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Protein Kinase C Is Required for p47^phox Phosphorylation and Translocation in Activated Human Monocytes¹

Erik A. Bey^*, Bo Xu^*, Ashish Bhattacharjee^*, Claudine M. Oldfield^*, Xiaoxian Zhao^*, Qing Li^*, Venkita Subbulakshmi^*, Gerald M. Feldman, Frans B. Wientjes and Martha K. Cathcart^2,*

^* Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195; Division of Monoclonal Antibodies, Office of Therapeutics, Research, and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892; and Department of Medicine, University College London, London, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKC, -, -, and -) and conventional PKC (including PKC and -), inhibited both phosphorylation and translocation of p47^phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKC, did not affect the phosphorylation or translocation of p47^phox, suggesting that PKC, -, or - is required. Furthermore, rottlerin (at doses that inhibit PKC activity) inhibited the phosphorylation and translocation of p47^phox. Rottlerin also inhibited O

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phagocytic cells of the blood, including monocytes, produce superoxide anion (O

The NADPH oxidase enzyme complex, also known as the respiratory burst oxidase, catalyzes the one-electron reduction of oxygen and, coupled with the oxidation of NADPH, results in the production of O

Compared with neutrophils, much less is understood about the organization and regulation of the monocyte NADPH oxidase. In our ongoing studies we are finding that the monocyte NADPH oxidase uses unique components and is regulated differently than the neutrophil NADPH oxidase complex (13, 14). For example, neutrophils, upon stimulation, produce a more immediate respiratory burst (peaking at 2–10 min depending on the stimulus), whereas monocytes gradually increase production of superoxide anion, with peak production at 1 h that wanes over time but is still detectable after several hours (15, 16). After stimulation of monocytes to activate NADPH oxidase, monocytes can mount an additional response after sufficient recovery and restimulation. This process is not observed in neutrophils. Additionally, agents that trigger NADPH oxidase activation in neutrophils do not necessarily trigger it in monocyte/macrophages, indicating alternative triggering mechanisms (17). These differences may account for the distinct roles of monocyte/macrophages and neutrophils in chronic vs acute inflammation (reviewed in Ref. 16).

Stimuli that activate the neutrophil NADPH oxidase cause extensive phosphorylation of p47^phox as well as other cytosolic components (9, 10, 18, 19). Phosphorylation of p47^phox in intact neutrophils is inhibited by general kinase inhibitors that also inhibit protein kinase C (PKC)³ (9, 10, 20, 21, 22, 23). In neutrophils and B lymphocytes, the translocation of p47^phox and other cytosolic oxidase components is blocked by general protein kinase inhibitors that inhibit PKC among other kinases, suggesting that phosphorylation precedes translocation (19, 24, 25). These studies demonstrated that the inhibition of phosphorylation and translocation correlated with a decrease in O

There are 12 known PKC isoforms divided among three families. The three families include classical PKCs (cPKC; including PKC, -I, -II, and -), novel PKCs (nPKC; including PKC, -, -, and -), and the atypical PKCs (aPKC; including PKC, -, -µ, and -). The isoforms are grouped according to their requirements for Ca²⁺ and phospholipids for maximal activity (26). The specific PKC isoforms regulating the phosphorylation and translocation of p47^phox in intact human monocytes and neutrophils have not been identified.

We have previously shown that PKC, but not PKC, is required for NADPH oxidase activity (13, 27). It appears that PKC is required for phosphorylating and activating cPLA₂. The purpose of this study was to determine whether other PKC isoforms are involved in regulating the activity of NADPH oxidase. In particular, we were interested in investigating the roles of PKC isoforms in regulating the phosphorylation and/or translocation of p47^phox in human monocytes activated by ZOP. Our results indicate that a calphostin C-sensitive, GF109203X-resistant PKC isoform(s) regulates p47^phox phosphorylation and translocation. We also show, in studies using a PKC-selective inhibitor (rottlerin) and specific PKC antisense oligodeoxyribonucleotides (ODN), that PKC regulates the phosphorylation and translocation of p47^phox. Additional studies demonstrate that p47^phox can serve as a substrate for monocyte-derived or recombinant PKC in vitro. Taken together, our data suggest that PKC expression and activity are required for monocyte NADPH oxidase activity through the regulation of phosphorylation and translocation of p47^phox.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of human monocytes and cell culture

