| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Phosphorylates a Subset of Selective Sites of the NADPH Oxidase Component p47phox and Participates in Formyl Peptide-Mediated Neutrophil Respiratory Burst
*
Institut National de la Santé et de la Recherche Médicale Unité 479, Centre Hospitalier Universitaire Xavier Bichat, Paris, France; and
Centre National de la Recherche Scientifique Unité Propre de la Recherche et des Études Scientifiques A 8068, Institut Cochin de Génétique Moléculaire, Département de Biologie Cellulaire, Hôpital Cochin, Paris, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(PKC ), an
atypical PKC isoform expressed abundantly in human polymorphonuclear
leukocytes (PMN), translocates to the PMN plasma membrane upon
stimulation by the chemoattractant fMLP. We investigated the role of
PKC in p47phox phosphorylation and in
superoxide anion production by human PMN. In vitro incubation of
recombinant p47phox with recombinant PKC
induced a time- and concentration-dependent phosphorylation of
p47phox with an apparent
Km value of 2 µM. Phosphopeptide mapping
analysis of p47phox showed that PKC
phosphorylated fewer selective sites in comparison to
"conventional" PKCs. Serine 303/304 and serine 315 were identified
as targets of PKC by site-directed mutagenesis. Stimulation of PMN
by fMLP induced a rapid and sustained plasma membrane translocation of
PKC that correlated to that of p47phox. A
cell-permeant-specific peptide antagonist of PKC inhibited both
fMLP-induced phosphorylation of p47phox and its
membrane translocation. The antagonist also inhibited the fMLP-induced
production of oxidant (IC50 of 10 µM), but not that
induced by PMA. The inhibition of PKC expression in HL-60
neutrophil-like cells using antisense oligonucleotides (5 and 10 µM)
inhibited fMLP-promoted oxidant production (27 and 50%, respectively),
but not that induced by PMA. In conclusion,
p47phox is a substrate for PKC and
participates in the signaling cascade between fMLP receptors and NADPH
oxidase activation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PKC comprises a family of isoenzymes that play a key role in signaling
events and cell functions (21, 22). PKC has been
classified into the following three subgroups on the basis of their
molecular structure and mode of activation: 1) "conventional" PKCs
(
, 
1, 2, and 
), which require phosphatidylserine (PS) and
are activated by calcium and diacylglycerol (DAG); 2) "novel" PKCs
(
, 
, µ, 
, and 
), which require PS and DAG but not
calcium for activation; and 3) "atypical" PKCs (,
), which
are calcium-independent and are not activated by DAG (21, 22). Among the atypical PKC, PKC can be stimulated by
phospholipids, including phosphatidic acid (PA), phosphatidylinositol
trisphosphates, and ceramides. Activation of PKCs in intact cells is
generally associated with the translocation of the enzyme from the
cytosol to particulate compartments (18, 23, 24, 25).
Immunochemical studies have shown that human PMN express five PKC
isoforms: , 1, II, , and (23, 24, 26, 27, 28, 29).
The respective contribution of each PKC isoform in regulating PMN
respiratory burst has not yet been elucidated, although it has been
shown that PKC may modulate the respiratory burst of both
electropermeabilized PMN (15) and HL-60 cells
differentiated into PMN-like cells (30). PKC , which
like PKC is expressed at high levels in human PMN
(23), was shown to regulate PMN adhesion and chemotaxis
(31). The contribution of PKC to the respiratory burst
has not been documented so far, although it has been shown that PKC
translocates to the plasma membrane and that its activity is increased
in fMLP-stimulated PMN (31, 32, 33).
In the present work we have studied the role of PKC in the
signaling pathway between stimulated fMLP receptors and NADPH oxidase
activation. The results indicate that PKC phosphorylates selective
residues of p47phox and plays a predominant role
in the stimulation of superoxide production in fMLP-stimulated
human PMN.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
[ -32P]ATP (sp. act., 6000 Ci/mmol)
and [32P]orthophophoric acid (9000 Ci/mmol)
were obtained from NEN Life Science Products (Boston, MA). Protease
inhibitors (PMSF, diisopropylfluorophosphate, leupeptin, pepstatin, and
aprotinin), cytochrome c, fMLP and PMA, PA, PS, and DAG were
obtained from Sigma-Aldrich (St. Quentin Fallavier, France). Lipofectin
reagent and Ab to PKC were obtained from Life Technologies (Cergy
Pontoise, France). Myristoylated peptide inhibitor of PKC
(Myr-SIYRRGARRWRKL) and PKC (Myr-TRKRQRAMRRRVHQING) were
obtained from BioSource International QCB (Camarillo, CA). The
nonmyristoylated PKC inhibitor peptide (RRGARRWRK) was synthesized
by Neosystem (Strasbourg, France). The purity of both peptides was
>97%. Trypsin was obtained from Boehringer Mannheim (Meylan, France).
Recombinant p47phox and Ab against
p47phox were kindly provided by Dr. B. M.
Babior (Scripps Research Institute, La Jolla, CA). Wild-type
p47phox and mutants (S303/304A, S315A, S320A)
were obtained as previously described (16, 34). The
proteins were expressed in Escherichia coli and purified by
one-step immunoaffinity chromatography (35). Recombinant
mouse PKC was kindly provided by Dr. P. J. Parker (London,
U.K.) (36, 37). Recombinant human PKC was purchased
from Biomol (Tebu, France). HL-60 cells were obtained from the European
Collection of Animal Cell Cultures (Salisbury, U.K.). Thiololated
oligodeoxynucleotides, antisense and sense, to PKC were synthesized
by ScienceTec (Les Ullis, France) and purified by HPLC. The sequence of
the oligodeoxynucleotides was derived from the published coding
sequence of PKC cDNA (38), i.e.,
5'-GGTCCTGCTGGGCATGCGAAAGC and 5'-ATGCCCAGCAGGACC for antisense
and sense, respectively.
