Roles for beta II-Protein Kinase C and RACK1 in Positive and Negative Signaling for Superoxide Anion Generation in Differentiated HL60 Cells*

Helen M. KorchakDagger and Laurie E. Kilpatrick

From the Departments of Pediatrics and Biochemistry/Biophysics, University of Pennsylvania School of Medicine, The Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104

Received for publication, September 12, 2000, and in revised form, November 26, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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beta -Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O&cjs1138;2). Neutrophils and neutrophilic HL60 cells contain both beta I and beta II-PKC, isotypes that are derived by alternate splicing. beta I-PKC-positive and beta I-PKC null HL60 cells generated equivalent amounts of O&cjs1138;2 in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of beta II-PKC from beta I-PKC null cells inhibited ligand-initiated O&cjs1138;2 generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47phox, with beta II-PKC but not with RACK1, a binding protein for beta II-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of beta -PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O&cjs1138;2 generation. Therefore, beta II-PKC but not beta I-PKC is essential for activation of O&cjs1138;2 generation and plays a positive role in signaling for NADPH oxidase activation in association with p47phox. In contrast, RACK1 is involved in negative signaling for O&cjs1138;2 generation. RACK1 binds to beta II-PKC but not with the p47phox·beta II-PKC complex. RACK1 may divert beta II-PKC to other signaling pathways requiring beta -PKC for signal transduction. Alternatively, RACK1 may sequester beta II-PKC to down-regulate O&cjs1138;2 generation.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Ligand-initiated activation of superoxide anion (O&cjs1138;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&cjs1138;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 beta -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 alpha -PKC, beta -PKC, and gamma -PKC are phosphatidylserine (PS)-, diglyceride (DG)-, and Ca2+-dependent; novel PKC isotypes delta -PKC, epsilon -PKC, and theta - and eta -PKC also require PS and DG but are Ca2+-independent. The atypical PKC isotypes, zeta -PKC, and lambda -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 alpha -PKC, beta I-PKC, beta II-PKC, delta -PKC, and zeta -PKC (8, 13-15).

Depletion of beta -PKC by antisense pretreatment was previously shown to inhibit phosphorylation of p47phox, translocation of p47phox to the membrane, and generation of O&cjs1138;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 beta -PKC specific inhibitor to reduce ligand-initiated O&cjs1138;2 generation also indicated that beta -PKC is essential for activation of O&cjs1138;2 generation (16). However, these studies did not distinguish between a role for beta I-PKC or beta 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 beta I-PKC and beta II-PKC (8, 16).

Formation of a signaling complex that can target beta -PKC to substrates such as p47phox and p47phox to the cell membrane is essential for specificity and efficiency of signal transduction (17). However beta -PKC plays a role in signaling for multiple cell responses. beta -PKC is essential for both proliferation (18) and for O&cjs1138;2 generation in HL60 cells (8), events that occur at the nucleus and plasmalemma, respectively. beta -PKC also associates with the cytoskeleton (19). Therefore spatial considerations are a key element in defining a role for beta -PKC in signal transduction for a particular response. beta -PKC must be directed to different locations in the cell for each function, suggesting a role for scaffold proteins or PKC-binding proteins in beta -PKC-based signaling for activation of O&cjs1138;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 alpha -, beta -, and gamma -PKC as well as phospholipase Cgamma (23-30). PKC isotypes possess a pseudo-Rack binding site in the Ca2+ binding domain of alpha , beta , and gamma -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 beta -PKC and oocyte maturation (25, 26). RACK1 is a binding protein for beta II-PKC (31).

In this study we have assessed the roles of the PKC isotypes beta I-PKC and beta II-PKC in O&cjs1138;2 generation and, secondly, the role of RACK1 in beta -PKC-based signaling for O&cjs1138;2 generation. A beta I-PKC null subclone of HL60 cells (32) and depletion of beta II-PKC by an antisense strategy was used to demonstrate that beta II-PKC, but not beta I-PKC, is necessary for activation of O&cjs1138;2 generation. rhbeta 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-beta II-PKC interaction enhanced fMet-Leu-Phe- and PMA-induced O&cjs1138;2 generation. Therefore RACK1 is not essential in signaling for activation of the NADPH oxidase but may down-regulate beta II-PKC-based signaling either by diverting the beta II-PKC to another signaling pathway or by sequestering beta II-PKC as part of a down-regulation step in O&cjs1138;2 generation.

