From the Department of Biochemistry, Kyushu
University School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka
812-82, Japan and the § Human Genome Center, Institute of
Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku,
Tokyo 108, Japan
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ABSTRACT |
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Anionic amphiphiles, such as arachidonate, activate the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane, which contains cytochrome b558 comprising gp91phox and p22phox, and three cytosolic proteins: p47phox and p67phox, each harboring two SH3 domains, and the small GTPase Rac. Here we show that, even without the amphiphiles, the oxidase is activated in vitro by a C terminally truncated p47phox, retaining the N-terminal and the two SH3 domains, and the N terminus of p67phox. When either truncated p47phox or p67phox is replaced by the respective full-length one, the activation absolutely requires the amphiphiles. The results indicate that both p47phox and p67phox are the primary targets of the amphiphiles, and that their C-terminal regions play negative regulatory roles. We also find that the truncated p47phox, but not the full-length one, can bind to p22phox, a binding required for the oxidase activation. The N-terminal SH3 domain of p47phox is responsible for the binding not only to p22phox, but also to the p47phox C terminus. Thus the SH3 domain is accessible in the active p47phox, but is normally masked in the full-length one probably via intramolecularly interacting with the C terminus. The present findings support our previous proposal of regulatory SH3 domain-mediated interactions.
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INTRODUCTION |
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Increasing attention has currently been paid to specific protein-protein interactions in intracellular signal transduction, which are mediated by modular binding domains of signaling proteins (1, 2). Among the domains to be characterized at an earlier stage is the Src homology 3 (SH3)1 domain found in various proteins including the Src family tyrosine kinases. The domain directly binds, via its target-binding surface, to a proline-rich region (PRR) of its partners, thereby mediating protein-protein interactions (1-4). Unlike the case of SH2 domains, whose interactions with tyrosine-containing peptides are promoted by phosphorylation of the SH2 domain-binding site, the regulatory mechanism for SH3 domain-mediated associations largely remains elusive.
Specific interactions via SH3 domains play a crucial role in assembly and activation of the phagocyte NADPH oxidase (5-15). The oxidase, dormant in resting cells, is activated during phagocytosis to catalyze reduction of molecular oxygen to superoxide, a precursor of microbicidal oxidants (16-20). The significance of this enzyme in host defense is made evident by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease, whose phagocytes lack the superoxide-producing system (16-20). Recent studies, furthermore, have suggested that oxidants produced by the NADPH oxidase may be also involved in Ras-mediated mitogenic signaling in fibroblasts (21), oxygen sensing in airway chemoreceptors (22), and activation of c-Jun N-terminal kinase in kidney epithelial cells (23). The catalytic core of the oxidase, which transfers electrons from NADPH to oxygen molecule, is the membrane-integrated flavocytochrome b558, composed of the two subunit gp91phox and p22phox (16-20). Activation of the oxidase requires translocation of three cytosolic proteins, p47phox, p67phox, and the small GTPase Rac1/2, to the membrane where they assemble with the cytochrome. Both p47phox and p67phox harbor two SH3 domains, which mediate specific interactions between the oxidase factors. The C-terminal SH3 domain of p67phox interacts with the PRR of p47phox (6, 8, 12), while the N-terminal one of p47phox does with p22phox (11, 15). The latter interaction is required for both translocation of p47phox and activation of the NADPH oxidase (5, 8-11, 15). A monoclonal antibody specific for the p47phox SH3 domains interacts with p47phox in the presence of arachidonic acid, an activator of the oxidase, but not with the resting form of p47phox (5). It is likely that the N-terminal SH3 domain of p47phox is normally inaccessible, and, upon activation, becomes unmasked to interact with p22phox (5, 11). In the oxidase system, thus, the SH3 domain-mediated interactions are apparently regulated in contrast with the Grb2/Sos SH3 domain-mediated contacts, which are constitutive (1, 2).
In addition to the whole cell activation by various phagocytic or non-phagocytic stimuli, the NADPH oxidase can be activated in a cell-free system reconstituted with cytochrome b558 and the three cytosolic proteins, p47phox, p67phox, and Rac in the GTP-bound form (17-20). In the system, the activation is totally dependent on the addition of such anionic amphiphile activators as arachidonic acid and sodium dodecyl sulfate (SDS) (24). Here we have developed an in vitro system in which the NADPH oxidase is activated without using the amphiphile activators. A mutant p47phox, allowing the N-terminal SH3 domain unmasked, is capable of both binding to p22phox and activating the oxidase without the amphiphiles when used with the N terminus of p67phox. These findings indicate the regulatory intramolecular association of the SH3 domain in p47phox, which is directly linked to the oxidase activation.
