Selective Role for β-Protein Kinase C in Signaling for O⨪2 Generation but Not Degranulation or Adherence in Differentiated HL60 Cells*

A role for protein kinase C (PKC) isotypes is implicated in the activation of phagocytic cell functions. An antisense approach was used to selectively deplete β-PKC, both βI- and βII-PKC, but not α-PKC, δ-PKC, or ζ-PKC in HL60 cells differentiated to a neutrophil-like phenotype (dHL60 cells). Depletion of β-PKC in dHL60 cells elicited selective inhibition of O⨪2generation triggered by fMet-Leu-Phe, immune complexes, or phorbol myristate acetate, an activator of PKC. In contrast, neither ligand-elicited β-glucuronidase (azurophil granule) release nor adherence to fibronectin was inhibited by β-PKC depletion. Ligand-induced phosphorylation of a subset of proteins was reduced in β-PKC-depleted dHL60 cells. Phosphorylation of p47 phox and translocation of p47 phox to the membrane are essential for activation of the NADPH oxidase and generation of O⨪2. β-PKC depletion had no effect on the level of p47 phox in dHL60 cells but did significantly decrease ligand-induced phosphorylation of this protein. Furthermore, translocation of p47 phox to the membrane in response to phorbol myristate acetate or fMet-Leu-Phe was reduced in β-PKC-depleted cells. These results indicate that β-PKC is essential for signaling for O⨪2 generation but not cell adherence or azurophil degranulation. Depletion of β-PKC inhibited ligand-induced phosphorylation of p47 phox , translocation of p47 phox to the membrane, and activation of O⨪2 generation.

Protein kinase C (PKC) 1 mediates signaling for multiple functions of phagocytic cells, neutrophils, monocytes, macrophages, and differentiated HL60 (dHL60) cells. Ligands such as the chemotactic peptide fMet-Leu-Phe and phagocytic stimuli (immune complexes) trigger responses that include O 2 . generation, degranulation, adherence, and actin filament assembly (1)(2)(3)(4)(5)(6)(7)(8). These functions are essential for the microbicidal activity of phagocytic cells and are also proinflammatory. PKC, a phospholipid-dependent family of serine/threonine kinases, acts in multiple signal transduction pathways. The cofactor requirements differ between different classes of PKC isotypes. Classical ␣-, ␤-, and ␥-PKC are acidic phospholipid, diglyceride (DG), and Ca 2ϩ -dependent; novel forms ␦-, ⑀-, -, and -PKC also require acidic phospholipid and DG, but are Ca 2ϩ independent. The atypical PKC isotypes,and -PKC, require PS but are DG and Ca 2ϩ independent (9 -14). PKC isotypes differ in their tissue distribution and localization within the cell, suggesting that each isotype plays a specific role in signal transduction. Neutrophils, monocytes/macrophages, and dHL60 cells contain multiple isotypes of PKC, including Ca 2ϩ -dependent isotypes ␣-PKC, ␤I-PKC, and ␤II-PKC, Ca 2ϩ -independent DG-dependent isotype, ␦-PKC, and atypical PS-dependent, Ca 2ϩ /DGindependent -PKC (3, 14 -16). PKC has been implicated in the signaling for several different responses of phagocytic cells because PMA triggers O 2 . generation and adherence but not the release of azurophil granules (1,17). The PKC substrates involved in these processes are largely unknown. The cytoskeleton and integrins are involved in cell adherence, and several cytoskeletal proteins, including MARCKS, are PKC substrates (8). Assembly of an active NADPH oxidase for generation of O 2 .
