granulocyte-macrophage colony-stimulating factor

Phosphorylation of p47(phox) is a key event in NADPH oxidase activation. We examined the ability of proinflammatory cytokines such as TNFalpha, IL-1, and G-CSF to induce this process compared with GM-CSF. Only TNF-alpha and GM-CSF induced a clear p47(phox) phosphorylation. This phosphorylation was time dependent and reached its maximum at 20 min. Two-dimensional phosphopeptide mapping of p47(phox) phosphorylated in neutrophils primed with TNF-alpha revealed partial phosphorylation of p47(phox) on the same peptide as for GM-CSF. Neutrophil incubation with TNF-alpha and subsequent addition of the chemotactic peptide fMLP resulted in more intense phosphorylation of p47(phox) sites than with each reagent alone. A neutralizing Ab against the p55 TNF receptor, contrary to a neutralizing Ab against the p75 TNF receptor, inhibited TNF-alpha-induced p47(phox) phosphorylation. Neutrophil treatment with both TNF-alpha and GM-CSF resulted in more intense phosphorylation of the same p47(phox) peptide observed with each cytokine alone, suggesting that they engaged pathways converging on common serines. This additive effect was also obtained on the priming of NADPH oxidase activity. The use of protein kinase inhibitors pointed to the involvement of a protein tyrosine kinase, but not protein kinase C. These findings show that TNF-alpha, via its p55 receptor, induces a protein tyrosine kinase-dependent selective phosphorylation of p47(phox) on specific serines. The ability of TNF-alpha and GM-CSF, two different cytokines with two different receptors to induce this specific p47(phox) phosphorylation, suggests that this event could be a common element of the priming of neutrophils by TNF-alpha and GM-CSF.     

Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.     

Expression of a p67(phox) homolog in Caco-2 cells giving O(2)(-)-reconstituting ability to cytochrome b(558) together with recombinant p47(phox)

Human normal and transformed (Caco-2) colon tissues as well as guinea pig gastric mucosal cells express Nox1, which is a homolog of the phagocyte NADPH oxidase subunit, gp91(phox) of membrane-bound cytochrome b(558). It was reported that Nox1-transfection to NIH 3T3 cells could provide O(2)(-)-generating ability, independently of regulatory cytosolic factors (Rac2, p67(phox), and p47(phox)) that are obligatory in the phagocyte oxidase system. Here, we detected and sequenced a p67(phox) homolog in Caco-2 almost identical to the neutrophil sequence, except for three nucleotide substitutions, two of which changed lysines 181 and 328 to arginines. Investigation of its ability to support O(2)(-)-generation in cell-free reconstitution experiments combining with neutrophil cytochrome b(558) showed O(2)(-)-generation, provided that recombinant p47(phox) was added. This result demonstrates that the intrinsic p67(phox) homolog of Caco-2 was able to function as a phagocyte p67(phox) for cytochrome b(558). The requirement of p47(phox) addition suggested that this component was absent in Caco-2 cells. Caco-2 membranes, used as a source of Nox1 in place of cytochrome b(558), did not show significant O(2)(-)-generation, which was mainly explained by their very little Nox1 expression.     

Trichinella spiralis infection affects p47(phox) protein expression in guinea-pig alveolar macrophages

To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.