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EXPERIMENTAL PROCEDURES |
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Glutathione S-Transferase (GST) Fusion Proteins--
The DNA
fragments encoding the full-length p47phox (p47-F; amino acid
residues 1-390), p47-N (1-153), p47-(SH3)2 (154-286),
p47-F(W193R) (the full-length p47phox with a substitution of
Trp193 for Arg), the full-length p67phox (p67-F,
1-526), p67-N (1-242), p67-SH3(C) (455-526), and p22-C (the
cytoplasmic domain of p22phox, residues 132-195) were obtained
as described previously (5, 11, 12). The DNAs for the p47-C (1-286)
and p47-
N (154-390) were amplified by polymerase chain reaction
from a cloned cDNA encoding human p47phox or
p67phox. For the mutant p47-F carrying the Trp263
Arg substitution, namely p47-F(W263R), the mutation was introduced into p47-F by polymerase chain reaction-mediated site-directed mutagenesis. All the polymerase chain reaction products were subcloned into the pGEX-2T expression vector (Pharmacia Biotech, Uppsala, Sweden). All the plasmids were subjected to DNA sequencing for the
confirmation of precise construction. The GST fusion proteins were
expressed in Escherichia coli and purified by
glutathione-Sepharose-4B beads (Pharmacia Biotech).
Cell-free Activation of the NADPH Oxidase-- Both membrane and cytosolic fractions of human neutrophils were prepared by sequential centrifugations as described previously (5). To prepare Rac2-enriched fractions, the cytosolic fraction was applied to a 2',5'-ADP Sepharose CL-6B column. The flow-through fraction was applied to a DEAE Sepharose CL-6B column, and the Rac2-enriched fraction was eluted with 0.2 M NaCl. The fraction contained Rac2 but was free of p47phox and p67phox as confirmed by immunodetection (11).
The assay mixture was composed of 100 mM potassium phosphate (pH 7.0), 75 µM cytochrome c, 10 µM FAD, 10 µM GTPFar Western Blotting--
In vitro interaction
between p47phox and p22phox was estimated by far
Western blot as described previously (11). Briefly, the GST-p22-C (1.0 µg) were subjected to SDS-PAGE and transferred to nitrocellulose membrane, which was incubated with 10 µg of GST-p47-F, GST-p47-C, or GST-p47-(SH3)2 in 20 mM Tris-HCl (pH 7.5),
150 mM NaCl, and 1% bovine serum albumin. The filter was
then probed with an anti-GST monoclonal antibody (25), a generous gift
from Drs. Yoichi Tachibana (Nippon Zeon Corp., Tokyo, Japan) and
Michiyuki Matsuda (International Medical Center of Japan, Tokyo,
Japan), rather than a polyclonal anti-p47phox antibody, because
the latter may block the interaction with p22phox. The
monoclonal anti-GST antibody did not recognize the GST fusion protein
transferred to nitrocellulose membrane after SDS-PAGE under the
condition used (25). Complexes were detected using alkaline
phosphatase-conjugated anti-mouse IgG antibodies.
Two-hybrid Experiments--
The p47-F (amino acid residues
1-390), p47-C (1-286), and the C-terminal region of
p47phox, namely p47-C (286-390), were cloned into a modified
GAL4 activation domain-fusion vector pGAD424g (12) to obtain
pGAD-p47-F, pGAD-p47-
C, and pGAD-p47-C, respectively. Deletion
mutants of pGAD-p47-C, namely p47-C
P1 and p47-C
P2, lacked amino
acid residues 299-346 and 360-390, respectively (12). The C-terminal
SH3 domain of p67phox (455-526), namely p67-SH3(C), and the
C-terminal cytoplasmic tail of p22phox (132-195), namely
p22-C, were cloned into a modified GAL4 DNA-binding domain fusion
vector pGBT9g (12) to obtain pGBT-p67-SH3(C) and pGBT-p22-C,
respectively. All plasmids were subjected to DNA sequencing for the
confirmation of precise construction.