Phosphorylation of p47phox in vitro by PKC
Phosphorylation of recombinant p47phox by
recombinant PKC was performed in 40 µl of a mixture containing 50
mM glycero-2 phosphate, 0.4 mM EGTA, 10 mM magnesium acetate, 35 mM
Tris-HCl buffer (pH 7.5), various concentrations of wild-type or mutant
p47phox, and 0.15 U/ml recombinant PKC (1 U
corresponding to 1 nmol phosphate incorporated in peptide /min)
without activators. This PKC preparation has been used previously
to phosphorylate peptide and myristoyl alanine-rich C kinase
substrate peptide (37). Phosphorylation was initiated with
50 µM [-32P]ATP (specific radioactivity,
150–300 Ci/mmol) and conducted at 30°C. After 30 min of incubation,
20 µl of the reaction mixture was spotted onto Whatman p81 paper
(Tewksbury, MA) followed by three washes with 5%
H3PO4, and the
radioactivity was counted. The remaining fraction (20 µl) was mixed
with 3x Laemmli sample buffer (Bio-Rad, Ivry/Seine, France) and
subjected to SDS-PAGE (10% acrylamide gel) (39), then
blotted (40) for autoradiography analysis with Amersham
Hyperfilm MP (Arlington Heights, IL).
Cyanogen bromide (CNBr) cleavage of p47phox and two-dimensional phosphopeptide mapping analysis
The phosphorylated p47phox was subjected to SDS/PAGE and transferred to nitrocellulose. The band of interest was excised and incubated in darkness with 12.5 mg/ml CNBr in 70% (v/v) formic acid for 16 h at room temperature. The reaction mixture was quenched with 1 vol of water and lyophilized in a Speed-Vac (Savant, Holbrook, NY). The digested peptides were then separated by Tris-N-[2-hydroxy-1,1bis(hydromethyl)ethyl]glycine (Tricine)-SDS-PAGE as previously described (16, 41, 42). For peptide mapping, the phosphorylated p47phox blotted onto a nitrocellulose membrane was cut out and resuspended in 200 µl of 50 mM (NH4)HCO3 buffer (pH 7.8) and digested overnight with 25 µg trypsin at 37°C. After drying, the resulting phosphopeptides were resolved on thin-layer cellulose plates by electrophoresis at pH 1.9 for 30 min at 1000 V and 4°C in the first dimension, followed by ascending chromatography developed with isobutyric acid buffer (isobutyric acid/butanol/pyridine/acetic acid/water, 25/1/2.5/1.8/10 (v/v)) (16, 43). 32P-labeled peptides were detected by autoradiography at -70°C.
Preparation of PMN
PMN were isolated from heparinized venous blood of healthy volunteers by two-step sedimentation on dextran and Ficoll-Hypaque (23). Red cells were removed by hypotonic lysis, and the leukocyte suspension (95–97% PMN) was washed and resuspended either in HBSS containing 1.2 mM calcium (pH 7.4) or in phosphate-free buffer (10 mM HEPES (pH 7.4), 137 mM NaCl, 5.4 mM KCl, 5.6 mM glucose, 0.8 mM MgCl2, and 0.025% BSA).
Chemiluminescence measurement
A suspension of 5 x 105 cells was incubated in 0.3 ml HBSS at 37°C in the absence (control) or presence of various concentrations of PKC antagonist for 15 min in the presence of 0.025% BSA. Cells were then incubated with 10 µM luminol and stimulated with 20 nM fMLP or PMA (100 ng/ml). Results are expressed as the percentage of the peak of chemiluminescence or in cpm.
Phosphorylation of p47phox in PMN and immunoprecipitation
Cells were incubated in phosphate-free buffer containing 0.5 mCi
of 32P-labeled phosphoric
acid/108 cells/ml for 60 min at 30°C as
previously reported (44). After washing, PMN (40 x
106/2 ml) were treated in the absence or presence
of PKC peptide antagonists for 30 min in the presence of 0.025% BSA,
then stimulated with either PMA (250 ng/ml) for 6 min or fMLP (20 nM)
for 3 min. The reaction was stopped with ice-cold buffer and
centrifugation at 4°C (400 x g, 6 min). The cells
were suspended at 1 x 108 cells/ml in
homogenization buffer containing 20 mM Tris-HCl (pH 7.5), 0.5% Triton
X-100, 250 mM sucrose, 2 mM EDTA, 5 mM EGTA, 1 mg/ml DNase, 1.5 mM NaF,
1 mM p-nitro-phenyl-phosphate, 1 mM -glycerophosphate, and an
antiprotease mixture consisting of 10 µg/ml leupeptin, pepstatin,
aprotinin, 1 mM diisopropylfluorophosphate, and 1 mM PMSF and disrupted
by sonication (3 x 10 s) at 4°C. Homogenates were
centrifuged for 1 h at 100,000 x g (TL100
Ultracentrifuge; Beckman Coulter, Fullerton, CA), and the supernatant
was incubated overnight with anti-p47phox
Abs (dilution 1/200). Proteins were immunoprecipitated with -bind
G-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ), washed,
and electrophoresed as described (44).