    MATERIALS AND METHODS
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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 alpha -PKC, beta I-PKC, beta II-PKC, delta -PKC, and zeta -PKC. A clone that was protein null for beta I-PKC but positive for alpha -PKC, beta II-PKC, delta -PKC, and zeta -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 beta -PKC, a 19-mer oligonucleotide having the sequence beta -PKC antisense (beta AS), 5'-AGC CGG GTC AGC CAT CTT G-3', and a scrambled missense oligonucleotide beta -PKC missense (beta MS) were used as previously described (8). Antisense and scrambled control oligonucleotides to RACK1 and beta -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, beta AS, or beta 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&cjs1138;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 beta 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 beta 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 rhbeta II-PKC to Endogenous dHL60 Cell Proteins-- The ability of rhbeta 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. rhbeta 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 beta -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 beta 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 alpha -PKC, beta I-PKC, beta II-PKC, gamma -PKC, and delta -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 delta -PKC, zeta -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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A Role for beta II-PKC in Ligand-initiated O&cjs1138;2 Generation-- A clone of HL60 cells that contained the beta II-PKC isotype of PKC as well as alpha -PKC, delta -PKC, and zeta -PKC was selected and probed for immunoreactivity to PKC antibodies (Fig. 1A). In comparison to the parent cell line, which contains beta I-PKC, the beta I-PKC protein null line contained no detectable amount of beta I-PKC (Fig. 1A). In contrast, both beta I-PKC-positive and beta I-PKC null cell lines contained equivalent amounts of alpha -PKC, delta -PKC, and zeta -PKC. Previous studies in which both beta I-PKC and beta II-PKC were depleted by an antisense strategy demonstrated a role for beta -PKC in activation of the NADPH oxidase in dHL60 cells (8). To discriminate between roles for beta I-PKC and beta II-PKC in ligand-initiated activation of O&cjs1138;2 generation, we compared fMet-Leu-Phe-triggered O&cjs1138;2 generation in beta I-PKC null and beta I-PKC-positive dHL60 cells. Generation of O&cjs1138;2 triggered by 1 µM fMet-Leu-Phe was 11.3 ± 1.9 (n = 12) nmol/106 cells/10 min in beta 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 beta I-PKC-positive dHL60 cells (Fig. 1B). Generation of O&cjs1138;2 in response to 1 µg/ml PMA was also similar in beta I-PKC null and beta I-PKC-positive dHL60 cells (Fig. 1B). In beta I-PKC-positive dHL60 cells, PMA triggered generation of 22.3 ± 4.1 (n = 5) nmol of O&cjs1138;2/106 cells/10 min, whereas in beta I-PKC cells, PMA triggered generation of 21.2 ± 1.7 (n = 6) nmol of O&cjs1138;2/106 cells/10 min (Fig. 1B). Therefore, beta I-PKC was not essential for optimal PMA- or fMet-Leu-Phe-initiated generation of O&cjs1138;2 in dHL60 cells.


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Fig. 1.   O&cjs1138;2 generation in beta I-PKC null and beta I-PKC positive dHL60 cells. A, selective loss of beta I-PKC in a beta I-PKC null dHL60 clone. Cell lysates were prepared from beta I-PKC null and beta 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&cjs1138;2 generation in beta I-PKC null and beta I-PKC-positive dHL60 cells. O&cjs1138;2 generation by beta I-PKC null dHL60 cells and by beta I-PKC-positive dHL60 cells was determined as the superoxide dismutase inhibitable reduction of cytochrome c. O&cjs1138;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 beta I-PKC null cells and 12 experiments for beta I-PKC-positive dHL60 cells and are expressed as nmol of O&cjs1138;2/106 cells/10 min.

