(Received for publication, June 6, 1996, and in revised form, February 6, 1997)
From the Department of Molecular and Experimental Medicine, The
Scripps Research Institute, La Jolla, California 92037, the
Department of Biochemistry, Kyungpook National
University, Taegu, Korea, and § INSERM
U-294 at CHU X. Bichat, Paris, France
The leukocyte NADPH oxidase catalyzes the
1-electron reduction of oxygen to
O2 at the expense of NADPH: 2 O2 + NADPH
2 O2
+ NADP+ + H+. The oxidase is dormant in resting
cells but acquires activity when the cells are stimulated with a
suitable agent. Activation in whole cells is accompanied by extensive
phosphorylation of p47PHOX, an oxidase subunit located in the
cytosol of resting cells that during oxidase activation migrates to the
plasma membrane to complex with cytochrome
b558, an oxidase-specific flavohemoprotein.
Oxidase activation can be mimicked in a cell-free system using an
anionic amphiphile as activating agent. We now report a cell-free
system in which the oxidase can be activated in two stages using
phosphorylated p47PHOX. The first stage, which effects a change
in the membrane, requires ATP and GTP and is blocked by the protein
kinase inhibitor GF-109203X, suggesting a protein kinase requirement.
The second stage requires phosphorylated p47PHOX and GTP, but
no ATP, and is unaffected by GF-109203X; assembly of the oxidase may
take place during this stage. Activation is accomplished by
p47PHOX phosphorylated by protein kinase C but not protein
kinase A or mitogen-activated protein kinase. We believe that
activation by phosphorylated p47PHOX is more physiological than
activation by amphiphiles, because the mutant p47PHOX S379A,
which is inactive in whole cells, is also inactive in this system but
works in systems activated by amphiphiles.
The leukocyte NADPH oxidase is an enzyme found in neutrophils and
certain other leukocytes that catalyzes the one-electron reduction of
oxygen to O2 at the expense of NADPH
(1): 2 O2 + NADPH
2 O2
+ NADP+ + H+. The
O2
produced by this enzyme is itself
weakly microbicidal but serves as the precursor of a complex battery of
highly reactive oxidants that act as powerful microbicidal agents.
These oxidants are major components of the system used to defend the
host against invading pathogens.
The NADPH oxidase consists of four polypeptides that have been
identified through their absence from phagocytes that are unable to
manufacture O2 (2-5) plus a fifth
polypeptide, p40PHOX (6, 7), whose function is unclear. The
oxidase is dormant in resting cells but acquires catalytic activity
when the cells are exposed to any of a variety of stimuli. Activation
involves the transfer to the plasma membrane of cytochrome
b558, a flavohemoprotein located in the
membranes of the secretory vesicles and specific granules in the
resting neutrophil (8-10). A cytosolic complex consisting of the
oxidase components p47PHOX, p67PHOX, and
p40PHOX then associates with the cytochrome to assemble the
active oxidase (6, 11-15).
The mechanism of activation of the oxidase is unclear. The
phosphorylation of p47PHOX is a well-recognized concomitant of
oxidase activation in whole cells (16-22), but to date a
cause-and-effect relationship between the phosphorylation of this
oxidase component and the activation of the enzyme has not been
definitively established. The oxidase can be activated in a cell-free
system, but the activating agent usually employed is an anionic
amphiphile such as arachidonic acid or SDS (23, 24). At least two
examples of phosphorylation-mediated oxidase activation in a cell-free
system have been reported, however. In 1985, Cox et al. (25,
26) reported that the phosphorylation of resting neutrophil membranes
with protein kinase C led to a low level of oxidase activity. More
recently, McPhail and associates (27) showed that ATP increased by a
factor of 2.5 the rate of O2
production in a cell-free system that had been activated by 0.1 mM each of phosphatidic acid and diacylglycerol, suggesting
the possibility that a phosphatidic acid-activated protein kinase participated in oxidase activation in their system. We describe here
the activation of the leukocyte NADPH oxidase in a cell-free system by
p47PHOX that had been pre-phosphorylated by protein kinase
C.