requires translocation of cytosolic factors p47 phox , p67 phox , and rac2 to the plasma membrane, where they interact with the integral membrane protein cytochrome b 558 (18 -25). p47 phox is phosphorylated in ligand-activated phagocytic cells. p47 phox contains multiple phosphorylation sites, including a number of classical PKC substrate sites (RXXS/TXRX) and is phosphorylated by ␤-PKC in vitro (3). In addition, p47 phox phosphorylated in vitro by PKC, but not cAMP-activated kinase or mitogenactivated kinase, was active in a cell-free system for activation of the NADPH oxidase (4 -7). However, in vitro activity does not necessarily predict a role for a particular PKC isotype in the intact cell, where access to both substrate and cofactors is critical in controlling signaling specificity. Specific functions for each PKC isotype remain to be established. In the present study, an antisense strategy was used to probe a role for a specific PKC isotype in signaling for the ligandactivated responses of O 2 . generation and cell adherence in dHL60 cells. ␤-PKC was selectively depleted by an antisense strategy in dHL60 cells. Selective depletion of ␤-PKC in dHL60 cells decreased O 2 . generation but not degranulation or adherence. Depletion of ␤-PKC reduced ligand-induced phosphorylation of a subset of proteins including p47 phox and reduced translocation of p47 phox to the membrane, concordant with a selective role for ␤-PKC in signaling for assembly of an active NADPH oxidase. Ringer buffer before use. fMet-Leu-Phe was stored as a stock solution in ethanol and diluted in buffer prior to use. BSA and anti-BSA IgG (Cappel, Durham, NC) were used to form an immune complex by the method of Ward and Zvaifler (26).
HL60 Cell Culture-Human promyelocytic HL60 leukemic cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in suspension culture in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% minimal essential medium vitamin solution, 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.
Oligonucleotide Synthesis and Sequences-An antisense oligonucleotide was designed against the translation start site of human ␤-PKC using the commercial primer analysis software Oligo (National Biosciences). Since ␤I-PKC and ␤II-PKC differ only in the 3Ј terminus, this oligonucleotide should target both forms of ␤-PKC. A 19-mer sequence was chosen, which was without significant self-complimentarity and did not dimerize. The chosen sequence was also optimized for maximal Tm to promote high affinity binding to mRNA; a T m of 70.0°C was calculated at 150 mM salt and 37°C. The 19-mer oligonucleotides had the following sequences: ␤-PKC antisense (␤AS), 5Ј-AGC CGG GTC AGC CAT CTT G-3Ј, ␤-PKC sense (␤SS), 5Ј-C AAG ATG GCT GAC CCG GCT-3Ј. The unique nature of these sequences was confirmed by searching the GenBank data base. Antisense, sense, and scrambled control oligonucleotides were synthesized by the PENN Nucleic Acid Facility as the phosphorothioate derivatives and purified by high pressure liquid chromatography. In all oligonucleotides, the internucleoside linkages were completely phosphorothioate-modified.
Treatment of Cells with Oligonucleotides-Delivery of the oligonucleotides was enhanced equally with cationic lipids DMRIE-C (1,2dimyristoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/ cholesterol (1:1 (M/M))) and Lipofectin (Life Technologies, Inc.); however, DMRIE-C was chosen since this lipid lacks dioctanoyl phosphatidylethanolamine, which can be toxic to phagocytic cells, and allowed optimal retention of cell functions (27). HL60 cells were cultured in the presence of 1.3% Me 2 SO for 4 days to initiate differentiation 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 ␤AS, ␤SS, or ␤MS (a scrambled AS oligonucleotide) were suspended in Opti-MEM at a final concentration of 100 -1000 nM and mixed with the cationic lipid DMRIE-C (2.5 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% heatinactivated fetal bovine serum plus Me 2 SO (1.3% final concentration) was then added, and the cells cultured for 20 h. On day 5, the cells were washed and resuspended in fresh Opti-MEM and treated again with the cationic lipid/oligonucleotide mixture. Following 4 h of 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 Krebs-Ringer phosphate-buffered saline, pH 7.4, containing 1.2 mM Mg 2ϩ and 1.3 mM Ca 2ϩ .
Western Blots-Differentiated HL60 cell lysates (1 ϫ 10 6 cells/sample) were prepared by heating the cells at 95°C for 5 min in 2 ϫ SDS-PAGE sample buffer. The samples were briefly sonicated (12 s) to reduce viscosity. The dHL60 cell lysates were run on a 4 -16% gradient SDS-PAGE, transferred to 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. Immunoreactive bands were visualized by Pierce SuperSignal ULTRA chemiluminescence substrate. The software SigmaProscan (Jandel/ SPSS) was used for densitometric analysis; rhPKC isotypes (Pan Vera) were used as standards for quantitation.
