From the Department of Infectology and Pediatric
Immunology, Medical and Health Science Center, University of
Debrecen, H-4012, Debrecen, Nagyerdei krt.98, Hungary,
§ Department of Biochemistry, Wake Forest University School
of Medicine, Winston-Salem, North Carolina 27157, and ¶ Department
of Microbiology, Montana State University, Bozeman, Montana 59717
Received for publication, August 24, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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The enzyme NADPH oxidase is regulated by
phospholipase D in intact neutrophils and is activated by phosphatidic
acid (PA) plus diacylglycerol (DG) in cell-free systems. We showed
previously that cell-free NADPH oxidase activation by these lipids
involves both protein kinase-dependent and -independent
pathways. Here we demonstrate that only the protein kinase-independent
pathway is operative in a cell-free system of purified and recombinant NADPH oxidase components. Activation by PA + DG was ATP-independent and
unaffected by the protein kinase inhibitor staurosporine, indicating
the lack of protein kinase involvement. Both PA and DG were required
for optimal activation to occur. The drug R59949 reduced activation of
NADPH oxidase by either arachidonic acid or PA + DG, with
IC50 values of 46 and 25 µM,
respectively. The optimal concentration of arachidonic acid or PA + DG
for oxidase activation was shifted to the right with R59949, indicating interference of the drug with the interaction of lipid activators and
enzyme components. R59949 inhibited the lipid-induced
aggregation/sedimentation of oxidase components
p47phox and p67phox,
suggesting a disruption of the lipid-mediated assembly process. The
direct effects of R59949 on NADPH oxidase activation complicate its use
as a "specific" inhibitor of DG kinase. We conclude that the
protein kinase-independent pathway of NADPH oxidase activation by PA
and DG involves direct interaction with NADPH oxidase components. Thus,
NADPH oxidase proteins are functional targets for these lipid
messengers in the neutrophil.
The NADPH oxidase (the respiratory burst enzyme) in phagocytic
cells produces superoxide
(O Previous studies examined NADPH oxidase activation by PA plus DG in a
cell-free system consisting of membrane and cytosolic fractions from
human neutrophils (16, 17, 24). Both lipids are required for optimal
activation (16, 17), but the individual roles of each are not clear. We
showed (24) that activation in this system is dependent on both protein
kinase activity and other undefined phosphorylation-independent
mechanisms. The protein kinase-dependent mechanism may
involve the phosphorylation of NADPH oxidase components
p47phox and p22phox by a novel PA-activated
protein kinase (10, 25, 26). Alternatively, Erickson et al.
(17) postulated that the phosphorylation-dependent mechanism involved the conversion of DG to PA by DG kinase. They found
that the DG kinase inhibitor R59949 blocked the formation of PA from DG
as well as the activation of NADPH oxidase by DG.
R59949 inhibits DG kinase with an IC50 value of 1.25 × 10 Here, we further analyzed the individual roles of PA and DG on NADPH
oxidase activation using purified and recombinant NADPH oxidase
components. Optimal activation still required both PA and DG, and
activation was independent of ATP and protein kinase activity. We used
this system to further study the mechanism of activation of NADPH
oxidase. We found that R59949 acts in a DG kinase-independent manner on
this process through competition between R59949 and lipids during the
activation process. This characteristic of R59949 should be considered
before it is used at higher concentrations (>10 µM) in
either in vivo or in vitro assays. These results
strongly suggest that both PA and DG interact directly with NADPH
oxidase components and that this interaction is responsible for the
protein kinase-independent mechanism of oxidase activation by these
lipid second messengers.
Materials--
Phosphatidylcholine (PC) (porcine liver, 99%
pure) was from Doosan-Serdary Research Laboratories (Englewood Cliffs,
NJ), and Type IV-S PC (soybean, 40% pure) was from Sigma. The PA used
was 1,2-dicapryl-sn-glycero-3-phosphate, and the DG used was
1-oleoyl-2-acetyl-sn-glycerol; both were from Avanti Polar
Lipids, Inc. (Alabaster, AL). Lipids were freshly prepared by
sonication in water (16). Arachidonic acid was from Nu Chek Prep. Inc.