Fractionation of PMN
PMN were treated in HBSS containing 0.025% BSA in the absence or presence of PKC antagonist for 30 min and stimulated with fMLP or PMA under the conditions described above. Membranes were prepared as described (44). Briefly, the PMN suspensions (108 cells/ml) were sonicated three times for 5 s on ice in 1 ml relaxation buffer containing 10 mM PIPES, (pH 7.3), 3 mM MgCl2, 100 mM KCl, and 5 mM NaCl, supplemented with 0.5 mM PMSF, 1 mM EGTA, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. Cytosolic and membrane fractions were prepared by centrifugation (200,000 x g, 30 min) of the homogenates on 15–34% sucrose gradient at 4°C. A membrane preparation equivalent of 5 x 106 cells was used for SDS-PAGE.
In vitro phosphorylation of PMN endogenous p47phox induced by PA
Phosphorylation of endogenous substrates was performed with 20
µl of cytosol fraction from unlabeled PMN (2 x
106 cell equivalent) in a final volume of 100
µl containing 50 mM glycero-2 phosphate, 0.4 mM EGTA, 10 mM magnesium
acetate, and 35 mM Tris-HCl buffer (pH 7.5) to which PA (100 µM) or
DAG/PS/calcium (0, 2/0, 1/1, 2 mM) was added. In some experiments,
cytosolic fractions were pretreated in the presence or absence of
nonmyristoylated PKC peptide antagonist. The reaction was initiated
with [-32P]ATP at a final concentration of
50 µM (specific radioactivity, 150–300 Ci/mmol). Incubation was
conducted at 30°C for 30 min, and the reactions were stopped with 3x
Laemmli buffer. Proteins were heat-denatured, subjected to SDS-PAGE
(10% acrylamide gel), and blotted for autoradiography and Western blot
analysis.
Culture of HL-60 cells and oligonucleotide treatment
Cells were grown in suspension in RPMI 1640 medium (Life
Technologies, Cergy Pontoise, France) supplemented with 10% (v/v)
heat-inactivated FCS, 10 mM HEPES, 2 mM L-glutamine,
streptomycin (50 µg/ml), and penicillin (50 U/ml) in a 5%
CO2 atmosphere. To induce myeloid
differentiation, cells were seeded at a density of 5 x
105–1.0 x 106/ml and
treated with 1.3% DMSO for 4–6 days. For antisense treatment, we
adapted a procedure described by Xu et al. (38), using an
oligonucleotide complementary to the 5' end of the PKC messenger,
starting at the translation start codon. Three days after cell
treatment with DMSO, HL-60 cells were washed, resuspended in DMEM, and
treated with 5 and 10 µM PKC oligonucleotides (antisense or
sense) in the presence of 15 µg/ml lipofectin for 8 h at 37°
in 5% CO2 following the manufacturer’s
instructions. The medium was then replaced by fresh culture medium
containing oligonucleotides with 1.3% DMSO without lipofectin. After
48 h, cells were washed and suspended in HBSS, and superoxide
anion production was continuously recorded by monitoring superoxide
dismutase-inhibitable reduction of cytochrome c, as
described in Ref. 45 , with minor modifications. Aliquots
of 5 x 105 cells in 500 µl of HBSS were
used to generate superoxide anion induced by fMLP (100 nM) or PMA (160
nM). Cells were also lysed in Laemmli sample buffer to analyze the
expression of PKC isoforms and p47phox in
immunoblotting experiments as described above. In control experiments,
lipofectine per se did not induce a marked cytotoxic effect (14 ±
2%) relative to untreated cells (6 ± 2%), as assessed by the
trypan blue exclusion test. The functional activity of differentiated
HL-60 was also decreased by 
15%, determined by measurement of PMA-
or fMLP-induced superoxide production.
Statistical analysis
Each experiment was performed in duplicate and repeated at least three times. Unless otherwise indicated, data represent means ± SEM. Statistically significant differences between means were identified using Student’s paired t test with a threshold of p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The phosphorylation of p47phox on multiple
serines is required for NADPH oxidase activation. Previous studies have
reported that p47phox is a good substrate for
PKCs. However, the contribution of each isoform to the
multiphosphorylation process of p47phox is not
known. We and others have reported that PKC is expressed in human
neutrophils and translocates to the plasma membranes upon PMN
activation (23, 24, 31, 32). To determine whether
p47phox is a potential substrate for PKC , we
analyzed the phosphorylation of recombinant
p47phox in vitro by a constitutively active
recombinant PKC . The results show (Fig. 1
, right) that PKC
phosphorylated p47phox as a function of the
incubation time. In this experiment, which was conducted at 30°C in
the absence of enzyme activators, phosphorylation of
p47phox began to be visible after 3 min and was
maximal at 30 min. In the absence of p47phox, a
weakly phosphorylated band of 80 kDa was detected (Fig. 1, left), which probably corresponded to autophosphorylated PKC
because the band was recognized by an anti-PKC Ab in
Western blotting experiments (results not shown). Phosphorylation
of p47phox by PKC was also dependent on the
concentration of the protein (Fig. 2A). The apparent
Km value of PKC for
p47phox, calculated from a Lineweaver-Burk
representation of the data, was 2.0 µM (Fig. 2B),
indicating that p47phox is a good substrate for
PKC .
|
|
in vitro
In PMN stimulated with PMA or fMLP, p47phox
is phosphorylated on multiple serine residues (42).
