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INTRODUCTION |
Ligand-initiated activation of superoxide anion (O
2)
generation by phagocytic cells such as neutrophils and
neutrophilic-differentiated HL60 cells (dHL60
cells),1 involves
translocation of cytosolic components p47phox and
p67phox to the membrane and interaction with
membrane-associated cytochrome b558 (1-2).
Protein kinase C (PKC), a phospholipid-dependent family of
serine/threonine kinases, acts in the signal transduction pathway for
O
2 generation and is critical for assembly of an active NADPH
oxidase (3-7). Phosphorylation of p47phox allows a
conformational change, translocation of the p47phox to the
membrane, and assembly of an active NADPH oxidase (7). p47phox
contains multiple phosphorylation sites including classical PKC substrate sites; p47phox is phosphorylated by
-PKC in
vitro and is phosphorylated in ligand-activated phagocytic cells
(1-3, 8).
PKC is a family of structurally related isotypes, with differing
cofactor requirements but similar substrate specificity (9-12). Classical PKC isotypes
-PKC,
-PKC, and
-PKC are
phosphatidylserine (PS)-, diglyceride (DG)-, and
Ca2+-dependent; novel PKC isotypes
-PKC,
-PKC, and
- and
-PKC also require PS and DG but are
Ca2+-independent. The atypical PKC isotypes,
-PKC, and
-PKC, require PS but are DG- and
Ca2+-independent (9-12). PKC isotypes differ in their
tissue distribution and localization within the cell, suggesting that
each isotype plays a specific role in specific signal transduction
pathways. dHL60 cells and neutrophils possess multiple PKC isotypes
including
-PKC,
I-PKC,
II-PKC,
-PKC, and
-PKC (8,
13-15).
Depletion of
-PKC by antisense pretreatment was previously shown to
inhibit phosphorylation of p47phox, translocation of
p47phox to the membrane, and generation of O
2 in
response to cell activation by ligands such as fMet-Leu-Phe or to the
PKC activator phorbol myristate acetate (PMA) (8). The ability of a
-PKC specific inhibitor to reduce ligand-initiated O
2
generation also indicated that
-PKC is essential for activation of
O
2 generation (16). However, these studies did not distinguish
between a role for
I-PKC or
II-PKC, isotypes that are identical
except for the C-terminal V5 variable region (9, 10). The antisense
oligonucleotide targeted the transcriptional start site, which is
common to both these isoforms, and the inhibitor inhibited both
I-PKC and
II-PKC (8, 16).
Formation of a signaling complex that can target
-PKC to substrates
such as p47phox and p47phox to the cell membrane is
essential for specificity and efficiency of signal transduction (17).
However
-PKC plays a role in signaling for multiple cell responses.
-PKC is essential for both proliferation (18) and for O
2
generation in HL60 cells (8), events that occur at the nucleus and
plasmalemma, respectively.
-PKC also associates with the
cytoskeleton (19). Therefore spatial considerations are a key element
in defining a role for
-PKC in signal transduction for a particular
response.
-PKC must be directed to different locations in the cell
for each function, suggesting a role for scaffold proteins or
PKC-binding proteins in
-PKC-based signaling for activation of
O
2 generation (20-22).
Receptor for Activated C
Kinase (RACKs) are a family of cytoskeleton and
membrane-associated anchor molecules that bind activated, Ca2+/DG-dependent PKC isotypes
-,
-, and
-PKC as well as phospholipase C
(23-30). PKC isotypes possess a
pseudo-Rack binding site in the Ca2+ binding domain of
,
, and
-PKC (28). The conformational change in PKC induced by
cofactors frees the RACK binding site and allows the PKC to bind to
RACK. Thus cofactors simultaneously activate and target PKC isotypes. A
peptide based on a sequence in annexin I disrupts the binding of PKC to
RACK (24-27). When peptide I is injected into Xenopus
oocytes, it inhibits insulin-induced translocation of
-PKC and
oocyte maturation (25, 26). RACK1 is a binding protein for
II-PKC
(31).
In this study we have assessed the roles of the PKC isotypes
I-PKC
and
II-PKC in O
2 generation and, secondly, the role of
RACK1 in
-PKC-based signaling for O
2 generation. A
I-PKC null subclone of HL60 cells (32) and depletion of
II-PKC by an
antisense strategy was used to demonstrate that
II-PKC, but not
I-PKC, is necessary for activation of O
2 generation.
rh
II-PKC bound to a number of endogenous HL60 proteins in a
cofactor-dependent manner, including RACK1 and
p47phox. Depletion of RACK1 by an antisense strategy and
electroporation of cells with a peptide that inhibits the
RACK1-
II-PKC interaction enhanced fMet-Leu-Phe- and PMA-induced
O
2 generation. Therefore RACK1 is not essential in signaling
for activation of the NADPH oxidase but may down-regulate
II-PKC-based signaling either by diverting the
II-PKC to another
signaling pathway or by sequestering
II-PKC as part of a
down-regulation step in O
2 generation.
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MATERIALS AND METHODS |
HL60 Cell Culture--
Human promyelocytic HL60 leukemia cells
were obtained from the American Type Culture Collection. The cells were
grown in suspension culture in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1% minimal essential
medium vitamin solution, 1% nonessential amino acids, 0.1%
gentamicin, and 10% heat-inactivated fetal bovine serum. The cell
cultures were maintained at 37 °C in a 5%
CO2-humidified atmosphere. The initial culture was positive
for
-PKC,
I-PKC,
II-PKC,
-PKC, and
-PKC. A clone that
was protein null for
I-PKC but positive for
-PKC,
II-PKC,
-PKC, and
-PKC was selected (32).