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RESULTS |
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Role of the p47phox Regions in the NADPH Oxidase Activation
in a Cell-free System--
The protein p47phox, an activating
factor of the phagocyte NADPH oxidase, comprises four portions: the
N-terminal region, the N-terminal and C-terminal SH3 domains, and the
C-terminal tail (Fig. 1A). To
investigate roles of the individual regions, we isolated deletion
mutant proteins as GST fusions, and tested their abilities to activate
the oxidase stimulated by SDS in a cell-free system reconstituted with
human neutrophil membrane, the full-length p67phox, and the
Rac2-enriched faction (Fig. 1A). In the cell-free system, the wild-type full-length p47phox (p47-F) activated the NADPH
oxidase in a dose-dependent manner (Fig. 1B),
and the maximal activity was obtained at the concentration of 13.0 µg/ml (about 0.2 nmol/ml). At the concentration of 0.1 nmol/ml, the
p47phox lacking the C terminus (p47-C) fully activated the
oxidase, while neither the N terminally deleted one (p47-
N) nor the
one without both termini (p47-(SH3)2) was capable of
supporting superoxide production (Fig. 1A). The latter two
proteins were completely inactive even at 4-fold higher concentrations
(data not shown). Thus the N-terminal region is essential for the
oxidase activation. To clarify the role of the C-terminal SH3 domain,
we introduced the substitution of Arg for Trp263, the most
conserved residue in SH3 domains that directly interacts with a proline
of target peptides (1, 2, 4). The mutation resulted in decreased
ability to support superoxide production at submaximal (Fig.
1A) and saturated (Fig. 1B) concentrations, indicating that the SH3 domain is not essential, but is required for
the full activation. This is in contrast with that the N-terminal SH3
domain was an absolute requisite for the oxidase activation (Fig.
1A), which agrees with previous results by us and others (11, 15). Taken together, the N-terminal region and both SH3 domains
are required for fully activating the oxidase.
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The N-terminal Region of p67phox Is Sufficient for the
NADPH Oxidase Activation--
Another oxidase activating protein
p67phox also harbors two SH3 domains (Fig.
2), both of which seem indispensable for
superoxide production in stimulated cells (7). However, the domains and the region between them are not required in a cell-free system: the
N-terminal region of p67phox is sufficient for the oxidase
activation (7), which was confirmed in our cell-free activation system
(Fig. 2). The region (amino acid residues 1-242) contains the
Rac-binding site (26), and the GTP-dependent interaction
between p67phox and Rac seems essential for the oxidase
activation (26, 27). The activation accomplished by the C terminally
deleted p47phox (p47-C) and the N terminus of
p67phox (p67-N) was essentially the same as that by their
respective full-length proteins, in both the maximal activity (Fig. 2)
and the dose dependence on the proteins (data not shown). Among the four portions of p67phox, the N-terminal region is thus
sufficient for the cell-free oxidase activation, in combination with
the C terminally deleted p47phox (p47-
C) as well as with
full-length one (p47-F).
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Anionic Amphiphile-independent Activation of the NADPH Oxidase in a
Cell-free System--
In the cell-free system with p47-F and p67-F,
the oxidase activation was totally dependent on the anionic amphiphile
SDS (Fig. 3A). On the other
hand, to our surprise, the NADPH-dependent superoxide production was observed even in the absence of the amphiphile, when
both p47-C and p67-N were used instead of the full-length ones (Fig.
3A). The activation required not only the truncated proteins
(Fig. 3B) but also the neutrophil membrane and Rac2, but was
diminished in the presence of GDP
S, an agent keeping Rac2 in an
inactive form (Fig. 3C). These properties confirm that the
superoxide production is indeed catalyzed by the phagocyte NADPH
oxidase.
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Inter- and Intra-molecular Interactions of the N-terminal SH3
Domain of p47phox--
To investigate the molecular event that
enables p47phox to activate the oxidase, we compared the nature
of the C terminally deleted p47phox (p47-C) with that of the
full-length one (p47-F). It is well established that, upon cell
stimulation, p47phox interacts with the C-terminal cytoplasmic
tail of p22phox (5, 8-11, 13-15). The induced interaction is
mediated by the N-terminal SH3 domain of p47phox and is
essential for the oxidase activation (11, 15). An in vitro
binding assay using purified proteins revealed that p47-
C directly
bound to p22phox as strongly as the p47phox composed
solely of the SH3 domains (p47-(SH3)2) did, whereas the
full-length p47phox could not (Fig.
5A). The result completely
agrees with that obtained by an in vivo binding assay in the
yeast two-hybrid system (Fig. 5B). In the C terminally
deleted protein, thus, the N-terminal SH3 domain exists in a state
accessible to the target p22phox. This raised a question how
the SH3 domain is normally masked. We have previously shown that the
SH3 domains of p47phox can interact with the C terminus of this
protein, an interaction which seems to keep the SH3 domains
inaccessible (5). To determine the precise regions involved in this
interaction, we performed binding experiments in the two-hybrid system.