Inhibition of O&cjs1138;2 Generation in beta II-PKC-depleted beta I-PKC Null dHL60 Cells-- beta I-PKC null dHL60 cells were treated for 2 days with 400 nM of an antisense oligonucleotide to beta -PKC (beta PKC AS) or with 400 nM control missense oligonucleotide to beta -PKC (beta PKC MS) as described under "Materials and Methods." Treatment with beta PKC AS resulted in a reduction in the level of beta II-PKC to 362 ± 47 (n = 4) density units (DU) as compared with a level of 603 ± 34 (n = 4) DU in control beta 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 beta -PKC had no significant effect on the levels of alpha -PKC, delta -PKC, or zeta -PKC (Fig. 2, A and B) and as previously shown (8).


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Fig. 2.   Selective depletion of beta II-PKC by a beta -PKC antisense oligonucleotide in beta I-PKC null dHL60 cells. beta I-PKC null dHL60 cells were treated with 400 nM beta -PKC antisense oligonucleotide (AS) or beta -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 beta PKC AS- and beta PKC MS-pretreated dHL60 cells was analyzed by ScanPro and plotted as density units (representative experiment of 5).

The effect of depletion of beta II-PKC on O&cjs1138;2 generation was determined in beta I-PKC null cells treated with beta PKC AS or beta PKC MS. fMet-Leu-Phe (1 µM) triggered generation of 5.3 ± 1.5 (n = 6) nmol of O&cjs1138;2/106 cells/10 min in beta PKC AS-treated beta 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 beta PKC MS (41.7 ± 9.5% control, p < 0.025) (Fig. 3A). The kinetics of fMet-Leu-Phe-activated O&cjs1138;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&cjs1138;2. The Vmax of fMet-Leu-Phe-induced O&cjs1138;2 generation, defined as the maximal rate of O&cjs1138;2 generation, was reduced in cells depleted of beta -PKC. Calculation of the Vmax demonstrated that in control beta 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 beta -PKC-depleted cells treated with beta PKC AS was significantly reduced to 1.41 ± 0.74 (n = 6) nmol/min/106 cells (53.0 ± 7.2% control beta PKC MS-treated cells (p < 0.01) (Fig. 3B).


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Fig. 3.   Effect of antisense depletion of beta -PKC on O&cjs1138;2 generation in beta I-PKC null dHL60 cells. O&cjs1138;2 generation by beta I-PKC null dHL60 cells depleted of beta II-PKC was determined in missense (beta PKC MS)- and antisense (beta PKC AS)-pretreated dHL60 cells as described in Fig. 1B. A, O&cjs1138;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&cjs1138;2/106 cells/10 min. B, Vmax of O&cjs1138;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&cjs1138;2/106 cells/min.

Similarly, activation of control beta PKC MS-pretreated cells by 1 µg/ml PMA triggered generation of 16.6 ± 1.5 (n = 7) nmol of O&cjs1138;2/106 cells/10 min, an amount that was significantly greater than the generation of 8.2 ± 1.3 (n = 7) nmol of O&cjs1138;2/106 cells/10 min observed in dHL60 cells depleted of beta -PKC by treatment with beta PKC AS (48.7 ± 6.2% control, p < 0.01) (Fig. 3A). The Vmax of PMA-induced O&cjs1138;2 generation was also significantly decreased in beta -PKC-depleted cells as compared with controls. The Vmax of O&cjs1138;2 generation decreased significantly from a rate of 3.12 ± 0.37 (n = 7) nmol/min/106 cells in control beta PKC MS-treated cells to a Vmax of 2.06 ± 0.27 (n = 8) nmol/min/106 cells in beta -PKC-depleted cells (66.1 ± 8.2% control, p < 0.01) (Fig. 3B). Thus, depletion of beta II-PKC in beta I-PKC null dHL60 cells resulted in inhibition of the rate of O&cjs1138;2 generation in response to both fMet-Leu-Phe and PMA, indicating an essential role for the beta II isotype but not the beta I isotype of PKC in signaling for O&cjs1138;2 generation.