Materials
Chemicals and enzymes were obtained from the following sources:
dextran and Ficoll-Hypaque from Pharmacia; luminol, phosphatidylserine, diacylglycerol, sucrose, isopropyl -D-thiogalactoside,
NADPH, ATP, guanosine 5
-O-(3-thiotriphosphate)
(GTP
S),1 guanosine
5
-O-(2-thiodiphosphate) (GDP
S),
:
-imidoguanosine 5
-triphosphate (GppNHp), glutathione-agarose, phenylmethylsulfonyl fluoride, cytochrome c, and hexokinase from Sigma; rat brain
protein kinase C (PKC), rat brain protein kinase C catalytic subunit
(PKM), bovine heart protein kinase A catalytic subunit (PKA),
horseradish peroxidase, calyculin A, okadaic acid, GF-109203X (GFX),
H-7, and horseradish peroxidase were from Calbiochem; mitogen-activated protein kinase p42 (MAP kinase) was from Santa Cruz Biotechnology; anti-pan PKC from Upstate Biotechnology Inc.; and the Bio-Rad protein
assay kit and electrophoresis and immunoblotting reagents were from
Bio-Rad.
Preparation of Neutrophil Fractions
Neutrophil cytosol and membrane were prepared as described
previously (8). Briefly, neutrophils were obtained from normal subjects
by dextran sedimentation and Ficoll-Hypaque fractionation of freshly
drawn citrate-coagulated blood. The neutrophils were suspended at a
concentration of 108 cells/ml in a modified relaxation
buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 10 mM PIPES buffer, pH
7.3), and plasma membrane and cytosol were prepared by nitrogen
cavitation and centrifugation through Percoll. Both cytosol and
membrane were divided into aliquots and stored at 70 °C until use.
Cytosol deficient in p47PHOX was obtained from neutrophils
isolated from p47PHOX-deficient chronic granulomatous disease
patients as described above.
Preparation of Recombinant GST-p47PHOX Fusion Proteins
Recombinant fusion proteins composed of an upstream glutathione
S-transferase (GST) linked to a downstream p47PHOX,
either the wild type protein or the inactive mutant
p47PHOXS379A, were isolated from Escherichia coli
that had been transformed with pGEX-1T plasmids containing cDNA
inserts encoding the downstream proteins, as previously reported (11).
The fusion proteins were purified by affinity chromatography on
glutathione-agarose as described elsewhere. Before use, excess
glutathione was removed from the solution of purified recombinant
protein by dialysis against relaxation buffer. The concentration of
proteins was determined with the Bio-Rad assay kit using bovine serum
albumin as a standard.
In Vitro Phosphorylation and Measurement of 32P Incorporation
Labeling of p47PHOX with PKM, PKA, MAP kinase, or
combinations of kinases was performed by incubating a reaction mixture
containing 1 µg of recombinant p47PHOX, 1 mM ATP,
5 µCi of [-32P]ATP (Amersham Corp.), 10 mM MgCl2, 0.1 µg of the indicated kinase(s), and relaxation buffer, pH 7.3, in a total volume of 30 µl for 30 min
at 37 °C. To label with PKC, 1 µg of recombinant p47PHOX
was incubated for 30 min at 37° with 0.1 µg of PKC in 10 mM magnesium acetate, 1 mM ATP, 5 µCi
[
-32P]ATP, 0.5 mM CaCl2, 50 µg/ml phosphatidylserine, and 5 µg/ml diolein in a total volume of
30 µl. After terminating the phosphorylation reactions by the
addition of 10 µl of 4 × SDS-sample buffer, the samples were
subjected to SDS-PAGE using an 8% running gel according to the method
of Laemmli (28). 32P-Labeled proteins on the dried gels
were detected by autoradiography, and 32P was quantified by
excising the labeled bands from the dried gel and measuring their
radioactivity using Cerenkov counting. To determine background, a piece
of nitrocellulose of similar size was excised from a
32P-free portion of the gel and counted.
Preparation of Phosphorylated GST-p47PHOX
Phosphorylation of recombinant GST-p47PHOX was typically
carried out as described above, except that radioactive ATP was omitted and 10-50 µg of fusion protein and a reaction volume of 100 µl were employed. Incubations were carried out in Eppendorf tubes for 30 min at 37 °C. Each incubation was terminated by the addition of 1.0 ml of ice-cold MTPBS (150 mM NaCl, 16 mM
Na2HPO4, 4 mM NaH2PO4, pH 7.3) and 100 µl of packed
MTPBS-washed GSH-agarose beads. The tubes were then rotated
end-over-end for 1 h at 4 °C and then spun for a few seconds at
maximum speed in an Eppendorf centrifuge to sediment the GSH-agarose
beads. After washing the beads with four 1-ml portions of ice-cold
MTPBS, the bound phosphorylated p47PHOX fusion protein was
eluted by incubating for 30 min at 4 °C with 200 µl of 50 mM Tris·HCl, pH 8.0, 5 mM GSH, 0.2 M NaCl. Before use, the eluted fusion protein was dialyzed
against relaxation buffer. To compare the phosphorylation of wild type
GST-p47PHOX and GST-p47PHOX S379A, 50 µg of each of
the two proteins were phosphorylated as described above except in the
presence of 10 µCi of [-32P]ATP, then analyzed by
SDS-PAGE, transferred to nitrocellulose, and detected and quantified by
autoradiography. The labeled bands were then excised from the blot and
analyzed by two-dimensional peptide mapping as described (29, 30). The
autoradiogram indicated that the levels of phosphorylation of the wild
type and mutant proteins were similar, and the two-dimensional peptide
maps of the wild type and mutant proteins were virtually identical
(Fig. 1).
