From the Department of Molecular and Experimental Medicine, The
Scripps Research Institute, La Jolla, California 92037, the

Department of Pediatrics, University of
Massachusetts School of Medicine, Worcester, Massachusetts 01655, and
INSERM U-294 at CHU X. Bichat, Paris, France
The leukocyte NADPH oxidase is an enzyme in
phagocytes and B lymphocytes that when activated catalyzes the
production of O
2 from oxygen and NADPH. During oxidase
activation, serine residues in the C-terminal quarter of the oxidase
component p47PHOX become extensively phosphorylated, the
protein acquiring as many as 9 phosphate residues. In a study of 11 p47PHOX mutants, each containing an alanine instead of a serine
at a single potential phosphorylation site, we found that all but S379A corrected the defect in O
2 production in Epstein-Barr virus
(EBV)-transformed p47PHOX-deficient B cells (Faust, L. P.,
El Benna, J., Babior, B. M., and Chanock, S. J. (1995)
J. Clin. Invest. 96, 1499-1505). In particular,
O
2 production was restored to these cells by the mutants S303A
and S304A. Therefore, apart from serine 379, whose state of
phosphorylation in the activated oxidase is unclear, no single
potential phosphorylation site appeared to be essential for oxidase
activation. We now report that the double mutant p47PHOX
S303A/S304A was almost completely inactive when expressed in EBV-transformed p47PHOX-deficient B cells, even though it was
expressed in normal amounts in the transfected cells and was able to
translocate to the plasma membrane when the cells were stimulated. In
contrast, the double mutant p47PHOX S303E/S304E was able
to support high levels of O
2 production by EBV-transformed
p47PHOX-deficient B cells. The surprising discovery that the
double mutant S303K/S304K was also able to support considerable
O
2 production suggests either that the effect of
phosphorylation is related to the increase in hydrophilicity around
serines 303 and 304 or that activation involves the formation of a
metal bridge between the phosphorylated serines and another region of
the protein.
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INTRODUCTION |
The leukocyte NADPH oxidase is a membrane-associated enzyme in
phagocytes and B lymphocytes that catalyzes the production of
O
2 from oxygen using NADPH as electron donor (1),
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(Eq. 1)
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Dormant in resting cells, it acquires catalytic activity when the
cells are exposed to appropriate stimuli. Activation involves the
transfer of cytosolic subunits designated p47PHOX and
p67PHOX to the plasma membrane, where they associate with a
flavocytochrome known as cytochrome b558 to
assemble the active oxidase (2).
During oxidase activation in whole cells, p47PHOX becomes
phosphorylated on numerous serine residues that lie between
Ser303 and Ser379 in the C-terminal quarter of
this 390-residue molecule (3-7). Studies conducted to date have
identified the phosphorylated serines (7-9), examined their
susceptibility to phosphorylation by various protein kinases that occur
in neutrophils (9, 10), and shown that in all likelihood no single
phosphorylated serine is indispensable for oxidase activity (8). The
present report is concerned with the phosphorylation of p47PHOX
Ser303 and Ser304 in relation to the activation
of the leukocyte NADPH oxidase.
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MATERIALS AND METHODS |
The mutants p47PHOX S303A/S304A, S303D/S304D,
S303E/S304E, and S315A/S320A were constructed by single strand
mutagenesis of the p47PHOX cDNA as described previously
(8). Mutagenesis was performed on cDNA cloned into pBluescript II
KS+. The mutated cDNAs were then subcloned into the mammalian
expression vector EBOpLPP. Mutations that altered the sequence of the
protein were accompanied by silent mutations of nearby restriction
sites, introduced to aid in screening. The mutations were confirmed by
sequencing in the departmental facility.
The S303K/S304K, S303A/S304E, and S303E/S304A mutations were
constructed by a PCR1
strategy. In each case, the 5' end of the "forward" primer spanned a unique NarI site 16 bases upstream of the 303 codon, the
appropriately altered 303 and 304 codons, and 18 additional bases of
native downstream sequence. A 19-base "reverse" primer that spanned
an NaeI site about 180 base pairs downstream from the 304 codon was used for all three PCR mutagenesis reactions. PCR was
performed with Pfu polymerase (Stratagene) using the wild-type
p47PHOX cDNA as template. The PCR products were isolated
from agarose gels using QIAEXII (Qiagen) and digested with
NarI and NaeI. The resulting fragments were
ligated into the NarI and NaeI sites of the
p47PHOX S303A/S304A plasmid, a procedure that introduced the
desired mutation into the product and destroyed a BssHII
site that was introduced during the construction of the p47PHOX
S303A/S304A cDNA. All PCR mutations were confirmed and the
constructs documented to be error-free by sequencing across the full
NarI-NaeI span. Table
I lists the primers and plasmids used in
preparing the p47PHOX mutants.
