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CAVEAT LECTOR
Jeanette H. W. Leusen1,2, Arthur J. Verhoeven1 and Dirk Roos1,3
1 Central Laboratory
of the Netherlands Red Cross Blood Transfusion Service and Laboratory
for Experimental and Clinical Immunology, University of Amsterdam,
Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
2Department
of Pediatrics, Emma's Children Hospital, Academic Medical Center,
Amsterdam, The Netherlands.
Received 04/15/96; Accepted 05/05/96; On-line 07/01/96
When microorganisms invade the body, they encounter a large asssortment of defense mechanisms. Among these, phagocytes play an important role in the process of killing pathogens. This event is mediated by two important processes, viz. activation of the NADPH oxidase enzyme, which leads to the production of toxic oxygen metabolites, and fusion of intracellular granules with the phagosome (the vesicle that contains the ingested micro-organisms), which causes release of the toxic granule contents into this vesicle. The human NADPH oxidase is a very complex enzyme, in two ways:
1. it exists of at least 6 components: cytochrome b558 (a heterodimer comprised of gp91-phox and p22-phox), p47-phox, p67-phox, p40-phox, rac and Rap1A, and
2. there are multiple signal transduction pathways leading to activation of the NADPH oxidase.
The most likely reason for this complexity is the toxicity of the oxygen radicals produced by the active NADPH oxidase; these compounds are not only harmful to the invading pathogens, but also to the surrounding tissues. This latter effect is enforced by the activation of metalloproteases released by neutrophils and by oxidation of protease inhibitors by oxygen metabolites (1). Therefore, an improper activation of the NADPH oxidase must be prevented at all costs and, when the infection has been cleared, a rapid deactivation mechanism is imperative.
In this review, the interaction between the different components of the NADPH oxidase and the activation of these proteins will be discussed.
The NADPH oxidase is a multi-component enzyme, localized in the plasma membrane of phagocytic leukocytes (2). It accepts electrons from NADPH at the cytosolic side of the membrane and donates these to molecular oxygen at the other side of the membrane, either on the outside of the cells or in the phagosome that contains ingested micro-organisms (3). In this way, a one-electron reduction of oxygen to superoxide anion is catalyzed, at the expense of NADPH.
2 O2 + NADPH --->. 2 O2· - + NADP++ H+
The superoxide produced is subsequently converted to hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and other microbicidal products.
In resting, non-phagocytosing leukocytes, the NADPH oxidase is inactive. In this form, the enzyme components are localized in different parts of the cell. Phagocyte activation, e.g. by binding of opsonized micro-organisms to cell surface receptors, leads to assembly of the active enzyme complex.
2.1. Mechanisms of activation of the oxidase
The oxidase can be activated by receptor-mediated and by receptor-independent mechanisms. Typical receptor-dependent stimuli are the complement fragment C5a, the chemotactic tripeptide N-formyl-Met-Leu-Phe (fMLP)(for reviews see (4, 5)), and immune complexes (for reviews, see (6-9)). Receptor-independent stimuli include long-chain unsaturated fatty acids and phorbol 12-myristate 13-acetate (PMA). Oxidase activation by receptor-mediated stimuli usually lasts less than 5 min, while receptor-independent stimuli activate the enzyme for a much longer period, but only when the stimulus remains present. It appears that in intact cells the activated oxidase is undergoing a continuous process of activation and deactivation.
3. Chronic Granulomatous Disease (CGD)
The importance of the NADPH oxidase in human host defense is exemplified by patients suffering from chronic granulomatous disease (CGD). CGD is an immunodeficiency syndrome characterized clinically by severe recurrent bacterial and fungal infections. These infections typically consist of pneumonia, lymphadenitis and abcesses that involve the liver, the subcutaneous tissues and the bones. Control of the infections can usually be achieved with appropriate antibiotic therapy, but eradication of the infecting organisms is often slow and attained with great difficulty. The persistence of micro-organisms, often within the phagosomal vacuoles of neutrophils or macrophages, is the stimulus to a chronic inflammatory state with granuloma formation.
Biochemically, CGD is characterized by the inability of phagocytic leukocytes (neutrophils, eosinophils, monocytes and macrophages) to activate the NADPH oxidase and to generate the reactive oxygen compounds needed for the killing of phagocytosed micro-organisms (10, 11). Therefore, the most common pathogens encountered in CGD patients are catalase-positive organisms, because catalase prevents the CGD phagocytes from using microbial hydrogen peroxide for killing these pathogens. Predominant are Staphylococcus aureus, Aspergillus species and a variety of gram-negative enteric bacilli. CGD is a rare disease, with an estimated incidence of 1 in about 250,000 individuals, without any ethnic preference. It usually manifests itself in early childhood and is predominantly found in boys (12). CGD is a very heterogeneous disorder; clinically because of many antimicrobial systems that can partially compensate for the defect in oxygen-dependent killing systems, and biochemically because of the complicated genetic origin of CGD (13). Characterizing the mutations that lead to CGD is important for improved diagnosis and treatment of CGD patients, but also provides a better understanding of the functional domains within the oxidase components.
4. Thecomponents of the NADPH oxidase.
Four oxidase components have been identified through studies with CGD cells (14-20): two membrane-bound components, gp91-phox and p22-phox, which together comprise cytochrome b558, and two cytosolic components called p47-phox and p67-phox. Upon cell activation, the latter two proteins, together with a third cytosolic p40-phox component, translocate to cytochrome b558 in the plasma membrane to form the active enzyme (21, 22). Also, two low-molecular-weight GTP-binding proteins are involved in the activation of the NADPH oxidase, viz. Rap 1A and rac. The components of the NADPH oxidase and the assembly of this enzyme in the plasma membrane of a phagocyte are depicted in Fig. 1.
Figure 1. Assembly of the NADPH oxidase in phagocytic cells. In resting phagocytes, the cytochrome b558 subunits p22-phox and gp91-phox, in association with Rap1A, are located in the membranes of specific granules and of secretory vesicles. Upon cell activation, these organelles fuse with the plasma membrane, which results in expression of cytochrome b558 in this membrane. At the same time, a complex of p47-phox, p67-phox and p40-phox translocates from the cytosol to the plasma membrane and forms a complex with cytochrome b558. This translocation is facilitated by simultaneous redistribution of rac to the plasma membrane.
The membrane-bound gp91-phox/p22-phox heterodimer is designated cytochrome b558 because of its optical spectrum with an a absorbance peak at 558 nm (23-25). This protein is also known as cytochrome b-245 for its unusual low midpoint potential at -245 mV (26). Both a flavin (27-29) and two heme redox centers (30) are contained within the cytochrome heterodimer. Electrons supplied by NADPH are transferred to oxygen as follows (25,31)
| e- | ||||||||
|---|---|---|---|---|---|---|---|---|
| Heme | ---> | O2 | ---> | O2· - | ||||
| 2e- | 2e- | |||||||
| NADPH | ---> | Flavin (FAD) | ---> | Heme | ---> | O2 | ---> | O2· - |
| e- | ||||||||
| cytochrome b558 | ||||||||
In resting neutrophils, this flavocytochrome is localized in the membranes of specific granules and of secretory vesicles (26, 32-34). During cell activation, fusion of these organelles with the plasma membrane leads to re-allocation of the oxidase (34, 35).
The cDNA's of both subunits of cytochrome b558 have been cloned and sequenced, and the genes encoding these proteins have been localized and characterized (15, 36-38). The alpha subunit p22-phox contains 195 amino acids, with hydrophobic helices in the Nterminal half of the protein that could serve as membrane-spanning domains (38). The CYBA gene encoding p22-phox is located on chromosome 16 (15).
The beta subunit gp91-phox of cytochrome b558 contains 570 amino acids, with four or five transmembrane helices and five potential N-linked glycosylation sites in the amino-terminal region (37, 39, 40). The topology of gp91-phox has been determined with anti-peptide antibodies and partial proteolysis (40). This study showed that residues 150-172 are exposed on the outside, and an 18-kD C-terminal fragment is cytosolic. Furthermore, there is evidence that residue 240 is glycosylated, thus extracellular (A.W. Segal, unpublished results). The CYBB gene for gp91-phox is located on the X chromosome (37, 39). Mutations in this gene account for all cases of X-linked CGD (X91 CGD), which is the most common form of the disease (about 70% of all patients). In most cases of X-linked CGD, gp91-phox cannot be detected on immunoblot; this is referred to as X910 CGD. In some cases, residual gp91-phox protein and oxidase activity are present (X91- CGD) (41), and in a few exceptional cases the (non-functional) protein is present in normal amounts (X91+ CGD) (42-44). In p22-phox-affected CGD patients, the protein is typically absent (15, 38, 45), except in one patient with A22+ CGD (46). The mutation in the CYBA gene of this last patient is predicted to lead to a proline-toglutamine substitution at residue 156 in p22-phox. This residue resides in a proline-rich region that could be an important counter structure for src homology region 3 (SH3) domains (47-49) (see Fig. 2). The functional defect in this patient has been studied in ref. (50). Both subunits of cytochrome b558 are usually missing in A220 CGD as well as in X910 CGD (24, 38, 51, 52). This indicates that these subunits stabilize each other (53). It is not known, however, which regions in p22-phox and gp91-phox are involved in this mutual stabilization.
A 22-kD low-molecular-weight GTP-binding protein co-purifies with cytochrome b558 and remains associated with the cytochrome even after immunoaffinity purification on matrices composed of antibodies to cytochrome b558 (54, 55). This protein was identified as Rap1A by immunostaining. Rap1A has been shown to bind specifically to cytochrome b558, with a one-to-one stoichiometry. In neutrophils, Rap1A becomes phosphorylated by the cAMP-dependent kinase PKA (56). A serine residue (180Ser) at the COOH-terminal region of Rap1A has been identified as a site of phosphorylation by PKA (56), and phosphorylation of Rap1A abrogates the interaction with cytochrome b558 (57). It is possible that (phosphorylation of) Rap1A regulates the deactivation of the NADPH oxidase.