Cell-free Activation of the Respiratory Burst Oxidase
The activity of protein kinase-activated NADPH oxidase was measured by chemiluminescence (31). Two types of assays were used as follows: a one-stage assay in which recombinant GST-p47PHOX was added at the start of the incubation, and a two-stage assay in which the oxidase was partly activated in an initial incubation carried out in the absence of added GST-p47PHOX and then fully activated and assayed for activity in a second incubation initiated by adding phosphorylated GST-p47PHOX, NADPH, and the detection system to the initial incubation mixture. The assays were conducted by the following procedures.
One-stage Chemiluminescence AssayThe complete reaction
mixture contained 2.5 × 107 cell eq cytosol, 1.5 × 107 cell eq membrane, 50 µM GTPS, and
the unphosphorylated GST-p47PHOX mixture (5 µg of recombinant
unphosphorylated GST-p47PHOX, 1 mM ATP, 5 units of
protein kinase C, 10 mM MgCl2, 0.5 mM CaCl2, 25 µg of phosphatidylserine, and
2.5 µg of diacylglycerol, adding the lipids as mixed liposomes
prepared by dissolving 1.0 mg of phosphatidylserine and 0.1 mg of
diacylglycerol in chloroform, removing the chloroform under a stream of
nitrogen, and then sonicating the dried lipids for 2 min on ice in 0.8 ml of 20 mM Tris buffer, pH 7.4) or
GST-p47PHOXP6 (3 µg GST-p47PHOX that had
been phosphorylated with protein kinase C) in 0.38 ml (final volume)
relaxation buffer. A chemiluminescence detection mixture containing 18 µg of horseradish peroxidase, 10 µM luminol, and 0.16 mM NADPH (final concentrations) was then added to the reaction mixtures, either immediately (for
GST-p47PHOXP6-containing reactions) or after
incubating for 5 min at 37 °C (for reactions containing the
unphosphorylated GST-p47PHOX mixture). Oxidase activity was
then determined by measuring chemiluminescence at room temperature in a
Monolight 2010 luminometer (Analytical Luminescence Laboratories, San
Diego) at successive 10-s intervals; the final volume of the assay was
0.5 ml.
An identical protocol was used for the one-stage cytochrome c assay, except that the assay mixture contained 1.5 × 107 cell eq solubilized membrane (prepared by mixing 100 µl (1.25 × 108 cell eq) of membrane suspension with 50 µl of glycerol, 50 µl of relaxation buffer, 25 µl of octylglucoside (10%, w/v), and 25 µl of sodium deoxycholate (10%, w/v) and incubating the mixture on ice for 15 min); the detection mixture contained 0.1 mM cytochrome c and 0.16 mM NADPH (final concentrations); the final volume was 0.75 ml; and cytochrome c reduction was followed at 550 nm for 5 min at room temperature in a Uvikon 941 dual-beam recording spectrophotometer (Kontron Instruments, Milan), reading against a reference containing the same components plus 45 µg of superoxide dismutase.
Two-stage Chemiluminescence AssayA reaction mixture
containing 2.5 × 107 cell eq cytosol and/or 1.5 × 107 cell eq membrane as indicated plus 50 µM GTPS in a total volume of 0.35 ml (the initial
incubation) was incubated for 20 min at 37 °C. The second incubation
was then started by adding the chemiluminescence detection mixture
(final total volume 0.5 ml), with or without 3 µg of
GST-p47PHOXP6, and oxidase activity was determined
by chemiluminescence as described above. In both the one-stage and
two-stage chemiluminescence assays, light emission was followed until
shortly past the point where it reached a maximum; this maximum,
expressed in relative light units/s (RLU/s), is the luminescence value
reported in the tables and figure legends. Deviations from these
general procedures are indicated in the legends to the figures and
tables.