Protein Kinase C Assay-Cytosol fractions were prepared by sonicating dHL60 cells in 50 mM Tris-HCl, pH 7.5, containing 2 mM PMSF, 33 M leupeptin, 35 M antipain, 24 g/ml chymostatin, 35 M pepstatin, and 0.48 TIU/ml aprotinin, 10 mM EGTA, and 50 mM mercaptoethanol, as described previously (2), and by centrifuging for 20 min at 115,000 ϫ g at 4°C. PKC activity of cytosol fractions was assayed in the presence of Ca 2ϩ , PS, and DG by measuring the incorporation of 32 P into histone type IIIS or into PKC substrate peptide [Ser 25 ]PKC(19 -31) (2). The net Ca 2ϩ -dependent activity was obtained by subtraction of the Ca 2ϩ -independent, PS/DG activity from the Ca 2ϩ /PS/DG-dependent activity.
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 (28) 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, or 300 g/ml BSA/anti-BSA immune complex in the absence of cytochalasin B.
Degranulation-The release of the azurophil granule-associated enzyme ␤-glucuronidase triggered by fMet-Leu-Phe from dHL60 cells was measured in the presence of 5 g/ml cytochalasin B to allow extracellular release of granule contents. Degranulation triggered by insoluble immune complex, BSA/anti-BSA, was measured in the absence of cytochalasin B. dHL60 cells (5 ϫ 10 6 cells/ml) were incubated for 5 min at 37°C with buffer, fMet-Leu-Phe (1 M) or BSA/anti-BSA (300 g/ml). ␤-Glucuronidase was determined by a microplate assay using phenolphthalein glucuronidate as substrate (29,30). Total enzyme activities were determined simultaneously in reaction mixtures containing the detergent Triton X-100 (0.2%) and using phenolphthalein to calibrate the nmoles of cleaved substrate.
Adherence-Adherence of dHL60 cells to fibronectin-coated plates was determined by a colorimetric assay according to the method of Aumailley et al. (31). 96-well microtiter plates were coated with fibronectin as described by Nathan et al. (32). dHL60 cells (4 ϫ 10 5 cells/well) were incubated for 30 min at 37°C with either buffer, fMet-Leu-Phe (1 M) or PMA (1 g/ml). Nonadherent cells were removed by washing with Krebs-Ringer phosphate-buffered saline buffer, and the adherent cells were fixed with 70% ethanol. The bound cells were stained with crystal violet (0.1% in dH 2 O). The plates were washed extensively to remove excess stain, and the cells were solubilized with 1% Nonidet P-40 in dH 2 O. Optical density was read at 550 nm.
Protein Phosphorylation in Activated dHL60 Cells-dHL60 cells (50 ϫ 10 6 cells/ml) were incubated for 60 min at 37°C with 32 P-P i (250 Ci of [ 32 P]orthophosphoric acid/ml). The 32 P-labeled cells were stimulated with either buffer alone or fMet-Leu-Phe (1 M) for 1 min in the presence of 5 g/ml cytochalasin B. The reaction was stopped by placing the samples on ice. Cell lysates were prepared and run on 4 -12% gradient SDS-PAGE. The gel was dried and subjected to autoradiography.
Immunoprecipitation of p47 phox -dHL60 cells (10 ϫ 10 6 cells/ml) were incubated for 60 min at 37°C with 32 P-P i (250 Ci of [ 32 P]orthophosphoric acid/ml). The 32 P-labeled cells were stimulated with either buffer alone, PMA (1 g/ml) or fMet-Leu-Phe (1 M), for 10 min. The reaction was stopped by the addition of cold immunoprecipitation (IP) buffer. IP buffer consisted of 10 mM HEPES (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 2 mM PMSF, 0.2% Nonidet P-40, 0.027 TIU/ml 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 microfuging for 5 min. A rabbit polyclonal antibody to p47 phox was added and incubated overnight at 4°C. Protein A-agarose was added and incubated for 1 h at 4°C with shaking. The reaction tubes were then microfuged for 30 s, and the supernatants were discarded. The protein A-agarose pellet was washed four times with IP buffer, and the sample was eluted by incubation for 20 min at 65°C in 2 ϫ SDS-PAGE sample buffer.