Phosphorylated residues were located in the carboxyl-terminal portion
of the protein resulting from CNBr cleavage of
p47phox (42), and previous studies
identified serine residues that were specifically phosphorylated by
"conventional" PKC, mitogen-activated protein kinase (MAP kinase),
and protein kinase A (PKA) (16). To map the
p47phox amino acid residues that were
phosphorylated by PKC , we analyzed the phosphorylated protein by
CNBr cleavage and tryptic peptide mapping. Cleavage of
p47phox by CNBr followed by Tricine-SDS-PAGE
showed that the phosphorylated sites were located in a single peptide
of 14 kDa (Fig. 3, autoradiography)
corresponding to the C-terminal portion of the protein because it was
recognized by an Ab directed against the 10 carboxyl aa 380–390 of
p47phox (Fig. 3, Western blot).
|
(Fig. 4A) showed the presence of
only four phosphopeptides, compared with the much larger number of
phosphorylated peptides found in the phosphopeptide map of the
p47phox phosphorylated by "conventional"
PKCs (Fig. 4B). This result suggests that PKC
specifically phosphorylates a subgroup of serine residues on
p47phox.
|
phosphorylation targets include one or more
of these critical residues, we analyzed the phosphomaps of
p47phox mutants in which the serine residues
mentioned above were replaced by alanine residues S303/304A-, S315A-,
S320A-, and S328A-p47phox. One of the major
phosphopeptides resulting from wild-type p47phox
phosphorylation by PKC was absent in the phosphopeptide map of
mutant S303/304A-p47phox (Fig. 4
C),
indicating that the peptide containing serine residues 303/304 is a
target of PKC . Interestingly, the labeling of the other
phosphopeptides of S303/304A-p47phox was
reduced, suggesting that phosphorylation by PKC may be sequential
and that phosphorylation of serine residues 303/304 occurs first.
Accordingly, the phosphorylation map of the S315A mutant showed the
disappearance of a weakly labeled peptide but not other changes in the
phosphorylation pattern (Fig. 4D). The phosphorylation map
of both the S320A (Fig. 4E) and S328A (not shown) mutants
were identical with that of wild-type p47phox,
indicating that corresponding serine residues do not constitute
phosphorylation targets for PKC .
In stimulated PMN, PKC and p47phox translocate to
plasma membrane fractions, and PKC phosphorylates
p47phox
Stimulation of superoxide production by fMLP in PMN is associated
with the phosphorylation and translocation of
p47phox to the plasma membrane (reviewed in Ref.
6). We reported previously that fMLP induced a
translocation of PKC to the plasma membrane of human PMN
(32). Here, we studied whether this PKC translocation
paralleled that of p47phox, which could be
consistent with a coordinated spatial redistribution of the two
proteins. In PMN stimulated by fMLP, PKC translocated to the plasma
membrane in a rapid and sustained manner (Fig. 5). The membrane translocation of
p47phox induced by fMLP was similar to that of
PKC . Densitometric analysis of the immunoblots showed a good
correlation in the membrane translocation of the two proteins
(r = 0.88, p < 0,01). In addition, a
myristoylated membrane-permeable peptide antagonist (10 µM)
corresponding to the pseudo-substrate region of PKC inhibited
membrane translocation of p47phox by 45%
(Fig. 6, upper
panel). This peptide, which was shown to inhibit protein
kinase activity in vitro (46, 47), was used to demonstrate
that PKC is involved in the regulation of integrin-dependent
adhesion and chemotaxis of intact human neutrophils (31).
A control myristoylated peptide directed against PKC , used at the
same concentration (10 µM), was ineffective on fMLP-induced
translocation of phosphorylated p47phox. This
peptide was chosen as a control because the PKC isoform seems not
to be expressed in human PMN, as determined by immunoblotting
experiments (23, 25). The PKC peptide antagonist did
not alter the translocation of p47phox induced
by PMA, indicating that this peptide did not interfere with
PMA-sensitive PKCs (i.e., "conventional" and "novel" PKCs). The
PKC antagonist (10 µM) also inhibited the fMLP-induced
phosphorylation of p47phox in
32P-loaded PMN, but not the
p47phox phosphorylation induced by PMA (Fig. 6, lower panel), whereas the control peptide was without
effect. These results are consistent with the hypothesis that
fMLP-activated PKC phosphorylates and activates
p47phox in intact PMN.
|
|
participates in the fMLP-dependent activation of NADPH oxidase
The data obtained above suggest that PKC regulates NADPH
oxidase activation. Incubation of PMN in the presence of the
myristoylated peptide antagonist of PKC for 15 min induced a
concentration-dependent inhibition of the fMLP-induced respiratory
burst, as assessed by a chemiluminescence assay (Fig. 7). This effect was not due to a
cytotoxic effect of the peptide because peptide-treated PMN excluded
trypan blue (data not shown). Unlike the PKC antagonist, the
control PKC peptide antagonist did not inhibit fMLP- and
PMA-induced PMN superoxide production, but rather slightly potentiated
their effect. The concentration of the PKC antagonist used here
(5–15 µM) did not appear to inhibit other PKC isoforms. This was
first suggested by the observation that the myristoylated peptide did
not alter PMA-induced superoxide production, in agreement with previous
data (31). In addition, measurement of in vitro
phosphorylation of endogeneous p47phox upon
stimulation of conventional PKC by diglycerides and calcium in a
cytosolic fraction of PMN was not altered by low peptide concentrations
(5–15 µM) (Fig. 8). By contrast, this
low peptide concentration did inhibit p47phox
phosphorylation induced by a PKC activator (PA). Higher peptide
concentrations (20–50 µM) inhibited the activation of conventional
PKC (Fig. 8). Taken together, our results indicate that the
down-regulation of PMN respiratory burst by the myristoylated PKC
-derived peptide results from inhibition of PKC function.
|
|
, we
down-regulated its expression with antisense phosphorothioate
oligonucleotides. For this purpose, HL-60 cells differentiated into
neutrophils were used rather than fresh blood PMN because this latter
cell type is inappropriate for the antisense strategy, given their low
transcriptional activity and short survival. After differentiation of
HL-60 cells into neutrophil-like cells with DMSO, cells were incubated
with 5 and 10 µM PKC sense (control) and antisense
oligonucleotides. Immunoblot experiments (Fig.