The two chemiluminescence assays are diagrammed in Scheme I.
[View Larger Version of this Image (12K GIF file)]Scheme I.
Depletion of ATP and Measurement of ATP Concentration
To deplete it of ATP, cytosol (1 × 108 cell eq) was supplemented with 0.1 M glucose, 6 mM MgCl2, and 0.1 mg of hexokinase (final concentrations) and incubated for 30 min at room temperature. ATP concentrations were measured by an ATP bioluminescent assay kit (FL-AA, Sigma), calibrating by comparison with the chemiluminescence signal from a standard curve established for known concentrations of ATP.
Separation of Membrane from Cytosol after Initial Incubation in the Two-stage Assay
Membranes from the initial incubation were reisolated by layering the incubation mixture over a discontinuous sucrose gradient composed of 1 ml of 15% (w/v) sucrose layered over 0.5 ml of 50% (w/v) sucrose, both in relaxation buffer, and centrifuging at 105,000 × g for 30 min at 4 °C. After centrifugation, the contents of the centrifugation tube were carefully withdrawn from the bottom, discarding the first 250 µl and saving the next 300 µl as the preincubated membrane.
Electrophoresis and Immunoblotting
Protein samples containing GST-p47PHOX were subjected to SDS-PAGE on 8% polyacrylamide gels using the Laemmli buffer system (28). The separated proteins were electrophoretically transferred onto a nitrocellulose sheet (32) and probed with a 1:5000 dilution of partially purified rabbit polyclonal antibody raised against C-terminal decapeptide from p47PHOX and finally detected with a 1:2000 dilution of alkaline phosphatase-labeled goat anti-rabbit Ig antibody (Sigma) using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as substrate (Bio-Rad). To establish whether purification over GSH-agarose was able to separate the PKC in the phosphorylation mixture from the newly phosphorylated GST-p47PHOXP6, blots prepared from the pass-through and eluate fractions obtained from the GSH-agarose beads were probed with a pan-PKC antibody (5 µg/ml), visualizing the bands as described for the anti-p47PHOX antibody.
Inhibitor Activity
The activities of inhibitors in the cell-free system were
investigated by evaluating the incorporation of 32P from
[-32P]ATP into proteins by gel electrophoresis and
autoradiography. Reaction mixtures contained 1.5 × 106 cell eq cytosol, 5 µCi of [
-32P]ATP,
and where indicated 2 × 106 cell eq membrane, 50 µM GTP
S, 0.25 µM calyculin A (an
inhibitor of protein phosphatases 1 and 2A (33)), and 5 µM GFX (an inhibitor of protein kinases (34)) in a final
volume of 30 µl. Incubations were conducted for 20 min at 37 °C.
The reactions were then terminated with 2 × sample buffer, and
the reaction mixtures were subjected to SDS-PAGE on a 10%
polyacrylamide gel. The gels were dried and the labeled proteins
detected by autoradiography. The autoradiogram (Fig. 2)
showed that in this system both inhibitors exerted their expected
effects.
In a cell-free system, the leukocyte NADPH
oxidase has customarily been activated by certain anionic amphiphiles,
including arachidonic acid and SDS. In an earlier study in which we
supplemented the cell-free system with extra p47PHOX (added as
the GST fusion protein) (35), we found that the enzyme can also be
activated by PKC. We have now employed the cytochrome c
assay to obtain better quantitation of
O2 production by the PKC-activated
system (36). The results obtained with this assay (Table
I) were qualitatively similar to those obtained with the
chemiluminescence assay. In particular, maximum rates of
O2
production were achieved only in
the presence of added PKC and lipids. The sustained maximum rate of
O2
production of 3.8 ± 0.4 nmol/min/107 cell eq membrane in the PKC-activated system
was
15-20% of the typical rate seen in the detergent-activated
system.
|
Activation of the cell-free oxidase by PKC may represent a more
physiological process than activation by anionic amphiphiles, because
in intact cells, as in the PKC-activated cell-free system, oxidase
activation is associated with a PKC-dependent event (in the
case of intact cells, the phosphorylation of the oxidase subunit p47PHOX). Further evidence that the PKC-activated system may
mimic the physiological route of oxidase activation was provided by
experiments with a p47PHOX mutant in which serine 379 was
replaced by alanine (GST-p47PHOX S379A). This mutant was shown
to support O2 production by a
cell-free system activated with anionic amphiphiles, but it did not
restore oxidase activity to intact p47PHOX-deficient B
lymphoblasts (31). We found that GST-p47PHOX S379A supported
only very low levels of oxidase activity in the PKC-activated cell-free
system (Fig. 3). In this respect, activation of the
oxidase by PKC resembled activation of the oxidase in intact cells,
supporting the physiological nature of the PKC-dependent activation process in the cell-free system.