MGDTFIRHIALLGFEKRFVPSQHYVYMFLVKWQDLSEKWYRRFTEIYEFHKTLKE
MFPIEAGAINPENRIIPHLPAPKWFDGQRAAENRQGTLTEYCSTLMSLPTKISRCP
HLLDFFKVRPDDLKLPTDNQTKKPETYLMPKDGKSTATDITGPIILQTYRAIADYEKT
SGSEMALSTGWVVEVVEKSESGWWFCQMKAKRGWIPASFLEPLDSPDETEDPE
PNYAGEPYVAIKAYTAVEGDEVSLLEGEAVEVIHKLLDGWWVIRKDDVTGYFPSMYL
303/304 320 328
QKSGQDVSQAQRQIKRGAPRRSSIRNAHSIHQRSRKRLSQDAYRRNSVRRFLQQR
345 348 370 379
RRQARPGPQSPGSPLEEERQTQRSKPQPAVPPRPSADLILNRCSESTKRKLASAV
|
Figure 2. Putative phosphorylation sites in p47-phox. The 390 amino-acid sequence of p47-phox is given. In the last quarter of the protein, six putative PKC phosphorylation sites are localised (marked in red) and two putative MAP kinase sites (marked in blue). The consensus sequence for a PKC site is R-X-S-X-(R-R), with R for arginine or another positively charged amino acid (i.e. lysine) and X for a non-charged amino acid. The last two positively charged amino acids are not essential for a PKC site. The consensus sequence for a MAP kinase site is P-X-S/T-P or even X-P, with P for proline, S/T for serine or threonine and X for any amino acid. Whether these sites actually become phosphorylated is discussed in the text. The importance of the C-terminal sequence (marked in purple) is to be discussed in the text.
The majority of patients with CGD due to a deficiency of cytosolic NADPH oxidase components lacks detectable levels of p47-phox by immunoblotting; this defectaccounts for about 30% of all patients with CGD (58). Segel et al. have shown that a 47-kD phosphoprotein is absent in the neutrophils from certain patients with autosomal recessive inheritance of CGD who have normal levels of cytochrome b558 (59). Subcellular fractionation techniques have shown that this 47-kD phosphoprotein (now recognized as p47-phox) is present in the cytosol and in the plasma membrane, but not in granule fractions of PMA-stimulated neutrophils (60, 61). An explanation for this dual location was provided by Heyworth et al., who showed that phosphorylation of p47-phox initially occurs in the cytosol, before transloction of p47-phox to the membrane, and continues after membrane association (62). Two-dimensional electrophoresis revealed that the two most acidic isoforms are not found in stimulated neutrophils from CGD patients lacking cytochrome b558 (63, 64). Normal cells, as well as cells that contain normal levels of dysfunctional cytochrome b558 (bearing a 415Pro--->His substitution in gp91-phox) contain all phosphorylated isoforms (64). Thus, the final phosphorylations of p47-phox occur only when an intact cytochrome b558 is present and are likely to take place after translocation of p47-phox to the membrane.
The cDNA for p47-phox has been cloned, and the gene has been localized and characterized (18, 19). The NCF1 gene encoding p47-phox is located on chromosome 7 (65); mutations in this gene cause the A47 type of CGD. Recombinant p47-phox as well as purified cytosol fractions containing p47-phox are able to restore oxidase-supporting activity in cytosol from A47 CGD neutrophils (17-19, 66-68). The deduced amino-acid sequence (390 residues) of p47-phox contains at least six potential serine phosphorylation sites for protein kinase C (PKC), in good agreement with the apparent number of phosphorylated isoforms seen on two-dimensional electrophoresis. However, the actual kinase responsible for p47-phox phosphorylation in intact cells is not yet known. A schematic representation of the phos-phorylation sites in p47-phox is shown in Fig. 2.
Furthermore, the amino-acid sequence of p47-phox contains two SH3 motifs and at least one proline-rich region (Fig. 3). These domains probably play a role in the interaction between the cytosolic components and in the assembly of the NADPH oxidase (see below).
A minority of patients with cytosol-deficient CGD are lacking p67-phox, as determined by immunoblotting; this defect accounts for less than 5% of all cases of CGD (58). A full-length cDNA has been obtained and sequenced (20); p67-phox is encoded by the NCF2 gene that is located on chromosome 1 (65). The predicted 526-amino-acid sequence of p67-phox also contains two SH3 domains an at least one proline-rich region, as depicted in Fig. 3. Translocation experiments have revealed that p67-phox is unable to translocate to the plasma membrane in cells of p47-phox-deficient patients (69). In contrast, p47-phox appeared to translocate normally in cells of a p67-phox-deficient donor (69-71). However, we observed otherwise (almost no translocation of p47-phox in p67-phox deficient patients, J. Leusen et al., manuscript submitted).
A third cytolic phox protein was shown to reside in a complex with p67-phox in the cytosol of resting neutrophils (72, 73). The cDNA of this 40-kD protein has also been cloned, and the predicted protein of 339 amino acids contains one SH3 domain (73). Although p40-phox is not required for activity in the cell-free assay (see Section 5.1 'Cell-free system'), it is thought to play a role in stabilizing p67-phox in intact cells.
The last cytosolic protein required for the activity of the NADPH oxidase is a rasrelated protein called rac (74-78). This protein family has a wide tissue distribution; in neutrophils p21-rac2 is the most abundant rac protein, but p21-rac1 is also present (79). Probably, rac functions by changing from an inactive, GDP-bound state to an active, GTP-bound state in which it can mediate the activation of the NADPH oxidase. Fine-tuning of this process may be mediated by the regulation of GTP/GDP exchange of rac by GDP-dissociation inhibitor (GDI) protein and GDP-dissociation stimulator (GDS) protein (75-78, 80).
Figure 3. SH3 domains and proline-rich regions in the components of the NADPH oxidase. The membrane-bound p22-phox, the light subunit of cytochrome b558, contains one proline-rich region, a putative counter structure for one of the SH3 domains in the cytosolic proteins. P47-phox and p67-phox each contain two SH3 domains and one proline-rich region, p40-phox contains only one SH3 domain.
CGD patients with decreased NADPH oxidase activity due to mutations in rac or GTP/GDP exchange-regulating proteins are not known, probably due to the fact that these proteins are involved in several other (essential) cellular functions, such as vesicular transport and cytoskeleton dynamics. Mutations in such proteins may be incompatible with life.
5. Activation of the NADPH oxidase
Many of the recent advances in our understanding of the components of the NADPH oxidase have been the result of studies employing a cell-free activation system, first developed by Heyneman and Vercauteren (81) and Bromberg and Pick (82), and later extended by other investigators (83-88). The cell-free NADPH system is comprised of neutrophil membranes (containing cytochrome b558), and three neutrophil cytosolic fractions containing p47-phox, p67-phox and rac (89). The system is activated by GTP or its non-hydrolysable derivative GTPgS and an amphiphilic agent (SDS or arachidonic acid). NADPH is needed as a substrate for the NADPH oxidase. Unlike in intact cells, phosphorylation of p47-phox is not required in this system: inhibitors of PKC have no inhibitory effect in the cell-free system.
5.2. Important domains within cytochrome b558
CGD patients with a single amino-acid substitution in one of the subunits of cytochrome b558 have provided useful information about the structure and function of the cytochrome. For the A22+ patient described in ref. (50) the functional defect was obvious: the 156Pro--->Gln substitution in the proline-rich region of p22-phox disrupts a putative counter structure for SH3 domains in p47-phox. Indeed, we found no translocation of p47-phox and p67-phox to the plasma membrane in either intact neutrophils or in the cell-free system of this patient (50). We also showed a normal electron flow within the cytochrome b558 of this patient when neutrophil membranes were tested in a cell-free system without p47-phox and p67-phox, in which negatively charged phospholipids were added as activators for cytochrome b558 (90, 91). This proves that 1) the mutation in p22-phox in the neutrophil membranes does not affect the transfer of electrons from NADPH via FAD and heme to oxygen, and 2) the cytosolic proteins act only as activators of the NADPH oxidase, as was already stated by Koshkin and Pick (90, 91). This means that the superoxide-generating capacity is fully contained within the cytochrome b558. Most likely, the cytosolic proteins p47-phox and p67-phox induce by their interaction with cytochrome b558 a conformational change in the cytochrome that permits binding of NADPH and start of the electron flow in this flavocytochrome. The neutrophil membranes from all four X91+ patients tested by us in this system (with negatively charged phospholipids but without p47-phox and p67-phox as activators of the oxidase) were incapable of generating any superoxide, regardless of the ability of the cytosolic proteins to bind to cytochrome b558 in the usual cell-free system.
Figure 4. Localization of X91+ CGD mutations in gp91-phox. Schematic representation of the gp91-phox protein. Indicated are the possible oritentation of the peptide in the membrane, the N and C termini, the possible glycosylation sites (Y), the putative binding regions for FAD and NADPH, and the positions of the mutations that induce the X91+ CGD phenotype (normal expression of gp91-phox protein, no oxidase activity).
This may implicate that any mutation in gp91-phox destroys the catalytic activity of the flavocytochrome under all conditions tested, suggesting that gp91-phox alone is the catalytic unit, and that p22-phox is only required for stabilization of gp91-phox. Indeed, sequence-homology studies between the C-terminal half of gp91-phox and the ferredoxin-NADP+ reductase flavoenzyme family imply that both FAD and NADPH can be bound by this part of gp91-phox, and the location of these putative FAD- and NADPH-binding domains within gp91-phox have been deduced (2729) (see Fig. 4).