(Elysian, MN), and was prepared in 25% ethanol (16, 32). R59949
[3-[2-[4-[bis(4-fluorophenyl) methylene]-1-piperidinyl]ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone] was from Alexis Corp. (San Diego, CA) and was dissolved in
Me2SO. N-Octyl- Isolation of Neutrophils and Subcellular
Fractions--
Heparinized venous blood was obtained from healthy
donors, and neutrophils were isolated by dextran sedimentation and
centrifugation through Isolymph (35). Isolated neutrophils were
treated with diisopropyl fluorophosphate (26), resuspended in
sonication buffer (11% sucrose, 50 mM
NaxPO4, pH 7.0, 120 mM NaCl, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride
(PMSF), 10 µM benzamidine, 1 µg/ml leupeptin, 1 µM pepstatin, 1 µg/ml aprotinin), and broken by
sonication (36). Sonicates were centrifuged (200 × g,
10 min) to remove unbroken cells and nuclei, layered onto a 15/40% (w/v) discontinuous sucrose gradient, and centrifuged (150,000 × g, 30 min) (36). Cytosolic fractions were collected from the top layer down to the 15% interface, and membrane fractions were collected from the 15/40% interface. Fractions were stored at Purification and Relipidation of Flavocytochrome
b558--
Human neutrophil membrane fractions were
prepared as described (37). NaCl was added to the suspended membranes
to a final concentration of 1 M, and membranes
were centrifuged at 100,000 × g for 30 min at
4 °C. The pellet was resuspended by transferring to a cell
homogenizer and grinding until milky in 1-2 ml of solubilization buffer (10 mM HEPES, 1 mM EDTA, 1 mM MgCl2, 40 mM OG, 100 mM KCl, 10 mM NaCl, and 2 mM
NaN3, pH 7.4, with 10 µg/ml chymostatin, 1 mM
dithiothreitol (DTT), 10 µM FAD, 100 µM
NADPH, and 1 mM PMSF added just before use). The homogenate
was reconstituted to its original volume in solubilization buffer,
sonicated briefly on ice using a probe-style sonicator, stirred on ice
for 1-2 h, and centrifuged at 100,000 × g for 45 min
at 4 °C. The supernatant was diluted to a salt concentration of 75 mM with dilution buffer (10 mM HEPES, 1 mM EGTA, 1 mM MgCl2, 40 mM OG, 2 mM NaN3, pH 7.4, supplemented with 10 µg/ml chymostatin, 0.1 mM DTT, 10 µM FAD, 100 µM NADPH, and 1 mM
PMSF just before use). The diluted supernatant was introduced to a
heparin-agarose affinity column (Sigma catalog number H0402) that had
previously been rinsed with 30 bed volumes of dilution buffer with 2 M NaCl added and equilibrated with ~50 bed volumes of
wash buffer (10 mM HEPES, 1 mM EGTA, 1 mM MgCl2, 40 mM OG, 75 mM NaCl, 2 mM NaN3, pH 7.4, supplemented with 10 µg/ml chymostatin, 0.1 mM DTT, 10 µM FAD, 100 µM NADPH, and 0.2 mM PMSF just before use); the flow rate was 20 ml/h. The
cytochrome was eluted using a 0.075-2 M NaCl gradient in
elution buffer (50 mM NaH2PO4, 1 mM EGTA, 1 mM MgCl2, 40 mM OG, 2 mM NaN3, pH 7.4, supplemented with 10 µg/ml chymostatin, 0.1 mM DTT, 10 µM FAD, 100 µM NADPH, and 0.2 mM PMSF just before use). One-ml fractions were collected,
and those containing flavocytochrome b absorbance were
pooled and concentrated to a final volume of 1 ml using a 50-kDa
nominal molecular mass centrifugal concentration device (Millipore).
The sample was passed through a 0.2-µm pore size syringe filter and
introduced to a gel filtration column (Amersham Pharmacia Biotech
Superdex 200 HR, 10-30) maintained at 4 °C and previously
equilibrated in high performance liquid chromatography column buffer
(50 mM NaH2PO4, 1 mM
EGTA, 1 mM MgCl2, 40 mM OG, 150 mM NaCl, 2 mM NaN3, pH 7.4, supplemented with 10 µg/ml chymostatin, 0.1 mM DTT, 10 µM FAD, and 0.2 mM PMSF just before use).
Peak fractions containing flavocytochrome b were collected
in 500-µl aliquots, and cytochrome concentrations were determined
using
Stored fractions were relipidated as described (19, 23), with
modifications. Briefly, PC (soybean, Type IV-S, 7 mg/ml), was prepared
by sonication in buffer A (50 mM
Na3PO4, pH 7.0, 1 mM EGTA, 1 mM MgCl2, 50 mM Na3N,
20% glycerol, 40 mM OG, 0.05 mM DTT, 1 µM FAD). The lipid solution (0.7 mg/ml final) was added to the purified flavocytochrome b558 (600 µl,
0.85-1.2 µM). The mixture was kept on ice for 20 min,
diluted 6-fold with phosphate buffer (50 mM
Na3PO4, pH 7.0, 1 mM EGTA, 1 mM MgCl2) and stored at NADPH Oxidase Activation and Assay--
Mixtures containing 50 mM NaxPO4 buffer, pH 7.0, 100 µM cytochrome c, 10 µM FAD, 1 mM EGTA, 5 mM MgCl2, zero or 12 µM ATP, zero or 0.16 mg/ml superoxide dismutase, 0.5 µg
of membrane protein (0.6 pmol of flavocytochrome
b558) (semi-recombinant system), or 2 pmol of
relipidated flavocytochrome b558
(purified-recombinant system) plus 40 pmol
p47phox, 15 pmol p67phox,
and 60 pmol Q61L Rac1 (hereafter referred to as Rac1) were incubated with various concentrations of PA, DG, or PA plus DG for 90 min or with
AA for 30 min in a final volume of 0.12 ml. For Figs. 4-6, R59949 or
0.1% Me2SO (solvent control) was added into the reaction
mixture before the addition of lipid activators. After the desired
incubation time, 200 µM NADPH was added, and the
reactions were stopped at 2 min by 1% (v/v) Triton X-100 (16, 24). In some experiments (described under "Results"), R59949 or 0.1% Me2SO4 (solvent control) was added with the
NADPH at the end of the incubation. The O ATP Determination--
The ATP concentrations present in
membrane fractions and stock solutions of recombinant
p47phox, p67phox, and
Rac1 were determined by using the ATP determination kit from Molecular
Probes (42). Samples were boiled for 5 min before analysis.
Luminescence was read on a Turner TD 20e Luminometer (Sunnyvale, CA),
and values were calculated based on an ATP standard curve.