The N-terminal SH3 domain seems responsible for the interaction with
the C terminus (p47-C; amino acid residues 286-390), since a mutation
in this domain (Trp193
Arg) abrogated the binding (Fig.
5C) but one in the other SH3 domain (Trp263
Arg) did not (data not shown). The results are consistent with the
observation that the N-terminal SH3 domain can interact with p47phox in vitro (15). As shown in Fig.
5C, the SH3 domain interacted with p47-C
P2 that lacked
the PRR
(Pro361-Gln-Pro-Ala-Val-Pro-Pro-Arg-Pro369),
the target for the C-terminal SH3 domain of p67phox (6, 8, 12).
Another deletion (the deleted residues 299-346), giving p47-C
P1,
abolished the interaction with the p47phox SH3 domain, but did
not affect the contact with p67phox (Fig. 5C). Thus
the intramolecular target of the p47phox N-terminal SH3 domain
seems different from the site for the p67phox SH3 domain. The
p67phox-binding site is likely to be exposed in the folded
inactive form of p47phox, as indicated by the two hybrid
interaction between the full-length p47phox and the C-terminal
SH3 domain of p67phox (Fig. 5B).
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DISCUSSION |
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Here we presented that the phagocyte NADPH oxidase is activated by
the C terminally deleted p47phox (p47-C) and the N terminus
of p67phox (p67-N), even in the absence of in vitro
activators, anionic amphiphiles, such as arachidonic acid and SDS. The
observation that the anionic amphiphile-independent activation requires
both active forms of p47phox and p67phox indicates that
the two proteins are the primary targets of the activators in the
cell-free system. On the other hand, the action of the amphiphiles on
either cytochrome b558 or Rac, if any, is not
essential for the oxidase activation. The extent of the oxidase activation using both truncated proteins is comparable to that elicited
by the amphiphiles: about a half of the superoxide producing activity
is obtained in the present system. To our knowledge, a similar level of
the activation has not been accomplished in any reported cell-free
systems using purified cytosolic factors without the amphiphiles. Two
recent studies have demonstrated arachidonic acid- or SDS-independent
cell-free activation of the NADPH oxidase using crude neutrophil
cytosol (28, 29): one reports that phosphatidic acid fully activates
the enzyme in a phosphorylation-dependent manner in a
system using cellular membranes and cytosol with diacylglycerol (28),
and the other shows that phosphorylated p47phox is capable of
activating the oxidase, but to a lesser extent, when membranes are
treated with cytosol and GTP
S (29).
The present system is considered quite useful for studying in detail
the activation mechanisms of the individual cytosolic proteins. In the
absence of the amphiphiles, the oxidase activation is dependent on the
state of p47phox, when p67-N (an active form of
p67phox) is used with the GTP-bound Rac2. Under the conditions,
the oxidase is activated by the addition of the active p47phox
(p47-C), but not the full-length one (Fig. 4). Similarly, with p47-
C, the state of p67phox is the determinant. When both
p47-
C and p67-N are present, the activation totally depends on
the state of Rac: little superoxide production was observed with Rac2
in the GDP-bound inactive form (Fig. 3C).
We also studied here the mechanism for activating p47phox. The
active p47phox (p47-C) interacted with p22phox both
in vivo and in vitro, while the full-length one
in the resting state did not (Fig. 5). This interaction is mediated via
binding of the p47phox N-terminal SH3 domain to the PRR of
p22phox (11, 15), an interaction which is indispensable for the
oxidase activation (11, 15). Thus the conformational change of
p47phox that is induced by the amphiphiles appears to culminate
in unmasking of the N-terminal SH3 domain, leading to the access to
p22phox. In the resting state, this domain is masked probably
by interacting with a C-terminal region of p47phox. The region
required for this interaction is different from the PRR, the target for
the C-terminal SH3 domain of p67phox. This implies that the
intramolecular interaction in p47phox and its intermolecular
binding to p67phox are not mutually exclusive, i.e.
both interactions can occur at the same time. Indeed the full-length
p47phox, in which the N-terminal SH3 domain is masked, can bind
to the p67phox SH3 domain (Fig. 5B). The
intramolecular interaction appears to require the target-binding
surface of the p47phox SH3 domain, since a mutation of the
surface (W193R) abrogates the binding (Fig. 5C). On the
other hand, the target region in p47phox does not contain a
typical PRR. In this context, it should be noted that the SH3 domain of
the tyrosine kinase Src interacts intramolecularly, via its
target-binding surface, with the region lacking a proline-rich sequence
(30). Taken together, the N-terminal SH3 domain of p47phox
likely undergoes an intramolecular interaction with the C-terminal region, which could restrict access of the domain to its target (Fig.