9, lower panel) showed that this
treatment efficiently inhibited the expression of PKC relative to
control cells (cells incubated with the sense oligonucleotide). The
antisense oligonucleotide did not alter the expression of other PKC
isoforms (, , and ) or of p47phox (Fig. 9, lower panel), indicating the specificity of the
oligonucleotide treatment toward PKC . Both concentrations of PKC
antisense oligonucleotide significantly inhibited the fMLP-promoted
production of superoxide anion relative to controls (Fig. 9, upper panel). In contrast, the PMN respiratory burst induced
by PMA was not altered. This fact is consistent with the observation
that the expression level of "conventional" and "novel" PKCs
was not altered by the oligonucleotide and shows that the NADPH oxidase
complex remained fully functional in these experiments. These results,
obtained with the two different approaches, strongly indicate that PKC
is a major effector of fMLP receptors in the signaling pathway
leading to NADPH oxidase activation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. PKC selectively targeted only a few serine residues on
p47phox, mainly serines 303/304 and 315. In
addition, using two different strategies based on the inhibition of PKC
activity or enzyme expression, we provide the first evidence that
PKC is indeed involved in the regulation of fMLP-induced PMN
respiratory burst.
In intact cells, p47phox is phosphorylated on
several serine residues located between positions 303 and 379. Previous
studies have shown that p47phox is
phosphorylated in vitro by different types of protein kinases,
including "conventional" PKC (14, 16) such as PKC
(17), MAP kinases (p42-ERK2 and p38) (16, 49), PKA (16, 50), p21-activated kinases
(51), undefined protein kinases (17, 52), and
PKC (53). Phosphopeptide mapping of
p47phox revealed that "conventional" PKC,
MAP kinase, and PKA phosphorylate different groups of serine residues,
suggesting that these different kinases may have different regulatory
effects on NADPH oxidase (16). The comparison of
phosphopeptide maps described here further indicates that some
specificity exists among members of the PKC family for the
phosphorylation of p47phox. Indeed, PKC
selectively phosphorylated serine residues at position 303/304, and, to
a lesser extent, at position 315, as well as two other residues that
remain to be determined. PKC did not phosphorylate the other
residues that were phosphorylated by "conventional" PKCs,
indicating that PKC and conventional PKCs may have discrete
specificities for p47phox functions. In
addition, the sites previously found to be phosphorylated by MAP kinase
(S345/348A) and by PKA (S320A and one or both peptides containing S328A
and S359/370A) (16) were not phosphorylated by PKC
.
Site-directed mutagenesis studies using EBV-transformed lymphocytes
expressing exogenous wild-type or p47phox
mutants in which serine residues were replaced by alanine residues
indicated that each individual serine residue so far identified is
phosphorylated independently (34). Our data regarding
phosphorylation of the three p47phox mutants
(S315A, S320A, and S328A) by PKC are in agreement with this
assumption. However, in the case of the p47phox
S303/304A mutant, the phosphorylation of some residues by PKC was
strongly blunted. The decreased phosphorylation might result from an
altered conformation of p47phox caused by the
mutation. However, this possibility can be ruled out because the
p47phox mutant S303/304A is still phosphorylated
in PMA-activated EBV-lymphoblasts (34) and by
"conventional" PKCs in vitro (data not shown). Alternatively, the
strong decrease in the phosphorylation of the
p47phox mutant suggests that phosphorylation of
S303/304A may facilitate the subsequent phosphorylation of other
residues. Finally, the double mutation S(303 + 304)A inhibited
superoxide production in B lymphoblasts (54), underlining
the importance of the phosphorylation of these two serine residues in
oxidase activation in intact cells.
p47phox is extensively phosphorylated during PMN
stimulation (42) and plays the main role in transporting
the cytosolic oxidase components to the plasma membrane
(13). fMLP and PMA, two major activators of NADPH oxidase,
induce the phosphorylation of several serine residues including
S303/304A and S315A (42). In human PMN stimulated by fMLP,
PKC was shown to be activated. To examine the contribution of PKC
to NADPH oxidase activity, we used a synthetic peptide antagonist
of PKC to down-regulate the enzyme activity. The sequence of this
peptide corresponds to the pseudosubstrate region of the N-terminal
regulatory domain that maintains the enzyme in an inactive form in the
absence of activator (55). This peptide, which was
previously found to block PKC -dependent signal transduction in
Xenopus oocytes and mouse fibroblasts (46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56),
inhibited 50% of the fMLP-induced translocation of
p47phox and PMN respiratory burst. The
specificity of this inhibition was suggested by the absence of
alteration of PMA-induced respiratory burst. Because PMA is a direct
activator of both "conventional" and "novel" PKCs (21, 57), these data further suggest that the peptide antagonist does
not interfere with oxidase activation mediated by these two classes of
PKC. From these experiments, it appears that PKC may contribute
substantially to the stimulation of PMN respiratory burst by
chemoattractants. Consistent with this assumption, the peptide
antagonist inhibited the phosphorylation and translocation of
p47phox induced by fMLP but not by PMA. Further
evidence for the involvement of PKC in NADPH oxidase regulation
comes from the antisense-mediated inhibition of PKC expression in
neutrophil-like cells from differentiated HL-60 cells. Again, this
treatment, which caused a significant inhibition of the respiratory
burst induced by fMLP, was ineffective on PMA-mediated superoxide
production. The oligonucleotide treatment did not alter the expression
of other PKC isoforms (, , and ), further indicating its
specificity for PKC . The sense sequence of PKC we used is also
shared by a recently cloned human serine/threonine kinase termed kpm
(58). However, the sense sequence within kpm is located
more than 1500 bp away from the initiation codon, which makes the
antisense oligonucleotides less effective than those targeting around
the translational start codon, as in the case of PKC . In addition,
kpm is expressed during mitosis and has been shown to regulate cell
cycle; there is no evidence that kpm can be activated by PKC
activators. Finally, an important contribution of PKC in
respiratory burst is further illustrated by the strong inhibition of
respiratory burst and p47phox phosphorylation by
the PKC peptide antagonist (pseudo-substrate region), the sequence
of which is not present in kpm.