Sedimentation of p47phox and p67phox by
PA + DG--
Mixtures (0.12 ml) containing 50 mM
NaxPO4 buffer, pH 7.0, 10 µM FAD, 1 mM EGTA, 5 mM MgCl2, zero or 0.5 µg of membrane protein (0.6 pmol of flavocytochrome
b558), 40 pmol of
p47phox, 15 pmol of
p67phox, and 60 pmol of Q61L Rac1 were incubated
with 20 µM PC or 10 µM PA + 10 µM DG for 90 min in the presence or absence of 50 µM R59949. Ten reaction mixtures for each condition were
mixed (1.2 ml) and layered on a 15/50% (w/v) (1 ml/0.5 ml)
discontinuous sucrose gradient and centrifuged in a SW55 rotor (Beckman
Instruments) at 150,000 × g for 30 min at room
temperature (43). Soluble fractions were collected from the top of the
gradient (0.9 ml); the pellet fractions were collected from the 15/50%
interface plus the 50% fraction (0.75 ml). Proteins from 140 µl of
each soluble and pellet fraction were separated by 10%
SDS-polyacrylamide gel electrophoresis (44), transferred to
nitrocellulose (45), and analyzed by Western blot with a 1:500 dilution
of a mixture of p47phox and
p67phox antibodies. Blots were developed using
SuperSignalTM enhanced chemiluminescence reagent
(Pierce) and analyzed by densitometry (PDI, Huntington Station, NY).
The percentage of each protein in the pellet fraction was calculated
using the sum of the soluble and pellet fractions as 100%. The pmol
amounts of each protein in the pellet fraction, after treatment with PA + DG, were estimated using the pmol added to the reaction mixture
(calculated after correcting for the purity of the added protein) as
100%.
Membrane NADPH Oxidase Assay--
Pellet fractions containing
neutrophil membrane from the sedimentation assay were analyzed for
their NADPH oxidase activity, as described previously (46). 80 µl of
each sample was mixed and incubated for 5 min at room temperature with
20 mM KxPO4, pH 7.0, 1 mM
EGTA, 7.6 mM MgCl2, 10 µM FAD, 75 µM cytochrome c, and 0.16 mM SDS
in a final volume of 220 µl. The mixture was divided into two
cuvettes, one containing superoxide dismutase at a final concentration
of 0.24 mg/ml. The reaction was started by the addition of NADPH (final
concentration, 0.2 mM) to both cuvettes, and absorbance changes were monitored continuously at 550 nm by a UV-2401PC Shimadzu spectrophotometer (Columbia, MD). O Characteristics of NADPH Oxidase Activation by PA + DG in a
Cell-free System Using Purified and Recombinant Oxidase
Components--
Previous studies (16, 17, 24) examined cell-free NADPH
oxidase activation by PA + DG using mixtures of membrane and cytosolic
fractions from human neutrophils. Results indicated that oxidase
activation required both PA and DG for optimal activity and involved
both phosphorylation-dependent and -independent mechanisms (17, 24). Our previous data (24-26) suggested that the
phosphorylation-dependent mechanism involved one or more
cytosolic protein kinases. Therefore, we hypothesized that the
substitution of recombinant NADPH oxidase components for the neutrophil
cytosolic fraction would eliminate the
phosphorylation-dependent activation pathway. To test this, we examined the ability of PA + DG to activate NADPH oxidase in a
"semi-recombinant" cell-free system consisting of the recombinant proteins p47phox,
p67phox, and Rac1 mixed with neutrophil membrane
fractions. As shown in Fig.
1A, PA + DG induced NADPH
oxidase activation in the semi-recombinant system. To test whether a
protein kinase was involved in NADPH oxidase activation in this system,
we examined the effect of a potent, nonselective protein kinase
inhibitor, staurosporine (47). Staurosporine reduced NADPH oxidase
activation by about 50% when neutrophil cytosol was used as the source
of cytosolic oxidase components (Fig. 1A, left
panel). In contrast, staurosporine had no effect on NADPH oxidase
activation by PA + DG in the semi-recombinant system (Fig.
1A, right panel).
To further address whether activation by PA + DG in the
semi-recombinant system was phosphorylation-independent, we tested the
effect of ATP. The endogenous ATP concentration in our reaction mixtures of membrane and recombinant oxidase proteins was determined to
be 1 ± 0.9 nM (mean ± S.E., n = 3), which is too low to support lipid or protein phosphorylation
reactions. We performed concentration curves with PA + DG in the
presence or absence of 12 µM added ATP (Fig.
1B). The curves were virtually superimposable, indicating that ATP had no effect on the level of activity or the EC50
for the lipids. Taken together, these data indicate that activation of
NADPH oxidase by PA + DG in the semi-recombinant system is phosphorylation- and protein kinase-independent.
We next addressed whether both PA and DG were required for activation
to occur in the semi-recombinant system. As shown in Fig.
2A, oxidase activity was
negligible with DG alone and was present at only low levels with PA
alone. In contrast, when both lipids were added, substantial levels of
oxidase activity were detected. AA induced similar levels of activation
as PA + DG. Both PA and DG could have direct activating effects on one
or more oxidase components. However, the neutrophil membrane fraction present in the semi-recombinant system might have other targets for one
or both of these lipids, which could influence oxidase activation.
Therefore, we tested the effect of PA and DG on oxidase activation when
purified, relipidated (with soybean PC) flavocytochrome b558 replaced neutrophil membrane. Levels of
oxidase activation were lower compared with the semi-recombinant
system. As shown in Fig. 2B, DG alone was ineffective in
this system, whereas PA showed slight activation of the enzyme. However
the combination of the two lipids induced a greater than additive
response, similar to that observed in the semi-recombinant system. AA
also could activate NADPH oxidase in this purified-recombinant system,
similar to observations by others (18, 19). These results strongly suggest that one or more NADPH oxidase components is a direct target(s)
of PA and DG for the activation of the enzyme.
As shown in Fig. 3, the activation of
NADPH oxidase by PA + DG in both the semi-recombinant and
purified-recombinant cell-free systems reached a maximum by 30 min and
did not decline for up to 90 min. The rate of activation was faster in
the purified-recombinant system, since ~40% of maximum activity was
achieved without preincubation (Fig. 3B) compared with
~25% of maximum in the semi-recombinant system at zero time (Fig.
