From the Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322
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ABSTRACT |
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Superoxide generation by the neutrophil respiratory burst oxidase (NADPH oxidase) can be reconstituted in a cell-free system using flavocytochrome b558 and the cytosolic proteins p47phox, p67phox, and Rac. p47phox functions as an adaptor protein; it increases the affinity of p67phox and Rac in the NADPH oxidase complex, but is not essential when high concentrations of these proteins are used (Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578-22582), implying that p67phox and/or Rac directly regulates enzyme activity. Herein, we describe an activation domain in p67phox that is essential for NADPH oxidase activity. A series of C-terminal truncation mutants of p67phox showed that residues 211 to the C terminus (residue 526) are not needed for cell-free activity. However, shorter truncations were inactive, pointing to an activation domain within the region spanning residues 199-210. p67phox mutated at single amino acid residues within this region showed diminished activity, and p67phox V204A was completely inactive. The effects of mutations on activity were independent of p47phox, and mutations did not affect the binding of p67phox to Rac. In the presence of wild-type p67phox, the V204A mutant was a potent inhibitor of superoxide generation, and inhibition was partially reversed by high concentrations of p67phox, but not by p47phox or Rac. The V204A mutant competed with native p67phox for translocation to neutrophil plasma membrane, indicating that p67phox V204A assembles to form an inactive complex. The data imply a direct activation of flavocytochrome b558 by an activation domain in p67phox.
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INTRODUCTION |
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During the respiratory burst, neutrophils generate
superoxide and secondarily generate reactive oxygen species
(H2O2, O2, and HOCl) that
together participate in killing invading microorganisms (1-4).
Superoxide generation is catalyzed by a multicomponent enzyme, the
respiratory burst oxidase (NADPH oxidase). The enzyme consists of a
plasma membrane-associated flavocytochrome b558 composed of two subunits, gp91phox (where phox is
phagocytic oxidase) and p22phox. The
former contains all of the electron-carrying groups (one FAD and two
hemes) needed to transfer electrons from NADPH to molecular oxygen as
well as a candidate NADPH-binding site (5-8). In addition, three
cytosolic factors, p47phox, p67phox, and Rac, are
needed for optimal activity. In resting cells, p47phox and
p67phox reside in the cytosol in a 240-kDa complex with a third
component, p40phox (9-11). Upon cell activation,
p47phox and p67phox associate with the membrane, where
they interact with flavocytochrome b558
(12-15). The small GTP-binding protein Rac is complexed in the cytosol
with an inhibitory protein, RhoGDI (Rho GDP
dissociation inhibitor), and translocates to
the plasma membrane independently of the other cytosolic components
(16-18). Assembly and activation can be reconstituted in a cell-free
system consisting of the protein components, phospholipid,
GTP
S,1 and an anionic
amphiphile such as arachidonate (19-26).
The molecular details of protein-protein interactions among the oxidase components have been the subject of recent investigations (27-32). p47phox contains tandem SH3 (Src homology 3) domains, and one or more of these bind to a proline-rich region in the C terminus of p22phox. p67phox also contains two SH3 domains in the C-terminal half of the molecule, and one or both of these mediate binding to proline-rich regions in p47phox in the assembled oxidase. p47phox and p67phox bind cooperatively in the active complex (33), and p47phox is needed for the stable assembly of p67phox (27, 33, 34). Rac interacts with the membrane through its isoprenylated C terminus (35), which in Rac1 contains a polybasic region (36). Membrane binding is important for stimulation of high rates of superoxide generation in a cell-free system (37). In addition, Rac contains an effector region (residues 26-45) that interacts in a GTP-dependent manner with a binding site in the N terminus of p67phox (38, 39). Another region (the "insert region," residues 124-135) is important for activation of superoxide generation by Rac (40); this region has been proposed to interact directly with the flavocytochrome (39). Thus, a complex set of interactions among multiple proteins governs assembly of the active NADPH oxidase.