Oligonucleotide Synthesis and Sequences--
An antisense
oligonucleotide was designed against the translation start site of
human RACK1 using the commercial primer analysis software Oligo
(National Biosciences). A 20-mer sequence was chosen that was without
significant self-complimentarity and was optimized for maximal
Tm to promote high affinity binding to mRNA; a
Tm of 64.6 °C was calculated at 150 mM salt and 37 °C. The 20-mer oligonucleotides had the
following sequences: RACK1 antisense (RACK1 AS): 5'-T GCC ACG AAG GGT
CAT CTG C-3'; RACK1 sense: 5'-G CAG ATG ACC CTT CGT GGC A-3'. A
scrambled missense oligonucleotide of the RACK1 AS was used as a
control. The unique nature of these sequences was confirmed by
searching the GenBankTM data base. For depletion of
-PKC, a 19-mer oligonucleotide having the sequence
-PKC antisense
(
AS), 5'-AGC CGG GTC AGC CAT CTT G-3', and a scrambled missense
oligonucleotide
-PKC missense (
MS) were used as previously
described (8). Antisense and scrambled control oligonucleotides to
RACK1 and
-PKC were synthesized by the PENN Nucleic Acid Facility as
the phosphorothioate derivatives and purified by high performance
liquid chromatography. In all oligonucleotides, the internucleoside
linkages were completely phosphorothioate-modified.
Treatment of Cells with Oligonucleotides--
Delivery of the
oligonucleotides was enhanced with the cationic lipid
1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium bromide/cholesterol (DMRIE-C) (1:1
(M/M)) (Life Technologies, Inc.). HL60
cells were cultured in the presence of 1.3% Me2SO for 4 days to initiate differentiation to a neutrophil-like phenotype before
treatment with the oligonucleotide. On day 4, the cells were washed and
resuspended in Opti-MEM I reduced serum medium (Life Technologies,
Inc.) at a cell concentration of 25 × 106 cells/well.
Oligonucleotides RACK1 AS, RACK1 MS,
AS, or
MS were suspended in
Optimem at a final concentration of 100-1000 nM and mixed
with the cationic lipid DMRIE-C (4 µg/ml). The cationic lipid/oligonucleotide mixture was added to the cells and incubated at
37 °C for 4 h. An equal volume of RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum plus Me2SO (1.3%
final concentration) was then added, and the cells were cultured for 20 h. On day 5, the cells were washed and resuspended in fresh Opti-MEM medium and treated again with the cationic
lipid/oligonucleotide mixture. After a 4-h incubation, an equal volume
of RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum
plus Me2SO (1.3% final concentration) was then added, and
the cells were cultured for an additional 24 h. The cells were
harvested and suspended in Hepes buffer (pH 7.5) having the composition 150 mM Na+, 5 mM K+,
1.29 mM Ca2+, 1.2 mM
Mg2+, 155 mM Cl
, and
10 mM Hepes (8).
Western Blots--
Lysates of dHL60 cells (1 × 106 cells/sample) were prepared by heating the cells at
95 °C for 5 min in 2× SDS-PAGE sample buffer. The samples were
briefly sonicated (12 s) to reduce viscosity. The dHL60 cell lysates
were run on a 4-12% gradient SDS-PAGE, transferred to a PVDF
membrane, and blocked for 1 h at room temperature with
Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 and 1% BSA,
3% casein. To identify the different PKC isotypes, the membrane was
incubated with a panel of PKC antibodies followed by incubation with
peroxidase-conjugated goat anti-rabbit IgG. For detection of RACK1, the
membrane was incubated with a monoclonal antibody to RACK1, followed by
incubation with peroxidase-conjugated goat anti-mouse IgM.
Immunoreactive bands were visualized by Pierce SuperSignal Ultra
chemiluminescence substrate. The software SigmaProscan (Jandel/SPSS)
was used for densitometric analysis.
Superoxide Anion Generation--
The generation of superoxide
anion (O
2) by dHL60 cells was measured as superoxide dismutase
inhibitable cytochrome c reduction by either a continuous
recording method (33) or end point analysis. Cells were activated by 1 µM fMet-Leu-Phe in the presence of 5 µg/ml cytochalasin
B or by 1 µg/ml PMA in the absence of cytochalasin B.
Immunoprecipitation of
II-PKC and p47phox--
dHL60
cells (50 × 106 cells/ml) were stimulated with either
buffer alone or fMet-Leu-Phe (1 µM) for 1 min. The
reaction was stopped by the addition of cold immunoprecipitation
buffer. Immunoprecipitation buffer consisted of 10 mM Hepes
(pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 2 mM
phenylmethanesulfonyl fluoride, 0.2% Nonidet P-40, 0.027 trypsin
inhibitory units/ml of aprotinin, 2 µg/ml leupeptin, and 5 mg/ml BSA. The samples were then vortexed for 20 min to solubilize the
membrane fraction, and the supernatant was collected after
microcentrifuging for 5 min. A rabbit polyclonal antibody to
p47phox or to
II-PKC was added, and the samples were
incubated for 2 h at 4 °C. Protein A-agarose was added, and the
samples were incubated for 1 h at 4 °C with shaking. The
reaction tubes were then microcentrifuged for 30 s, and the
supernatants were discarded. The protein A-agarose pellet was washed
four times with immunoprecipitation buffer, and the sample was eluted
by incubation for 20 min at 65 °C in 2× SDS-PAGE sample buffer.
Binding of rh
II-PKC to Endogenous dHL60 Cell
Proteins--
The ability of rh
II-PKC to bind to endogenous
proteins from dHL60 cells was assayed by an overlay procedure (30).
