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

β-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O⨪2). Neutrophils and neutrophilic HL60 cells contain both βI and βII-PKC, isotypes that are derived by alternate splicing. βI-PKC-positive and βI-PKC null HL60 cells generated equivalent amounts of O⨪2 in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of βII-PKC from βI-PKC null cells inhibited ligand-initiated O⨪2generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47 phox , with βII-PKC but not with RACK1, a binding protein for βII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of β-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O⨪2 generation. Therefore, βII-PKC but not βI-PKC is essential for activation of O⨪2generation and plays a positive role in signaling for NADPH oxidase activation in association with p47 phox . In contrast, RACK1 is involved in negative signaling for O⨪2 generation. RACK1 binds to βII-PKC but not with the p47 phox ·βII-PKC complex. RACK1 may divert βII-PKC to other signaling pathways requiring β-PKC for signal transduction. Alternatively, RACK1 may sequester βII-PKC to down-regulate O⨪2 generation.

Depletion of ␤-PKC by antisense pretreatment was previously shown to inhibit phosphorylation of p47 phox , translocation of p47 phox 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 p47 phox and p47 phox 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, Ca 2ϩ /DG-dependent PKC isotypes ␣-, ␤-, and ␥-PKC as well as phospholipase C␥ (23)(24)(25)(26)(27)(28)(29)(30). PKC isotypes * 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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% CO 2 -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 T m to promote high affinity binding to mRNA; a T m 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): 5Ј-T GCC ACG AAG GGT CAT CTG C-3Ј; RACK1 sense: 5Ј-G CAG ATG ACC CTT CGT GGC A-3Ј. A scrambled missense oligonucleotide of the RACK1 AS was used as a control. The unique nature of these sequences was confirmed by searching the GenBank TM data base. For depletion of ␤-PKC, a 19-mer oligonucleotide having the sequence ␤-PKC antisense (␤AS), 5Ј-AGC CGG GTC AGC CAT CTT G-3Ј, 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 (DM-RIE-C) (1:1 (M/M)) (Life Technologies, Inc.). HL60 cells were cultured in the presence of 1.3% Me 2 SO 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 ϫ 10 6 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 g/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 Me 2 SO (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 Me 2 SO (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 ϩ , 5 mM K ϩ , 1.29 mM Ca 2ϩ , 1.2 mM Mg 2ϩ , 155 mM Cl Ϫ , and 10 mM Hepes (8).
Western Blots-Lysates of dHL60 cells (1 ϫ 10 6 cells/sample) were prepared by heating the cells at 95°C for 5 min in 2ϫ 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 Su-perSignal Ultra chemiluminescence substrate. The software Sigma-Proscan (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 M fMet-Leu-Phe in the presence of 5 g/ml cytochalasin B or by 1 g/ml PMA in the absence of cytochalasin B. Immunoprecipitation of ␤II-PKC and p47 phox -dHL60 cells (50 ϫ 10 6 cells/ml) were stimulated with either buffer alone or fMet-Leu-Phe (1 M) 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 g/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 p47 phox 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 2ϫ 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 g/ml leupeptin, 10 g/ml soy bean trypsin inhibitor, 20 mM phenylmethanesulfonyl fluoride in the presence or absence of 0.1 mM CaCl 2 , 50 g/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 Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 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 Ͻ1000 Da and transient passage of molecules Ͼ1000Da. 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 M peptide I. Previous studies using the fluorescent probe Dextran-Indo1 conjugate indicated that the attained intracellular concentration of peptides under these electroporation conditions is ϳ10 M (15).
Statistical Analysis-Results are expressed as mean Ϯ 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 Me 2 SO 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 p47 phox were purchased from Transduction Laboratories. Protein A-agarose was obtained from Life Technologies, Inc.