The model described above does not predict the position of the heme groups in cytochrome b558, because ferridoxin reductases do not contain heme moieties. Also, the model only describes the C-terminal part of gp91-phox, and the hemes are most probably located in the N-terminal half of gp91-phox. In fact, thereare indications for two hemes for each cytochrome b558 unit; one liganded by two histidines of gp91-phox (92) and another shared between the two cytochrome b558 subunits (30), possibly involving histidine-239 of gp91-phox and histidine-94 of p22-phox (C.D. Porter et al., in press). A recent report on an X91+ CGD patient with an 54Arg--->Ser substitution has substantiated the two-heme hypothesis (93, 94). This mutation abolishes the electron attraction of the positively charged arginine and perhaps disrupts a hydrogen bond between the arginine and a heme propionate side chain. As a result, the mutant form of the cytochrome contains two non-identical hemes with midpoint potentials of Em7 = -220 and Em7 = -300 mV. In the light of this information, the investigators reanalyzed the wild-type cytochrome b558 and concluded that it also contains two separate heme centers, with midpoint potentials of Em7 = -225 mV and Em7 = -265 mV (94).
It is generally believed that the cytosolic components p47-phox and p67-phox have to interact with the membane-bound p22-phox and gp91-phox to induce NADPH oxidase activity. In the neutrophils of the patient with the 54Arg--->Ser substitution in the Nterminal part of gp91-phox the cytosolic proteins translocated normally (93). Two other X91+ patients are known thus far with normal translocation of p47-phox and p67-phox: patient R.C. (42, 44, 64, 69) and a patient described in ref. (95). Patient R.C. carries a 415Pro--->His substitution in a putative NADPH-binding region in gp91-phox (Fig. 4), which leads to decreased binding of NADPH to the oxidase (27). The second patient has a deletion of five nucleotides and an insertion of eight nucleotides in exon 12 of the CYBB gene, resulting in a replacement of 507Gln-508Lys-509Thr by His-Ile-Trp-Ala in gp91-phox. This mutation also resides in a putative NADPH-binding region (Fig. 4).
We have characterized three X91+ patients with a defective translocation of p47phox and p67-phox: patient D.S., described in ref. (44), patient C.E. and patient N.K. (J. Leusen et al., manuscript in preparation). All three patients had point mutations leading to an amino-acid substitution in the cytosolic, C-terminal part of gp91-phox. In two of the patients, the substitutions are situated in protein loops exposed to the cytosol, as predicted by a computer model of the C-terminal half of gp91-phox (96), viz. 500Asp--->Gly in patient D.S. and 369Cys--->Arg in patient C.E. (Figs. 4 and 5). We have established the domain around residue 500 as a binding region for either p47-phox or p67phox by blocking the translocation of both cytosolic proteins in a cell-free assay with a synthetic peptide with the sequence of this domain (44).
Previously, we have postulated (44) that p47-phox is the most likely candidate for interaction with the domain surrounding 500Asp of gp91-phox, because this protein has been described to translocate without p67-phox (69, 71), while the opposite (translocation of p47-phox independently of p67-phox) did not seem to occur (69). Because we found no translocation of either cytosolic protein to the neutrophil plasma membranes of patient D.S., it seemed logical to suppose a disturbed interaction of this gp91-phox domain with p47-phox. Furthermore, p47-phox contains several basic regions that might act as counter structures for the negatively charged 500Asp; it was observed that positively charged peptides also block NADPH oxidase assembly (97). However, upon neutrophil activation, p47-phox becomes phosphorylated at 6-8 serine residues, and only the two most acidic isoforms are associated with the membrane. Also, we found recently that p47-phox did not translocate in an A670 patient (J. Leusen et al., submitted). Furthermore, there is good evidence that p47-phox interacts with p22-phox after activation of the oxidase, mediated via the SH3 domains of p47-phox (see next section) and the proline-rich region in p22-phox mutated in the A22+ patient (50). These observations render p67-phox a better candidate for binding to the domain in gp91-phox surrounding residue 500 (see also next paragraph). The loop containing residue 369 is involved in this interaction, because the two domains are quite close in the 3D model of gp91-phox (96) (Fig. 5).
Recently, it has been shown that p47-phox and p67-phox have distinct roles in the regulation of electron flow in cytochrome b558 (98, 99). In the absence of p47-phox, p67phox alone can facilitate electron flow from NADPH to the flavin center, resulting in the reduction of FAD, whereas the presence of p47-phox is required for electron transfer to proceed beyond the flavin center to the hemes in cytochrome b558 and then to oxygen. This fits with the idea suggested above that p67-phox is the cytosolic component that binds to the protein loops mutated in patients D.S. and C.E., because p67-phox would then be in close proximity to the NADPH-binding regions and the flavin center of gp91phox, enabling it to perform a regulatory function in this part of the protein.
The 408Gly, substituted for Glu in patient N.K., is localized between the two protein loops containing residue 500 and 369, and predicted to be buried in the protein (Fig. 5). Therefore, it is not a likely binding site for one of the cytosolic proteins. However, it is possible that the mutation changes the folding of the protein, perhaps leading to another orientation of the two loops with respect to each other. If this is true and one protein (p67-phox) binds to both loops, this interaction might be disturbed by the 408Gly--->Glu substitution.
A number of additional regions within gp91-phox have been identified as binding regions of p47-phox by inhibition studies with synthetic peptides in the cell-free system (oxidase activity or translocation) (70, 100) or by phage display and panning system with purified or recombinant proteins (101, 102). To substantiate the importance of these domains, synthetic peptides were used to assess inhibition of oxidase activation. However, the IC50 of some of these peptides was sometimes relatively high (>10 µn;M), and scrambled peptides were not always used as a control. It is known that positively charged peptides are inhibitory under cell-free activation conditions with IC50 values <2 µn;M (97, 103). Possibly, the effect of positively charged peptides is specific in the sense that they block the binding of p47-phox to domain 86TRVRRQL93 of gp91-phox (101). This domain, however, has been postulated by others to reside in an extracellular loop of the protein (see Fig. 4). Therefore, the specificity of these interactions remains to be proven, and genetic studies still provide the most convincing evidence for physiologically relevant interactions within the NADPH oxidase system.
5.3. Interactions between the cytosolic components
The cytosolic components of the NADPH oxidase appear to reside in a complex of 240-300 kD in the cytosol of resting neutrophils (104-106). Both p47-phox and p67-phox contain two SH3 domains, known to be involved in protein-protein interactions (47, 48, 107). P40-phox contains one SH3 domain (38). SH3 domains bind to proline-rich regions (49). Both p47-phox and p67-phox contain at least one such putative SH3-binding domain. As described in (108), p47-phox binds to p67-phox blotted onto nitrocellulose, and p67-phox binds to blotted p47-phox and p40-phox. We found that the proline-rich region in p67-phox is not required for interaction with either p47-phox or p40-phox. In contrast, the proline-rich region of p47-phox is essential for the binding to p67-phox, also when both proteins are in solution. In addition, the last 13 amino acids of p47-phoxseem to be involved in exposing this proline-rich region, because an antibody directed against the C-terminal 13 amino acids of p47-phox blocks the interaction between p47-phox and p67-phox, but only when p47-phox is in solution. A truncated p47-phox confirmed these findings. In Fig. 6, a hypothetical model is given of the interactions between the oxidase components, based on the data described in this section.
Figure 5. Model of the cytosolic part of gp91-phox in the activated, NADPH-bound state. The protein is modelled on Spinach ferrodoxin (157) as described by Taylor et al. (96). The figure was drawn with MOLSCRIPT (158), and kindly provided by dr Nicholas Keep of prof. Segal's group (University College, London, G.B.). The position of the substitutions in patients C.E. (369Cys--->Arg), patient N.K. (408Gly--->Glu) and patient D.S. (500Asp--->Gly) are shown. Residues 369 and 500 are situated in protein loops exposed to the cytosol, but residue 408 is buried in the protein between these loops.
It is of interest to note that a synthetic peptide mimicking the last 13 amino acids of p47-phox becomes phosphorylated in vitro by PKC and other serine kinases (109). In combination with our findings, this indicates that the phosphorylation of the C-terminus could be an important switch for repositioning the cytosolic phox proteins, leading to their interaction with the membrane-bound oxidase components and thus to an active NADPH oxidase. Other investigators have shown that the C-terminal SH3 domain of p67-phox is the counter structure for the proline-rich region in p47-phox (110, 111) and that the SH3 domains of p47-phox can bind to proline-rich sequences in p47-phox itself and to the proline-rich sequence in p22-phox (111, 112). Addition of SDS or arachidonic acid, activators of the cell-free system, leads to exposure of the SH3 domains in p47-phox (112). In the cell-free system, the p47-phox without its proline-rich region (108) and p67phox without its SH3 domains (113) can still induce superoxide production. Hence, the interaction between p47-phox and p67-phox is not required for cell-free oxidase activity (108,113)). However, in intact cells (EBV transformed B-cell lines of CGD patients transfected with cDNA encoding the missing cytosolic component), the second SH3 domain of p67-phox that interacts with p47-phox is indispensable for oxidase activity (113), suggesting that a complex between p47-phox and p67-phox is required in intact cells.
With the use of the two-hybrid system the binding domains of p40-phox have also been mapped: the SH3 domain of p40-phox interacts with p47-phox, and its last 36 amino acids interact with p67-phox (114). The same method revealed that the C-terminal SH3 domain of p67-phox is crucial for the interaction with p40-phox and p47-phox (A. Fuchs, unpublished results). Thus far, there are no domains of p67-phox known to interact with cytochrome b558 after activation.