3A). The zero time point includes a 2-min incubation for
measurement of NADPH oxidase activity, indicating that the rate of
activation in the first 2 min is almost twice as fast in the
purified-recombinant cell-free system.
Effect of R59949 on the Activation of NADPH Oxidase in the
Semi-recombinant Cell-free System--
The above data indicated that
oxidase activation in the semi-recombinant system was independent of
phosphorylation reactions and suggested that both PA and DG were
required for the activation process. Previously, Erickson et
al. (17) used the DG kinase inhibitor R59949 to suggest that a
phosphorylation-dependent mechanism of oxidase activation
by PA + DG involved the conversion of DG to PA by DG kinase. Since our
results suggest that DG has direct effects on NADPH oxidase
component(s), we hypothesized that R59949 might exert its inhibitory
effect in a DG kinase-independent way. Thus, we studied the effect of
R59949 on oxidase activation in the semi-recombinant system, where
phosphorylation-dependent reactions are not involved. NADPH
oxidase activation was examined in the presence of various
concentrations of R59949 using either AA or PA + DG as lipid
activators. Assays were performed in the presence of 12 µM ATP to maximize the ability of any DG kinase present
in the membrane to convert DG to PA. As shown in Fig. 4A, R59949 had no inhibitory
effect on oxidase activation until the concentration was above 10 µM, and inhibition was nearly complete at 100 µM. The IC50 for inhibition by R59949 was 25 µM with PA + DG and 46 µM with AA. R59949
(50 µM) was not inhibitory when added after the
preincubation with lipids (PA + DG: 2608 ± 260 versus
PA + DG + R59949: 2521 ± 260; AA: 3130 ± 434 versus AA + R59949: 4196 ± 262 mol O R59949 Shifts to the Right the Optimal Concentration of Lipid
Activators for the Activation of NADPH Oxidase in the Semi-recombinant
Cell-free System--
To address the mechanism of inhibition by
R59949, we further characterized the effect of R59949 on the
concentration of AA (0-70 µM) needed to induce NADPH
oxidase activation (Fig. 4B). The presence of 50 µM R59949 shifted the optimal concentration of AA to the
right, from 15-25 to 40-50 µM AA, and slightly reduced the maximal level of activation. These data indicate that inhibition by
R59949 of NADPH oxidase activation is likely due to competition with AA
for binding to and/or interaction with components of the enzyme.
Next we addressed whether R59949 might exert a similar effect on the
amount of PA + DG needed for NADPH oxidase activation. As observed with
AA, 50 µM R59949 shifted the optimal concentration of PA
plus DG to the right (Fig.
5A). R59949 increased the EC50 of PA plus DG from 17 ± 7 to 72 ± 22 µM (mean ± S.E., n = 3). We next
asked whether R59949 exerted its effect on both PA and DG or on just
one of these lipids. The concentration of DG (Fig. 5B) or PA
(Fig. 5C) was varied, keeping the other lipid concentration
at 10 µM. R59949 (50 µM) shifted the
optimal concentration of DG to the right, increasing the
EC50 of DG from 3 ± 2 to 24 ± 15 µM (mean ± S.E., n = 3). The drug
also slightly shifted the optimal concentration of PA, and it also
decreased the maximal NADPH oxidase activation under these conditions.
Taken together, these results indicate that the effect of R59949 on PA + DG-mediated oxidase activation is similar to that in the AA-activated
system, with a shift to the right in the lipid concentration required
for optimal NADPH oxidase activation. Furthermore, increasing the
concentration of DG was better able to overcome the inhibitory effect
of the drug compared with PA. These data suggest that R59949 is acting
in a competitive manner with the lipid activators, with selectivity for
DG over PA.
R59949 Inhibits NADPH Oxidase Activation in the
Purified-recombinant Cell-free System--
We tested whether R59949
would have similar inhibitory effects on NADPH oxidase activation in
the purified-recombinant cell-free system. R59949 was slightly less
effective in this series of studies, inhibiting NADPH oxidase
activation by PA + DG in the semi-recombinant system by 45% at 100 µM (30 µM PA + 30 µM DG, data
not shown). As illustrated in Fig.
6A for the
purified-recombinant cell-free system, 100 µM R59949
reduced NADPH oxidase activation induced by either AA or PA + DG by 66 and 40%, respectively. We also examined whether R59949 was competitive
with PA + DG in this cell-free system. As shown in Fig. 6B,
the presence of R59949 during NADPH oxidase activation shifted the
optimal concentration for the lipid activators to the right. These data
indicate that the mechanism of inhibition of NADPH oxidase activation
by R59949 is the same when only NADPH oxidase components are present.
Thus, the drug must directly interfere with the ability of lipid
activators to interact with NADPH oxidase components.
R59949 Inhibits the Lipid-induced Sedimentation of Recombinant
p47phox and p67phox--
To further support
the direct interference of R59949 with lipid activators, we tested the
effect of the drug on the ability of PA + DG to induce aggregation and
sedimentation of cytosolic NADPH oxidase components. Previously, AA was
shown to induce the presumed aggregation of
p47phox and p67phox in
either the presence or absence of neutrophil membrane fractions, resulting in the sedimentation of these proteins during high speed centrifugation (48). In the presence of neutrophil membrane, this
process aids in the assembly of the active NADPH oxidase enzyme. Here,
we determined whether PA + DG induced a similar effect and whether
R59949 interfered with the aggregation/sedimentation process.