Cofactor-dependent Binding of rhbeta II-PKC to Endogenous Proteins from dHL60 Cells-- beta -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 beta II-PKC in the presence of the cofactors PS, DG, and Ca2+. To assess the presence of binding proteins for beta II-PKC in dHL60 cells, we tested the ability of rhbeta II-PKC to bind to endogenous dHL60 proteins using an overlay assay. Lysates of beta I-PKC null dHL60 cells were separated on SDS-PAGE and transferred to PVDF membranes, and the membranes were incubated with rhbeta II-PKC in the presence and absence of the PKC cofactors PS, DG, and Ca2+. Western blotting with an antibody to beta II-PKC demonstrated that the exogenous rhbeta II-PKC bound to several endogenous proteins in a cofactor-dependent manner (Fig. 4A). In the absence of exogenous rhbeta II-PKC and cofactors, a band at 80 kDa corresponding to the endogenous beta II-PKC was observed (Fig. 4A, lane 1). rhbeta II-PKC in the absence of cofactors bound strongly only to a protein of 19 kDa (Fig. 4A, lane 2). However when rhbeta II-PKC was added in the presence of the cofactors PS, DG, and Ca2+, additional binding of rhbeta 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 beta 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 rhbeta 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 rhbeta II-PKC to a band of 47 kDa, a candidate for p47phox, was enhanced by the presence of cofactors. Binding of rhbeta 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 beta II-PKC can bind to numerous HL60 proteins in a cofactor-dependent fashion.


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Fig. 4.   Binding of rhbeta II-PKC to endogenous proteins from beta 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 rhbeta 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 beta II-PKC and RACK1, and densitometry was performed. Molecular mass markers are indicated on the left margin (K, ×1000), and bands corresponding to beta II-PKC and RACK1 are indicated on the right margin. Data shown are representative of three experiments. A, Western blot of binding of rhbeta II-PKC to endogenous dHL60 proteins. B, scanning densitometry of the immunoreactivity to anti beta II-PKC was analyzed by ScanPro and plotted as density versus molecular weight.

Ligand-initiated Association of p47phox with beta II-PKC, but Not with RACK1-- Coimmunoprecipitation was next used as a tool to determine whether beta II-PKC, RACK1, and p47phox formed a signaling complex in activated dHL60 cells. To determine whether p47phox and RACK1 were associated with beta II-PKC in activated cells, beta 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 beta II-PKC followed by densitometry of the immunoprecipitates demonstrated that approximately equivalent amounts of beta 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 beta 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 beta 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 beta 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 beta II-PKC with the scaffold protein RACK1 as well as with p47phox.


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Fig. 5.   Ligand-initiated association of beta II-PKC with p47phox. beta I-PKC null dHL60 were pre-treated with buffer or 1 µM fMet-Leu-Phe for 1 min. beta 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 beta II-PKC, p47phox, or RACK1. Densitometry was performed on the Western blots, and results are expressed as density units. A, immunoprecipitation of beta 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.

To determine whether fMet-Leu-Phe triggered enhanced association of RACK1 with p47phox, concomitant with the enhanced association of RACK1 with beta 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 beta II-PKC associated with the p47phox in these immunoprecipitates demonstrated a significant increase in association of beta II-PKC with p47phox (Fig. 5B). The level of beta 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 beta II-PKC demonstrated that cell activation by fMet-Leu-Phe triggered enhanced association of beta 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 beta II-PKC with the substrate p47phox, no increase in association of RACK1 with the p47phox or p47phox-associated beta II-PKC was observed. We therefore questioned whether RACK1 played a role in signaling for activation of O&cjs1138;2 generation, a process that is dependent on beta II-PKC.