Lysates of dHL60 cells were separated by SDS-PAGE and transferred to
PVDF membranes. Membrane strips were incubated with overlay block
buffer consisting of 50 mM Tris-HCl (pH 7.5), 0.1%
polyethylene glycol, 0.2 M NaCl, and 3% BSA for 1 h.
rh
II-PKC (0.1 ng) was bound to the membrane strip for 30 min at room
temperature in an overlay incubation buffer consisting of 50 mM Tris-HCl (pH 7.5), 0.1% polyethylene glycol, 0.2 M NaCl, 12 mM
-mercaptoethanol, 0.1% BSA, 5 µg/ml leupeptin, 10 µg/ml soy bean trypsin inhibitor, 20 mM phenylmethanesulfonyl fluoride in the presence or
absence of 0.1 mM CaCl2, 50 µg/ml PS, and 1 mg/ml DG. The membranes were washed three times for 15 min with
phosphate-buffered saline/Tween (140 mM NaCl, 8 mM Na2HPO4, 1.5 mM
KH2PO4, 3 mM KCl, 0.05% Tween 20 (pH 7.0)) and probed with antibodies to
II-PKC or RACK1.
Electroporation of HL60 Cells--
A method of electroporation
was chosen that allows efficient incorporation of molecules of
molecular mass <1000 Da and transient passage of molecules >1000Da.
We chose a one-pulse protocol to optimize preservation of intracellular
metabolites. The cells were electroporated once in a Bio-Rad Gene
Pulser at 400 V and a capacitance of 500 microfarads. dHL60 cells were
suspended in 800 ml of ice-cold Hepes buffer containing 1 mM ATP and 1 mM NADPH in the presence or
absence of 200 µM peptide I. Previous studies using the
fluorescent probe Dextran-Indo1 conjugate indicated that the attained
intracellular concentration of peptides under these electroporation
conditions is ~10 µM (15).
Statistical Analysis--
Results are expressed as mean ± S.E. (n). Data were analyzed by Student's t test.
Reagents--
Cytochalasin B, cytochrome c, protease
inhibitors (leupeptin, soy bean trypsin inhibitor, and aprotinin), BSA,
PMA, fMet-Leu-Phe, and phenylmethanesulfonyl fluoride were purchased
from Sigma. PMA was stored as a concentrated stock solution in
Me2SO and diluted with Hepes Buffer before use.
fMet-Leu-Phe was stored as a stock solution in ethanol and diluted in
buffer before use. Peptide I, KGDYEKILVALCGGN, was purchased from Coast Scientific.
Anti-peptide polyclonal antibodies to
-PKC,
I-PKC,
II-PKC,
-PKC, and
-PKC and peroxidase-conjugated goat anti-rabbit IgG and
peroxidase-conjugated goat anti-mouse IgG were obtained from Santa Cruz
Biotechnology. Peroxidase-conjugated anti-mouse IgM was obtained from
Kirkegaard and Perry Laboratories. Mouse monoclonal antibodies to
-PKC,
-PKC, and RACK1 and a rabbit polyclonal antibody to
p47phox were purchased from Transduction Laboratories. Protein
A-agarose was obtained from Life Technologies, Inc.
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RESULTS |
A Role for
II-PKC in Ligand-initiated O
2
Generation--
A clone of HL60 cells that contained the
II-PKC
isotype of PKC as well as
-PKC,
-PKC, and
-PKC was selected
and probed for immunoreactivity to PKC antibodies (Fig.
1A). In comparison to the
parent cell line, which contains
I-PKC, the
I-PKC protein null
line contained no detectable amount of
I-PKC (Fig. 1A). In contrast, both
I-PKC-positive and
I-PKC null cell lines
contained equivalent amounts of
-PKC,
-PKC, and
-PKC. Previous
studies in which both
I-PKC and
II-PKC were depleted by an
antisense strategy demonstrated a role for
-PKC in activation of the
NADPH oxidase in dHL60 cells (8). To discriminate between roles for
I-PKC and
II-PKC in ligand-initiated activation of O
2
generation, we compared fMet-Leu-Phe-triggered O
2 generation
in
I-PKC null and
I-PKC-positive dHL60 cells. Generation of
O
2 triggered by 1 µM fMet-Leu-Phe was 11.3 ± 1.9 (n = 12) nmol/106 cells/10 min in
I-PKC null dHL60 cells, a rate that was not significantly different
from the rate of 11.2 ± 1.8 (n = 8)
nmol/106 cells/10 min observed in
I-PKC-positive dHL60
cells (Fig. 1B). Generation of O
2 in response to 1 µg/ml PMA was also similar in
I-PKC null and
I-PKC-positive
dHL60 cells (Fig. 1B). In
I-PKC-positive dHL60 cells, PMA
triggered generation of 22.3 ± 4.1 (n = 5) nmol of O
2/106 cells/10 min, whereas in
I-PKC cells,
PMA triggered generation of 21.2 ± 1.7 (n = 6)
nmol of O
2/106 cells/10 min (Fig. 1B).
Therefore,
I-PKC was not essential for optimal PMA- or
fMet-Leu-Phe-initiated generation of O
2 in dHL60 cells.

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Fig. 1.
O 2 generation in
I-PKC null and I-PKC
positive dHL60 cells. A, selective loss of I-PKC in a I-PKC
null dHL60 clone. Cell lysates were prepared from I-PKC null and
I-PKC-positive dHL 60 cells by adding Laemmli buffer, and the
samples were subjected to 4-12% SDS-PAGE followed by Western blotting
(representative experiment of five). Molecular mass markers are
indicated on the left (K, ×1000), and PKC isotypes are indicated by
arrows on the right. B, ligand-initiated
O 2 generation in I-PKC null and I-PKC-positive dHL60
cells. O 2 generation by I-PKC null dHL60 cells and by
I-PKC-positive dHL60 cells was determined as the superoxide
dismutase inhibitable reduction of cytochrome c. O 2
generation was triggered by 1 µM fMet-Leu-Phe plus 5 µg/ml cytochalasin B or by 1 µg/ml PMA. Data shown are the mean of
8 experiments for I-PKC null cells and 12 experiments for
I-PKC-positive dHL60 cells and are expressed as nmol of
O 2/106 cells/10 min.