EBV-transformed p47PHOX-deficient B lymphocytes were
co-transfected with SV40 plus wild-type or mutant p47PHOX
expression vectors as indicated, and expanded under hygromycin selection as described previously (8), except that the
p47PHOX-deficient cells were maintained at 0.5-1.0 × 106/ml before transfection and 106 cells/ml
after transfection. Transfected cells were assayed only when fewer than
10% of the cells took up trypan blue. The cell line used for these
experiments contained an uncharacterized mutation in the
p47PHOX gene that prevented the expression of
p47PHOX.
Leukocyte NADPH oxidase activity was measured by chemiluminescence.
Assays using whole cells were carried out as described elsewhere (11),
except that 4 × 106 cells and 10 IU of horseradish
peroxidase were used in a final volume of 0.35 ml. The cell suspensions
were placed in a 96-well microplate, warmed to 37 °C, then activated
at the same temperature with phorbol myristate acetate (1 µg/ml).
Chemiluminescence was then measured at 1-min intervals using a
Luminoskan luminometer (Labsystems Research, Finland) at 37 °C. For
measurement of leukocyte NADPH oxidase activity in a cell-free system,
reaction mixtures contained 1.6 × 106 cell
equivalents of neutrophil membranes, 9 × 106 cell
equivalents of B lymphocyte cytosol, 1 mM luminol, 5 IU of
horseradish peroxidase, 90 µM SDS, 160 µM
NADPH, and Hanks' balanced salt solution containing 0.5 mM
CaCl2 and 1 mM MgCl2, with or
without 50 IU of superoxide dismutase, in final volume of 1 ml. Cytosol
was prepared by sonicating a suspension of lymphoblasts in Dulbecco's
phosphate-buffered saline for three 10-s intervals at 4 °C, then
removing particles by centrifugation for 15 min at the same temperature
in an Eppendorf Microfuge. Initially, the assay mixture contained all
the components except SDS and NADPH. The oxidase was then activated by
adding SDS and incubating for 1 min at room temperature. O
2
production was then initiated with NADPH, and chemiluminescence was
measured at successive 10-s interval using a Luminoskan luminometer at
room temperature.
The expression of p47PHOX in the transfected cells and the
translocation of p47PHOX from cytosol to membranes was
determined by immunoblotting (8). Fractions were subjected to
SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels
using the Laemmli buffer system. The separated proteins were
electrophoretically transferred onto a nitrocellulose membrane, which
was blocked with Blotto and probed with a 1:5000 dilution of an
antibody against the C-terminal decapeptide of WT p47PHOX. In
measurements of p47PHOX expression in whole cells,
p47PHOX on the immunoblots was visualized with alkaline
phosphatase-labeled goat anti-rabbit immunoglobulin antibodies followed
by visualization with the 5-bromo-4-chloro-3-indolyl phosphate/nitro
blue tetrazolium detection system (12). Expression was measured by
densitometry with a Zeinieh laser scanner, determining relative
quantities of p47PHOX from peak heights. In the translocation
experiments, peroxidase-labeled goat anti-rabbit immunoglobulin
antibodies and the ECL chemiluminescence detection system (Renaissance;
DuPont, Boston, MA) were used for the visualization of
p47PHOX.
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RESULTS |
O
2 Production--
In prior studies, we have shown that
the activation of the leukocyte NADPH oxidase in neutrophils and
EBV-transformed B lymphocytes is accompanied by the phosphorylation of
serines 303 and 304 in the cytosolic oxidase component p47PHOX
(7). Consistent with the possibility that the phosphorylation of these
serines is involved in oxidase activation, we reported that the
conversion of either Ser303 or Ser304 to
alanine resulted in a >50% decrease in O
2 production by
EBV-transformed lymphocytes expressing the mutant p47PHOX
proteins (8). For the individual serines these decreases were not
statistically significant (p > 0.05), but a level of
significance of p < 0.004 was achieved when the
results were reanalyzed under the hypothesis that the loss of either
one of these serines resulted in a decrease in O
2 production
(Wilcoxon rank sum test). This analysis suggested that the
phosphorylation of at least one of these serines was necessary for
oxidase activation in EBV-transformed B lymphocytes.