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-PKCpositive dHL60 cells. Generation of O 2 . triggered by 1 M fMet-Leu-Phe was 11.3 Ϯ 1.9 (n ϭ 12) nmol/10 6 cells/10 min in ␤I-PKC null dHL60 cells, a rate that was not significantly different from the rate of 11.2 Ϯ 1.8 (n ϭ 8) nmol/10 6 cells/10 min observed in ␤I-PKC-positive dHL60 cells (Fig. 1B). Generation of O 2 . in response to 1 g/ml PMA was also similar in 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 Ca 2ϩ . 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 Ca 2ϩ . 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 Ca 2ϩ , 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 Ϯ 3.1 (n ϭ 3) DU in the absence of cofactors and of 82.7 Ϯ 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 Ϯ 3.1 (n ϭ 3) DU in the absence of cofactors and significantly enhanced to 77.3 Ϯ 4.7 (n ϭ 3) DU in the presence of cofactors (p Ͻ 0.005 paired Student's t test). In addition, binding of rh␤II-PKC to a band of 47 kDa, a candidate for p47 phox , was enhanced by the presence of cofactors. Binding of rh␤II-PKC to the 47-kDa band was 23.0 Ϯ 1.4 (n ϭ 3) DU in the absence of cofactors and 60.7 Ϯ 6.9 DU in the presence of cofactors (p Ͻ 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.
Ligand-initiated Association of p47 phox with ␤II-PKC, but Not with RACK1-Coimmunoprecipitation was next used as a tool to determine whether ␤II-PKC, RACK1, and p47 phox formed a signaling complex in activated dHL60 cells. To determine whether p47 phox 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 M 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 Ϯ 207 (n ϭ 4) DU from resting cells as compared with 1895 Ϯ 142 DU in fMet-Leu-Phe-activated dHL60 cells (Fig. 5A). Probing of the immunoprecipitates with an antibody to p47 phox demonstrated a significant increase in the association of p47 phox with ␤II-PKC, from a level of 424 Ϯ 204 (n ϭ 4) DU in resting cells to a level of 1133 Ϯ 314 DU in cells activated by fMet-Leu-Phe (p Ͻ 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 Ϯ 115 (n ϭ 3) DU in resting cells to 1105 Ϯ 224 DU in fMet-Leu-Pheactivated cells (p Ͻ 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 p47 phox .
To determine whether fMet-Leu-Phe triggered enhanced association of RACK1 with p47 phox , concomitant with the enhanced association of RACK1 with ␤II-PKC, p47 phox was immunoprecipitated from resting and activated dHL60 cells. Western blots of immunoprecipitates of p47 phox followed by densitometry demonstrated equivalent levels of p47 phox in resting cells and in cells stimulated for 1 min with 1 M fMet-Leu-Phe (Fig. 5B). Densitometry of the Western blots showed a level of p47 phox of 1588 Ϯ 293 (n ϭ 3) DU in resting cells as compared with a level of 1519 Ϯ 284 (n ϭ 3) DU in cells activated by 1 M fMet-Leu-Phe. Measurement of the level of ␤II-PKC associated with the p47 phox in these immunoprecipitates demonstrated a significant increase in association of ␤II-PKC with p47 phox (Fig.   5B). The level of ␤II-PKC observed in resting cells was 451 Ϯ 154 (n ϭ 3) DU as compared with an enhanced level of 912 Ϯ 323 (n ϭ 3) DU in cells activated by fMet-Leu-Phe (213 Ϯ 36% control, p Ͻ 0.05) (Fig. 5B). Therefore, immunoprecipitation of p47 phox or of ␤II-PKC demonstrated that cell activation by fMet-Leu-Phe triggered enhanced association of ␤II-PKC with its substrate p47 phox . In contrast, probing the p47 phox immunoprecipitates with an antibody to RACK1 demonstrated no significant association of RACK1 with p47 phox (Fig. 5B). The level of RACK1 associated with the p47 phox immunoprecipitate from resting cells was 21 Ϯ 11 (n ϭ 3) DU, whereas the level in fMet-Leu-Phe-activated cells was 42 Ϯ 26 (n ϭ 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 p47 phox , no increase in association of RACK1 with the p47 phox or p47 phox -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. 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 Ca 2ϩ and in the presence or absence of 10 M peptide I. In the presence of 10 M 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 Ϯ 11.4 (n ϭ 4) DU in the absence of peptide I and significantly decreased to 25.0 Ϯ 7.2 (n ϭ 4) DU in the presence of 10 M peptide I (61. 0 Ϯ 7.1% control, p Ͻ 0.005). In contrast, the 80-kDa band, which represents endogenous ␤II-PKC, was 67.0 Ϯ 3.6 (n ϭ 4) DU in the absence of peptide I and 66.8 Ϯ 3.8 (n ϭ 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 M peptide I, which gives a final intracellular concentration of peptide I of ϳ10 M, caused a significant increase in O 2 . generation triggered by 1 M fMet-Leu-Phe (Fig. 6). fMet-Leu-Phe-triggered O 2 . generation of 13.4 Ϯ 3.0 (n ϭ 6) nmol/10 6 cells/10 min in buffer-treated cells; in peptide I-treated cells fMet-Leu-Phe-triggered O 2 . generation was increased to 170.2 Ϯ 35.3% control (n ϭ 4, p Ͻ 0.02) (Fig. 6). Similarly, when O 2 . generation was triggered by 1 g/ml PMA, peptide I enhanced O 2 . generation from a rate of 21.7 Ϯ 3.5 (n ϭ 6) nmol/10 6 cells/10 min in buffer-treated cells to a rate that was 121.7 Ϯ 14.6 (n ϭ 6) % control in peptide I-treated cells (p Ͻ 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. 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 Ϯ 273 (n ϭ 8) as compared with a level of 1949 Ϯ 452 (n ϭ 8) in control RACK1 MS-treated cells (57.9 Ϯ 5.5% control, p Ͻ 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 MSpretreated cells (Fig. 7). Therefore, the RACK1 AS treatment selectively depletes dHL60 cells of RACK1.