Figure 6. Hypothetical model of the interactions between the NADPH oxidase components. The interactions between the cytosolic components are represented for the resting situation in panel A. For the activated state, the interactions between the cytosolic components and cytochrome b558 are depicted in panel B. It is postulated that in the resting state, the C-terminus of p47-phox is folded backwards, binding or covering its SH3 domains and exposing the C-terminal proline-rich region, allowing it to bind to the C-terminal SH3 domain of p67-phox. The other two binding domains (one proline-rich region and one SH3 domain) of p67-phox are not involved in binding to one of the cytosolic proteins, but possibly interact with the cytoskeleton. It is known that the SH3 domain of p40-phox interacts with p47-phox (114), perhaps to its N-terminal proline-rich region, as is shown here. Upon cell activation, p47-phox becomes phosphorylated, and the phosphorylation of 379Ser has been shown to be crucial for activity and translocation (133). Therefore, we suggest here that phosphorylation of this site leads to uncovering of the SH3 domains of p47-phox, making them available for interaction with the proline-rich region of p22-phox (the idea that phosphorylation of p47-phox leads to exposure of SH3 domains was already suggested by Leto et al. (111)). This also disrupts the interaction with p67-phox. However, via binding to p40-phox, p67-phox is still retained in the complex. We also hypothesize that p67-phox binds to two protein loops containing residues 369 and 500 of gp91-phox, although the binding sites on p67-phox are still unknown. Rac can only bind after dissociation from rho-GDI and loading with GTP. To which domain of cytochrome b558 rac binds, is unknown.
5.4. Role of phosphorylations in activation of the oxidase
Neutrophils stimulated with PMA or fMLP exhibit rapid phosphorylation of p47phox on multiple serine residues (115), which accompanies translocation of this protein to the submembraneous cytoskeleton. Protein kinase antagonists that inhibit phosphorylation of p47-phox (e.g. H-7, staurosporine or 1-O-hexadecyl-2-O-methylglycerol (AMG)) block translocation of this protein to the detergent-insoluble fraction (116, 117), and, concomitantly, block superoxide anion release (62, 118120). Six to eight isoforms of p47-phox can be observed in stimulated cells that result from different numbers of phosphorylated serine residues (64, 121). Only the most heavily phosphorylated species are observed in the cytoskeletal fraction, which suggests that extensive phosphorylation of this protein is required for superoxide anion production when PMA is the stimulus (122). Interestingly, p47-phox undergoes continuous cycling between phosphorylation and dephosphorylation in PMA-stimulated cells, with the phosphorylation reaction predominating. Interruption of the phosphorylation of p47-phox results in rapid dephosphorylation of p47-phox and in dissociation of this protein from the cytoskeletal fraction (117, 123, 124). Thus, the activity of p47-phox can be regulated by a phosphorylation-dephosphorylation cycle that governs association-dissociation of this protein with cytochrome b558 (125). Protein kinase C may form a stable complex with p47-phox when PMA, but not when fMLP is the stimulus (117). Dephosphorylation of p47-phox is catalyzed by a phosphatase type 1 and/or 2A (124, 126).
All of the phosphorylations of p47-phox in neutrophils stimulated with PMA or fMLP occur in the C-terminal region between 303Ser and 379Ser (122). Edman degradation of 32P-labeled p47-phox revealed that 303Ser, 304Ser, 320Ser, 328Ser, 345Ser, 348Ser and at least one of the three 359Ser, 370Ser, and 379Ser were phosphorylated. The first four serine residues listed are flanked by basic groups and exhibit consensus sequences for phosphorylation by PKC (123) or one or more novel protein kinases described in neutrophils (127, 128). These novel kinases appear to function in a stimulatory pathway downstream of the phosphatidylinositol (PI) 3-kinase (129). The sequence around 345Ser and 348Ser (PGPQSPGSP) constitutes a consensus sequence for proline-directed protein kinases (e.g. MAP kinases) (130, 131). However, we tested whether recombinant p47phox was a substrate for the ERK2 MAP kinase, immunoprecipitated from 3T3 fibroblasts transfected with the insulin receptor and stimulated with insulin, and observed no difference in labeling between wild-type p47-phox and a deletion mutant of p47-phox lacking the putative MAP kinase phosphorylation site. Moreover, a classical substrate for MAP kinase, myelin basic protein (MBP), was phosphorylated about 100 times better (J. Leusen et al., unpublished results). Besides, the putative MAP kinase phosphorylation site is not conserved in the mouse sequence of p47-phox (132).
Only recently, Faust et al. (133) found an answer to the question which residue(s) in p47-phox need to become phosphorylated for NADPH oxidase activation. This was performed by mutating all the putative serine phosphorylation sites of p47-phox (between 303Ser and 379Ser) and monitoring these mutants in intact, transfected cells (EBVtransformed B lymphocytes of a CGD patient deficient for p47-phox) for superoxide-inducing capacity and for translocation to the plasma membrane upon cell activation. In this study it was demonstrated that only the phosphorylation of 379Ser is essential for oxidase activity and membrane association (133). This is a very intriguing result, because apparently all phosphorylations of p47-phox can be reduced to one essential phosphorylation, and this phosphorylation happens to be in the C-terminal domain that we postulate to be an important switch for NADPH oxidase activation.
5.5. Role of rac and Rap1A in activating the oxidase
The absolute requirement for rac in the NADPH oxidase was shown by cell-free studies in which cytosol could be replaced with recombinant p47-phox, p67-phox, and rac1 or rac2 (134-136); similar studies in which the plasma membrane was replaced by purified cytochrome b558 demonstrated that these four components were the minimum proteins required to obtain a fully active NADPH oxidase (28, 89). The cytosolic p40-phox and the cytochrome-associated Rap1A protein, absent from these purified preparations, appeared not to be necessary for oxidase function under the cell-free conditions utilized. Also, recombinant Rap1A was unable to support NADPH oxidase activity in the cell-free system (135). Transfection studies of dominant-negative (GDP-bound) and dominantpositive (GTP-bound) Rap1A in human B lymphocytes (137) or in differentiated HL60 cells (138) revealed that overexpression of either of these mutant forms of Rap1A suppressed oxidase activity. GTP-bound Rap1A is associated with purified cytochrome b558 and GDP-bound Rap1A is not (56). Perhaps GTP-rap1A activates cytochrome b558, by inducing a phosphorylation or a conformational change. As mentioned earlier, also phosphorylation of Rap1A disrupts the association with cytochrome b558. Taken together, it is likely that Rap1A in its GTP-bound state activates and in its GDP-bound state deactivates the NADPH oxidase in a dynamic cycle in intact cells.
Rac is normally found in the cytosolic fraction of phagocytes, in a complex with the GDP dissociation inhibitor Rho-GDI (75-77, 139). Rac is maintained in its inactive form in this complex, but upon cell activation, biologically active lipids, such as arachidonic acid, phosphatidic acid and phosphatidylinositols, can disrupt the binding of rac to Rho-GDI (139, 140). In fibroblasts, it has been shown that PI 3-kinase acts upstream of rac to mediate PDGF-induced lamellipodia formation (141). In neutrophils, PI 3-kinase and the NADPH oxidase are simultaneously activated by several stimuli; furthermore, wortmannin, an inhibitor of PI 3-kinase, can prevent NADPH oxidase activation (142, 143). PI 3-kinase may therefore act upstream of rac in this system as well.
Two potential targets for rac1 have been discovered: p67-phox and the serine kinase p68-pak, related to the brain p65-pak (144,145). The GTP-bound rac activates p68-pak, which in turn phosphorylates p47-phox on 328Ser (146). However, according to the findings of Faust et al. (133), this phosphorylation is not essential for oxidase activation. In contrast to p47-phox, p67-phox is recovered predominantly in the Triton X100 insoluble fraction of both stimulated and unstimulated neutrophils (116, 117). This property is consistent with p67-phox being bound to the cytoskeleton (147). Activated rac is known to be involved in cytoskeletal reorganization and could thus play a role in the function of p67-phox or mediate the interaction between p67-phox and other components of the NADPH oxidase. Some investigators have shown that rac associates with the plasma membrane upon cell activation (148, 149). However, we and others (150) could not reproduce these results. Rac translocation has also been reported to occur independently of p47-phox and p67-phox (151, 152). However, we have demonstrated with neutrophils from a CGD patient bearing a deletion of a lysine at position 58 in p67-phox that p47-phox and p67-phox failed to translocate in intact cells, whereas they did interact with cytochrome b558 in the cell-free system, but still without inducing superoxide production. The disease in this patient was caused by a disrupted interaction of p67phox-delta58Lys with rac (J. Leusen et al, submitted). This indicates that the binding of rac to p67-phox is essential for the activity of the NADPH oxidase in both intact cells and the cell-free system; it is possible that rac induces a conformational change or phosphorylation of p67-phox. Furthermore, a rac/p67-phox interaction is needed for the translocation of p47-phox and p67-phox in intact cells. Most likely, rac functions as a 'shuttle protein', carrying p67-phox to the membrane in its GTP-bound state, and dissociating in its GDP-bound state and returning to the cytosol. Conversely, rac might induce a change in the submembraneous cytoskeleton (like membrane ruffling (153)) and create a docking site for p67-phox. Thus, rac probably mediates translocation of p67-phox and, as a consequence, also the translocation of p47-phox.
5.6. NADPH oxidase: cell-free system vs intact cells
It is evident from several observations discussed above that there are some important differences between the cell-free system and intact cells concerning the activation of the NADPH oxidase. In intact cells, the NADPH oxidase is a dynamic enzyme that needs incessant stimulation to remain active (continuous phosphorylation of p47-phox, GTP loading of rac and cycling of Rap1A). In contrast, once the NADPH oxidase is assembled under cell-free conditions with SDS or arachidonic acid and with GTPgS, it remains active. For instance, the sulfhydryl reagent N-ethylmaleimide (NEM) inhibits ongoing oxidase activity in activated neutrophils, whereas in the cell-free system it has no effect once the oxidase has been assembled (154). Also synthetic peptides that interfere with the assembly of the NADPH oxidase are only effective in the cell-free system when added prior to activation (44, 70, 97, 101, 102, 155, 156).