Recombinant oxidase proteins were incubated with or without neutrophil
membrane fractions in the presence or absence of lipid activators
and/or 50 µM R59949, followed by separation of pellet and
soluble fractions over a discontinuous sucrose gradient. PA + DG
induced the appearance of both p47phox and
p67phox in the recovered pellet fraction either
in the presence or absence of neutrophil membrane (Fig.
7A). This indicates that PA + DG, like AA (48), induces presumed aggregation of these soluble proteins. The presence of 50 µM R59949 inhibited the
sedimentation of p47phox and
p67phox (Fig. 7A). The sedimentation
of p47phox appeared to be more sensitive to
R59949, since the drug inhibited the appearance in the pellet fraction
of 98 ± 2% of p47phox compared with
57 ± 2% of p67phox (Fig. 7B).
Parallel to the inhibition of p47phox and
p67phox sedimentation, R59949 also markedly
reduced the level of NADPH oxidase activity appearing in the pellet
fractions containing neutrophil membrane (Fig. 7B). These
results suggest that R59949 interferes with the ability of lipid
activators to bind to one or more soluble NADPH oxidase components and
induce the aggregation process, which might be an important step for
transporting p47phox and
p67phox to the membrane components in cell-free
assays (48).
Here, we provide evidence that the second messenger lipids PA and
DG interact directly with components of NADPH oxidase to induce
assembly and activation of the enzyme. This evidence includes the
following. 1) NADPH oxidase activation by PA plus DG in the semi-recombinant cell-free system was ATP-independent and insensitive to the nonselective protein kinase inhibitor staurosporine, indicating a phosphorylation-independent mechanism; 2) PA + DG induced NADPH oxidase activation when only oxidase components were present (the purified-recombinant system); 3) PA and DG were both required for
optimal activation, suggesting that each lipid may have one or more
oxidase protein targets; and 4) the drug R59949 competitively inhibited
the interaction of PA and DG with oxidase components and prevented the
assembly of p47phox and p67phox with
flavocytochrome b558.
Previously (16, 24), using mixtures of membrane and cytosolic fractions
from human neutrophils, we found that NADPH oxidase activation by PA + DG involved both protein kinase-dependent and -independent
pathways. A novel cytosolic PA-activated protein kinase capable of
phosphorylating two NADPH oxidase components (p47phox and
p22phox) was implicated as responsible for the
phosphorylation-dependent pathway (10, 24-26). In
addition, Erickson et al. (17) suggested that DG kinase
contributes to a phosphorylation-dependent pathway in
mixtures of membrane and cytosolic fractions. These pathways are
clearly not operative in the semi-recombinant cell-free system, where
recombinant cytosolic oxidase components replace neutrophil cytosol.
Based on the results reported here, it is evident that the
phosphorylation-independent mechanism of NADPH oxidase activation by PA + DG involves direct interaction between the lipids and oxidase
components. This direct mechanism is likely analogous to that used by
AA, since the drug R59949 caused a similar shift to the right in the
concentration curves of either AA or PA + DG. AA has been shown to
directly induce conformational changes in both flavocytochrome
b558 and p47phox (20-22), resulting in
the SH3 domain-mediated binding of p47phox with the
p22phox subunit of the flavocytochrome (49). PA has been shown
to induce partial activation of purified flavocytochrome
b558 in the absence of cytosolic components
(23). The inhibition of the aggregation/sedimentation of
p47phox and p67phox by R59949 implies
that the lipids bind to one or both of these components. A likely
target for lipid binding is p47phox, since a change in
conformation of p47phox appears to initiate the
translocation/assembly process (49-51). Indeed, we have shown in a
separate study2 that PA
selectively binds to p47phox. DG can enhance the
binding of PA to the enzyme CTP:phosphocholine cytidylyltransferase
through a proposed mechanism involving effects of DG on the clustering
of PA molecules in the lipid bilayer (52). The synergy between PA and
DG for NADPH oxidase activation could involve a similar mechanism.
Studies to address these issues are under way.
Our results also clearly show that the DG kinase inhibitor R59949
directly interferes with the ability of lipids to activate NADPH
oxidase. Because of its lipophilic nature, R59949 may exert its effect
by interaction with the lipids, preventing their binding to NADPH
oxidase components. Alternatively, R59949 may compete for the lipid
binding site(s) on oxidase proteins. Binding sites on DG kinase or
other proteins for R59949 have not been identified. It is possible that
R59949 competes with DG for binding to the active site of certain DG
kinase isoforms; however, the catalytic binding site for DG is not
known (53, 54), and kinetic studies to address this possibility have
not been published. Our results show that increasing the concentration of DG was more effective than increasing the concentration of PA at
overcoming the inhibition by the drug. Increasing the concentration of
AA could also overcome the effect of the drug. This suggests that, in
the oxidase system, R59949 is primarily competitive with DG or AA.
The direct inhibitory effect on NADPH oxidase activation by R59949
complicates interpretation of previous results (17) using this
inhibitor to implicate DG kinase in cell-free NADPH oxidase activation.
Erickson et al. (17) showed that R59949 inhibited the
conversion of DG to PA and also blocked the ability of DG alone to
activate NADPH oxidase. Our results suggest that the inhibition of
NADPH oxidase by R59949 in those studies could be due to competition
with lipids for oxidase activation. However, since in our hands DG
alone cannot activate NADPH oxidase, it is likely that the conversion
of some of the added DG to PA by DG kinase was involved in the
activation observed by Erickson et al. (17). DG kinase may
play a regulatory role in intact neutrophils, since a number of studies
(55-57) show that DG kinase inhibitors enhance O Activation of NADPH oxidase in intact neutrophils is complexly
regulated and incompletely understood. A wealth of data (reviewed in
Refs. 2 and 10)) implicates the activation of protein kinases and
phospholipases, particularly phospholipase D and phospholipase A2, but how these signaling pathways converge on NADPH
oxidase activation is unclear. The phospholipase-generated lipid
messengers could activate protein kinase C or other protein kinases
(58-62), contributing to the phosphorylation of NADPH oxidase
components. Phosphorylation of at least one oxidase component,
p47phox, is required for oxidase activation by the
nonphysiological agonist phorbol myristate acetate, which activates
protein kinase C isoforms (2, 8). Phosphorylation of p47phox by
protein kinase C can disrupt the "closed conformation" of the
protein, allowing it to initiate NADPH oxidase assembly (50, 51).