PKC can be activated in vitro by various lipidic second messengers
that are generated by different signaling effectors. Among these
messengers, phosphatidylinositol-3,4,5 trisphosphate is formed by
phosphatidylinositol 3-kinase (PI3-kinase); PA is produced by
phospholipase D (PLD) or DAG kinase; and ceramide is generated by
sphingomyelinase. These signaling effectors are all stimulated in PMN
by fMLP and may have a potential role in regulating the respiratory
burst (48), although their respective contribution to the
stimulation of PKC remains unknown. It was shown recently that in
PMN from PI3-kinase- knockout mice the PMN respiratory burst induced
by fMLP was markedly inhibited (59, 60). Interestingly, in
this model the translocation of p47phox as well
as Rac to the plasma membrane was not decreased (59),
indicating that signaling pathways other than PI3-kinase- may be
required for redistribution of p47phox and Rac.
The PLD pathway appears to be a possible candidate for this regulation.
This hypothesis is supported by the observation that PLD is a major
source of PA and diglycerides in stimulated PMN (45, 61)
and may thus contribute to the activation of PKC and other PKC
isoforms. In addition, the PLD-derived PA production was associated
with a priming of PMN respiratory burst (45, 62, 63), and
this priming was accompanied by an enhanced phosphorylation of
p47phox (19, 64). This study
supports the hypothesis that several pathways might participate in
p47phox phosphorylation. In PMA-activated cells,
"conventional" PKCs, and possibly "novel" PKCs, are activated
and phosphorylate p47phox. However, in more
physiological conditions such as fMLP stimulation of neutrophils, other
pathways, and so other PKC isoforms (such as PKC ), are activated
and participate in p47phox phosphorylation. The
cooperativity of different protein kinases or multiple isoforms of PKC
in phosphorylating p47phox is explained by the
fact that these kinases phosphorylate specific targets on
p47phox.
In conclusion, this study shows that p47phox is
a substrate for PKC in vitro. Phosphorylation of
p47phox occurs on the serine residue 303/304, on
serine 315, and on two unidentified residues. The inhibition of PKC
activity or enzyme expression in human PMN by the use of a specific
antagonist or antisense oligonucleotide markedly inhibited fMLP-induced
superoxide production, membrane translocation of PKC , and
phosphorylation of p47phox. These data provide
evidence of the involvement of PKC in the signaling pathways
leading to NADPH oxidase activation by fMLP receptors, but not by PMA,
and suggest a role for PKC in regulating the bactericidal function
of PMN.
|
Acknowledgments |
|---|
and careful suggestions. We also thank
S. Marullo and M. Scott for critically reading the
manuscript, and M. Lenoir for technical assistance. |
Footnotes |
|---|
2 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; PKC, protein kinase C; PKA, protein kinase A; PLD, phospholipase D; MAP kinase, mitogen-activated protein kinase; p47phox, the 47-kDa protein component of phagocyte oxidase; DAG, diacylglycerol; Tricine, N-[2-hydroxy-1,1bis(hydromethyl)ethyl]glycine; PS, phosphatidylserine; PA, phosphatidic acid; CNBr, cyanogen bromide; PI3-kinase, phosphatidylinositol 3-kinase.