Human monocytes were isolated and purified from whole blood as described previously (28). Briefly, PBS-diluted whole blood was layered over a Ficoll-Paque density solution and centrifuged. The mononuclear layer was collected and washed twice with PBS, and contaminating platelets were removed by centrifugation through bovine calf serum (BCS) after overlaying the serum with the mononuclear cells. This serum spin was repeated twice. Monocytes were isolated from the platelet-free mononuclear cells by adherence to flasks precoated with BCS and containing DMEM and 10% BCS (BCS/DMEM). The flasks were incubated for 2 h at 37°C in 10% CO₂. Nonadherent cells were removed by washing the flasks with BCS/DMEM. Adherent cells were detached with PBS containing 5 mM EDTA. The monocytes were collected, washed three times with BCS/DMEM, resuspended in BCS/DMEM, and incubated at 37°C in 10% CO₂ for at least 2 h before their use in experiments. In some of the experiments monocytes were isolated from human peripheral blood using a countercurrent centrifugal elutriation method (29, 30). Monocyte preparations purified by this procedure are consistently >95% CD14⁺. We obtained similar results using monocytes isolated by these two different protocols. Opsonized zymosan (ZOP; 2 mg/ml) was used to activate the monocytes by protocols previously described (27). Activation was for 1 h unless indicated otherwise.

Treatment of cells with pharmacological inhibitors

Monocytes were plated at a concentration of 5.0 x 10⁶/2 ml/well in six-well plates. The cells were treated for 30–60 min, as indicated, with or without one of the following inhibitors: HA-1004, H-7, GF109203X, calphostin C, or rottlerin, at specified concentrations. Cell viability, as assessed by the [¹⁴C]adenine release assay, was not affected by these inhibitors at the specified doses and times of treatment. After inhibitor treatment, monocytes were left unactivated or were activated with ZOP. Cells were collected and lysed as described below.

Treatment of cells with antisense ODN

Human monocytes were plated in six-well plates as described above. Monocytes were treated with PKC antisense or sense ODN as previously described (13) or with PKC antisense or sense ODN (31). ODN were HPLC-purified and were obtained from Sigma-Genosys (The Woodlands, TX) and Invitrogen Life Technologies (San Diego, CA). For PKC ODN treatment, human monocytes were treated with equivalent molar concentrations of two different PKC antisense or sense ODN sequences to yield final concentrations of 5 and 10 µM for 48 h with one refeeding at 24 h. Cells were then lysed or used for additional experimentation. The PKC antisense ODN sequences were 5'-GAAGGCGATGCGCAGGAA-3' and 5'-AGGAACGGCGCCATGGTGGG-3'. Complementary PKC sense ODN sequences were used as controls. The antisense ODN sequences were selected based on previous findings (32) and using our recently described protocol for identifying optimal mRNA regions for antisense ODN design (28).

Phosphorylation experiments

In these studies monocytes were plated as described above. The cells were radiolabeled with 100 µCi/ml [³²P]orthophosphate for 4–12 h, then treated with pharmacological inhibitors and activated as described above. The cells were collected and washed three times by centrifugation at 200 x g in PBS containing 1 mM sodium orthovanadate. The monocytes were lysed as previously described (33). After 30 min on ice, extracts were centrifuged at 9300 x g for 15 min. Postnuclear extracts were incubated with goat anti-human p47^phox for 2–12 h at 4°C with constant rotation. The immune complexes were collected by incubation with 20 µl of Sepharose A beads for 2 h. The beads were washed three times in lysis buffer and centrifuged at 1000 x g for 10 min at 4°C. After washing, the immune complexes were released from the beads by boiling in SDS sample buffer. The immune complexes were resolved by 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then placed in a phosphorimage screen for analysis.

Isolation of monocyte membrane fractions

Monocyte cytosol and membrane fractions were prepared by methods previously described (12). Briefly, drug-pretreated, ODN-pretreated cells or controls were left unactivated or were activated with ZOP. The monocytes were washed and resuspended in a relaxation buffer (12), then disrupted by two 15-s cycles of sonication at 4°C using a microprobe sonicator. After removal of unbroken cells and nuclei, the supernatant was subjected to centrifugation at 100,000 x g. The pellet was suspended and repelleted. This washed pellet is defined as the membrane fraction. The total membrane fraction was analyzed.