6).
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The anionic amphiphile activators seem to disrupt the intramolecular interaction in p47phox, thereby liberating the SH3 domain to engage p22phox. When cells are stimulated, arachidonic acid released from the membrane may promote the conformational change of p47phox, as well as p67phox, leading to the superoxide production. It is known that, during activation, p47phox becomes phosphorylated at multiple sites in the C-terminal region (31), accompanied by its translocation to the membrane, and it has recently been reported that the phosphorylated p47phox is active in a cell-free activation system (29). Future studies should test whether the phosphorylation converts the intramolecular interaction of the p47phox SH3 domain to the intermolecular one with p22phox, thereby activating the oxidase. In addition, the present finding that the truncated versions of p47phox and p67phox are both active, also raises a possibility that these proteins might be proteolytically activated in stimulated cells. Some protease inhibitors are shown to inhibit the superoxide production by phagocytes (32).
This study unequivocally shows that the amphiphile activators directly interact not only with p47phox but also with p67phox, the latter interaction which is previously suggested (33, 34). The precise event evoked in p67phox, however, is presently unknown. The oxidase activation is repressed by the p67phox region containing both SH3 domains. Pinpointing the responsible region will help our understanding of the mechanism, and such studies are now in progress in our laboratory.
On the basis of the studies on the NADPH oxidase system, we have previously proposed (5) and advanced here the "masking-unmasking" model for a regulatory mechanism of SH3 domain-mediated interactions: an SH3 domain, that is normally masked via its intramolecular interaction, becomes exposed to intermolecularly interact with the target. It should be strengthened that, in the oxidase system, the induced intermolecular interaction is specific and required for the activation both in vivo and in vitro (5, 8, 9, 11, 15): p22phox is the bona fide target for the N-terminal SH3 domain of p47phox. A similar molecular event has recently been postulated in the T-cell specific tyrosine kinase Itk, a member of the Tec family of non-receptor tyrosine kinase, that is required for signaling via the T-cell antigen receptor (35). The PRR adjacent to the SH3 domain of Itk interacts with the domain intramolecularly. Formation of this complex prevents the SH3 domain and the PRR from interacting with their respective putative substrates, Sam68 and Grb2 (35). In p120 Ras-GAP (GTPase-activating protein), containing an SH3 domain flanked by two SH2 domains, it undergoes a conformational change that leads to increased accessibility of the target binding surface of its SH3 domain, when two closely linked phosphotyrosine-containing peptides bind simultaneously to the two SH2 domains (36). Furthermore, recent structural studies have revealed that the SH3 domain of the tyrosine kinases Src and Hck interacts intramolecularly not only with the linker region between the SH2 and kinase domains, but also simultaneously with the kinase domain, resulting in inhibition of the enzymatic activity (30, 37). Thus such regulatory intramolecular association of SH3 domains, as occurred in p47phox, is currently considered to be much more general than previously expected (38). This type of the regulation would be found in a variety of signaling proteins that carry both an SH3 domain and a PRR, such as the p85 subunit of phosphoinositide 3-kinase (39) and the yeast protein Bem1p (40).
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ACKNOWLEDGEMENTS |
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We thank Drs. Y. Tachibana (Nippon Zeon Corp.) and M. Matsuda (International Medical Center of Japan) for providing the anti-GST monoclonal antibody, Prof. Y. Sakaki ( University of Tokyo) for encouragement, and Drs. D. Kang (Kyushu University) and T. Muta (Kyushu University) for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Uehara Memorial Foundation, the Kato Memorial Bioscience Foundation, and the Fukuoka Cancer Society.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.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Kyushu University School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan. Tel.: 81-92-642-6101; Fax: 81-92-642-6202; E-mail: hsumi{at}mailserver.med.kyushu-u.ac.jp.
1
The abbreviations used are: SH3, Src homology 3;
PRR, proline-rich region; GST, glutathione S-transferase;
GTPS, guanosine 5'-3-O-(thio)triphosphate; GDP
S,
guanosine 5'-2-O-(thio)diphosphate; PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
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