An understanding of the mechanism of oxidase activation by PKC in this system is confounded to a certain extent by the lipid and calcium requirements of the kinase. To eliminate these from consideration, we examined the activation of the cell-free oxidase by PKM, an active fragment of PKC that no longer requires lipids (phosphatidylserine and diacylglycerol) or calcium. We found that the oxidase was activated by PKM (not shown), indicating that the exogenous lipids and calcium were required only to support PKC function and not to activate the oxidase per se.
In the foregoing experiments, the protein kinase was added directly to
the assay mixtures. It was therefore hard to know whether O2 production in those experiments was
related to the phosphorylation of p47PHOX or to the
phosphorylation of other proteins in the assay mixture. To address this
question, we conducted experiments in which we replaced the combination
of unphosphorylated GST-p47PHOX plus PKC by GST-p47PHOX
that had been phosphorylated in isolation and then purified away from
the phosphorylating kinase (i.e. PKC). Preliminary
experiments showed that under the conditions used in our experiments,
GST-p47PHOX was completely phosphorylated by a 20-min
incubation with PKC and could be cleanly separated from PKC by
purification over glutathione-agarose (not shown). When the
phosphorylated GST-p47PHOX (henceforth designated
GST-p47PHOXP6) was added to the assay mixture, the
oxidase was activated without the need of additional PKC
(Fig. 4). Furthermore, the extent of activation
increased with increasing amounts of GST-p47PHOXP6.
These results strongly suggest that at least one of the events required
for oxidase activation in this system is the phosphorylation of
p47PHOX.
Earlier studies showed that besides PKC, both mitogen-activated protein
kinase (MAP kinase) and the catalytic subunit of protein kinase A (PKA)
were able to phosphorylate p47PHOX (37). These findings raised
questions as to whether phosphorylation of p47PHOX by either of
these two kinases affected the subsequent phosphorylation of
p47PHOX by PKC or the ability of phosphorylated p47PHOX
to activate the leukocyte NADPH oxidase. The stoichiometry of phosphorylation of GST-p47PHOX by the three kinases is shown in
Fig. 5. As we found
previously,2 treatment of
GST-p47PHOX by PKC led to the incorporation of 6 phosphates/mol
of protein. Treatment with PKM resulted in the same stoichiometry. Only
2 phosphates/mol of protein, however, were incorporated by
GST-p47PHOX treated with PKA, whereas MAP kinase treatment
resulted in the incorporation of slightly less than 1.5 phosphates/mol
of protein. Pretreatment of GST-p47PHOX with PKA had no effect
on the final amount of phosphate incorporated into the protein after
subsequent treatment with PKM, consistent with earlier results showing
that the serine residues phosphorylated by PKA were also phosphorylated
by PKM (37). Pretreatment with MAP kinase, however, increased by 1
mol/mol of protein the final amount of phosphate incorporated into
GST-p47PHOX by subsequent treatment with PKM. As to the
abilities of GST-p47PHOX phosphorylated by various kinases to
support oxidase activity in this system, the results are those expected
if the protein had only been exposed to PKC (or PKM)
(Fig. 6). GST-p47PHOX phosphorylated with either
MAP kinase or PKA was no more active than unphosphorylated
GST-p47PHOX, whereas the activity of GST-p47PHOX
phosphorylated by PKM was the same as that of PKC-treated
GST-p47PHOX regardless of whether or not the protein had been
previously treated with MAP kinase or PKA. In summary, phosphorylation
of GST-p47PHOX by MAP kinase or PKA had little effect on
oxidase activation in this system. Furthermore, other kinases in the
incubation mixture did not appear to catalyze the incorporation of
additional phosphate into GST-p47PHOX once it had been fully
phosphorylated by PKM (i.e. converted to
GST-p47PHOXP6), because no radioactivity was found
in GST-p47PHOXP6 reisolated on glutathione-agarose
and counted after incubation for 30 min in an oxidase assay mixture
containing 5 µCi of [
-32P]ATP (data not shown).