Received for publication August 2, 2000. Accepted for publication October 19, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-PKC and novel calcium-independent nPKC. Biochim. Biophys. Acta 1176:276.[Medline]
in human neutrophils. FEBS Lett. 349:338.[Medline]
in human neutrophils. J. Immunol. 157:4641.[Abstract]
-protein kinase C and a phospholipid-dependent protein kinase which is inhibited by long chain fatty acyl coenzyme A. J. Biol. Chem. 266:9285.-protein kinase C in signaling for O-2 generation but not degranulation or adherence in differentiated HL60 cells. J. Biol. Chem. 273:27292. protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis. J. Biol. Chem. 273:30306.2 integrin gene expression is mediated by protein kinase C-. J. Cell Biol. 134:1301. subspecies in maturation of Xenopus laevis oocytes. Mol. Cell Biol. 12:3776. isoform is critical for mitogenic signal transduction. Cell 74:555.[Medline]
in inflammation. Science 287:1049.2 and -3 and PI3K in chemoattractant-mediated signal transduction. Science 287:1046. priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation. J. Biol. Chem. 266:4173.This article has been cited by other articles:
|
|
 |
|
  R. D. Ye, F. Boulay, J. M. Wang, C. Dahlgren, C. Gerard, M. Parmentier, C. N. Serhan, and P. M. Murphy International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the Formyl Peptide Receptor (FPR) Family Pharmacol. Rev., June 1, 2009; 61(2): 119 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Huang, L.-Y. Chen, A. M. Doerner, W. W. Pan, L. Smith, S. Huang, T. J. Papadimos, and Z. K. Pan An Atypical Protein Kinase C (PKC{zeta}) Plays a Critical Role in Lipopolysaccharide-Activated NF-{kappa}B in Human Peripheral Blood Monocytes and Macrophages J. Immunol., May 1, 2009; 182(9): 5810 - 5815. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Brechard, A. Salsmann, and E. J. Tschirhart OAG induces an additional PKC-, PI3K-, and Rac2-mediated signaling pathway up-regulating NOX2 activity, independently of Ca2+ entry J. Leukoc. Biol., April 1, 2009; 85(4): 638 - 647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Raad, M.-H. Paclet, T. Boussetta, Y. Kroviarski, F. Morel, M. T. Quinn, M.-A. Gougerot-Pocidalo, P. M.-C. Dang, and J. El-Benna Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox FASEB J, April 1, 2009; 23(4): 1011 - 1022. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Brechard and E. J. Tschirhart Regulation of superoxide production in neutrophils: role of calcium influx J. Leukoc. Biol., November 1, 2008; 84(5): 1223 - 1237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Reinehr, A. Sommerfeld, V. Keitel, S. Grether-Beck, and D. Haussinger Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes J. Biol. Chem., January 25, 2008; 283(4): 2211 - 2222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. T. Huang, C. J. Paredes, E. T. Papoutsakis, and W. M. Miller Gene expression analysis illuminates the transcriptional programs underlying the functional activity of ex vivo-expanded granulocytes Physiol Genomics, September 11, 2007; 31(1): 114 - 125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Qian, K. S. Tan, S.-J. Wei, H.-M. Wu, Z. Xu, B. Wilson, R.-B. Lu, J.-S. Hong, and P. M. Flood Microglia-Mediated Neurotoxicity Is Inhibited by Morphine through an Opioid Receptor-Independent Reduction of NADPH Oxidase Activity J. Immunol., July 15, 2007; 179(2): 1198 - 1209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kuribayashi, K. Nakamura, M. Tanaka, T. Sato, J. Kato, K. Sasaki, R. Takimoto, K. Kogawa, T. Terui, T. Takayama, et al. Essential role of protein kinase C {zeta} in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP J. Cell Biol., March 26, 2007; 176(7): 1049 - 1060. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Lavigne, J. E. Albina, and J. S. Reichner beta-Glucan Is a Fungal Determinant for Adhesion-Dependent Human Neutrophil Functions J. Immunol., December 15, 2006; 177(12): 8667 - 8675. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, Z. Xie, R. Reece, D. Pimental, and M.-H. Zou Uncoupling of Endothelial Nitric Oxidase Synthase by Hypochlorous Acid: Role of NAD(P)H Oxidase-Derived Superoxide and Peroxynitrite Arterioscler Thromb Vasc Biol, December 1, 2006; 26(12): 2688 - 2695. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Reinehr, S. Becker, J. Braun, A. Eberle, S. Grether-Beck, and D. Haussinger Endosomal Acidification and Activation of NADPH Oxidase Isoforms Are Upstream Events in Hyperosmolarity-induced Hepatocyte Apoptosis J. Biol. Chem., August 11, 2006; 281(32): 23150 - 23166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. D. Ellson, K. Davidson, G. J. Ferguson, R. O'Connor, L. R. Stephens, and P. T. Hawkins Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing J. Exp. Med., August 7, 2006; 203(8): 1927 - 1937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Frey, X. Gao, K. Javaid, S. S. Siddiqui, A. Rahman, and A. B. Malik Phosphatidylinositol 3-Kinase {gamma} Signaling through Protein Kinase C{zeta} Induces NADPH Oxidase-mediated Oxidant Generation and NF-{kappa}B Activation in Endothelial Cells J. Biol. Chem., June 9, 2006; 281(23): 16128 - 16138. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lendemans, M. Rani, C. Selbach, E. Kreuzfelder, F. U. Schade, and S. Flohe GM-CSF priming of human monocytes is dependent on ERK1/2 activation Innate Immunity, February 1, 2006; 12(1): 10 - 20. [Abstract] [PDF]
|
|
|
|
|
|