Although knowledge about the protein interactions within the NADPH oxidase complex is becoming increasingly detailed, specific roles for individual cytosolic components have remained obscure. According to one hypothesis, p67phox regulates electron transfer in flavocytochrome b558 from NADPH to FAD, whereas p47phox controls electron flow from FAD through the heme groups to oxygen (41, 42). However, normal or near-normal rates of NADPH-dependent superoxide generation can be reconstituted in the absence of p47phox using high concentrations of p67phox and Rac (43, 44). p47phox acts as an adaptor protein to enhance the binding of the other two components by 50-100-fold (43). Thus, p47phox does not seem to function in regulating electron transfer, and either p67phox or Rac (or possibly both) is directly involved in activating the flavocytochrome. Herein, an activation domain in p67phox is identified, and a model is proposed in which binding of p67phox to both p47phox and Rac orients this domain in such a way as to interact with and activate flavocytochrome b558.
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EXPERIMENTAL PROCEDURES |
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Materials--
NADPH, FAD, arachidonic acid, cytochrome
c (type IV, horse heart), thrombin, glutathione,
n-octyl glucoside, cholic acid, GTPS, Gpp(NH)p,
phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine,
and sphingomyelin were purchased from Sigma. Hespan (6.2% hetastarch
in 0.9% NaCl) was obtained from American Hospital Supply Corp.
Lymphocyte separation medium (6.2% Ficoll and 9.4% sodium
diatrizoate) was purchased from Organon Tekniker. N-Methylisotoic anhydride was obtained from Molecular
Probes, Inc. Glutathione-Sepharose, heparin-Sepharose CL-6B,
DEAE-Sepharose CL-6B, CM-Sepharose CL-6B,
-aminooctyl-agarose, and
pGEX-2T vector were purchased from Amersham Pharmacia Biotech. Affi-Gel
10 was purchased from Bio-Rad. An ECL Western blotting analysis kit was purchased from Amersham Pharmacia Biotech. Oligonucleotide primers were
synthesized and purified by the Emory Microchemical Facility.
Preparation of Plasma Membrane, Cytochrome b558, and
Recombinant Proteins--
Human neutrophils were isolated from
peripheral blood from healthy donors as described previously (45).
Plasma membranes were isolated as described (46). Purification of
cytochrome b558 from plasma membrane was carried
out as described previously (5). Recombinant p47phox and
wild-type p67phox were expressed in Sf9 insect cells and
purified according to Uhlinger et al. (26, 33). Rac was
expressed in DH5 cells as a glutathione S-transferase
fusion protein and purified using glutathione-Sepharose followed by
thrombin cleavage (37). A series of truncated and point mutated forms
of p67phox were expressed in Escherichia coli,
purified with glutathione-Sepharose followed by glutathione elution as
described previously (38), and dialyzed to remove free glutathione.
Protein concentrations were determined according to Bradford (47).
Truncations and Site-directed Mutagenesis--
A series of
truncated p67phox clones were obtained by PCR using
p67phox DNA cloned in pGEX-2T as the template. For all PCRs,
the forward primer (CGTG GATCCATG TCC
CTGGTGGAGGCC) was designed to anneal to the 5'-end of the
p67phox sequence and to introduce a BamHI site
(shown in boldface) followed by an initiation codon (underlined). For
each truncation, the reverse primer (e.g. for
p67phox-(1-210), GATG
AATTCTTAATCCACCACAGATGC) was designed to anneal to the
p67phox sequence immediately 5' to the region to be truncated
and to introduce the stop codon (underlined) and an EcoRI
site (shown in boldface). For each mutation, the same forward primer as
above was used along with the reverse primer as above, except that the desired mutation codon replaced the wild-type codon, thus encoding a
point mutation in the carboxyl terminus of p67phox-(1-210).
These PCR products were ligated into the BamHI and
EcoRI sites of the pGEX-2T vector and transformed into
DH5 for expression of the protein. The PCR products were sequenced
to verify that no unexpected mutations were introduced and to confirm
the truncations.
Binding of p67phox to Rac-- Rac1 (1.2 µM) was preincubated with 0.85 µM mant-Gpp(NH)p for 15-20 min to form the Rac·mant-Gpp(NH)p complex as described previously (39). The complex was then titrated with either intact or truncated p67phox, and the fluorescence intensity at 440 nm was recorded after excitation at 355 nm. Fluorescence titrations were fit to a single site binding equation as described previously (48), solving for Kd and Fmax (maximal fluorescence change).