Another difference between the cell-free system and intact neutrophils is that in the cellfree system the oxidase components p40-phox and Rap1A are not required, but Rap1A is responsible for at least a four-fold increase in superoxide production when overexpressed in differentiated HL-60 cells (138). Also, a complex between p47-phox and p67-phox is not mandatory for cell-free oxidase activity, as shown with mutated p47-phox proteins in (108). In addition, interaction of rac with p67-phox is not necessary for translocation of the cytosolic components in the cell-free system (but it is for oxidase activity in this system) (J.Leusen et al.,submitted). We can conclude from these data that although the cell-free system is very useful for a number of applications, results obtained in this system cannot be directly extrapolated to intact cells.
To recapitulate: the human NADPH oxidase is a very intriguing enzyme; although its catalytic unit is retained within cytochrome b558, various additional proteins are required for activity of the NADPH oxidase. In the past few years, substantial progress has been made to elucidate the protein-protein interactions and the activation events involved. It has become evident that 1) activation of rac and subsequent interaction with p67-phox is crucial for the interaction of p67-phox with cytochrome b558, probably with gp91-phox, 2) p47-phox interacts with p22-phox, and phosphorylation of 379Ser of p47-phox is obligatory for this event, and 3) p47-phox and p67-phox regulate each others translocation in a positive sense (see also ref. (71)). To put it differently: it is vital to gain insight in the intrigues within the phox family and associated characters to fully understand NADPH oxidase activation.
This study was supported by grants from the Netherlands Organization for the Advancement of Scientific Research (NWO) and from the Netherlands Fund for Preventive Medicine (Praeventiefonds).
1. S. J. Weiss: Tissue destruction by neutrophils. N Engl J Med 320, 365-76 (1989)
2. A. J. Verhoeven, B.G.J.M. Bolscher & D. Roos: The superoxidegenerating enzyme in phagocytes: physiology, protein composition, and mechanism of activation.In: Membrane Lipid Oxidation. VigoPelfrey, C. editor. CRC press, Boston, pp. 42-59 (1991)
3. R. A. Clark: The human neutrophil respiratory burst oxidase. J Infect Dis 161, 1140-7 (1990)
4. L. T. Williams, R. Snyderman, M. C. Pike, & R. J. Lefkowitz: Specific receptor sites for chemotactic peptides on human polymorphonuclear leukocytes. Proc Natl Acad Sci USA 74, 1204-8 (1977)
5. J. E. Lehmeyer, R. Snyderman & R. B. Johnston: Stimulation of neutrophil oxidative metabolism by chemotactic peptides: influence of calcium ion concentration and cytochalasin B and comparison with stimulation by phorbol myristate acetate. Blood 54, 35-45 (1979)
6. F. Rossi: The O2-forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta 853, 65-89 (1986)
7. A. R. Cross & O. T. Jones. Enzymic mechanisms of superoxide production. Biochim Biophys Acta 1057, 281-98 (1991)
8. F. Morel, J. Doussière & P. V. Vignais: The superoxidegenerating oxidase of phagocytic cells. Physiological, molecular and pathological aspects. Eur J Biochem 201, 523-46 (1991)
9. M. Baggiolini, F. Boulay, J. A. Badwey & J. T. Curnutte: Activation of neutrophil leukocytes: Chemoattractant receptors and respiratory burst. FASEB J 7, 1004-10 (1993)
10. P. G. Quie, J. G. White, P. G. Holmes & R. A. Good: In vitro bactericidal capacity of human polymorphonuclear leucocytes: diminished activity in chronic granulomatous disease of childhood. J Clin Invest 46, 668-79 (1967)
11. A. W. Segal: Chronic granulomatous disease. Clin.Exp.Allergy 21 Suppl 1, 195-8 (1991)
12. C. B. Forrest, J. R. Forehand, R. A. Axtell, R. L. Roberts & R. B. Johnston: Clinical features and current management of chronic granulomatous disease. Hematol Oncol Clin North Am 2, 253-67 (1988)
13. R. M. Smith & J. T. Curnutte: Molecular basis of chronic granulomatous disease. Blood 77, 673-86 (1991)
14. L. M. Kunkel, A. P. Monaco, W. Middlesworth, H. D. Ochs & S. A. Latt: Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. Proc Natl Acad Sci USA 82, 4778-82 (1985)
15. M. C. Dinauer, E. A. Pierce, G. A. Bruns, J. T. Curnutte & S. H. Orkin: Human neutrophil cytochrome b light chain (p22phox). Gene structure, chromosomal location, and mutations in cytochromenegative autosomal recessive chronic granulomatous disease. J Clin Invest 86, 1729-37 (1990)
16. B. D. Volpp, W. M. Nauseef & R. A. Clark: Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease. Science 242, 1295-7 (1988)
17. H. Nunoi, D. Rotrosen, J. I. Gallin & H. L. Malech: Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors. Science 242, 1298-301 (1988)
18. K. J. Lomax, T. L. Leto, H. Nunoi, J. I. Gallin & H. L. Malech: Recombinant 47kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease [erratum appeared in Science 246, 987 (1989)
]. Science 245, 409-12 (1989)
19. B. D. Volpp, W. M. Nauseef, J. E. Donelson, D. R. Moser & R. A. Clark: Cloning of the cDNA and functional expression of the 47kilodalton cytosolic component of human neutrophil respiratory burst oxidase [erratum appeared in Proc.Natl.Acad.Sci.U.S.A. 1989; 86, 9563]. Proc Natl Acad Sci U S A 86, 7195-9 (1990)
20. T. L. Leto, K. J. Lomax, B. D. Volpp, H. Nunoi, J. M. Sechler, W. M. Nauseef, R. A. Clark, J. I. Gallin & H. L. Malech: Cloning of a 67kD neutrophil oxidase factor with similarity to a noncatalytic region of p60csrc. Science 248, 727-30 (1990)
21. D. R. Ambruso, B. G. J. M. Bolscher, P. M. Stokman, A. J. Verhoeven & D. Roos: Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate [erratum appeared in J.Biol.Chem. 265, 19370-19371 (1990) J Biol Chem 265, 924-30 (1990)
22. R. A. Clark, B. D. Volpp, K. G. Leidal & W. M. Nauseef: Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J Clin Invest 85, 714-21 (1990)
23. A. W. Segal: Absence of both cytochrome b-245 subunits from neutrophils in X-linked chronic granulomatous disease. Nature 326, 88-91 (1987)
24. C. A. Parkos, R. A. Allen, C. G. Cochrane & A. J. Jesaitis: Purified cytochrome b from human plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. J Clin Invest 80, 732-42 (1987)
25. A. W. Segal & O. T. Jones: Subcellular distribution and some properties of the cytochrome b component of the microbicidal oxidase system of human neutrophils. Biochem J 182, 181-8 (1979)
26. A. R. Cross, T. G. Jones, A. M. Harper & A. W. Segal: Oxidationreduction properties of the cytochrome b found in the plasmamembrane fraction of human neutrophils. Biochem J 194, 599-606 (1981)
27. A. W. Segal, I. West, F. Wientjes, J. H. Nugent, A. J. Chavan, B. Haley, R. C. Garcia, H. Rosen & Scrace: Cytochrome b-245 is a flavocytochrome containing FAD and the NADPHbinding site of the microbicidal oxidase of phagocytes. Biochem J 284, 781-8 (1992)
28. D. Rotrosen, C. L. Yeung, T. L. Leto, H. L. Malech & C. H. Kwong: Cytochrome b558: the flavinbinding component of the phagocyte NADPH oxidase. Science 256, 1459-62 (1992)
29. H. Sumimoto, N. Sakamoto, M. Nozaki, Y. Sakaki, K. Takeshige & S. Minakami: Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein. Biochem Biophys Res Commun 186, 1368-75 (1992)
30. M. T. Quinn, M. L. Mullen & A. J. Jesaitis: Human neutrophil cytochrome b contains multiple hemes. Evidence for heme associated with both subunits. J Biol Chem 267, 7303-9 (1992)
31. A. W. Segal: The electron transport chain of the microbicidal oxidase of phagocytic cells and its involvement in the molecular pathology of chronic granulomatous disease. J Clin Invest 83, 1785-93 (1989)
32. O. W. Bjerrum & N. Borregaard: Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils [erratum appeared in Eur. J.Haematol. 8, 270 (1989)]. Eur J Haematol 43, 67-77 (1989)
33. L. A. Ginsel, J. J. Onderwater, J. A. Fransen, A. J. Verhoeven & D. Roos: Localization of the lowMr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy. Blood 76, 2105-16 (1990)
34. J. Calafat, T. W. Kuijpers, H. Janssen, N. Borregaard, A. J. Verhoeven & D. Roos: Evidence for small intracellular vesicles in human blood phagocytes containing cytochrome b558 and the adhesion molecule CD11b/CD18. Blood 81, 3122-9 (1993)
35. N. Borregaard, J. M. Heiple, E. R. Simons & R. A. Clark: Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol 97, 52-61 (1983)
36. B. Royer-Pokora, L. M. Kunkel, A. P. Monaco, S. C. Goff, P. E. Newburger, R. L. Baehner, F. S. Cole, J. T. Curnutte & S. H. Orkin: Cloning the gene for an inherited human disorderchronic granulomatous diseaseon the basis of its chromosomal location. Nature 322, 32-8 (1986)