Effect of Inhibitor Peptide, Peptide I, on fMet-Leu-Phe Triggered O&cjs1138;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 beta II-PKC in activation of O&cjs1138;2 generation. Peptide I inhibits the binding of RACK to PKC and the translocation of beta -PKC to the membrane in other cell types (24-27). We first tested the ability of peptide I to inhibit the binding of rhbeta II-PKC to endogenous dHL60 proteins using an overlay assay (see Fig. 4). Lysates of beta I-PKC null dHL60 cells were separated on SDS-PAGE and transferred to PVDF membranes, and the membranes were incubated with rhbeta 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 beta 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 beta 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 rhbeta II-PKC to a 36-kDa band that was immunoreactive to a RACK1 antibody.

Peptide I was then used to probe a role for beta II-PKC binding to RACK1 in signaling for O&cjs1138;2 generation. To probe a possible role for RACK1 interaction with beta II-PKC in activation of O&cjs1138;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&cjs1138;2 generation triggered by 1 µM fMet-Leu-Phe (Fig. 6). fMet-Leu-Phe-triggered O&cjs1138;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&cjs1138;2 generation was increased to 170.2 ± 35.3% control (n = 4, p < 0.02) (Fig. 6). Similarly, when O&cjs1138;2 generation was triggered by 1 µg/ml PMA, peptide I enhanced O&cjs1138;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 beta -PKC and translocation of beta -PKC to the membrane, enhanced rather than inhibited ligand-initiated O&cjs1138;2 generation in beta I-PKC null dHL60 cells.


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Fig. 6.   Effect of the inhibitory peptide I on O&cjs1138;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&cjs1138;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&cjs1138;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.

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. beta 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 beta II-PKC, no difference in immunoreactivity to beta 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. beta 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 beta II-PKC (representative experiment of 8).

Depletion of RACK1 Enhances O&cjs1138;2 Generation by dHL60 Cells-- A role for RACK1 in signaling for activation of the NADPH oxidase and generation of O&cjs1138;2 was examined using beta I-PKC null dHL60 cells depleted of RACK1 by antisense pretreatment. O&cjs1138;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&cjs1138;2 generation in RACK1-depleted dHL60 cells was observed when the cells were activated by 1 µg/ml PMA. O&cjs1138;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&cjs1138;2 generation in beta I-PKC null dHL60 cells. O&cjs1138;2 generation by beta 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 beta I-PKC null dHL60 cells. A, O&cjs1138;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&cjs1138;2/106 cells/10 min. B, Vmax of O&cjs1138;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&cjs1138;2/106 cells/min.

In addition, the Vmax of ligand-induced O&cjs1138;2 generation, defined as the maximal rate of O&cjs1138;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&cjs1138;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 beta -PKC also resulted in enhanced O&cjs1138;2 generation (Fig. 6).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Phagocytic cells, such as HL60 cells differentiated to a neutrophilic phenotype, and neutrophils possess multiple forms of PKC isotypes including Ca2+-dependent alpha -PKC, beta I-PKC, and beta II-PKC, Ca2+-independent delta -PKC, and atypical zeta -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 beta -PKC in activation of the NADPH oxidase and generation of O&cjs1138;2 has previously been demonstrated using an antisense strategy (8) and by studies with a beta -PKC-selective inhibitor (16).