NADPH Oxidase Activity--
Superoxide generation was measured
by superoxide dismutase-inhibitable reduction of cytochrome
c as described (46) using a Thermomax Kinetic Microplate
reader (Molecular Devices, Menlo Park, CA). Rac was preloaded with
5-fold molar excess of GTPS for 15 min at room temperature in the
absence of MgCl2 (37). For standard assay conditions, the
cell-free reaction mixtures included 10 µg of plasma membrane
protein, 850 nM p47phox, 900 nM
p67phox, 950 nM Rac, 10 µM GTP
S,
and 200-240 µM arachidonate in a total volume of 50 µl. Three 10-µl aliquots of each reaction mixture were transferred
to 96-well assay plates and preincubated for 5 min at 25 °C. For
each well, 240 µl of substrate mixture containing 200 µM NADPH and 80 µM cytochrome c
in buffer A (100 mM KCl, 3 mM NaCl, 4 mM MgCl2, 1 mM EGTA, and 10 mM PIPES, pH 7.0) was added to initiate the assay.
Cytochrome c reduction was quantified by monitoring the
absorbance increase at 550 nm using an extinction coefficient of 21 mM
1 cm
1 (49).
Translocation of p67phox--
Experiments were
carried out by a modification of the procedure of Uhlinger et
al. (26). The reaction mixture included 10 µg of plasma membrane
protein, 1.3 µM p47phox, 1.3 µM
p67phox, varying amounts of p67phox V204A, 1.2 µM Rac, 10 µM GTPS, and 200-240
µM arachidonate in 50 µl of buffer A. The reaction
mixture was incubated at 25 °C for 5 min and layered onto a
discontinuous sucrose gradient (0.5 ml each of 60 and 20% (w/v) in
buffer A). The reactions were then centrifuged for 15 min at 55,000 rpm
(259,000 × g) in a TLS-55 swinging bucket rotor at
25 °C in a Beckman TL100 ultracentrifuge. Immediately after
centrifugation, the upper 400 µl containing soluble components was
removed. 400 µl spanning the interphase and containing the plasma
membrane (50) was then removed. Proteins from each phase were
precipitated with trichloroacetic acid (10% final concentration) using
lysozyme (2 mg/ml) as a carrier protein. The volume of each aliquot was
adjusted to 850 µl with deionized water, and 50 µl of lysozyme (2 mg/ml) and 100 µl of trichloroacetic acid (100% (w/v)) were added.
The mixture was incubated on ice for 5 min and microcentrifuged for 5 min at 4 °C. The pellet was briefly washed with deionized water and
resuspended in 80 µl of buffer A, and the pH was adjusted to 7.0 with
1 M NaOH. The samples were run on a 10% SDS-polyacrylamide
gel and transferred to nitrocellulose. For Western blots, a rabbit
polyclonal antibody raised against holo-p67phox (50) was
further purified using purified p67phox bound to Affi-Gel 10. Purified p67phox antibody was used for detecting native
p67phox, whereas unpurified antiserum to p67phox was
used for detecting p67phox-(1-210) V204A because of poor
detection by the purified antibody. Western blotting was carried out
according to the Amersham Pharmacia Biotech ECL Western blotting
protocol.
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RESULTS |
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p67phox Contains an Activation Domain within the Region from Amino Acid Residues 199 to 210-- Previously, we showed that an N-terminal glutathione S-transferase fusion form of truncated p67phox-(1-246) lacking both SH3 domains partially substituted for full-length p67phox to activate the respiratory burst oxidase, whereas another truncated mutant, p67phox-(1-198), failed support superoxide generation (43). Based on these observations, a series of truncated p67phox mutants were generated and expressed (Fig. 1, A and B) to identify the minimal size p67phox fragment that could activate cell-free superoxide generation. In Fig. 1C, a saturating concentration of p67phox-(1-246) activated superoxide generation to ~50% of the rate seen with full-length p67phox, as noted earlier (43). Surprisingly, shorter truncated forms of the protein, p67phox-(1-235), p67phox-(1-221), p67phox-(1-216), and p67phox-(1-210), supported superoxide generation at the same rate as wild-type p67phox (Fig. 1C). Thus, the region from residues 235 to 246 is moderately inhibitory to the function of p67phox. Also of interest, the proline-rich region (residues 226-234; underlined in Fig. 1A), which has been implicated in binding to p47phox and translocation of p67phox to the membrane (27, 30), did not influence the ability of p67phox to support superoxide generation (Fig. 1C). Truncated forms of the protein shorter than 210 amino acid residues failed to support detectable levels of superoxide generation. Thus, p67phox contains an activation domain within the region spanning amino acid residues 199-210.