Although further phosphorylation of GST-p47PHOXP6 did not take place during oxidase activation, there was an additional ATP-dependent step in the activation sequence. This was shown in experiments using cytosol that had been depleted of ATP by hexokinase plus glucose. Treatment of cytosol with the hexokinase-glucose combination reduced the ATP concentration in the treated cytosol to 67.3 ± 12.5 nM (mean ± S.D., n = 3), a value 20-40-fold below the Km for PKC (38). This ATP-depleted cytosol was as active as untreated cytosol when used in the standard SDS-activated cell-free system (data not shown). In the GST-p47PHOXP6-activated system, however, the depleted cytosol was only 13.0 ± 1.2% (mean ± range, n = 2) as active as untreated control. These findings suggest that at least two kinase-dependent reactions participate in protein kinase-dependent oxidase activation, one involving the phosphorylation of p47PHOX and the other the phosphorylation of a substrate (or substrates) yet to be identified.
ATP-dependent Activation of the Leukocyte NADPH Oxidase Is a Multistep ProcessMeasurements of activity as a function of
time showed that the activation of the oxidase by phosphorylation
occurs with a very long lag, peak activity not being achieved until
half an hour has passed (Fig. 7). This is in contrast to
oxidase activation in whole cells and in the amphiphile-activated
cell-free system, in which the lag is less than 5 min in length. The
lag seen in the protein kinase-dependent activation system
was not shortened by preincubating GTPS (39-41) with membranes
alone, cytosol alone, or the combination of cytosol plus
GST-p47PHOXP6 and PKM. The lag fell to less than 8 min, however, when cytosol and membranes together were preincubated
with GTP
S before starting O2
production with GST-p47PHOXP6 and NADPH. The fall
in the lag was even sharper when cytosol plus membranes were
preincubated with GTP
S in the presence of calyculin A, a protein
phosphatase inhibitor, O2
production
in this reaction being started as before by the addition of
GST-p47PHOXP6 and NADPH to the preincubation
mixture. Okadaic acid, another protein phosphatase inhibitor (33, 42),
had the same effect in this system as calyculin A (not shown). In the
calyculin A-containing assay, the maximum rate of
O2
production was 25-30% that seen
in a similar assay mixture activated by SDS, the latter amounting to
379,000 ± 41,800 S.D. relative luminescence units/s
(n = 3). These results indicate that the protein
kinase-dependent activation of the cell-free system can be
divided into three distinct sets of events as follows: 1) events that
occur during the initial incubation (i.e. the
preincubation), 2) the phosphorylation of p47PHOX, and finally,
3) events that occur during the second incubation (i.e.
after the addition of GST-p47PHOXP6 and NADPH).
To determine which of the two fractions, membrane or cytosol, is
altered by the initial incubation, experiments were carried out in
which membranes and cytosol were incubated together in the presence of
GTPS, then separated, combined with their unincubated complementary
fractions plus GST-p47PHOXP6 and NADPH, and
immediately assayed for O2
production.
Activity was only seen in assay mixtures containing preincubated
membranes (Table II). An assay mixture containing preincubated cytosol plus unincubated membranes showed no
O2
production. The initial incubation
therefore causes a modification of some kind affecting the
membranes.
|
Earlier results (40, 41) have indicated that a guanine nucleotide
binding protein (GNBP) is required for the assembly of the oxidase, an
event that probably does not occur in this experimental system until
GST-p47PHOXP6 is added (13, 43). To identify the
stage(s) where the protein kinase-dependent activation
process requires a GNBP and to determine whether activation depends on
transfer of the -phosphate of GTP, experiments were conducted in
which incubation protocols were varied and analogs of GTP were
employed. The results are shown in Table III. Oxidase
activity was nil when the two incubations were conducted in the absence
of GTP
S or in the presence of GDP
S, confirming that a GNBP is
required for oxidase activity. A GNBP appeared to be required in both
the initial and second incubations, because the addition of GTP
S
after the initial incubation was complete (i.e. at the same
time as the addition of GST-p47PHOXP6), although
leading to O2
production, resulted in
a much lower activity than when GTP
S was present for both the
initial and second incubations. The activity generated when GTP
S was
added at the same time as GST-p47PHOXP6 probably
reflected the extent to which the events of the initial incubation took
place during the second incubation. A role for a GNBP in the second
incubation was further supported by an experiment in which activated
membranes reisolated from an initial GTP
S-containing incubation were
used in a second incubation with fresh cytosol containing either
GTP
S or GDP
S. O2
production was
brisk in the second incubation that contained GTP
S, but nil in the
second incubation that contained GDP
S. Finally, the ability of
GppNHp to support oxidase activation indicates that activation does not
require the cleavage of the bond between the
and
phosphates of
GTP.