However, the phosphorylation-dependent mechanisms used by
physiological agonists have not been elucidated with any certainty. Shiose and Sumimoto (49) recently demonstrated that AA can synergize with phosphorylation of p47phox to activate NADPH oxidase. This
is an attractive model that may help to explain the complex regulation
of oxidase activation in intact cells by physiological agonists. In
addition, we recently showed (63) that phospholipase
D-dependent pathways are involved in the phosphorylation of
p22phox in intact cells induced by physiological agonists. The
use of the semi-recombinant and purified-recombinant cell-free
activation systems described here should allow careful dissection of
the functional effects of phosphorylation (by added protein kinases) and lipids on NADPH oxidase components during the activation of the
enzyme by PA and DG. Mechanisms identified in cell-free systems would
then need to be studied in intact cells to determine their physiological relevance.
In conclusion, we have identified a protein kinase-independent
pathway for NADPH oxidase activation by the lipid second messengers PA
and DG. The pathway clearly involves direct interaction of the lipids
with NADPH oxidase components. Thus, PA and DG have multiple potential
protein targets in human neutrophils, including a novel PA-activated
protein kinase (10, 25), protein kinase C isoforms (58, 59), and now,
NADPH oxidase proteins. Interaction of PA and DG with all of these
proteins may contribute to the regulation of NADPH oxidase in
neutrophils and other cell types.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2)1 by catalyzing
electron transfer from NADPH to molecular oxygen upon cell stimulation
(1-3). This enzyme plays important roles in host defense against
infection and in tissue damage due to inflammation (1-5). In addition,
NADPH oxidase-like enzymes are present in a variety of other cell
types, where the oxygen radicals formed may have signaling roles (5,
6). The enzyme in phagocytes consists of the membrane-bound
heterodimeric flavocytochrome b558 (gp91phox and p22phox)
and four cytosolic proteins (p47phox,
p67phox, p40phox, Rac1/2)
(2-4, 7). Components must assemble in the membrane for the enzyme to
become active (2-4, 7). The activation of NADPH oxidase is initiated
by receptor-ligand interaction and involves complex intracellular
signaling events. These include the activation of protein kinases to
phosphorylate cellular proteins and NADPH oxidase components (2, 8) and
the generation of various lipid second messengers (AA by phospholipase
A2 (9); DG by phospholipase C or PA phosphohydrolase; PA by
phospholipase D or DG kinase (10, 11)). In cell-free systems, these
lipids can induce activation of the enzyme (12-17). AA exerts its
effect by directly acting on enzyme components (18-22). PA has been
shown to partially activate purified flavocytochrome
b558 (23), suggesting it interacts with this
protein. It is not known whether DG has any direct effect(s) on NADPH
oxidase components.
7 M in isolated platelet
membranes and in intact platelets (27). However, at concentrations
above 10
5 M, the drug has
nonspecific effects on overall lipid and protein metabolism (28). Since
then, R59949 has been widely used (29, 30). Jiang et al.
showed (31) that R59949 is selective for Ca2+-activated DG
kinases and that the drug interacts with the catalytic subunit of the
enzyme. These observations raise the possibility that the effect of
R59949 on cell-free NADPH oxidase activation (17) is unrelated to
inhibition of DG kinase, since high concentrations were used, and
Ca2+ was not present in the activation system.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside
(OG) was from Calbiochem-Novabiochem. SDS-polyacrylamide gel
electrophoresis reagents were from Bio-Rad. NADPH and superoxide
dismutase were from Roche Molecular Chemicals. Glutathione beads,
dextran T-500, and Q-Sepharose Fast Flow were from Amersham Pharmacia
Biotech. Isolymph was from Gallard-Schlesinger Industries (Carle Place,
NY). The ATP determination kit was from Molecular Probes (Eugene, OR).
The p47phox and p67phox
vector-containing baculoviruses were generous gifts from Dr. David
Lambeth (Emory University, Atlanta, GA). The
p47phox and p67phox
proteins were produced in Sf9 cells and partially purified on CM-cellulose (p47phox) or Q-Sepharose Fast Flow
(p67phox) (33). The goat
anti-p47phox and
anti-p67phox antibodies were generous gifts from
Dr. Tom Leto (NIAID, NIH, Bethesda, MD). The Q61L active conformational
mutant of Rac1 (a generous gift from Dr. Tom Leto) was produced in
Escherichia coli as a glutathione S-transferase
fusion protein and purified on glutathione-Sepharose beads by thrombin
cleavage (34). All other reagents were from Sigma.
70 °C. Protein concentration was determined using the Coomassie Plus Protein protocol from Pierce using bovine serum albumin as a standard.
414 = 130.8 mM
1 cm
1
(38). The preparation was greater than 90% pure cytochrome, the major
contaminant being CD11b/CD18. Glycerol was added to each fraction to a
final concentration of 20%, and fractions were stored at 4 °C,
wrapped in foil, and relipidated within 2 days. The entire purification
process, beginning at the salt wash, was completed in 1 day.