To test this possibility, O
2 production was measured in
p47PHOX-deficient EBV-transformed B lymphocytes expressing
p47PHOX with a number of double mutations at positions
Ser303 and Ser304. Time courses for O
2
production by untransformed B lymphocytes and by cells expressing
wild-type or mutant forms of p47PHOX are illustrated in Fig.
1; peak chemiluminescence values for the
same cells are shown in Fig. 2.
Inspection of the results obtained with the mutant in which serines 303 and 304 were converted to alanines showed that O
2 production
was nearly abolished by these mutations, supporting the idea that the
phosphorylation of at least one of these serines is required for
oxidase activation. A second double Ser
Ala mutant, p47PHOX
S310A/S315A, was fully active, indicating that the mere replacement with alanines of 2 serines in the region of the phosphorylation targets
was not enough to abolish the activity of p47PHOX.

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Fig. 1.
Time course of oxidase activity in
untransfected p47PHOX-deficient B cells and cells expressing WT
and mutant p47PHOX. Chemiluminescence assays were
performed as described in the text. This time course is representative
of three or more separate experiments, depending on the mutant. ,
WT; , Ser Ala; , Ser Glu; , Ser Asp; , Ser Lys; , CGD, untransfected B cells.
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Fig. 2.
Oxidase activity of phorbol-activated
p47PHOX-deficient B cells expressing WT and mutant forms of
recombinant p47PHOX. Experiments were carried out as
described in the text. The results represent peak luminescence by
transfected p47PHOX-deficient B cells expressing different
forms of p47PHOX. The results are expressed as % of WT
control, and are presented as the mean ± 1 S.D. of three or more
separate sets of transfections. Peak chemiluminescence for cells
expressing the WT protein was 6.2 ± 0.4 Luminoskan units.
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Further support for this idea was obtained when these two serines were
replaced, not with alanines, but with negatively charged residues. One
of the effects of serine phosphorylation is the introduction of
negative charge in a region of a polypeptide that was originally
uncharged. Negative charge can also be introduced by replacing a serine
with an aspartate or glutamate residue, and in fact studies on other
proteins activated by serine phosphorylation have shown that the
replacement of a phosphorylated serine with an aspartate residue can
have the same functional consequences as the phosphorylation of that
serine (13). To ascertain whether the introduction of negative charge
in the vicinity of p47PHOX
Ser303-Ser304 had the same effect on oxidase
activity as the phosphorylation of serines 303 and 304, we measured
O
2 production in EBV-transformed B lymphocytes expressing the
p47PHOX double mutants S303D/S304D and S303E/S304E. The results
showed that, in contrast to the double p47PHOX mutant
S303A/S304A, the glutamate mutant was able to support O
2
production. In cells that expressed p47PHOX S303E/S304E,
oxidase activation (Fig. 1) and maximum rates of O
2 production
(Fig. 2) were both similar to their counterparts in cells transfected
with WT p47PHOX. On the other hand, the results obtained with
the cells transfected with p47PHOX S303D/S304D were equivocal,
not an unexpected outcome given that in steric terms, p47PHOX
S303S/S304S-OPO3= is much more similar
to p47PHOX S303E/S304E than to p47PHOX S303D/S304D.
Replacement of one of the two serines with glutamate and the other with
alanine led to p47PHOX mutants that supported O
2
production to the extent of about 25% of wild-type levels, a finding
consistent with earlier work showing that mutants in which one of the
two serines was replaced by alanine and the other was left alone were
able to support O
2 production, but at reduced levels (8).
Expression and Translocation of the p47PHOX
Mutants--
One possible explanation for the inability of cells
transfected with the S303A/S304A mutant to generate O
2 is that
the EBV-transformed p47PHOX-deficient B cells were unable to
express p47PHOX S303A/S304A, the mutant that failed to support
O
2 production. The findings presented in Fig.