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 generation triggered by 1 g/ml PMA in RACK1 AS-reated cells was significantly increased to a level of 26.3 Ϯ 1.9 (n ϭ 5) nmol/10 6 cells/10 min as compared with a level of 22.3 Ϯ 2.5 (n ϭ 5) nmol/10 6 cells/10 min in control RACK1 MS-treated dHL60 cells (119.4 Ϯ 6.4% control, p Ͻ 0.03) (Fig. 8A).
In addition, the V max 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 V max demonstrated that in RACK1 AS-treated cells activated by 1 M fMet-Leu-Phe, the V max was significantly enhanced to a rate of 7.4 Ϯ 0.8 (n ϭ 5) nmol/min/10 6 cells as compared with a rate of 5.1 Ϯ 0.6 (n ϭ 5) nmol/min/10 6 in control cells treated with RACK1 MS (150.0 Ϯ 45.8% control, p Ͻ 0.05) (Fig. 8B).
Similarly, an increase in V max was observed in RACK1-depleted dHL60 cells activated by 1 g/ml PMA; however, the increase was not statistically significant. The V max in RACK1 AS-treated cells activated by 1 g/ml PMA was 5.1 Ϯ 6.2 (n ϭ 5) nmol/min/10 6 cells as compared with a V max of 3.6 Ϯ 6.2 (n ϭ 5) nmol/min/10 6 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).

DISCUSSION
Phagocytic cells, such as HL60 cells differentiated to a neutrophilic phenotype, and neutrophils possess multiple forms of PKC isotypes including Ca 2ϩ -dependent ␣-PKC, ␤I-PKC, and ␤II-PKC, Ca 2ϩ -independent ␦-PKC, and atypical -PKC. Assembly of an active NADPH oxidase is tightly controlled and involves association of the cytosolic components p47 phox and p67 phox with the plasma membrane-associated cytochrome b 558 (1-3). Phosphorylation of p47 phox on multiple sites is an essential step in triggering translocation of p47 phox to the plasmale-mma, where it binds to the cytochrome b 558 (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. 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 Ca 2ϩ 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 p47 phox . In dHL60 cells, immunoprecipitated ␤II-PKC associated with p47 phox and with RACK1 upon cell activation by fMet-Leu-Phe. In contrast, when p47 phox was immunoprecipitated from dHL60 cells, fMet-Leu-Phe triggered association of ␤II-PKC with p47 phox but no association of RACK1 with p47 phox . Therefore, although ␤II-PKC associated with RACK1 in fMet-Leu-Phe-activated cells, the signaling complex of p47 phox and ␤II-PKC was not associ- ated with RACK1, indicating that RACK1 might not be involved in promoting activation of the NADPH oxidase through ␤II-PKC-based phosphorylation of p47 phox . 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 p47 phox with ␤II-PKC, but not with RACK1, indicating that RACK1 was not a part of the signaling complex of p47 phox 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.