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Inhibition of O
2 Generation in
II-PKC-depleted
I-PKC
Null dHL60 Cells--
I-PKC null dHL60 cells were treated for 2 days with 400 nM of an antisense oligonucleotide to
-PKC
(
PKC AS) or with 400 nM control missense oligonucleotide
to
-PKC (
PKC MS) as described under "Materials and Methods."
Treatment with
PKC AS resulted in a reduction in the level of
II-PKC to 362 ± 47 (n = 4) density units (DU)
as compared with a level of 603 ± 34 (n = 4) DU
in control
PKC MS-treated cells (62.2 ± 10.0% control,
p < 0.02) (Fig. 2,
A and B). Treatment of the dHL60 cells with the
antisense or missense oligonucleotides to
-PKC had no significant
effect on the levels of
-PKC,
-PKC, or
-PKC (Fig. 2,
A and B) and as previously shown (8).

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Fig. 2.
Selective depletion of
II-PKC by a -PKC antisense
oligonucleotide in I-PKC null dHL60
cells. I-PKC null dHL60 cells were treated with 400 nM -PKC antisense oligonucleotide (AS) or
-PKC missense oligonucleotide (MS) in the presence of 4 µg/ml DMRIE-C (see "Materials and Methods:). Cell lysates were
prepared by adding Laemmli buffer, and the samples were subjected to
4-12% SDS-PAGE followed by Western blotting and densitometry
(representative experiment of 5). A, Western blots of PKC
isotypes in AS- and MS-pretreated dHL60 cells. PKC isotypes are
indicated on the right. B, scanning densitometry of the
Western blot of PKC isotypes in PKC AS- and PKC MS-pretreated
dHL60 cells was analyzed by ScanPro and plotted as density units
(representative experiment of 5).
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The effect of depletion of
II-PKC on O
2 generation was
determined in
I-PKC null cells treated with
PKC AS or
PKC MS. fMet-Leu-Phe (1 µM) triggered generation of 5.3 ± 1.5 (n = 6) nmol of O
2/106
cells/10 min in
PKC AS-treated
I-PKC null cells, which was significantly less than the level of 12.2 ± 2.7 (n = 6) nmol/106 cells/10 min observed in
control cells pretreated with
PKC MS (41.7 ± 9.5% control,
p < 0.025) (Fig.
3A). The kinetics of
fMet-Leu-Phe-activated O
2 generation are characterized by a
rapid initial rate continuing for ~2 min followed by a slow rate of
generation that ceases by 5-10 min. In contrast, PMA triggers a
sustained generation of O
2. The Vmax of
fMet-Leu-Phe-induced O
2 generation, defined as the maximal
rate of O
2 generation, was reduced in cells depleted of
-PKC. Calculation of the Vmax demonstrated
that in control
PKC MS-treated cells activated by 1 µM
fMet-Leu-Phe, the Vmax was 2.70 ± 0.44 (n = 6) nmol/min/106 cells; the
Vmax of
-PKC-depleted cells treated with
PKC AS was significantly reduced to 1.41 ± 0.74 (n = 6) nmol/min/106 cells (53.0 ± 7.2% control
PKC MS-treated cells (p < 0.01) (Fig. 3B).

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Fig. 3.
Effect of antisense depletion of
-PKC on O 2 generation in
I-PKC null dHL60 cells. O 2 generation
by I-PKC null dHL60 cells depleted of II-PKC was determined in
missense ( PKC MS)- and antisense ( PKC
AS)-pretreated dHL60 cells as described in Fig. 1B.
A, O 2 generation triggered by 1 µM
fMet-Leu-Phe plus 5 µg/ml cytochalasin B (n = 6) and
1 µg/ml PMA (n = 7). Data shown are expressed as nmol
of O 2/106 cells/10 min. B,
Vmax of O 2 generation triggered by 1 µM fMet-Leu-Phe plus 5 µg/ml cytochalasin B and 1 µg/ml PMA. Data shown are expressed as nmol of
O 2/106 cells/min.
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Similarly, activation of control
PKC MS-pretreated cells by 1 µg/ml PMA triggered generation of 16.6 ± 1.5 (n = 7) nmol of O
2/106 cells/10 min, an amount that
was significantly greater than the generation of 8.2 ± 1.3 (n = 7) nmol of O
2/106 cells/10
min observed in dHL60 cells depleted of
-PKC by treatment with
PKC AS (48.7 ± 6.2% control, p < 0.01) (Fig.
3A). The Vmax of PMA-induced
O
2 generation was also significantly decreased in
-PKC-depleted cells as compared with controls. The
Vmax of O
2 generation decreased
significantly from a rate of 3.12 ± 0.37 (n = 7)
nmol/min/106 cells in control
PKC MS-treated cells to a
Vmax of 2.06 ± 0.27 (n = 8) nmol/min/106 cells in
-PKC-depleted cells (66.1 ± 8.2% control, p < 0.01) (Fig. 3B).
Thus, depletion of
II-PKC in
I-PKC null dHL60 cells resulted in
inhibition of the rate of O
2 generation in response to both
fMet-Leu-Phe and PMA, indicating an essential role for the
II
isotype but not the
I isotype of PKC in signaling for O
2 generation.
Cofactor-dependent Binding of rh
II-PKC to Endogenous
Proteins from dHL60 Cells--
-PKC is capable of phosphorylating
multiple proteins in vitro (15). However, in the intact
cell, scaffold proteins may provide added substrate specificity by
targeting the kinase to a particular cellular location. RACK1 is a
scaffold or escort protein that selectively binds to
II-PKC in the
presence of the cofactors PS, DG, and Ca2+. To assess the
presence of binding proteins for
II-PKC in dHL60 cells, we tested
the ability of rh
II-PKC to bind to endogenous dHL60 proteins using
an overlay assay. Lysates of
I-PKC null dHL60 cells were separated
on SDS-PAGE and transferred to PVDF membranes, and the membranes were
incubated with rh
II-PKC in the presence and absence of the PKC
cofactors PS, DG, and Ca2+. Western blotting with an
antibody to
II-PKC demonstrated that the exogenous rh
II-PKC bound
to several endogenous proteins in a cofactor-dependent
manner (Fig. 4A). In the
absence of exogenous rh
II-PKC and cofactors, a band at 80 kDa
corresponding to the endogenous
II-PKC was observed (Fig.