Preparation of Particulate Fractions from fMet-Leu-Phe-stimulated dHL60 Cells and Translocation of p47 phox and PKC Isotypes to Cell Membranes-dHL60 cells (2.5 ϫ 10 7 cells/ml) were incubated at 37°C in the presence of buffer, 1 M fMet-Leu-Phe (1 min) or 1 g/ml PMA (5 min). At the end of the incubation period, the cell suspension was transferred to an ice bath. The suspension was then centrifuged for 10 min at 300 ϫ g, and the cell pellet was resuspended in buffer containing 131 mM NaCl, 1 mM EGTA, 100 mM potassium phosphate buffer, pH 7.0, 2 mM PMSF, 20 g/ml leupeptin, 20 g/ml aprotinin, and 20 g/ml pepstatin (buffer A). (24). The cells were disrupted using three 5-s bursts of a microprobe sonicator at low power at 4°C and centrifuged at low speed (500 ϫ g, 5 min) to remove unbroken cells and nuclei. The supernatant was layered over a 15% sucrose cushion made up in buffer A and centrifuged at 4°C for 20 min at 115,000 ϫ g. The supernatant (cytosol) was mixed 4:1 with 4 ϫ SDS sample buffer. The pellet was solubilized in hot Laemmli buffer; pellet (10 ϫ 10 6 cell equivalents/lane) and cytosol (3 ϫ 10 6 cell equivalents/lane) were run on 4 -12% SDS-Selective Role for ␤-PKC in O 2 . Generation PAGE and probed by Western blotting. Statistical Analysis-Results are expressed as mean Ϯ S.E. (n). Data were analyzed by Student's one-tailed t test for paired samples.

RESULTS
Depletion of ␤-PKC Isotypes by Antisense Treatment-To deplete ␤-PKC, dHL60 cells were first treated with 1.3% Me 2 SO for 4 days to initiate differentiation, followed by two treatments with ␤-PKC antisense oligonucleotide (␤AS) and the cationic lipid DMRIE-C (2.5 g/ml) at 24-h intervals (see "Materials and Methods"). Preliminary experiments demonstrated that cationic lipids were essential to enhance the potency of the oligonucleotides (33,34). Phosphorothioate oligonucleotides were synthesized since these have a greater intracellular halflife than the native oligonucleotides. Since the half-life of PKC is long, reported to vary from 6.7 h to over 24 h, prolonged treatment with ␤AS was used (35). The cells were treated twice with the ␤AS/DMRIE-C mixture to prolong the exposure to ␤AS and to achieve decreased protein expression of ␤-PKC (35); a single treatment with ␤AS/DMRIE-C was not effective. The effect of antisense treatment on dHL60 cells following differentiation was examined over a concentration range of 0 -1000 nM ␤AS (Fig. 1). Treatment with ␤AS resulted in decreased expression of ␤-PKC (Fig. 1). Depletion of ␤-PKC followed a bell-shaped concentration response curve; maximal depletion of ␤-PKC immunoreactivity occurred at 250 and 500 nM ␤AS (Fig. 1). For this experiment, ␤-PKC was reduced to 23 and 31% of control levels by 250 and 500 nM ␤AS, respectively. At higher concentrations of ␤AS oligonucleotide, there was increased ␤-PKC expression. Attempts to enhance the depletion of ␤-PKC by more prolonged incubation or by increases in cationic lipid levels were not successful due to a loss of cell viability.