|
GFX is a powerful inhibitor of protein kinase C
and other protein kinases (34). As expected from the ATP requirement
shown in previous experiments, GFX was able to inhibit the activation of the oxidase in this system. O2
production was also inhibited by H-7, another protein kinase inhibitor
(44) (not shown). Table IV shows that GFX inhibited oxidase activation considerably if present during the entire activation process but had no effect if added after the initial incubation, suggesting that ATP was required during the initial incubation but not
during the second incubation. The possibility that GFX was acting
exclusively against the phosphorylation of p47PHOX was ruled
out by the use of GST-p47PHOXP6, the fully
phosphorylated fusion protein. This result confirms a requirement for
ATP beyond that necessary for the phosphorylation of p47PHOX
and strongly suggests that the action of one or more protein kinases is
required during the initial incubation but that if phosphorylated
p47PHOX is supplied, further phosphorylation during the second
incubation is unnecessary.
|
The kinase requirement during the initial incubation reflected more
than just the preliminary phosphorylation of the p47PHOX
already present in the cytosol. This is indicated by the finding that
cytosol deficient in p47PHOX was able to function in the
initial incubation, as indicated by the short lag and the high level of
oxidase activity seen during the second incubation in experiments using
the deficient cytosol (Fig. 8).
The use of GFX made it possible to ask whether unphosphorylated
p47PHOX could function in the second stage of the oxidase
activation reaction. For this experiment, the initial incubation was
carried out as described under "Experimental Procedures," but the
second incubation received either no protein, unphosphorylated
GST-p47PHOX, or GST-p47PHOXP6 as indicated
in Table V. GFX was added to the second incubations as
indicated, to inhibit protein phosphorylation during the second stage
of the reaction. The results showed that as compared with O2 production in a reaction mixture
containing no added p47PHOX,
O2
production was more than doubled by
the addition of unphosphorylated GST-p47PHOX to the second
incubation. This effect of unphosphorylated GST-p47PHOX,
however, was abolished by the simultaneous presence of GFX in the
second incubation, suggesting that before it could stimulate O2
production, GST-p47PHOX had
to be phosphorylated. Confirming this conclusion were the results
obtained with GST-p47PHOXP6 (Table V). These
results showed that the fully phosphorylated protein led to an even
greater augmentation of O2
production
than was seen with the unphosphorylated protein. Furthermore, in
contrast to the results with unphosphorylated p47PHOX, the
increase seen with p47PHOXP6 was unaffected by the
protein kinase inhibitor. These results indicate that 1) the form of
p47PHOX that was active in the protein
kinase-dependent oxidase activating system was the
phosphorylated protein, and 2) the phosphorylation of p47PHOX
was the only second stage phosphorylation required for the activity of
that system.
|
In most of the work
described above, recombinant p47PHOXP6 was used in
the assays. Neutrophil cytosol, however, contains an amount of
p47PHOX comparable to the amount of recombinant protein added
to the incubation mixtures in the foregoing experiments, so to further evaluate the physiological significance of the protein
kinase-dependent oxidase activation system, it was
necessary to determine whether this endogenous p47PHOX could
serve as a element of this system. For this purpose, cell-free oxidase
activation by endogenous p47PHOX phosphorylated with PKC was
compared with cell-free oxidase activation by recombinant
p47PHOXP6. The detergent-activated cell-free system
was also examined. The results (Fig. 9) showed that the
peak rate of O2 production by the
unsupplemented kinase-activated system was 60% greater than the rate
seen in the GST-p47PHOX-supplemented system, confirming that
endogenous p47PHOX could participate in the
kinase-dependent oxidase activation system.
O2
production by the
detergent-activated system, however, was 5-fold greater than
O2
production by the PKC-activated
system (not shown), suggesting that additional facets of the
kinase-dependent oxidase activation system remain to be
investigated.