4A, lane 1). rh
II-PKC in the absence of
cofactors bound strongly only to a protein of 19 kDa (Fig.
4A, lane 2). However when rh
II-PKC was added
in the presence of the cofactors PS, DG, and Ca2+,
additional binding of rh
II-PKC to bands of 29, 32, 36, 39, 47, and
55 kDa was observed (Fig. 4A, lane 3). Probing
with an antibody to RACK1 (Fig. 4A, lane 4)
showed a strong band at 36 kDa, demonstrating the presence of RACK1 in
dHL60 cells. Quantitation by densitometry demonstrated that the band at
80 kDa corresponding to endogenous
II-PKC was not affected by the
presence of cofactors and had a density of 81.3 ± 3.1 (n = 3) DU in the absence of cofactors and of 82.7 ± 0.7 DU in the presence of cofactors (Fig. 4B). In contrast, binding of rh
II-PKC to the 36-kDa band, which had the same
molecular mass as RACK1, was significantly enhanced in the presence of
cofactors (Fig. 4B). The density of the 36-kDa band was
34.7 ± 3.1 (n = 3) DU in the absence of cofactors
and significantly enhanced to 77.3 ± 4.7 (n = 3)
DU in the presence of cofactors (p < 0.005 paired
Student's t test). In addition, binding of rh
II-PKC to a
band of 47 kDa, a candidate for p47phox, was enhanced by the
presence of cofactors. Binding of rh
II-PKC to the 47-kDa band was
23.0 ± 1.4 (n = 3) DU in the absence of cofactors
and 60.7 ± 6.9 DU in the presence of cofactors (p < 0.05, paired t test). These results demonstrated that
RACK1 is present in dHL60 cells and that
II-PKC can bind to numerous
HL60 proteins in a cofactor-dependent fashion.

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Fig. 4.
Binding of rh II-PKC
to endogenous proteins from I-PKC null dHL60
cells. dHL60 cell lysates were run on SDS-PAGE and transferred to
PVDF membranes. Membrane strips were incubated for 30 min at room
temperature in an overlay buffer containing rh II-PKC in the presence
and in the absence of the cofactors PS, DG, and Ca2+ (see
"Materials and Methods"). Membrane strips were probed with
antibodies to II-PKC and RACK1, and densitometry was performed.
Molecular mass markers are indicated on the left margin (K, ×1000),
and bands corresponding to II-PKC and RACK1 are indicated on the
right margin. Data shown are representative of three experiments.
A, Western blot of binding of rh II-PKC to endogenous
dHL60 proteins. B, scanning densitometry of the
immunoreactivity to anti II-PKC was analyzed by ScanPro and plotted
as density versus molecular weight.
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Ligand-initiated Association of p47phox with
II-PKC, but
Not with RACK1--
Coimmunoprecipitation was next used as a tool to
determine whether
II-PKC, RACK1, and p47phox formed a
signaling complex in activated dHL60 cells. To determine whether
p47phox and RACK1 were associated with
II-PKC in activated
cells,
II-PKC was immunoprecipitated from resting buffer-treated
dHL60 cells and from cells activated for 1 min with 1 µM
fMet-Leu-Phe. Western blots with an antibody to
II-PKC followed by
densitometry of the immunoprecipitates demonstrated that approximately
equivalent amounts of
II-PKC were derived from resting and activated
cells, 1987 ± 207 (n = 4) DU from resting cells
as compared with 1895 ± 142 DU in fMet-Leu-Phe-activated dHL60
cells (Fig. 5A). Probing of
the immunoprecipitates with an antibody to p47phox demonstrated
a significant increase in the association of p47phox with
II-PKC, from a level of 424 ± 204 (n = 4) DU
in resting cells to a level of 1133 ± 314 DU in cells activated
by fMet-Leu-Phe (p < 0.03) (Fig. 5A).
Probing the
II-PKC immunoprecipitates with an antibody to RACK1 also
revealed that activation of the dHL60 cells with fMet-Leu-Phe triggered
a significant increase in association of RACK1 with the
II-PKC, from
201 ± 115 (n = 3) DU in resting cells to
1105 ± 224 DU in fMet-Leu-Phe-activated cells (p < 0.04) (Fig. 5A). Therefore activation of dHL60 cells by
fMet-Leu-Phe triggers enhanced association of
II-PKC with the
scaffold protein RACK1 as well as with p47phox.

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Fig. 5.
Ligand-initiated association of
II-PKC with p47phox. I-PKC null
dHL60 were pre-treated with buffer or 1 µM fMet-Leu-Phe
for 1 min. II-PKC or p47phox was immunoprecipitated from
each sample, and the immune complexes run on 4-12% gradient SDS-PAGE,
blotted to PVDF membrane, and probed with antibodies to II-PKC,
p47phox, or RACK1. Densitometry was performed on the Western
blots, and results are expressed as density units. A,
immunoprecipitation of II-PKC from dHL60 cells treated with buffer
or fMet-Leu-Phe. B, immunoprecipitation of p47phox
from dHL60 cells treated with buffer or fMet-Leu-Phe.
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To determine whether fMet-Leu-Phe triggered enhanced association of
RACK1 with p47phox, concomitant with the enhanced association
of RACK1 with
II-PKC, p47phox was immunoprecipitated from
resting and activated dHL60 cells. Western blots of immunoprecipitates
of p47phox followed by densitometry demonstrated equivalent
levels of p47phox in resting cells and in cells stimulated for
1 min with 1 µM fMet-Leu-Phe (Fig. 5B).