70 °C. The
flavocytochrome b558 concentration in the
relipidated mixture was determined spectrophotometrically from the
reduced minus oxidized spectrum using
428 nm=106
mM
1 cm
1
(39).
2 production was
determined by measuring the absorbance of cytochrome c at
550 nm using a Thermomax® kinetic microplate reader
(Molecular Devices Corp., Menlo Park, CA) and correcting for the
absorbance of samples containing superoxide dismutase. Activity
was linear over the 2-min assay period and was expressed as mol of
O
2/min/mol of flavocytochrome b558
using an extinction coefficient of 21 mM
1 cm
1
for cytochrome c (40). IC50 values were
determined in SigmaPlot (Jandel Scientific Software, San Rafael, CA) by
fitting the data to the hyperbolic decay equation (competitive
inhibition model) (41), activity = max activity × IC50/[R59949]+ IC50. EC50 values
were determined in SigmaPlot using the single rectangular three-parameter nonlinear regression model.
2 production was calculated using the linear slopes and an extinction coefficient for cytochrome c of 21 mM
1
cm
1 (40). Activity was expressed as nmol of
O
2/min/ml of pellet fraction, since the amount of protein and
flavocytochrome b558 present in the fractions
were too low to measure.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
Activation of NADPH oxidase by PA + DG in a
semi-recombinant cell-free system is protein kinase-independent.
A, effect of staurosporine. Reaction mixtures contained the
following. Left panel, 0.25 µg of membrane protein (0.3 pmol of flavocytochrome b558) and 12.5 µg of
cytosolic protein and were incubated with H2O or 100 µM PA + 100 µM DG; right panel,
0.5 µg of membrane protein (0.6 pmol of flavocytochrome
b558), 40 pmol of
p47phox, 15 pmol of
p67phox, and 60 pmol of Rac1 and were incubated
with 30 µM PC or 30 µM PA + 10 µM DG. Assays were performed in the presence of 12 µM added ATP and in the presence of either
Me2SO (DMSO, light gray bars) or 100 nM staurosporine (dark gray bars). NADPH oxidase
activity was measured as described under "Experimental Procedures"
and expressed as mol of O 2/min/mol of flavocytochrome
b558. The data shown are mean ± S.E. of
three (left panel) or seven (right panel)
experiments. B, effect of ATP. Reaction mixtures contained
0.5 µg of membrane protein (0.6 pmol of flavocytochrome
b558), 40 pmol of
p47phox, 15 pmol of
p67phox, and 60 pmol of Rac1 and were incubated
with various amounts of PA + DG in the presence (
) or absence (
)
of 12 µM added ATP. PA and DG were present at equal
concentrations, each representing half of the total value shown at any
given concentration. NADPH oxidase activity was measured as described
under "Experimental Procedures" and expressed as mol of
O
2/min/mol of flavocytochrome b558. The
data shown are mean ± S.E. of three experiments.
View larger version (31K):
[in a new window]
Fig. 2.
Both PA and DG are required for optimal NADPH
oxidase activation in the semi-recombinant and purified-recombinant
cell-free systems. A, semi-recombinant system. Reaction
mixtures contained 0.5 µg of membrane protein (0.6 pmol of
flavocytochrome b558), 40 pmol of
p47phox, 15pmol of
p67phox, and 60 pmol of Rac1 and were incubated
with 30 µM PC, 10 µM PA, 10 µM DG, or PA + DG (10 µM each) for 90 min
or ethanol (Eth) or 25 µM AA for 30 min. ATP
(12 µM) was present in all reaction mixtures except those
containing AA. NADPH oxidase activity was measured as described under
"Experimental Procedures." Data shown are the mean ± S.E. of
three experiments. B, purified-recombinant system. Reaction
mixtures were as above, except that 2 pmol of purified, relipidated
flavocytochrome b558 was substituted for the
membrane protein. Mixtures were incubated with either H2O,
30 µM PA, 30 µM DG, or 30 µM
PA + 30 µM DG for 90 min or with ethanol (Eth)
or 10 µM AA for 30 min. NADPH oxidase activity was
measured as described under "Experimental Procedures." Data shown
are mean ± S.E. (n = 3).
View larger version (22K):
[in a new window]
Fig. 3.
Time-dependent activation of
NADPH oxidase by PA + DG in semi-recombinant and purified-recombinant
cell-free systems. A, reaction mixtures as in Fig.
2A were incubated with 30 µM PA + 30 µM DG ( ) or H2O (
) for 0-90 min.
B, reaction mixtures as in Fig. 2B were incubated
with 30 µM PA + 30 µM DG (
) or
H2O (
) for 0-90 min. ATP was omitted from all reaction
mixtures. NADPH oxidase activity was measured as described under
"Experimental Procedures." Activity is expressed as mol of
O
2/min/mol of flavocytochrome b558.
Values with PA + DG are the mean ± S.E. of three experiments
(A) and the average of two closely agreeing experiments
(B). H2O control values are the average of two
experiments.
2/min/mol
of flavocytochrome b558, mean ± S.E.,
n = 3). AA is documented to induce NADPH oxidase activation through direct interaction with NADPH oxidase components (18, 19). Our results in Figs. 1-3 indicate that PA and DG do the same
in the semi-recombinant cell-free system. Therefore, these data suggest
that R59949 directly interferes with the ability of lipids to induce
activation of NADPH oxidase.
View larger version (25K):
[in a new window]
Fig. 4.
R59949 inhibits the activation of NADPH
oxidase in the semi-recombinant cell-free system. A,
inhibition by R59949 is concentration-dependent. Reaction
mixtures as in Fig. 2A were incubated with 15 µM AA for 30 min ( ), 10 µM PA + 10 µM DG for 90 min (
), or H2O (
) in the
presence of 0-100 µM R59949. 12 µM ATP was
present in the PA + DG-activated system. B, R59949 shifts
the AA concentration curve to the right. Reaction mixtures as in Fig.