Western blots

Samples were resolved by 10% SDS-PAGE and transferred to a PVDF membrane (0.2 µm; Bio-Rad, Richmond, CA). Nonspecific binding sites were blocked with 5% milk in TBS (20 mM Tris-base (pH 7.4), 1.5 M NaCl, and 1% Nonidet P-40) at 4°C for 24 h. Human p47^phox was detected with either goat anti-human p47^phox polyclonal Abs (provided by Drs. H. Malech and T. Leto and diluted 1/1000 in PBS/Tween 20 (0.1%) (PBST) with 3% BSA), with rabbit anti-human p47^phox (34) or with a p47^phox mAb (1/1000 in PBST with 3% BSA; BD Transduction Laboratories, Lexington, KY), followed by incubation with an HRP-conjugated rabbit anti-goat, goat anti-rabbit, or goat anti-mouse IgG (1/1000 in PBST with 3% BSA; respectively from Kirkegaard & Perry (Gaithersburg, MD), Pierce (Rockford, IL), and BD Transduction Laboratories). The membrane was developed using Enhanced Chemiluminescence Detection reagents (Pierce) and quantified using the National Institutes of Health Image program (version 1.6; http:/rsb.info.nih.gov/nih-image) (35).

Superoxide anion assay

The method used to determine O

Preparation of GST fusion p47^phox

The full-length cDNA for p47^phox was cloned into the 5' end in-frame after the GST gene in pGEX.2T as previously described (37). The plasmid was transformed into Escherichia coli BL-21. The fusion protein was incubated with isopropylthiogalactoside (0.5 mM) at 37°C. After harvest, the bacteria were washed twice with ice-cold PBS containing protease inhibitors (500 µM PMSF and 1/100 dilution of protease inhibitor mixture (Sigma-Aldrich)). Cells were resuspended in PBS with protease inhibitors as described above, then treated with lysozyme (Sigma-Aldrich) in the presence of DNase I, followed by five cycles of freezing/thawing. After removing the cell debris by centrifugation, the GST-p47^phox fusion protein was purified using a MicroSpin GST Purification Module. To remove the fused GST tag, the fusion protein was digested with thrombin. The purity and stability of the purified recombinant p47^phox protein were evaluated by SDS/PAGE. All materials, except where indicated otherwise, were purchased from Amersham Biosciences (Piscataway, NJ).

In vitro phosphorylation of recombinant GST-p47^phox by PKC

The GST-p47^phox fusion protein or GST alone (20 pmol) was kinased by 2 pmol of PKC human recombinant protein (Upstate Biotechnology, Lake Placid, NY) in a 20-µl final reaction volume containing 20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 400 µM MgCl₂, 10 µM CaCl₂, lipid activator (consisting of 10 µM PMA and 0.28 mg/ml phosphatidylserine in a 0.3% Triton X-100 mixed micelle suspension), 1 µM ATP, 0.00025% Triton X-100, 1/100 protease inhibitor mixture (Sigma Genosys), 1/10 phosphatase inhibitor mixture, and 1 µCi of [-³²P]ATP for 30 min at 37°C. The kinase reaction was stopped by adding 5x sample buffer and boiling for 5 min. Controls using no substrate or 20 pmol of PKC substrate peptide (ERMRPRKRQGSVRRRV; Upstate Biotechnology) were treated in the same manner. Boiled samples were then resolved by 10% SDS-PAGE, transferred to a PVDF membrane, and visualized with a phosphorimager.

Immune complex PKC kinase assay

The immune complex PKC kinase assay was performed as described previously (31), using histone H1 (10 µg) or 5 µg of thrombin-digested GST-p47^phox fusion protein as substrates for PKC. The phosphorylated forms of histone H1 or p47^phox were detected by analysis using a phosphorimager. The blots were also subjected to Western blot analysis to examine the amount of PKC in the immune complexes.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Superoxide anion production, inhibited by GF109203X, can be restored by arachidonic acid in ZOP-activated human monocytes

We have previously shown that PKC is required for monocyte-mediated production of O

View larger version (19K): [in this window] [in a new window]   FIGURE 1. O2 production is restored by addition of arachidonic acid in monocytes inhibited by GF109203X, but not by calphostin C. Human monocytes (1 x 106/ml) were activated by ZOP in the presence or the absence of calphostin C (A; at the indicated doses) or GF109203X (B; 10 µM) with or without pretreatment for 30 min with arachidonic acid (1 µg/ml). Superoxide anion production was measured during the first hour of activation. Data represent the mean ± SD from three experiments.

Previous studies of the effect of kinase inhibitors on the phosphorylation of p47^phox in intact cells have been conducted using neutrophils and monocytic cell lines. To date, no studies have examined the effect of kinase inhibitors on the ZOP-activated phosphorylation of p47^phox in intact human monocytes. We recently reported that monocyte p47^phox protein was phosphorylated after ZOP activation (38). In that same study we detected translocation of p47^phox from the cytosol to the membrane at 1 h. We therefore used the 1 h time point to investigate the effect of kinase inhibitors on the phosphorylation and translocation of p47^phox.