Effect of ␤-PKC Depletion on Superoxide Anion Generation by dHL60 Cells-Activation of dHL60 cells by a variety of stimuli elicits the assembly of an active NADPH oxidase enzyme complex, which generates superoxide anion (O 2 . ). Previous studies suggested that ␤-PKC may play an important role in the activation of the NADPH oxidase (3,20). Therefore, the effect of ␤-PKC depletion on O 2 . generation triggered by different ligands was evaluated. The PKC activator PMA (1 g/ml) triggered continuous generation of O 2 . by dHL60 cells following a lag period of approximately 1-3 min (Fig. 3A). The mean lag period for control ␤MS-treated cells was 205 Ϯ 55 s (n ϭ 5); the lag period was significantly increased to 222 Ϯ 43% (n ϭ  generation by dHL60 cells in the presence of cytochalasin B (Fig. 3B). The onset of O 2 . generation was rapid but ceased after 3-4 min. (Fig. 3B). Control (␤MS-treated) cells activated by 1 M fMet-Leu-Phe generated 16.88 Ϯ 2.6 nmol/10 6 cells/15 min (n ϭ 8) (Fig. 3C). Treatment of dHL60 cells with ␤AS plus DMRIE-C, at a concentration that significantly depleted levels of ␤-PKC and inhibited O 2 . production in response to fMet-Leu-Phe, had no significant effect on degranulation (Fig. 4); 10.5 Ϯ 2.7% (n ϭ 4) of total ␤-glucuronidase was released in response to fMet-Leu-Phe in control (␤MS-treated) cells, whereas 12.2 Ϯ 2.5% (n ϭ 4) of total ␤-glucuronidase was released from ␤-PKC-depleted cells. Similarly, degranulation triggered by BSA/anti-BSA in the absence of cytochalasin B was not affected by depletion of ␤-PKC; 7.5 Ϯ 0.2% (n ϭ 4) of total ␤-glucuronidase was released in response to BSA/anti-BSA in control (␤MS-treated) cells, whereas 8.3 Ϯ 0.6% (n ϭ 4) of total ␤-glucuronidase was released from ␤-PKC-depleted cells. Furthermore, total cell content of ␤-glucuronidase, a marker for HL60 cell differentiation, was not altered in ␤-PKC-depleted cells (Fig. 4). Therefore, depletion of ␤-PKC with a ␤-PKC antisense oligonucleotide selectively inhibits ligand-elicited O 2 . generation but not ␤-glucuronidase release.
Effect of ␤PKC Depletion on Adherence of dHL60 Cells to Fibronectin-A role for PKC in integrin-mediated adherence to fibronectin has been suggested since phorbol esters activate adherence mechanisms in phagocytic cells (7,36). To ascertain whether ␤-PKC plays a role in integrin-mediated adherence in dHL60 cells, the effect of ␤-PKC depletion on basal and stimulated adherence to fibronectin was examined in dHL60 cells. Activation of control (␤MS-treated) cells by PMA (1 g/ml) or fMet-Leu-Phe (1 M) triggered increased cell adherence to fibronectin-coated wells (Fig. 5). PMA produced the greatest increase in adherence, to 242. Effect of ␤-PKC Depletion on Protein Phosphorylation Triggered by 1 M fMet-Leu-Phe-␤AS-and ␤MS-treated dHL60 cells were labeled with 32 P inorganic phosphate for 1 h at 37°C and treated with buffer or fMet-Leu-Phe. Cell lysates were prepared, the phosphorylated proteins were separated on SDS-PAGE, and the gels were subjected to autoradiography. Activation of the cells for 1 min with 1 M fMet-Leu-Phe triggered phosphorylation of multiple proteins, including 18-, 24-, 38-, 43-, 47-, 54-, 68-, and 80-kDa proteins (Fig. 6A). Phosphorylation of prominent 14-, 97-, and 105-kDa bands was not significantly altered in fMet-Leu-Phe-activated cells (Fig. 6A). Densitometry of the autoradiograph demonstrated that treatment of cells with antisense to ␤-PKC reduced the fMet-Leu-Pheinduced phosphorylation of the 18-, 38-, 43-, 47-, 54-, and 68-kDa proteins (Fig. 6B); the 47-kDa band is a candidate for p47 phox . In contrast, phosphorylation of a band at 105 kDa was enhanced in ␤-PKC-depleted cells. The effect of ␤-PKC depletion on fMet-Leu-Phe-induced protein phosphorylation was selective, since treatment with ␤AS had no significant effect on the fMet-Leu-Phe-induced phosphorylation of the 24-or 80-kDa bands. These results suggest that depletion of ␤-PKC inhibits fMet-Leu-Phe-induced phosphorylation of a discrete number of proteins including a 47-kDa band, which is a candidate for Depletion of ␤-PKC and Reduced Ligand-initiated Phosphorylation of p47 phox -Phosphorylation of p47 phox and its translocation to the membrane-associated cytochrome b 558 is essential for assembly of the NADPH oxidase. ␤-PKC-depleted dHL60 cells were used to determine whether ␤-PKC was essential for phosphorylation of p47 phox in cells activated by fMet-Leu-Phe. dHL60 cells were pretreated with ␤AS or ␤MS and stimulated for 1 min with 1 M fMet-Leu-Phe; the p47 phox was then immunoprecipitated. Western blotting and densitometry analysis demonstrated that control (␤MS) and ␤-PKCdepleted (␤AS) dHL60 cells contained equivalent amounts of p47 phox immunoreactivity (Fig. 7A). However, phosphorylation of the p47 phox band was reduced in the ␤AS-treated dHL60 cells as compared with the control (␤MS-treated) cells; 32 P counts eluted from the ␤AS band were 56% of control levels measured in the ␤MS band (Fig. 7B). ␤-PKC-depleted cells contained equivalent protein levels of p47 phox as compared with control cells. However, fMet-Leu-Phe-induced phosphorylation of p47 phox was decreased in the ␤-PKC-depleted cells, concordant with a role for ␤-PKC in phosphorylation of p47 phox in ligandinitiated signaling and in the assembly of an active NADPH oxidase.