37. C. G. Teahan, P. Rowe, P. Parker, N. Totty & A. W. Segal: The Xlinked chronic granulamotous disease gene codes for the beta chain of cytochrome b-245. Nature 327, 720-1 (1987)
38. C. A. Parkos, M. C. Dinauer, L. W. Walker, R. A. Allen, A. J. Jesaitis & S. H. Orkin: Primary structure and unique expression of the 22kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci U S A 85, 3319-23 (1988)
39. M. C. Dinauer, S. H. Orkin, R. Brown, A. J. Jesaitis & C. A. Parkos: The glycoprotein encoded by the Xlinked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. Nature 327, 717-20 (1987)
40. S. Imajoh Ohmi, K. Tokita, H. Ochiai, M. Nakamura & S. Kanegasaki: Topology of cytochrome b558 in neutrophil membrane analyzed by antipeptide antibodies and proteolysis. J Biol Chem 267, 180-4 (1992)
41. D. Roos, M. de Boer, N. Borregaard, O. W. Bjerrum, N. H. Valerius, R. A. Seger, T. Muhlebach, B. H. Belohradsky & R. S. Weening: Chronic granulomatous disease with partial deficiency of cytochrome b558 and incomplete respiratory burst: variants of the X-linked, cytochrome b558-negative form of the disease. J Leukoc Biol 51, 164-71 (1992)
42. M. C. Dinauer, J. T. Curnutte, H. Rosen & S. H. Orkin: A missense mutation in the neutrophil cytochrome b heavy chain in cytochromepositive Xlinked chronic granulomatous disease. J Clin Invest 84, 2012-6 (1989)
43. B. L. Schapiro, P. E. Newburger, M. S. Klempner & M. C. Dinauer: Chronic granulomatous disease presenting in a 69yearold man. N Engl J Med 325, 1786-90 (1991)
44. J. H. W. Leusen, M. de Boer, B. G. J. M., Bolscher, P. M. Hilarius, R. S. Weening, H. D. Ochs, D. Roos & A. J. Verhoeven: A point mutation in gp91phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox. J Clin Invest 93, 2120-6 (1994)
45. M. de Boer, A. de Klein, J. P. Hossle, R. Seger, L. Corbeel, R. S. Weening & D. Roos: Cytochrome b558-negative, autosomal recessive chronic granulomatous disease: two new mutations in the cytochrome b558 light chain of the NADPH oxidase (p22-phox)
. Am J Hum Genet 51, 1127-35 (1992)
46. M. C. Dinauer, E. A. Pierce, R. W. Erickson, T. J. Muhlebach, H. Messner, S. H. Orkin, R. A. Seger & J. T. Curnutte: Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease. Proc Natl Acad Sci U S A 88, 11231-5 (1991)
47. C. A. Koch, D. Anderson, M. F. Moran, C. Ellis & T. Pawson: SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science 252, 668-74 (1991)
48. A. Musacchio, T. Gibson, V. P. Lehto & M. Saraste: SH3: an abundant protein domain in search of a function. FEBS Lett 307, 55-61 (1992)
49. R. Ren, B. J. Mayer, P. Cicchetti & D. Baltimore: Identification of a tenamino acid prolinerich SH3 binding site. Science 259, 1157-61 (1993)
50. J. H. W. Leusen, B. G. J. M. Bolscher, P. M. Hilarius, R. S. Weening, W. Kaulfersch, R. A. Seger, D. Roos & A. J. Verhoeven: 156Pro--->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22phox)
leads to defective translocation of the cytosolic proteins p47-phox and p67-phox. J Exp Med 180, 2329-34 (1994)
51. C. A. Parkos, M. C. Dinauer, A. J. Jesaitis, S. H. Orkin & J. T. Curnutte: Absence of both the 91kD and 22kD subunits of human neutrophil cytochrome b in two genetic forms of chronic granulomatous disease. Blood 73, 1416-20 (1989)
52. A. J. Verhoeven, B. G. J. M. Bolscher, L. J. Meerhof, R. van Zwieten, J. Keijer, R. S. Weening & D. Roos: Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils. Blood 73, 1686-94 (1989)
53. C. D. Porter, M. H. Parkar, A. J. Verhoeven, R. J. Levinsky, M. K. L. Collins & C. Kinnon: p22-phox-Deficient chronic granulomatous disease: Reconstitution by retrovirus-mediated expression and identification of a boisynthetic intermediate of gp91-phox. Blood 84, 2767-75 (1994)
54. M. T. Quinn, C. A. Parkos, L. Walker, S. H. Orkin, M. C. Dinauer & A. J. Jesaitis: Association of a Rasrelated protein with cytochrome b of human neutrophils. Nature 342, 198-200 (1989)
55. M. T. Quinn, M. L. Mullen, A. J. Jesaitis & J. G. Linner: Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559. Blood 79, 1563-73 (1992)
56. L. A. Quilliam, H. Mueller, B. P. Bohl, V. Prossnitz, L. A. Sklar, C. J. Der & G. M. Bokoch: Rap1A is a substrate for cyclic AMPdependent protein kinase in human neutrophils [erratum appeared in J.Immunol. 147, 3660 (1991)
]. J Immunol 147, 1628-35 (1991)
57. G. M. Bokoch, L. A. Quilliam, B. P. Bohl, A. J. Jesaitis & M. T. Quinn: Inhibition of Rap1A binding to cytochrome b558 of NADPH oxidase by phosphorylation of Rap1A. Science 254, 1794-6 (1991)
58. R. A. Clark, H. L. Malech, J. I. Gallin, H. Nunoi, B. D. Volpp, D. W. Pearson, W. M. Nauseef & J. T. Curnutte: Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system. N Engl J Med 321, 647-52 (1989)
59. A. W. Segal, P. G. Heyworth, S. Cockcroft & M. M. Barrowman: Stimulated neutrophils from patients with autososomal recessive chronic granulomatous disease fail to phosphorylate a Mr-44,000 protein. Nature 316, 547-9 (1985)
60. P. C. Andrews & B. M. Babior: Phosphorylation of cytosolic proteins by resting and activated human neutrophils. Blood 64, 883-90 (1984)
61. T. Hayakawa, K. Suzuki, S. Suzuki, P. C. Andrews & B. M. Babior: A possible role for protein phosphorylation in the activation of the respiratory burst in human neutrophils. Evidence from studies with cells from patients with chronic granulomatous disease. J Biol Chem 261, 9109-15 (1986)
62. P. G. Heyworth, C. F. Shrimpton & A. W. Segal: Localization of the 47 kDa phosphoprotein involved in the respiratoryburst NADPH oxidase of phagocytic cells. Biochem J 260, 243-8 (1989)
63. N. Okamura, J. T. Curnutte, R. L. Roberts & B. M. Babior: Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 48kDa proteins in two forms of chronic granulomatous disease. J Biol Chem 263, 6777-82 (1988)
64. N. Okamura, S. E. Malawista, R. L. Roberts, H. Rosen, H. D. Ochs, B. M. Babior & J. T. Curnutte: Phosphorylation of the oxidaserelated 48K phosphoprotein family in the unusual autosomal cytochromenegative and Xlinked cytochromepositive types of chronic granulomatous disease. Blood 72, 811-6 (1988)
65. U. Francke, C. L. Hsieh, B. E. Foellmer, K. J. Lomax, H. L. Malech & T. L. Leto: Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2)
and 7q11.23 (NCF1). Am J Hum Genet 47, 483-92 (1990)
66. J. T. Curnutte, P. J. Scott & L. A. Mayo: Cytosolic components of the respiratory burst oxidase: resolution of four components, two of which are missing in complementing types of chronic granulomatous disease. Proc Natl Acad Sci U S A 86, 825-9 (1989)
67. B. G. J. M. Bolscher, R. van Zwieten, I. M. Kramer, R. S. Weening, A. J. Verhoeven & D. Roos: A phosphoprotein of Mr 47,000, defective in autosomal chronic granulomatous disease, copurifies with one of two soluble components required for NADPH:O2 oxidoreductase activity in human neutrophils. J Clin Invest 83, 757-63 (1989)
68. C. G. Teahan, N. Totty, C. M. Casimir & A. W. Segal: Purification of the 47 kDa phosphoprotein associated with the NADPH oxidase of human neutrophils. Biochem J 267, 485-9 (1990)
69. P. G. Heyworth, J. T. Curnutte, W. M. Nauseef, B. D. Volpp, D. W. Pearson, H. Rosen & R. A. Clark: Neutrophil nicotinamide adenine dinucleotide phosphate (reduced form) oxidase assembly: Translocation of p47-phox and p67-phox requires interaction between p47-phox and cytochrome b558. J Clin Invest 87, 352-6 (1991)
70. M. E. Kleinberg, D. Mital, D. Rotrosen & H. L. Malech: Characterization of a phagocyte cytochrome b558 91-kilodalton subunit functional domain: identification of peptide sequence and amino acids essential for activity. Biochemistry 31, 2686-90 (1992)
71. D. J. Uhlinger, K. L. Taylor & J. D. Lambeth: p67-phox enhances the binding of p47-phox to the human neutrophil respiratory burst oxidase complex. J Biol Chem 269, 22095-8 (1994)
72. F. B. Wientjes, J. J. Hsuan, N. F. Totty & A. W. Segal: p40-phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J 296, 557-61 (1993)
73. S. Tsunawaki, H. Mizunari, M. Nagata, O. Tatsuzawa & T. Kuratsuji: A novel cytosolic component, p40-phox, of respiratory burst oxidase associates with p67-phox and is absent in patients with chronic granulomatous disease who lack p67-phox. Biochem Biophys Res Commun 199, 1378-87 (1994)
74. U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte & G. M. Bokoch: Regulation of phagocyte oxygen radical production by the GTPbinding protein Rac 2. Science 254, 1512-5 (1991)