3 and Table
II, however, show that all the mutant
forms of p47PHOX examined in these experiments were expressed
to approximately the same extent as the WT protein. The inability of
p47PHOX S303A/S304A to support O
2 production therefore
could not be attributed to a failure of expression by the transfected
cells. A second possible explanation is that, although fully expressed, p47PHOX S303A/S304A could not fold into a native conformation,
and was therefore unable to participate in the activation of the
leukocyte NADPH oxidase. To investigate this possibility, we examined
the ability of the mutant forms of p47PHOX to support
O
2 production in a cell-free system in which activation was
accomplished by an anionic detergent (in this case, SDS), not
phosphorylation. Fig. 4 shows that there
was little difference in O
2 production among cell-free systems
containing the various forms of p47PHOX. In particular, the
cell-free system that contained p47PHOX S303A/S304A produced
O
2 at the same rate as the system that contained WT
p47PHOX. Therefore the conformation of p47PHOX
S303A/S304A was sufficiently similar to the native conformation that
the mutant protein functioned normally in the cell-free oxidase activating system, even though it was inactive in whole cells.

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Fig. 3.
Expression of p47PHOX in
untransfected p47PHOX-deficient B cells and in cells
transfected with plasmids expressing WT and mutant recombinant
p47PHOX. Each lane contained 2 × 105
cell equivalents of protein. Molecular weight markers were included in
each gel. The results are representative of p47PHOX expression
in two or more separate sets of transfections. The arrow
indicates the position of p47PHOX.
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Table II
Expression of p47PHOX mutants in transfected
p47PHOX-deficient B lymphocytes relative to the expression of
the wild-type protein
Results are expressed as the mean ± 1 S.E. n is the
number of separate transfections assayed.
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Fig. 4.
Oxidase activity in a cell-free oxidase
activating system containing WT and mutant forms of recombinant
p47PHOX. Assays were carried out as described under
"Materials and Methods." Results shown are the differences between
luminescence values measured in the absence and presence of 50 units of
superoxide dismutase; the latter values never exceeded 5% of the
former. The results were calculated from the peak luminescence values
observed with each form of p47PHOX, and are shown as % of WT
control (mean ± range of duplicate experiments). Control values
were 24.5 ± 3.4 Luminoskan units.
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Activation of the leukocyte NADPH oxidase is accompanied by the
translocation of the oxidase components p47PHOX and
p67PHOX from the cytosol to the plasma membrane, where they
associate with the flavocytochrome b558 (2, 14).
This association is thought to result at least in part from the
appearance of a membrane-binding site on p47PHOX when the
oxidase is activated (12, 15). We found that when p47PHOX-deficient cells expressing the double mutants
p47PHOX S303A/S304A, p47PHOX S303D/S304D, or
p47PHOX S303E/S304E were activated with phorbol ester, the
mutant p47PHOX polypeptides were transferred to the plasma
membrane as efficiently as the WT protein (Fig.
5). The finding that p47PHOX
S303A/S304A translocates normally suggests that a negative
electrostatic potential in the vicinity of residues 303-304 is not
needed for the binding of p47PHOX to the flavocytochrome,
although it is required for the enzyme to express its catalytic
activity.

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Fig. 5.
Translocation of WT and mutant
p47PHOX during oxidase activation. Experiments were
carried out as described previously (8), except for changes in the
immunoblotting procedure as described under "Materials and
Methods." Results are representative of two experiments, each carried
out with a separate transfection. The track labeled CTRL contained
1.5 × 106 cell eq of cytosol. The remaining tracks
each contained 1.25 × 107 cell eq of membrane. Plus
(+) and minus ( ) indicate cells activated with phorbol or resting
cells, respectively.
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p47PHOX S303K/S304K, an Active Mutant--
Besides
adding local negative charge to p47PHOX, the phosphorylation of
serines 303 and 304 causes a large increase in the polarity of the
polypeptide in the vicinity of those residues. To determine whether the
effect of phosphorylation on oxidase activity was due to the negative
electrostatic potential or the increase in polarity, we examined the
activity of the mutant p47PHOX S303K/S304K, in which residues
303 and 304 carry positive, not negative, charges. The translocation of
this mutant was normal (Fig. 6), and its
activity was normal as well, somewhat to our surprise (Figs. 1 and 2).