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Effect of Point Mutations in the Cytochrome Activation Domain of p67phox on Superoxide Generation-- Ten mutant forms of p67phox-(1-210) were made, and mutant proteins were purified on glutathione-Sepharose (Fig. 2A). Non-alanine residues within amino acids 201-210 were individually mutated to alanines, whereas the two alanine residues were converted to leucines. Mutant proteins were then evaluated for their abilities to support NADPH oxidase activity in the cell-free system. The experiment was carried out using the complete cell-free system, which contained p47phox, Rac1, and plasma membrane (as the source of cytochrome b558) (Fig. 2B), and in a modified system, which lacked p47phox and utilized purified FAD-reconstituted cytochrome b558 (Fig. 2C). In both assay systems, mutations within the region from residues 201 to 210 resulted in a significant reduction in the ability of p67phox to support superoxide generation. In particular, p67phox V204A showed little or no ability to support superoxide generation, and the V205A mutation showed only ~20-30% of the wild-type activity. This pattern was independent of p47phox, indicating that the effects of mutating these sites are not a result of altered binding of p67phox to p47phox.
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Binding of Truncated Forms of p67phox to Rac1-- p67phox was reported to contain a Rac-binding site within the region spanned by residues 1-199 (38). To test whether the presence of the activation domain was influencing Rac binding to p67phox, we utilized the fluorescence binding method described previously (39). This method utilizes a fluorescent analog of GTP (mant-Gpp(NH)p) that binds tightly to Rac1 and serves as a reporter group, undergoing an increase in fluorescence upon p67phox binding (39). The concentrations of full-length p67phox, p67phox-(1-246), and p67phox-(1-198) were varied as indicated in Fig. 3, and the increase in fluorescence was monitored. All three types of p67phox bound Rac with about the same affinity. Wild-type p67phox and p67phox-(1-198) bound with respective Kd values of 31 and 43 nM. Thus, the absence of the activation domain did not affect the ability of p67phox to bind to Rac.
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p67phox V204A Inhibits Activation of Superoxide Generation by Wild-type p67phox-- Superoxide generation was measured in the complete cell-free system (i.e. containing wild-type (full-length) p67phox). As shown in Fig. 4, increasing the concentration of p67phox V204A to 1.5 µM or greater resulted in complete inhibition of superoxide generation. Thus, in an in vitro system, p67phox V204A functions as a "dominant negative" for superoxide generation.
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p67phox V204A Competes with Wild-type p67phox in the NADPH Oxidase Complex-- We previously used a discontinuous sucrose gradient to demonstrate that p47phox and p67phox are recovered in the plasma membrane fraction after cell-free activation, that this "translocation" is dependent upon the presence of cytochrome b558, and that binding occurs with a 1:1:1 stoichiometry among p47phox, p67phox, and cytochrome b558 (26). We utilized this method to test directly whether p67phox V204A competes with wild-type p67phox for binding in the active NADPH oxidase complex. p67phox V204A and p67phox were recovered from the plasma membrane fraction, visualized by Western blotting, and distinguished by their different sizes. As shown in Fig. 5, increasing concentrations of p67phox V204A resulted in decreased recovery of wild-type p67phox in the plasma membrane fraction, which paralleled increased recovery of p67phox V204A in the plasma membrane fraction. The amount of wild-type p67phox seen in the plasma membrane fraction at higher concentrations of p67phox V204A was similar to the amount recovered in this fraction in the absence of activators (data not shown), indicating that most of the residual band was due to nonspecific binding. Based on comparison with standards, we estimate that 40-50 pmol (about half) of wild-type p67phox translocates to the plasma membrane fraction. In the presence of the highest concentration of p67phox V204A, this value is reduced to ~3-5 pmol, whereas ~50 pmol of p67phox V204A is associated with the membrane. Thus, the total amount of wild-type plus mutant p67phox associated with the membrane is relatively constant, indicating that p67phox V204A competes with wild-type p67phox for binding in the assembled NADPH oxidase complex.