2A were incubated with 0-70 µM AA for 30 min
in the presence (
) or absence (
) of 50 µM R59949.
For both panels, NADPH oxidase activity was measured as
described under "Experimental Procedures" and expressed as mol
of O
2/min/mol of flavocytochrome
b558. Values with lipid activators are the
mean ± S.E. of three experiments. Water controls are the average
of two experiments. DMSO, Me2SO.
View larger version (20K):
[in a new window]
Fig. 5.
R59949 shifts the concentration curves of PA
and DG for NADPH oxidase activation to the right in the
semi-recombinant cell-free system. A, varying PA + DG.
Reaction mixtures as in Fig. 2A were incubated with various
amounts of PA + DG in the presence ( ) or absence (
) of 50 µM R59949. PA and DG were present at equal
concentrations, each representing half of the total value shown at any
given concentration. B, varying DG. Reaction mixtures as in
panel A were incubated with 10 µM PA plus
0-300 µM DG. C, varying PA. Reaction mixtures
as in panel A were incubated with 10 µM DG
plus 0-300 µM PA. NADPH oxidase activity was measured as
described under "Experimental Procedures" and expressed as mol of
O
2/min/mol of flavocytochrome b558.
Values shown are the mean ± S.E. of three experiments.
DMSO, Me2SO.
View larger version (21K):
[in a new window]
Fig. 6.
R59949 inhibits activation of NADPH oxidase
in the purified-recombinant system. A, inhibition by
R59949. Reaction mixtures containing purified, relipidated
flavocytochrome b558 and recombinant cytosolic
oxidase components as in Fig. 2B were incubated with ethanol
(Eth) or 10 µM AA or with H2O or
30 µM PA + 30 µM DG in the presence
(white bars) or absence (black bars) of 100 µM R59949. DMSO, Me2SO.
B, varying PA + DG. Reaction mixtures as in Fig.
2B were incubated with various amounts of PA + DG in the
presence ( ) or absence (
) of 100 µM R59949. PA and
DG were present at equal concentrations, each representing half of the
total value shown at any given concentration. Data represent the
average of two closely agreeing experiments.
View larger version (44K):
[in a new window]
Fig. 7.
R59949 inhibits the aggregation/sedimentation
of p47phox and p67phox
induced by PA + DG. Reaction mixtures containing zero or 0.5 µg of membrane protein (0.6 pmol flavocytochrome
b558), 40 pmol of
p47phox, 15 pmol of
p67phox, and 60 pmol of Rac1 were incubated with
20 µM PC (no activator control) or 10 µM PA + 10 µM DG for 90 min in the presence or absence of 50 µM R59949. Ten reaction mixtures were combined, and
pellet and soluble fractions were separated as described under
"Experimental Procedures." NADPH oxidase activity in the pellet
fractions containing neutrophil membrane was measured as described
under "Experimental Procedures." Proteins from the soluble and
pellet fractions were separated on a 10% polyacrylamide gel,
transferred to nitrocellulose, and analyzed by Western blot for
p47phox and p67phox.
Panel A, shown is a representative Western blot of pellet
fractions. Panel B, left side, Western blots of
recovered pellet and soluble fractions were analyzed by densitometry.
The distributions of p47phox and
p67phox were determined, and the amount (pmol)
of each protein present in the pellet fraction was calculated as
described under "Experimental Procedures." Right side,
NADPH oxidase activity was expressed as nmol of O 2/min/ml of
pellet fraction. Values shown in panel B are mean ± S.E. of three experiments. DMSO, Me2SO.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 release
during agonist stimulation. These studies used drug concentrations too
low to have direct effects on lipid-mediated oxidase activation.
However, caution should be used when interpreting the effects of R59949
on intact cell functions when concentrations above 10-20
µM are used. NADPH oxidase-like enzymes are now being
found in a variety of cell types, where the resulting O
2 and
H2O2 may be involved in signaling pathways (5,
6). Thus, the direct inhibition of NAD(P)H oxidase activation also may
confound the use of R59949 to implicate a role for DG kinase in
functional responses in cell types other than neutrophils.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Tom Leto for the antibodies to p47phox and p67phox and the E. coli containing recombinant Q61L Rac1, Dr. David Lambeth for the baculovirus stocks used to express p47phox and p67phox proteins, Drs. Shabnam Motalebi and Dr. Reidar Wallin for advice during expression and purification of p47phox and p67phox proteins, Dr. Roy Hantgan for help with statistical analysis, Dr. Susan Sergeant, Dr. Debra Regier, Dianne Greene, Dr. Lu Fan, and WenXiao Zhang for technical help and insightful discussion. We also thank Dr. László Fésüs for support.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant RO1-AI-22564 and March of Dimes Research Foundation Grants FY99-0561 and FY00-463 (to L. C. M.), National Institutes of Health Grant RO1-AI26711 (to A. J. J.), and Hungarian Research Fund Grant OTKA T25780 (to L. M.).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, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel: 336-716-2621; Fax: 336-716-7671; E-mail: lmcphail@wfubmc.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M007759200
2 W.-X. Zhang, J. B. Nixon, T. L. Leto, and L. C. McPhail, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
O2, superoxide;
AA, arachidonic acid;
DG, diacylglycerol;
DTT, dithiothreitol;
OG, N-octyl-
-D-glucopyranoside;
PA, phosphatidic
acid;
PC, phosphatidylcholine;
phox, phagocyte
oxidase;
PMSF, phenylmethylsulfonyl fluoride.
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REFERENCES |
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