Therefore the function of the Ser303 and Ser304
phosphates in the activation of the oxidase must be related to more
than simply the acquisition of negative charge by the serine 303-304
region of p47PHOX.

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Fig. 6.
Translocation of p47PHOX S303K/S304K
during oxidase activation. Experiments were carried out as
described previously (8), except for changes in the immunoblotting
procedure as described under "Materials and Methods." Results are
representative of two experiments, each carried out in duplicate with a
separate transfection. The track labeled CTRL contained 1.5 × 106 cell eq of cytosol. The remaining tracks each contained
1.25 × 107 cell eq of membrane. Plus (+) and minus
( ) indicate cells activated with phorbol or resting cells,
respectively.
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DISCUSSION |
In an earlier study of 11 p47PHOX mutants containing an
alanine instead of a serine at a single potential phosphorylation site, we found that all but p47PHOX S379A were active when expressed
in EBV-transformed p47PHOX-deficient B cells (8). Therefore,
apart from serine 379, no single phosphorylation site appeared to be
essential for oxidase activation. As to Ser379, its state
of phosphorylation in the activated oxidase is unclear: it became
phosphorylated only to a very limited extent when neutrophils were
activated, and p47PHOX S379D was no more active than
p47PHOX S379A in the p47PHOX-deficient
cells.2 Consequently, it was
not possible to be absolutely certain from those results whether or not
the phosphorylation of p47PHOX, either at Ser379 or
at any other potential phosphorylation site, was required for the
activation of the oxidase. Unlike Ser379, serines 303 and
304 are extensively phosphorylated during oxidase activation (7, 9). In
earlier work, we found that the oxidase could still be activated if
either serines 303 or 304 were converted to alanine (8). The present
results, however, show that if both of these serines are converted to
alanine, the activation of the oxidase is almost completely abolished.
These findings provide direct evidence that the phosphorylation of at
least one of the serines of p47PHOX, specifically, serines 303 or 304, is necessary for the activation of the leukocyte NADPH oxidase.
Our results support similar conclusions drawn from earlier studies
showing that p47PHOX is phosphorylated when the oxidase is
activated by a variety of stimuli (3, 5-7); that various protein
kinase inhibitors can diminish or prevent O
2 production
(16-18) while protein phosphatase inhibitors augment O
2
production (19-23); and that recombinant p47PHOX
phosphorylated by protein kinase C can activate the oxidase in the
cell-free system in the absence of added detergents (24-26).
The effect of the serine-to-alanine mutations is to substitute methyl
groups for two of the hydroxymethyl groups known to be phosphorylated
during oxidase activation in whole cells (7). The conversion of these
serines to alanines destroyed the activity of p47PHOX. In
contrast, the conversion of the same serines to glutamate, a mutation
that substituted negatively charged carboxymethyl groups for the two
hydroxymethyl groups, gave rise to mutant protein with considerable
activity. Since the phosphorylation of other serines in p47PHOX
was unaffected by the elimination of serines 303 and 304 (9), the
simplest explanation for all these findings is that oxidase activation
in whole cells requires a negative electrostatic potential in the
vicinity of serines 303 and 304 of p47PHOX, and that this
potential is generated through the phosphorylation of one or both of
those serines. The results with the S303K/S304K mutant, however,
indicate that the acquisition of a negative electrostatic potential is
not a sufficient explanation. They suggest instead that the purpose of
the phosphorylation of Ser303 and Ser304 may be
to change the polarity of the protein in the neighborhood of
those serines. Alternatively, they may indicate that the phosphates are
there to bind divalent metals such as Mg2+, an action that
would confer on the serine 303-304 region a net positive charge
similar to that in the S303K/S304K mutant.
How can these local effects promote the activity of the leukocyte NADPH
oxidase? Because the mutants all translocate normally, it is clear that
phosphorylation at Ser303-Ser304 is not an
obligatory precursor of translocation. In principle, then,
phosphorylation at these positions can take place either before or
after the translocation of p47PHOX to the membrane. Earlier
evidence showing that the final 1 or 2 phosphorylations take place
after translocation (6) suggests that at least some p47PHOX is
phosphorylated after it is transferred from the cytosol to the
membrane. It seems likely that the phosphorylation of
Ser303 and Ser304 converts the membrane-bound
subunit into a form that is able to render the oxidase catalytically
active.