HL60 cells and neutrophils contain two beta -PKC isotypes, beta I-PKC and beta 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 beta -PKC-selective inhibitor could discriminate between roles for these beta -PKC isotypes. The present study demonstrated a specific role for beta II-PKC in signaling for activation of O&cjs1138;2 generation using a beta I-PKC null subclone of dHL60 cells. The beta I-PKC HL60 cell subclone was negative for immunoreactivity to beta I-PKC but contained equivalent amounts of alpha -PKC, beta II-PKC, delta -PKC, and zeta -PKC. Generation of O&cjs1138;2 generation triggered by the chemotactic peptide fMet-Leu-Phe or the PKC activator PMA was not significantly different in beta I-PKC null cells as compared with the beta I-PKC-positive cells. Therefore the presence of beta I-PKC was not necessary for activation of O&cjs1138;2 generation, and beta II-PKC was implicated in signaling for O&cjs1138;2 generation. A role for beta II-PKC in signaling for the activation of O&cjs1138;2 generation was demonstrated by antisense depletion of beta II-PKC. Both fMet-Leu-Phe and PMA-induced O&cjs1138;2 generation were significantly inhibited in beta I-PKC null cells depleted of beta II-PKC. Therefore, beta II-PKC but not beta 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 beta -PKC has been shown in ligand-initiated O&cjs1138;2 generation of dHL60 cells and also in proliferation of HL60 cells (8, 16, 18). Indeed, beta II-PKC can translocate to the nucleus in K562 erythroleukemia cells (38); the V5 region of beta II-PKC binds to phosphatidylglycerol, a PKC activator in the nuclear membrane. In addition, beta -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&cjs1138;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 Cgamma and activated forms of alpha /beta -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 rhbeta II-PKC to a 36-kDa band that was immunoreactive to RACK1 as well as binding of rhbeta II-PKC to a 47-kDa band, a candidate for the substrate p47phox. In dHL60 cells, immunoprecipitated beta 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 beta II-PKC with p47phox but no association of RACK1 with p47phox. Therefore, although beta II-PKC associated with RACK1 in fMet-Leu-Phe-activated cells, the signaling complex of p47phox and beta II-PKC was not associated with RACK1, indicating that RACK1 might not be involved in promoting activation of the NADPH oxidase through beta 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 beta -PKC to RACK1 (24-27). Peptide I enhanced O&cjs1138;2 generation triggered by fMet-Leu-Phe or by PMA in electroporated dHL60 cells. Therefore, the interaction of RACK1 with beta -PKC was not essential for generation of O&cjs1138;2, since inhibition of beta -PKC interaction with RACK by peptide I increased O&cjs1138;2 generation.

Depletion of RACK1 in beta I-PKC null dHL60 cells by an antisense strategy also produced enhanced O&cjs1138;2 generation triggered by fMet-Leu-Phe or by PMA. This finding confirmed that the interaction of RACK1 with beta -PKC was not essential in the signaling pathway for activation of NADPH oxidase. The enhanced O&cjs1138;2 generation upon RACK1 depletion indicates that RACK1 might serve to remove beta -PKC from the signaling complex required specifically for signaling activation of the NADPH oxidase. RACK1 might be viewed as diverting the beta -PKC to another pathway requiring beta -PKC as a signaling element. Indeed RACKs have been shown to associate with the cytoskeleton, to bind selective PH domains (43), the beta -integrin subunit (44), phospholipase Cgamma (29), and to inhibit src (45). The binding of RACK1 to beta II-PKC might act as a mechanism for down-regulation of signaling for O&cjs1138;2 generation.

beta II-PKC, but not beta I-PKC, plays a selective role in the activation of O&cjs1138;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 beta II-PKC, but not with RACK1, indicating that RACK1 was not a part of the signaling complex of p47phox with beta II-PKC for assembly of the NADPH oxidase. Inhibition of association of beta -PKC with RACK1 by inhibitory peptides or depletion of RACK1 by an antisense strategy enhanced O&cjs1138;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&cjs1138;2 generation and may act to sequester beta -PKC as a mechanism to down-regulate O&cjs1138;2 generation or may divert beta -PKC to other pathways requiring beta -PKC for signal transduction.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AI 24840.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.

Dagger To whom correspondence should be addressed: Immunology Section, Room 1208C Abramson Bldg., Children's Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-2136; Fax: 610-525-1190; E-mail: korchak@email.chop.edu.

Published, JBC Papers in Press, December 18, 2000, DOI 10.1074/jbc.M008326200

    ABBREVIATIONS

The abbreviations used are: dHL60 cells, HL60 cells differentiated to a neutrophil phenotype; fMet-Leu-Phe, N-formyl-methionyl-leucyl-phenylalanine; O&cjs1138;2, superoxide anion; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol myristate acetate; DMRIE-C, 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammoniumbromide/cholesterol; PS, phosphatidylserine; DG, diglyceride; PVDF, polyvinylidene difluoride; BSA, bovine serum albumin; DU, density units; AS, anti-sense; MS, missense; rhbeta II, recombinant human beta II.

    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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