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DISCUSSION |
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A large number of signal transduction mechanisms involve the reversible formation of multimeric protein complexes, frequently at a membrane surface such as the plasma membrane. Often these complexes contain an effector enzyme plus additional proteins whose functions are incompletely defined. Two rationales are generally offered for the role of these protein complexes: approximation and activation. Approximation involves bringing two or more protein components together within a small volume or on a surface, thus increasing the efficiency of their interactions, as is the case for the recruitment of the Grb2·mSOS (son of sevenless) complex to the plasma membrane by binding to tyrosine phosphate, e.g. on the epidermal growth factor receptor. The SOS protein acts an exchange factor for Ras, which is already located in the plasma membrane. Activation involves allosteric regulation of the effector enzyme activity by association with other proteins. The interaction of Ras·GTP with the protein kinase Raf is usually thought of as an example of this type of mechanism and may involve not only Ras and Raf, but also additional proteins/factors including 14-3-3 protein, membrane, and other unknown proteins (51, 52). The respiratory burst oxidase provides an example of activation, and the present studies help to define individual roles for each of the regulatory cytosolic factors.
Herein, we have described an activation domain in p67phox that we propose is directly involved in regulating NADPH oxidase enzymatic function. The domain was initially localized to amino acid residues 199-210 based on a series of truncated mutants of p67phox. Forms of p67phox containing the N-terminal 210 amino acids retained their ability to activate the respiratory burst oxidase, whereas forms shorter than this were no longer functional. The activation domain was further defined using a series of point mutations within amino acids 201-210. Although mutation of most of the residues within this range reduced activity, the valine residue at position 204 was particularly important since its mutation resulted in a complete loss of function.
The mechanism by which valine 204 and surrounding residues affects function is not yet clear, although several potential mechanisms can be ruled out. This region is not involved in binding interactions with the other cytosolic factors. Effects of mutations in this region were the same whether or not p47phox was present, ruling out interaction with p47phox as an explanation for the role of the activation domain. Similarly, the binding of p67phox to Rac was the same whether or not the activation domain of p67phox was present. Thus, it is likely that this region of p67phox directly interacts with and regulates the electron transfer from NADPH to oxygen within flavocytochrome b558. The specific electron transfer step(s) that are regulated by the cytochrome activation domain are not yet known. This question is being investigated in our laboratory.
The present studies suggest a model for the roles of the cytosolic factors in the respiratory burst oxidase. In response to upstream signals that presumably involve phosphorylation, p47phox interacts directly with the proline-rich region in the small subunit of cytochrome b558 (see the Introduction). We have previously provided evidence that p47phox functions as a regulated adaptor protein, the function of which is to enhance the binding of the other two cytosolic components (43). p47phox increased the apparent binding (based on EC50 values) of p67phox by 100-fold and that of Rac by 50-fold. Thus, minimally, p47phox provides a binding site for p67phox when it associates with the cytochrome. Translocation of Rac2 (the major isoform in neutrophil) to the plasma membrane occurs in the absence of p47phox (17) and with distinct kinetics (16, 53). Translocation to the plasma membrane is regulated in part by guanine nucleotide exchange and involves dissociation from the inhibitor protein RhoGDI in the cytosol. We have shown that its interaction with the plasma membrane is important for activity (37) and that the effector region (residues 26-45) in Rac serves as a binding site for p67phox (39). Rac contains an additional region, the insert region, that is important for assembly (54) and that was proposed to interact directly with the cytochrome (39). Thus, both Rac and p47phox are the targets of "assembly signals," including guanine nucleotide exchange and phosphorylation, and both provide binding sites for p67phox. We propose that these regions bind and orient p67phox in such a way as to juxtapose the cytochrome activation domain in p67phox with a target region on the cytochrome. The interaction of this domain with cytochrome b558 then activates electron flow from NADPH to molecular oxygen. Such a model is attractive in that it assigns unique roles to each of the cytosolic factors and provides a paradigm by which effector enzymes are reversibly regulated by complex formation with multiple regulatory proteins.
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ACKNOWLEDGEMENT |
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We thank Alan Hall for providing the p67phox-(1-246) and p67phox-(1-198) recombinant vectors.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AI 22809.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Howard Hughes Medical Inst., Dept. of Medicine,
Duke University School of Medicine, Durham, NC 27710.
§ To whom correspondence should be addressed. Tel.: 404-727-5875; Fax: 404-727-2738; E-mail: dlambe{at}bimcore.emory.edu.
1
The abbreviations used are: GTPS, guanosine
5'-O-(3-thiotriphosphate); Gpp(NH)p, guanosine
5'-(
,
-imido)triphosphate; PCR, polymerase chain reaction; mant,
methylanthraniloyl; PIPES, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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