Effect of ␤PKC Depletion on p47 phox Translocation-Phosphorylation and translocation of p47 phox to the membrane and association of phosphorylated p47 phox with cytochrome b 558 are essential steps in the assembly of an active NADPH oxidase complex. Activation of dHL60 cells by fMet-Leu-Phe or PMA elicited translocation of p47 phox from the cytosol to the membrane fraction (Fig. 8). Depletion of ␤-PKC by ␤AS treatment was associated with decreased translocation of p47 phox from the cytosol to the membrane in both fMet-Leu-Phe-and PMAactivated cells as compared with ␤MS-treated controls (Fig. 8).
In contrast, the membrane-associated gp91 phox subunit of cytochrome b 558 was not affected by depletion of ␤-PKC (Fig. 8).
In resting cells, ␤I-PKC and ␤II-PKC were predominantly in the cytosol (Fig. 8). Activation of dHL60 cells by 1 M fMet-Leu-Phe for 1 min elicited translocation of both ␤I-PKC and ␤II-PKC from the cytosol to the membrane (Fig. 8). Activation of the cells with 1 g/ml PMA for 5 min triggered an almost total disappearance of ␤I-PKC and ␤II-PKC from the cytosol and translocation of both isotypes to the membrane (Fig. 8). In the ␤-PKC-depleted cells, less ␤I and ␤II-PKC was associated with the membrane in fMet-Leu-Phe-and PMA-treated cells actin assembly, and cell adherence. PKC is a family of structurally related isotypes that have different cofactor and substrate specificities (9,10). It has therefore been suggested that different isotypes of PKC transduce different signals, although there is little evidence to date to indicate specific functions for each isotype of PKC.
Multiple isotypes of PKC were demonstrated in dHL60 cells including ␣-, ␤I-, ␤II-, ␦-, and -PKC, in agreement with other studies (15); the expression of these PKC isotypes is similar to the PKC isotypes observed in neutrophils (3,14). In addition, the cofactors required for activation of PKC, calcium, DG and PIP 3 , are elevated in ligand-activated phagocytic cells, indicating a potential role for these PKC isotypes in activating responses such as O 2 . generation and adherence (37)(38)(39)(40).
A role for ␤-PKC in ligand-initiated responses was probed by selective depletion of the ␤-PKC isotypes. Selective depletion by antisense was used since difficulty in distinguishing primary and secondary effects may be seen in overexpression studies either of the wild-type isotype or of a dominant negative mutant (41). Selective depletion of ␤-PKC but not depletion of ␣-, ␦-, or -PKC was achieved using an antisense oligonucleotide designed against the translocation start site of ␤-PKC.  (17). These findings also demonstrate that ␤-PKC depletion did not have a nonspecific effect on signaling for cell activation. dHL60 cells adhere to fibronectin by a ␤1 integrin (36); adherence of dHL60 cells to a fibronectin-coated surface was triggered by PMA and fMet-Leu-Phe. Depletion of ␤-PKC in dHL60 cells did not inhibit either PMA or fMet-Leu-Phe-induced adherence to fibronectin. Thus, signaling for adherence must use other isotypes of PKC such as ␣or ␦-PKC that, like ␤-PKC, are also activated by PMA. FIG. 7. Effect of depletion of ␤-PKC on phosphorylation of p47 phox in dHL60 cells activated by fMet-Leu-Phe. dHL60 cells treated with ␤AS or ␤MS oligonucleotides were prelabeled with 32 P-P i (300 Ci) for 60 min at 37°C. fMLP (1 M) was added (zero time), and the reaction was stopped after 1 min. p47 phox was immunoprecipitated from each sample, and the immune complexes were run on 4 -12% gradient SDS-PAGE and blotted to PVDF membrane. Representative experiment of two. A, Western blot using anti p47 phox . B, autoradiogram of membrane. Molecular weight markers are indicated on the left margin and p47 phox on the right margin.