75. A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan & A. W. Segal: Activation of the NADPH oxidase involves the small GTPbinding protein p21rac1. Nature 353, 668-70 (1991)
76. A. Abo & E. Pick: Purification and charac-terization of a third cytosolic component of the superoxidegenerating NADPH oxidase of macrophages. J Biol Chem 266, 23577-85 (1991)
77. U. G. Knaus, P. G. Heyworth, B. T. Kinsella, J. T. Curnutte, & G. M. Bokoch: Purification and characterization of Rac 2. A cytosolic GTPbinding protein that regulates human neutrophil NADPH oxidase. J Biol Chem 267, 23575-82 (1992)
78. O. Dorseuil, A. Vazquez, P. Lang, J. Bertoglio, G. Gacon & G. Leca: Inhibition of superoxide production in B lymphocytes by rac antisense oligonucleotides. J Biol Chem 267, 20540-2 (1992)
79. S. Dusi, M. Donini & F. Rossi: Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes. Biochem J 308, 991-4 (1995)
80. T. Mizuno, K. Kaibuchi, S. Ando, T. Musha, K. Hiraoka, K. Takaishi, M. Asada, H. Nunoi, I. Matsuda & Y. Takai: Regulation of the superoxidegenerating NADPH oxidase by a small GTPbinding protein and its stimulatory and inhibitory GDP/GTP exchange proteins. J Biol Chem 267, 10215-8 (1992)
81. R. A. Heyneman & R. E. Vercauteren: Activation of a NADPH oxidase from horse polymorphonuclear leukocytes in a cellfree system. J Leukocyte Biol 36, 751-9 (1984)
82. Y. Bromberg & E. Pick: Activation of NADPHdependent superoxide production in a cellfree system by sodium dodecyl sulfate. J Biol Chem 260, 13539-45 (1985)
83. J. T. Curnutte: Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (trisphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cellfree system. J Clin Invest 75, 1740-3 (1985)
84. T. G. Gabig, D. English, L. P. Akard & M. J. Schell: Regulation of neutrophil NADPH oxidase activation in a cellfree system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxininsensitive G protein. J Biol Chem 262, 1685-90 (1987)
85. R. A. Clark, K. G. Leidal, D. W. Pearson & W. M. Nauseef: NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonateactivatable superoxidegenerating system. J Biol Chem 262, 4065-74 (1987)
86. S. E. Caldwell, C. E. McCall, C. L. Hendricks, P. A. Leone, D. A. Bass, D.A. & L. C. McPhail: Coregulation of NADPH oxidase activation and phosphorylation of a 48kD protein(s)
by a cytosolic factor defective in autosomal recessive chronic granulomatous disease. J Clin Invest 81, 1485-96 (1988)
87. J. Doussière, M. C. Pilloud & P. V. Vignais: Activation of bovine neutrophil oxidase in a cell free system. GTPdependent formation of a complex between a cytosolic factor and a membrane protein. Biochem Biophys Res Commun 152, 993-1001 (1988)
88. B. G. J. M. Bolscher, S. W. Denis, A. J. Verhoeven & D. Roos: The activity of one soluble component of the cellfree NADPH:O2 oxidoreductase of human neutrophils depends on guanosine 5'O(3thio)
triphosphate. J Biol Chem 265, 15782-7 (1990)
89. A. Abo, A. Boyhan, I. West, A. Thrasher & A. W. Segal: Reconstitution of neutrophil NADPH oxidase activity in the cellfree system by four components: p67phox, p47phox, p21rac1 and cytochrome b245. J Biol Chem 267, 16767-70 (1992)
90. V. Koshkin & E. Pick: Generation of superoxide by purified and relipidated cytochrome b559 in the absence of cytosolic activators. FEBS Lett 327, 57-62 (1993)
91. V. Koshkin & E. Pick: Superoxide production by cytochrome b559. Mechanism of cytosolindependent activation. FEBS Lett 338, 285-9 (1994)
92. J. K. Hurst, T. M. Loehr, J. T. Curnutte & H. Rosen: Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558. J Biol Chem 266, 1627-34 (1991)
93. A. R. Cross, P. G. Heyworth, J. Rae & J. T. Curnutte: A variant Xlinked chronic granulomatous disease patient (X91+)
with partially functional cytochrome b. J Biol Chem 270, 8194-200 (1995)
94. A. R. Cross, J. Rae & J. T. Curnutte: Cytochrome b245 of the neutrophil superoxidegenerating system contains two nonidentical hemes. J Biol Chem 270, 17075-7 (1995)
95. H. Azuma, H. Oomi, K. Sasaki, I. Kawabata, T. Sakaino, S. Koyano, T. Suzutani, H. Nunoi & A. Okuno: A new mutation in exon 12 of the gp91phox gene leading to cytochrome bpositive Xlinked chronic granulomatous disease. Blood 85, 3274-7 (1995)
96. W. R. Taylor, D. T. Jones & A. W. Segal: A structural model for the nucleotide binding domains of the flavocytochrome b245 betachain. Protein Sci 2, 1675-85 (1993)
97. A. J. Verhoeven, J. H. W. Leusen, G. C. R. Kessels, P. M. Hilarius, D. B. De Bont & R. M. Liskamp: Inhibition of neutrophil NADPH oxidase assembly by a myristoylated pseudosubstrate of protein kinase C. J Biol Chem 268, 18593-8 (1993)
98. A. R. Cross, J. L. Yarchover & J. T. Curnutte: The superoxidegenerating system of human neutrophils possesses a novel diaphorase activity. Evidence for distinct regulation of electron flow within NADPH oxidase by p67phox and p47phox. J Biol Chem 269, 21448-54 (1994)
99. A. R. Cross & J. T. Curnutte: The cytosolic activating factors p47phox and p67phox have distinct roles in the regulation of electron flow in NADPH oxidase. J Biol Chem 270, 6543-8 (1995)
100. A. Nakanishi, S. Imajoh-Ohmi, T. Fujinawa, H. Kikuchi & S. Kanegasaki: Direct evidence for interaction between COOH-terminal regions of cytochrome b558 subunits and cytosolic 47-kDa protein during activation of an O2--generating system in neutrophils. J Biol Chem 267, 19072-4 (1992)
101. F. R. DeLeo, L. Yu, J. B. Burritt, L. R. Loetterle, C. W. Bond, A. J. Jesaitis & M. T. Quinn: Mapping sites of interaction of p47-phox and flavocytochrome b with random sequence peptide phage display libraries. Proc Natl Acad Sci USA 92, 7110-4 (1995)
102. F. R. DeLeo, W. M. Nauseef, A. J. Jesaitis, J. B. Burritt, R. A. Clark & M. T. Quinn: A domain of p47phox that interacts with human neutrophil flavocytochrome b558. J Biol Chem 270, 26246-51 (1995)
103. G. Joseph, Y. Gorzalczany, V. Koshkin & E. Pick: Inhibition of NADPH oxidase activation by synthetic peptides mapping within the carboxyl-terminal domain of small GTP-binding proteins. Lack of amino acid sequence specificity and importance of polybasic motif. J Biol Chem 269, 29024-31 (1994)
104. J. W. Park, M. Ma, J. M. Ruedi, R. M. Smith & B. M. Babior: The cytosolic components of the respiratory burst oxidase exist as a M(r)
approximately 240,000 complex that acquires a membranebinding site during activation of the oxidase in a cellfree system. J Biol Chem 267, 17327-32 (1992)
105. J. W. Park, J. El Benna, K. E. Scott, B. L. Christensen, S. J. Chanock & B. M. Babior: Isolation of a complex of respiratory burst oxidase components from resting neutrophil cytosol. Biochemistry 33, 2907-11 (1994)
106. S. S. Iyer, D. W. Pearson, W. M. Nauseef & R. A. Clark: Evidence for a readily dissociable complex of p47phox and p67phox in cytosol of unstimulated human neutrophils. J Biol Chem 269, 22405-11 (1994)
107. T. Pawson: SH2 and SH3 domains in signal transduction. Adv Cancer Res 64, 87-110 (1994)
108. J. H. W. Leusen, K. Fluiter, P. M. Hilarius, D. Roos, A. J. Verhoeven & B. G. J. M. Bolscher: Interactions between the cytosolic components p47phox and p67phox of the human neutrophil NADPH oxidase that are not required for activation in the cellfree system. J Biol Chem 270, 11216-21 (1995)
109. D. J. Uhlinger & D. K. Perry: A carboxyterminal peptide from p47phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase. Biochem Biophys Res Commun 187, 940-8 (1992)
110. P. Finan, Y. Shimizu, I. Gout, J. Hsuan, O. Truong, C. Butcher, P. Bennett, M. D. Waterfield & S. Kellie: An SH3 domain and prolinerich sequence mediate an interaction between two components of the phagocyte NADPH oxidase complex. J Biol Chem 269, 13752-5 (1994)
111. T. L. Leto, A. G. Adams & I. de Mendez: Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to prolinerich targets. Proc Natl Acad Sci U S A 91, 10650-4 (1994)
112. H. Sumimoto, Y. Kage, H. Nunoi, H. Sasaki, T. Nose, Y. Fukumaki, M. Ohno, S. Minakami & K. Takeshige: Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. Proc Natl Acad Sci U S A 91, 5345-9 (1994)
113. I. de Mendez, M. C. Garrett, A. G. Adams & T. L. Leto: Role of p67phox SH3 domains in assembly of the NADPH oxidase system. J Biol Chem 269, 16326-32 (1994)
114. A. Fuchs, M.- C. Dagher & P. V. Vignais: Mapping the domains of interaction of p40phox with both p47phox and p67phox of the neutrophil oxidase complex using the two hybrid system. J Biol Chem 270, 5695-7 (1995)