C. Kim and M. C. Dinauer Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid J. Leukoc. Biol., January 1, 2006; 79(1): 223 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Dey, A. Sarkar, N. Majumder, S. Bhattacharyya (Majumdar), K. Roychoudhury, S. Bhattacharyya, S. Roy, and S. Majumdar Regulation of Impaired Protein Kinase C Signaling by Chemokines in Murine Macrophages during Visceral Leishmaniasis Infect. Immun., December 1, 2005; 73(12): 8334 - 8344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. R. Sheppard, M. R. Kelher, E. E. Moore, N. J. D. McLaughlin, A. Banerjee, and C. C. Silliman Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation J. Leukoc. Biol., November 1, 2005; 78(5): 1025 - 1042. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Omiyi, R. J. Brue, P. Taormina II, M. Harvey, N. Atkinson, and L. H. Young Protein Kinase C {beta}II Peptide Inhibitor Exerts Cardioprotective Effects in Rat Cardiac Ischemia/Reperfusion Injury J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 542 - 551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Phillipson, E. E. Peterman, P. Taormina Jr., M. Harvey, R. J. Brue, N. Atkinson, D. Omiyi, U. Chukwu, and L. H. Young Protein kinase C-{zeta} inhibition exerts cardioprotective effects in ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H898 - H907. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Reinehr, S. Becker, A. Eberle, S. Grether-Beck, and D. Haussinger Involvement of NADPH Oxidase Isoforms and Src Family Kinases in CD95-dependent Hepatocyte Apoptosis J. Biol. Chem., July 22, 2005; 280(29): 27179 - 27194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-Y. Chen, A. Doerner, P. F. Lehmann, S. Huang, G. Zhong, and Z. K. Pan A Novel Protein Kinase C (PKC{epsilon}) Is Required for fMet-Leu-Phe-induced Activation of NF-{kappa}B in Human Peripheral Blood Monocytes J. Biol. Chem., June 10, 2005; 280(23): 22497 - 22501. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sasamoto, M. Nagino, S. Kobayashi, K. Naruse, Y. Nimura, and M. Sokabe Mechanotransduction by integrin is essential for IL-6 secretion from endothelial cells in response to uniaxial continuous stretch Am J Physiol Cell Physiol, May 1, 2005; 288(5): C1012 - C1022. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Rask-Madsen and G. L. King Proatherosclerotic Mechanisms Involving Protein Kinase C in Diabetes and Insulin Resistance Arterioscler Thromb Vasc Biol, March 1, 2005; 25(3): 487 - 496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Bey, B. Xu, A. Bhattacharjee, C. M. Oldfield, X. Zhao, Q. Li, V. Subbulakshmi, G. M. Feldman, F. B. Wientjes, and M. K. Cathcart Protein Kinase C{delta} Is Required for p47phox Phosphorylation and Translocation in Activated Human Monocytes J. Immunol., November 1, 2004; 173(9): 5730 - 5738. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Tuluc, A. Garcia, O. Bredetean, J. Meshki, and S. P. Kunapuli Primary granule release from human neutrophils is potentiated by soluble fibrinogen through a mechanism depending on multiple intracellular signaling pathways Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1264 - C1272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Quinn and K. A. Gauss Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases J. Leukoc. Biol., October 1, 2004; 76(4): 760 - 781. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ueyama, M. R. Lennartz, Y. Noda, T. Kobayashi, Y. Shirai, K. Rikitake, T. Yamasaki, S. Hayashi, N. Sakai, H. Seguchi, et al. Superoxide Production at Phagosomal Cup/Phagosome through {beta}I Protein Kinase C during Fc{gamma}R-Mediated Phagocytosis in Microglia J. Immunol., October 1, 2004; 173(7): 4582 - 4589. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tsunawaki, L. S. Yoshida, S. Nishida, T. Kobayashi, and T. Shimoyama Fungal Metabolite Gliotoxin Inhibits Assembly of the Human Respiratory Burst NADPH Oxidase Infect. Immun., June 1, 2004; 72(6): 3373 - 3382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lenoir, B. Djerdjouri, and A. Perianin Stroma Cell-Derived Factor 1{alpha} Mediates Desensitization of Human Neutrophil Respiratory Burst in Synovial Fluid from Rheumatoid Arthritic Patients J. Immunol., June 1, 2004; 172(11): 7136 - 7143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-S. Kim, B. A. Diebold, J.-I. Kim, J. Kim, J.-Y. Lee, and J.-B. Park Rho Is Involved in Superoxide Formation during Phagocytosis of Opsonized Zymosans J. Biol. Chem., May 14, 2004; 279(20): 21589 - 21597. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Oshizawa, T. Yamaguchi, K. Suzuki, Y. Yamamoto, and T. Hayakawa Possible Involvement of Optimally Phosphorylated L-Plastin in Activation of Superoxide-Generating NADPH Oxidase J. Biochem., December 1, 2003; 134(6): 827 - 834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Dewas, P. M.-C. Dang, M.-A. Gougerot-Pocidalo, and J. El-Benna TNF-{alpha} Induces Phosphorylation of p47phox in Human Neutrophils: Partial Phosphorylation of p47phox Is a Common Event of Priming of Human Neutrophils by TNF-{alpha} and Granulocyte-Macrophage Colony-Stimulating Factor J. Immunol., October 15, 2003; 171(8): 4392 - 4398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. G. Shenoy, G. J. Gleich, and L. L. Thomas Eosinophil Major Basic Protein Stimulates Neutrophil Superoxide Production by a Class IA Phosphoinositide 3-Kinase and Protein Kinase C-{zeta}-Dependent Pathway J. Immunol., October 1, 2003; 171(7): 3734 - 3741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.M. Touyz, G. Yao, and E.L. Schiffrin c-Src Induces Phosphorylation and Translocation of p47phox: Role in Superoxide Generation by Angiotensin II in Human Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 981 - 987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Chen, D. W. Powell, M. J. Rane, S. Singh, W. Butt, J. B. Klein, and K. R. McLeish Akt Phosphorylates p47phox and Mediates Respiratory Burst Activity in Human Neutrophils J. Immunol., May 15, 2003; 170(10): 5302 - 5308. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Siflinger-Birnboim and A. Johnson Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury Am J Physiol Lung Cell Mol Physiol, March 1, 2003; 284(3): L435 - L451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-J. Cheng, Y.-J. Chao, and D. L. Wang Cyclic Strain Activates Redox-sensitive Proline-rich Tyrosine Kinase 2 (PYK2) in Endothelial Cells J. Biol. Chem., December 6, 2002; 277(50): 48152 - 48157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-J. Rabiet, M. Tardif, L. Braun, and F. Boulay Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells Blood, August 13, 2002; 100(5): 1835 - 1844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Kindzelskii and H. R. Petty From the Cover: Apparent role of traveling metabolic waves in oxidant release by living neutrophils PNAS, July 9, 2002; 99(14): 9207 - 9212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Frey, A. Rahman, J. C. Kefer, R. D. Minshall, and A. B. Malik PKC{zeta} Regulates TNF-{alpha}-Induced Activation of NADPH Oxidase in Endothelial Cells Circ. Res., May 17, 2002; 90(9): 1012 - 1019. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