Identifying key substrate(s) is important in defining a role for ␤-PKC in signal transduction. Addition of fMet-Leu-Phe to dHL60 cells triggered phosphorylation of numerous proteins. In ␤-PKC-depleted cells, phosphorylation of a subset of proteins was reduced in response to fMLP, indicating a selective effect of ␤-PKC depletion on ligand-induced phosphorylation. Phosphorylation of a cytosolic component of the NADPH oxidase, p47 phox , and translocation and binding of p47 phox to cytochrome b 558 are essential steps in ligand-initiated activation of the NADPH oxidase. Indeed, depletion of ␤-PKC reduced the fMet-Leu-Phe-induced phosphorylation of a 47-kDa band and phosphorylation of immunoprecipitated p47 phox . However, the level of p47 phox , which is a differentiation marker in HL60 cells, was not altered by depletion of ␤-PKC. Thus, phosphorylation of p47 phox is triggered by ␤-PKC in activated dHL60 cells.
Phosphorylation of p47 phox is required for a conformational change in p47 phox , which releases binding of p47 phox to itself and to p40 phox (43), and allows translocation and binding of p47 phox to membrane-associated cytochrome b 558 (43)(44)(45)(46). In the present study, fMet-Leu-Phe and PMA triggered phosphorylation and translocation of p47 phox from the cytosol to the membrane in dHL60 cells. In ␤-PKC-depleted dHL60 cells, the ligand-induced translocation of p47 phox to the membrane was reduced, concordant with a role for ␤-PKC in the phosphorylation and translocation of p47 phox to the membrane-associated cytochrome b 558 and activation of the NADPH oxidase. Several serines in the C terminus of p47 phox , including Ser-303, Ser-304, and Ser-379, are consensus sequences for phosphorylation by PKC (47), and we have demonstrated that in vitro ␤-PKC phosphorylates p47 phox (3). The importance of a role for phosphorylation of p47 phox by PKC for assembly of an active NADPH oxidase was demonstrated in a neutrophil cell-free system (4). Phosphorylation of Ser-379 was essential for translocation of p47 phox to the membrane and activation of the NADPH oxidase, whereas a double mutation of Ser-303 and Ser-304 inhibited oxidase activity but not translocation of p47 phox (5,6). The present finding that ␤-PKC depletion reduced both phosphorylation and translocation of p47 phox suggests that Ser-379 was a target for phosphorylation by ␤-PKC.
Previous studies demonstrated translocation of ␤-PKC from cytosol to the membrane or cytoskeleton in PMA-activated neutrophils (3,20,48). Such translocation of PKC would allow ␤-PKC to access the activating lipid cofactors PS and DG, which are located in the membrane, as well as membraneassociated substrates. In the present study, both ␤I-PKC and ␤II-PKC were translocated to the membrane in dHL60 cells activated by fMet-Leu-Phe and by PMA. The amount of ␤I-PKC and ␤II-PKC recruited to the membrane in response to ligand was reduced in ␤-PKC-depleted cells. Thus, it is presently not possible to distinguish whether ␤I-PKC or ␤II-PKC is essential for activation of O 2 . generation. Although these studies demonstrated a role for ␤-PKC in ligand-induced phosphorylation and translocation of p47 phox , the findings do not preclude role(s) for other kinases in activation of O 2 . generation, either in phosphorylation of p47 phox , which has multiple phosphorylation sites, or in other aspects of signaling. Roles for ␦-PKC and a phosphatidate-activated kinase have been suggested in activation of the NADPH oxidase (16,49). Selective depletion of ␤-PKC by an antisense strategy demonstrated a selective role for ␤-PKC in signaling for fMet-Leu-Phe, immune complex, and PMA-induced O 2 . generation but not for ligand-initiated cell adherence to fibronectin or for azurophil degranulation. Depletion of ␤-PKC decreased ligand-induced phosphorylation of p47 phox , translocation of p47 phox and ␤-PKC to the membrane, and inhibited O 2 . generation.