115. IJ. M. Kramer, A. J. Verhoeven, R. L. van der Bend, R. S. Weening & D. Roos: Purified protein kinase C phosphorylates a 47kDa protein in control neutrophil cytoplasts but not in neutrophil cytoplasts from patients with the autosomal form of chronic granulomatous disease. J Biol Chem 263, 2352-7 (1988)
116. W. M. Nauseef, B. D. Volpp, S. McCormick, K. G. Leidal & R. A. Clark: Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components. J Biol Chem 266, 5911-7 (1991)
117. J. T. Curnutte, R. W. Erickson, J. Ding & J. A. Badwey: Reciprocal interactions between protein kinase C and components of the NADPH oxidase complex may regulate superoxide production by neutrophils stimulated with a phorbol ester. J Biol Chem 269, 10813-9 (1994)
118. IJ. M. Kramer, R. L. van der Bend, A. T. J. Tool, W. J. van Blitterswijk, D. Roos & A. J. Verhoeven: 1Ohexadecyl2Omethylglycerol, a novel inhibitor of protein kinase C, inhibits the respiratory burst in human neutrophils. J Biol Chem 10, 5876-84 (1989)
119. J. M. Robinson, P. G. Heyworth & J. A. Badway: Utility of staurosporine in uncovering differences in the signal transduction pathways for superoxide production in neutrophils. Biochim Biophys Acta 1055, 55-62 (1990)
120. G. C. Kessels, K. H. Krause & A. J. Verhoeven: Protein kinase C activity is not involved in Nformylmethionylleucylphenylalanineinduced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C. Biochem J 292, 781-5 (1993)
121. D. Rotrosen & T. L. Leto: Phosphorylation of neutrophil 47kDa cytosolic factor. Translocation to membrane is associated with distinct phosphorylation events. J Biol Chem 265, 19910-5 (1990)
122. J. El Benna, L. P. Faustm & B. M. Babior: The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by prolinedirected kinases. J Biol Chem 269, 23431-6 (1994)
123. P. G. Heyworth & J. A. Badwey: Continuous phosphorylation of both the 47 and the 49 kDa proteins occurs during superoxide production by neutrophils. Biochim Biophys Acta 1052, 299-305 (1990)
124. J. Ding & J. A. Badwey: Effects of antagonists of protein phosphatases on superoxide release by neutrophils. J Biol Chem 267, 6442-8 (1992)
125. J. M. Robinson & J. A. Badwey: The NADPH oxidase complex of phagocytic leukocytes: A biochemical and cytochemical view. Histochemistry 103, 163-80 (1995)
126. D. J. Lu, A. Takai, T. L. Leto & S. Grinstein: Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor. Am J Physiol 262, C39-49 (1992)
127. J. Ding & J. A. Badwey: Stimulation of neutrophils with a chemoattractant activates several novel protein kinases that can catalyze the phosphorylation of peptides derived from the 47kDa protein component of the phagocyte oxidase and myristoylated alaninerich C kinase substrate. J Biol Chem 268, 17326-33 (1993)
128. S. Grinstein, W. Furuya, J. R. Butler & J. Tseng: Receptormediated activation of multiple serine/threonine kinases in human leukocytes. J Biol Chem 268, 20223-31 (1993)
129. J. Ding & J. A. Badwey: Wortmannin and 1butanol block activation of a novel family of protein kinases in neutrophils. FEBS Lett 348, 149-52 (1994)
130. F. A. Gonzales, D. L. Raden & R. J. Davis: Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases. J Biol Chem 266, 22159-63 (1991)
131. P. J. Kennely & E. G. Krebs: Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem 266, 15555-8 (1991)
132. S. H. Jackson, H. L. Malech, C. A. Kozak, K. J. Lomax, J. I. Gallin & S. M. Holland: Cloning and functional expression of the mouse homologue of p47phox. Immunogenetics 39, 272-5 (1994)
133. L. P. Faust, J. El Benna, B. M. Babior & S. J. Chanock: The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by sitedirected mutagenesis. J Clin Invest 96, 1499-505 (1995)
134. C. H. Kwong, H. L. Malech, D. Rotrosen & J. L Leto: Regulation of the human neutrophil NADPH oxidase by rhorelated Gproteins. Biochemistry 32, 5711-7 (1993)
135. P. G. Heyworth, U. G. Knaus, X. Xu, D. J. Uhlinger, L. Conroy, G. M. Bokoch & J. T. Curnutte: Requirement for posttranslational processing of Rac GTPbinding proteins for activation of human neutrophil NADPH oxidase. Mol Biol Cell 4, 261-9 (1993)
136. S. Ando, K. Kaibuchi, T. Sasaki, K. Hiraoka, T. Nishiyama, T. Mizuno, M. Asada, H. Nunoi, I. Matsuda, Y. Matsuura, P. Polakis, F. McCormick & Y. Takai: Posttranslational processing of rac p21s is important both for their interaction with the GDP/GTP exchange proteins and for their activation of NADPH oxidase. J Biol Chem 36, 25709-13 (1995)
137. F. E. Maly, L. A. Quilliam, O. Dorseuil, C. J. Der & G. M. Bokoch: Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of EpsteinBarr virustransformed human B lymphocytes. J Biol Chem 269, 18743-6 (1994)
138. T. G. Gabig, C. D. Crean, P. L. Mantel & R. Rosli: Function of wildtype or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation. Blood 85, 804-11 (1995)
139. T. H. Chuang, B. P. Bohl & G. M. Bokoch: Biologically active lipids are regulators of Rac.GDI complexation. J Biol Chem 268, 26206-11 (1993)
140. N. Bourmeyster, M. -J. Stasia, J. Garin, J. Gagon, P. Boquet & P. V. Vignais: Copurification of Rho protein and the RhoGDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on Rho ADPribosylation by the C3 exoenzyme of Clostridium botulinium. Biochemistry 31, 12863-9 (1992)
141. P. T. Hawkins, A. Eguinoa, R. -G. Qiu, D. Stokoe, F. T. Cooke, R. Walters, S. Wennström, L. ClaessonWelsh, T. Evans, M. Symons & L. Stephens: PDGF stimulates an increase in GTPRac via activation of phosphoinositide 3kinase. Curr Biol 5, 393-403 (1995)
142. B. Dewald, M. Thelen & M. Baggiolini: Two transduction sequences are necessary for neutrophil activation by receptor agonists. J Biol Chem 263, 16179-84 (1988)
143. T. Okada, L. Sakuma, Y. Fukui, O. Hazeki & M. Ui: Blockage of chemotactic peptideinduced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3kinase. J Biol Chem 269, 3563-7 (1994)
144. D. Diekmann, A. Abo, C. Johnston, A. W. Segal & A. Hall: Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity. Science 265, 531-3 (1994)
145. E. Prigmore, S. Ahmed, A. Best, R. Kozma, E. Manser, A. W. Segal & L. Lim: A 68kDa kinase and NADPH oxidase component p67phox are targets for Cdc42Hs and Rac1 in neutrophils. J Biol Chem 270, 10717-22 (1995)
146. U. G. Knaus, S. Morris, H. - J. Dong, J. Chernoff & G. M. Bokoch: Regulation of human leukocyte p21activated kinases through G proteincoupled receptors. Science 269, 221-3 (1995)
147. J. R. White, P. H. Naccache & R. I. Sha'afi: Stimulation by chemotactic factor of actin association with the cytoskeleton in rabbit neutrophils. J Biol Chem 258, 14041-7 (1983)
148. M. T. Quinn, T. Evans, L. R. Loetterle, A. J. Jesaitis & G. M. Bokoch: Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components. J Biol Chem 268, 20983-7 (1993)
149. J. El Benna, J. M. Ruedi & B. M. Babior: Cytosolic guanine nucleotidebinding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation. J Biol Chem 269, 6729-34 (1994)
150. V. Le Cabec, H. Möhn, G. Gacon & I. MaridonneauParini: The small GTPbinding protein rac is not recruited to the plasma membrane upon NADPH oxidase activation in human neutrophils. Biochem Biophys Res Commun 198, 1216-24 (1994)
151. P. G. Heyworth, B. P. Bohl, G. M. Bokoch & J. T. Curnutte: Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. Evidence for its interaction with flavo-cytochrome b558. J Biol Chem 269, 30749-52 (1994)
152. O. Dorseuil, M. T. Quinn & G. M. Bokoch: Dissociation of rac translocation from p47phox/p67phox movements in human neutrophils by tyrosine kinase inhibitors. J Leukoc Biol 58, 108-13 (1995)
153. A. J. Ridley, H. F. Paterson, C. L. Johnston, D. Diekmann & A. Hall: The small GTPbinding protein rac regulates growth factorinduced membrane ruffling. Cell 70, 401-10 (1992)
154. L. P. Akard, D. English & T. G. Gabig: Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst. Blood 72, 322-7 (1988)
155. M. Y. Park, S. Imajoh Ohmi, H. Nunoi & S. Kanegasaki: Peptides corresponding to the region adjacent to His94 in the small subunit of cytochrome b558 inhibit superoxide generation in a cellfree system from human neutrophils. Biochem Biophys Res Commun 204, 924-9 (1994)
156. D. Tisch, Y. Sharoni, M. Danilenko & I. Aviram: The assembly of neutrophil NADPH oxidase: effect of mastoparan and its synthetic analogues. Biochem J 310, 715-9 (1995)
157. P. A. Karplus, M. J. Daniels & J. R. Herriott: Atomic structure of ferredoxinNADP+ reductase: prototype for a structurally novel flavoenzyme family. Science 251, 60-6 (1991)
158. P. Kraulis: A program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24, 946-50 (1991)