Densitometry of the Western blots showed a level of p47phox of
1588 ± 293 (n = 3) DU in resting cells as
compared with a level of 1519 ± 284 (n = 3) DU in
cells activated by 1 µM fMet-Leu-Phe. Measurement of the
level of
II-PKC associated with the p47phox in these
immunoprecipitates demonstrated a significant increase in association
of
II-PKC with p47phox (Fig. 5B). The level of
II-PKC observed in resting cells was 451 ± 154 (n = 3) DU as compared with an enhanced level of
912 ± 323 (n = 3) DU in cells activated by
fMet-Leu-Phe (213 ± 36% control, p < 0.05)
(Fig. 5B). Therefore, immunoprecipitation of p47phox
or of
II-PKC demonstrated that cell activation by fMet-Leu-Phe triggered enhanced association of
II-PKC with its substrate
p47phox. In contrast, probing the p47phox
immunoprecipitates with an antibody to RACK1 demonstrated no significant association of RACK1 with p47phox (Fig.
5B). The level of RACK1 associated with the p47phox
immunoprecipitate from resting cells was 21 ± 11 (n = 3) DU, whereas the level in fMet-Leu-Phe-activated
cells was 42 ± 26 (n = 3) DU, a difference that
was not statistically different. Therefore, although fMet-Leu-Phe
triggered an increase in association of
II-PKC with the substrate
p47phox, no increase in association of RACK1 with the
p47phox or p47phox-associated
II-PKC was observed.
We therefore questioned whether RACK1 played a role in signaling for
activation of O
2 generation, a process that is dependent on
II-PKC.
Effect of Inhibitor Peptide, Peptide I, on fMet-Leu-Phe Triggered
O
2 Generation--
Peptide I, KGDYEKILVALCGGN, which is
derived from the annexin I and 14-3-3 sequences, was used to probe a
possible role for RACK1 interaction with
II-PKC in activation of
O
2 generation. Peptide I inhibits the binding of RACK to PKC
and the translocation of
-PKC to the membrane in other cell types
(24-27). We first tested the ability of peptide I to inhibit the
binding of rh
II-PKC to endogenous dHL60 proteins using an overlay
assay (see Fig. 4). Lysates of
I-PKC null dHL60 cells were separated
on SDS-PAGE and transferred to PVDF membranes, and the membranes were
incubated with rh
II-PKC in the presence of the PKC cofactors PS, DG
and Ca2+ and in the presence or absence of 10 µM peptide I. In the presence of 10 µM
peptide I, there was a selective inhibition of the ability of
II-PKC
to bind to a band of 36 kDa that was immunoreactive to RACK1 antibody.
The density of the 36-kDa band was 40.3 ± 11.4 (n = 4) DU in the absence of peptide I and significantly decreased to
25.0 ± 7.2 (n = 4) DU in the presence of 10 µM peptide I (61. 0 ± 7.1% control,
p < 0.005). In contrast, the 80-kDa band, which represents endogenous
II-PKC, was 67.0 ± 3.6 (n = 4) DU in the absence of peptide I and 66.8 ± 3.8 (n = 4) DU in the presence of peptide I. Therefore,
Peptide I inhibits binding of rh
II-PKC to a 36-kDa band that was
immunoreactive to a RACK1 antibody.
Peptide I was then used to probe a role for
II-PKC binding to RACK1
in signaling for O
2 generation. To probe a possible role for
RACK1 interaction with
II-PKC in activation of O
2
generation, cells were electroporated in the presence of buffer or
peptide I. Electroporation of dHL60 cells in the presence of 200 µM peptide I, which gives a final intracellular
concentration of peptide I of ~10 µM, caused a
significant increase in O
2 generation triggered by 1 µM fMet-Leu-Phe (Fig. 6).
fMet-Leu-Phe-triggered O
2 generation of 13.4 ± 3.0 (n = 6) nmol/106 cells/10 min in
buffer-treated cells; in peptide I-treated cells fMet-Leu-Phe-triggered
O
2 generation was increased to 170.2 ± 35.3% control
(n = 4, p < 0.02) (Fig. 6). Similarly,
when O
2 generation was triggered by 1 µg/ml PMA, peptide I
enhanced O
2 generation from a rate of 21.7 ± 3.5 (n = 6) nmol/106 cells/10 min in
buffer-treated cells to a rate that was 121.7 ± 14.6 (n = 6) % control in peptide I-treated cells
(p < 0.05) (Fig. 6). Thus peptide I, which inhibits
the binding of RACK1 to
-PKC and translocation of
-PKC to the
membrane, enhanced rather than inhibited ligand-initiated O
2
generation in
I-PKC null dHL60 cells.

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Fig. 6.
Effect of the inhibitory peptide I on
O 2 generation in electroporated dHL60 cells. The effect
of electroporation of dHL60 cells in the presence or absence of 200 µM peptide I on O 2 generation triggered by 1 µM fMet-Leu-Phe plus 5 µg/ml cytochalasin B and 1 µg/ml PMA. dHL60 cells (10 × 106) were
electropermeabilized in the presence of ATP and NADPH, and O 2
generation was measured as the superoxide dismutase inhibitable
reduction of cytochrome c (see "Materials and Methods").
Data shown are mean of four experiments and are expressed as % of
control.
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Depletion of RACK1 by Antisense Treatment--
The use of peptides
to probe a role for RACK1, particularly in electroporated cells, has
the potential for nonspecific effects. Depletion of RACK1 by an
antisense strategy is a more specific probe in assessing a role for
RACK1 in signaling for activation of the NADPH oxidase.
I-PKC null
dHL60 cells were treated for 2 days with 500 nM
phosphorothioate antisense oligonucleotide to RACK1 (RACK1 AS) or with
500 nM control missense phosphorothioate oligonucleotide to
RACK1 (RACK1 MS) as described under "Materials and Methods."