It has been known for many years that p47PHOX, one of the cytosolic subunits of the leukocyte NADPH oxidase, becomes heavily phosphorylated on serine when the oxidase is activated (16-22). More recent studies showed that the phosphates are confined to the C-terminal quarter of the molecule, identified many of the phosphorylation sites (37, 45), and suggested a number of kinases, including PKC (22, 46-53), the p21rac/cdc42-activated kinase (PAK kinase) (54), and other kinases yet to be characterized (55-58), as being potentially responsible for the phosphorylation of p47PHOX. The question is still open, however, whether the phosphorylation of p47PHOX actually plays a role in oxidase activation or is just an epiphenomenon; the only serine in the phosphorylated portion of the molecule that has been shown to be essential for oxidase activity is Ser-379, and it is not certain that Ser-379 is a functionally significant phosphorylation target (31). The experiments reported here have shown that the oxidase can be activated in a cell-free system by the addition of phosphorylated GST-p47PHOX but not by the unphosphorylated molecule. They show further that p47PHOX phosphorylated by PKC is competent to activate the oxidase but that p47PHOX phosphorylated by PKA or MAP kinase is inactive. These findings strongly suggest that the phosphorylation of p47PHOX that occurs during the activation of the leukocyte NADPH oxidase is of functional significance and that PKC is at least one of the enzymes capable of converting p47PHOX into a functionally active molecule by phosphorylation. They further suggest that the kinase-dependent activation mechanism described here may reflect at least in part an oxidase activating system that operates in the whole cell, because of the extensive literature implicating phosphorylation in the activation of the oxidase and because a p47PHOX mutant that is nonfunctional in the whole cell (i.e. p47PHOX S379A (31)) is also nonfunctional in the kinase-dependent cell-free system, even though the phosphorylation of the p47PHOX mutant appears to be normal both in vitro and in whole cells (37).
The assay mixtures used in these experiments contained 2.5 × 107 cell equivalents of cytosol. Assuming that the volume
of a neutrophil is 500 fl, that its cytosol contains 20% protein
(w/v), and that p47PHOX represents 0.4% of this protein (43),
it can be calculated that the cytosol contributed 10 µg of
unphosphorylated p47PHOX to the reaction mixture. From this
calculation and the above results, it appears that the oxidase activity
elicited by GST-p47PHOXP6 is roughly comparable to
that which would be observed in a system containing a similar amount of
(unphosphorylated) p47PHOX. Therefore on a weight-for-weight
basis, GST-p47PHOXP6 seems to be as active as
p47PHOX that has been activated by endogenous posphorylation.
This conclusion is supported by the results in Fig. 9, which shows that
the activity achieved in an assay containing only endogenous
p47PHOX is comparable to that obtained in an assay that was
supplemented with GST-p47PHOXP6.
The foregoing results also show that the protein kinase-dependent activation of leukocyte NADPH oxidase in a cell-free system is a multi-step process that can be divided into three distinct stages: 1) the activation of the membrane, which appears to take place during the initial incubation; 2) the phosphorylation of p47PHOX; and finally 3) the assembly of the active oxidase on the activated membrane. The activation of the membrane seems to be the most complicated of these events. The participation of a GNBP in membrane activation is suggested by the finding that GTP is required in the initial incubation, and the involvement of one or more protein kinases is suggested by the ATP requirement in the initial incubation and by the ability of GFX and H-7, antagonists of protein kinase C, to inhibit membrane activation.
All these observations suggest the following as a possible route of activation for the oxidase: 1) processing of the membrane, a complex series of events involving a GNBP-dependent step and a protein kinase; 2) the activation of PKC, which phosphorylates p47PHOX; and finally 3) the assembly of the active NADPH oxidase on a membrane that somehow has been rendered capable of supporting oxidase activation by virtue of its newly acquired protein phosphate. With regard to membrane processing, the transfer to the membrane of Rac2, a GNBP, is already known to participate in oxidase activation (41, 59), but many other possibilities can be considered, as for example the activation of the p21rac/cdc42-activated kinase (54), a single event that could explain the dependence of membrane processing on both a GNBP and a protein kinase; phosphorylation of a membrane-associated oxidase subunit; the participation of phosphatidic acid in ATP-dependent oxidase activation as described by McPhail and associates (27); and others. It is clear, however, that protein kinase-dependent oxidase activation requires an alteration in the membrane, at least in this system and perhaps in the intact cell as well.
In the activation of the cell-free system by amphiphiles, a GNBP (specifically, Rac1 or Rac2) is needed for the assembly of the oxidase (40, 41). The GTP requirement in the second incubation implies that a GNBP is necessary for the events taking place in that incubation, most likely the assembly of the oxidase, although the possibility of other reactions taking place during this incubation cannot be excluded on the basis of the present results. With regard to ATP, the experiments with ATP-depleted cytosol and the protein kinase inhibitor GFX suggest that, apart from its role in the phosphorylation of p47PHOX, this nucleotide is not required in the second incubation. A requirement for ATP in the second incubation cannot be conclusively ruled out, however, because it is conceivable that the very low level of ATP in the depleted cytosol is sufficient to support any (hypothetical) ATP-dependent reactions that may take place during the second incubation and that such hypothetical reactions may not be affected by GFX.