Treatment with RACK1 AS resulted in a reduction in the level of RACK1
to 1240 ± 273 (n = 8) as compared with a level of
1949 ± 452 (n = 8) in control RACK1 MS-treated
cells (57.9 ± 5.5% control, p < 0.01) (Fig.
7). In contrast, when the blots were
probed with an antibody to
II-PKC, no difference in immunoreactivity
to
II-PKC was observed between the RACK1 AS- and RACK1 MS-pretreated
cells (Fig. 7). Therefore, the RACK1 AS treatment selectively depletes
dHL60 cells of RACK1.

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Fig. 7.
Selective depletion of RACK1 by a RACK1
antisense oligonucleotide. I-PKC null dHL60 cells were treated
with 500 nM RACK1 antisense oligonucleotide (AS)
or RACK1 missense oligonucleotide (MS) in the presence of 4 µg/ml DMRIE-C (see "Materials and Methods"). Cell lysates were
prepared by adding Laemmli buffer, and the samples were subjected to
4-12% SDS-PAGE followed by Western blotting using antibodies to RACK1
and II-PKC (representative experiment of 8).
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Depletion of RACK1 Enhances O
2 Generation by dHL60
Cells--
A role for RACK1 in signaling for activation of the NADPH
oxidase and generation of O
2 was examined using
I-PKC null
dHL60 cells depleted of RACK1 by antisense pretreatment. O
2
generation triggered by 1 µM fMet-Leu-Phe in
RACK1-depleted cells treated with RACK1 AS was significantly increased
to a level of 16.7 ± 3.4 (n = 5)
nmol/106 cells/10 min as compared with a level of 10.7 ± 2.5 (n = 5) nmol/106 cells/10 min in
control RACK1 MS-treated dHL60 cells (175.0 ± 24.9% control,
p < 0.03) (Fig.
8A). A similar increase in
O
2 generation in RACK1-depleted dHL60 cells was observed when
the cells were activated by 1 µg/ml PMA. O
2 generation
triggered by 1 µg/ml PMA in RACK1 AS-reated cells was significantly
increased to a level of 26.3 ± 1.9 (n = 5)
nmol/106 cells/10 min as compared with a level of 22.3 ± 2.5 (n = 5) nmol/106 cells/10 min in
control RACK1 MS-treated dHL60 cells (119.4 ± 6.4% control,
p < 0.03) (Fig. 8A).

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Fig. 8.
Effect of antisense depletion of RACK1 on
O 2 generation in I-PKC null dHL60
cells. O 2 generation by I-PKC null dHL60 cells
depleted of RACK1 was determined as the superoxide dismutase
inhibitable reduction of cytochrome c (see "Materials and
Methods") in missense (RACK1 MS)- and antisense
(RACK1 AS)-pretreated I-PKC null dHL60 cells.
A, O 2 generation was triggered by 1 µM fMet-Leu-Phe plus 5 µg/ml cytochalasin B or by 1 µg/ml PMA and monitored for 10 min. Data shown are the mean of five
experiments and are expressed as nmol of O 2/106
cells/10 min. B, Vmax of O 2
generation triggered by 1 µM fMet-Leu-Phe plus
cytochalasin B and by 1 µg/ml PMA. Data shown are the mean of five
experiments and are expressed as nmol of O 2/106
cells/min.
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In addition, the Vmax of ligand-induced
O
2 generation, defined as the maximal rate of O
2
generation, was enhanced in cells depleted of RACK1 (Fig.
8B). Calculation of the Vmax
demonstrated that in RACK1 AS-treated cells activated by 1 µM fMet-Leu-Phe, the Vmax was
significantly enhanced to a rate of 7.4 ± 0.8 (n = 5) nmol/min/106 cells as compared with a rate of 5.1 ± 0.6 (n = 5) nmol/min/106 in control
cells treated with RACK1 MS (150.0 ± 45.8% control, p < 0.05) (Fig. 8B). Similarly, an increase
in Vmax was observed in RACK1-depleted dHL60
cells activated by 1 µg/ml PMA; however, the increase was not
statistically significant. The Vmax in RACK1 AS-treated cells activated by 1 µg/ml PMA was 5.1 ± 6.2 (n = 5) nmol/min/106 cells as compared with
a Vmax of 3.6 ± 6.2 (n = 5) nmol/min/106 cells in control RACK1 MS-treated dHL60
cells. Therefore, ligand-initiated O
2 generation was enhanced
in cells depleted of RACK1. These findings are in agreement with
studies using the inhibitory peptide, peptide 1, where inhibition of
the interaction of RACK1 with
-PKC also resulted in enhanced
O
2 generation (Fig. 6).
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DISCUSSION |
Phagocytic cells, such as HL60 cells differentiated to a
neutrophilic phenotype, and neutrophils possess multiple forms of PKC
isotypes including Ca2+-dependent
-PKC,
I-PKC, and
II-PKC, Ca2+-independent
-PKC, and
atypical
-PKC. Assembly of an active NADPH oxidase is tightly
controlled and involves association of the cytosolic components
p47phox and p67phox with the plasma membrane-associated
cytochrome b558 (1-3). Phosphorylation of
p47phox on multiple sites is an essential step in triggering
translocation of p47phox to the plasmalemma, where it binds to
the cytochrome b558 (34, 35). A selective role
for
-PKC in activation of the NADPH oxidase and generation of
O
2 has previously been demonstrated using an antisense
strategy (8) and by studies with a
-PKC-selective inhibitor
(16).
HL60 cells and neutrophils contain two
-PKC isotypes,
I-PKC and
II-PKC, which are derived by alternate splicing at the C terminus.
Neither the antisense strategy, which targeted a sequence at the
transcriptional start site, nor the
-PKC-selective inhibitor could
discriminate between roles for these
-PKC isotypes. The present
study demonstrated a specific role for
II-PKC in signaling for
activation of O
2 generation using a
I-PKC null subclone of
dHL60 cells. The
I-PKC HL60 cell subclone was negative for immunoreactivity to
I-PKC but contained equivalent amounts of
-PKC,
II-PKC,
-PKC, and
-PKC. Generation of O
2
generation triggered by the chemotactic peptide fMet-Leu-Phe or the PKC
activator PMA was not significantly different in
I-PKC null cells as
compared with the
I-PKC-positive cells. Therefore the presence of
I-PKC was not necessary for activation of O
2 generation,
and
II-PKC was implicated in signaling for O
2 generation. A
role for
II-PKC in signaling for the activation of O
2
generation was demonstrated by antisense depletion of
II-PKC. Both
fMet-Leu-Phe and PMA-induced O
2 generation were significantly
inhibited in
I-PKC null cells depleted of
II-PKC. Therefore,
II-PKC but not
I-PKC is essential for signaling in the activation
of the NADPH oxidase.
Spatial regulation of signaling elements is critical in the regulation
of NADPH oxidase assembly and activation. Differential localization of
PKC isotypes reflecting the many roles of PKC has been demonstrated in
multiple cell types; PKC has been demonstrated in microfilaments,
Golgi, endoplasmic reticulum, and nuclear and cell membranes (36-38).
A role for
-PKC has been shown in ligand-initiated O
2
generation of dHL60 cells and also in proliferation of HL60 cells (8,
16, 18). Indeed,
II-PKC can translocate to the nucleus in K562
erythroleukemia cells (38); the V5 region of
II-PKC binds to
phosphatidylglycerol, a PKC activator in the nuclear membrane. In
addition,
-PKC can translocate from cytosol to plasmalemma in
response to elevated Ca2+ levels or to activation by
fMet-Leu-Phe or PMA (3, 8, 39), where it could participate in signaling
for cell membrane-associated events such as O
2 generation.
Scaffold or PKC binding proteins can localize PKC isotypes to discrete
cell locations and to particular signaling cascades.
Scaffold proteins such as RACK, AKAP (A kinase
anchor protein), and adducins (20-22, 40-42)
are proteins that bind to PKC isotypes and provide localization for
greater specificity and efficiency of signaling. Scaffold proteins have
been identified by (a) overlay assays, which use PKC to
probe protein bands, (b) interaction cloning, and
(c) the yeast two-hybrid genetic screen for protein-protein interactions. RACKs are cytoskeleton and membrane-associated proteins that bind phospholipase C
and activated forms of
/
-PKC in
other cell types. Particular binding proteins may differentially target protein kinase C isotypes to defined cellular locations. In the present
study, an overlay assay demonstrated cofactor-dependent binding of rh
II-PKC to a 36-kDa band that was immunoreactive to
RACK1 as well as binding of rh
II-PKC to a 47-kDa band, a candidate for the substrate p47phox. In dHL60 cells, immunoprecipitated
II-PKC associated with p47phox and with RACK1 upon cell
activation by fMet-Leu-Phe. In contrast, when p47phox was
immunoprecipitated from dHL60 cells, fMet-Leu-Phe triggered association
of
II-PKC with p47phox but no association of RACK1 with
p47phox. Therefore, although
II-PKC associated with RACK1 in
fMet-Leu-Phe-activated cells, the signaling complex of p47phox
and
II-PKC was not associated with RACK1, indicating that RACK1 might not be involved in promoting activation of the NADPH oxidase through
II-PKC-based phosphorylation of p47phox.
Inactive PKC isotypes contain a RACK binding domain that binds to a
pseudo-RACK sequence in the regulatory domain; the presence of
cofactors causes a conformational change in the PKC to allow stable
binding of the RACK binding domain on the PKC molecule to RACK. Peptide
I, a peptide based on a sequence in annexin I/14-3-3, interferes with
the binding of
-PKC to RACK1 (24-27). Peptide I enhanced
O
2 generation triggered by fMet-Leu-Phe or by PMA in
electroporated dHL60 cells. Therefore, the interaction of RACK1 with
-PKC was not essential for generation of O
2, since
inhibition of
-PKC interaction with RACK by peptide I increased
O
2 generation.
Depletion of RACK1 in
I-PKC null dHL60 cells by an antisense
strategy also produced enhanced O
2 generation triggered by fMet-Leu-Phe or by PMA. This finding confirmed that the interaction of
RACK1 with
-PKC was not essential in the signaling pathway for
activation of NADPH oxidase. The enhanced O
2 generation upon RACK1 depletion indicates that RACK1 might serve to remove
-PKC from
the signaling complex required specifically for signaling activation of
the NADPH oxidase. RACK1 might be viewed as diverting the
-PKC to
another pathway requiring
-PKC as a signaling element. Indeed RACKs
have been shown to associate with the cytoskeleton, to bind selective
PH domains (43), the
-integrin subunit (44), phospholipase C
(29), and to inhibit src (45). The binding of RACK1 to
II-PKC might
act as a mechanism for down-regulation of signaling for O
2 generation.
II-PKC, but not
I-PKC, plays a selective role in the activation
of O
2 generation in dHL60 cells. Spatial regulation is important in activation of PKC and in assembly of the NADPH oxidase. Activation by fMet-Leu-Phe triggered association of p47phox
with
II-PKC, but not with RACK1, indicating that RACK1 was not a
part of the signaling complex of p47phox with
II-PKC for
assembly of the NADPH oxidase. Inhibition of association of
-PKC
with RACK1 by inhibitory peptides or depletion of RACK1 by an antisense
strategy enhanced O
2 generation by dHL60 cells. Therefore,
RACK1 is not implicated in positive signaling for assembly of the NADPH
oxidase. RACK1 is a negative regulator of O
2 generation and
may act to sequester
-PKC as a mechanism to down-regulate
O
2 generation or may divert
-PKC to other pathways requiring
-PKC for signal transduction.