We next investigated whether the calphostin C-sensitive, GF109203X-resistant kinase was involved in regulating the phosphorylation and/or translocation of p47^phox in activated monocytes. The effects of the general Ser/Thr kinase inhibitor H-7 and the PKC inhibitor calphostin C on the phosphorylation of p47^phox are shown in Fig. 2A. Even though lane 3 was underloaded, as indicated in the lower panel of Fig. 2A, the normalized percent inhibition of phosphorylation by calphostin C was 81%, and that by H-7 was 50%. Similar results were obtained in numerous experiments. These results suggest the involvement of Ser/Thr kinases, and particularly PKC, in p47^phox phosphorylation in ZOP-activated human monocytes. We then investigated the effect of GF109203X on the phosphorylation of p47^phox (Fig. 2B). GF109203X did not inhibit ZOP-activated p47^phox phosphorylation. These data suggest that a calphostin C-sensitive, GF109203X-resistant PKC isoform is involved in the regulation of p47^phox phosphorylation. Levels of p47^phox protein are not modulated by ZOP activation (28, 38); therefore, Western blots were performed to assess equal loading of the samples. These results are shown in the lower panels of Fig. 2, A and B.

View larger version (54K): [in this window] [in a new window]   FIGURE 2. PKC inhibitors affect p47phox phosphorylation and translocation. A, Calphostin C and H-7 inhibit p47phox phosphorylation. Human monocytes were plated in six-well plates at a concentration of 5 x 106 cells/2 ml/well. Monocytes were radiolabeled with [32P]orthophosphate as described in Materials and Methods. The cells were then left untreated or were treated with calphostin C (10 µM) or H-7 (100 µM) for 1 h, followed by activation with ZOP. After activation, cells were lysed, and lysates were immunoprecipitated using a goat anti-human polyclonal p47phox Ab as described in Materials and Methods. Immune complexes were resolved by SDS-PAGE and electrophoretically transferred to a PVDF membrane. Phosphorylation of p47phox was detected by phosphorimage analysis. In the lower panel, the same PVDF membrane was subjected to Western blot analysis using a rabbit polyclonal p47phox Ab. Near equal loading of p47phox protein was observed. B, GF109203X does not inhibit p47phox phosphorylation. Human monocytes, plated as described in Fig. 1A, were radiolabeled with [32P]orthophosphate as previously described, either left untreated or treated with GF109203X (10 µM) for 1 h, and activated with ZOP. After activation, cells were lysed and handled as described in A. The lower panel shows the same PVDF membrane after Western blot analysis. The p47phox protein was detected using a rabbit polyclonal p47phox Ab. C, Calphostin C and H-7 inhibit p47phox translocation. Monocytes were treated as described in A with calphostin C (10 µM), GF109203X (10 µM), HA-1004 (100 µM), or H-7 (100 µM) for 1 h. Cells were then left unactivated or were activated with ZOP. After activation, cells were fractionated as described in Materials and Methods, and the membrane fractions were resolved by SDS-PAGE. The p47phox protein was detected by Western blotting using a goat anti-human polyclonal p47phox Ab. The data in this figure are from representative experiments of three similar experiments that were performed.

Rottlerin inhibits both the phosphorylation and the translocation of p47^phox

Based on the results presented in Figs. 1 and 2, we hypothesized that an nPKC, other than PKC, was involved in p47^phox phosphorylation and translocation. We recently reported (13) that among nPKCs, the expressions of only PKC and PKC are induced upon ZOP activation of monocytes. We therefore proceeded to test the potential involvement of PKC in NADPH oxidase activity and, more specifically, in p47^phox phosphorylation. Rottlerin, a drug previously shown to be a selective inhibitor of PKC enzyme activity (39), caused a dose-dependent inhibition of p47^phox phosphorylation (Fig. 3A). Phosphorylation was inhibited 41.1% by 5 µM rottlerin and 89.0% by 10 µM in this experiment. Similar results were obtained in several experiments. These results suggest that PKC regulates the phosphorylation of p47^phox.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Rottlerin inhibits p47phox phosphorylation, translocation, and NADPH oxidase activity. A, Rottlerin inhibits p47phox phosphorylation. Monocytes were plated as described previously. The cells were radiolabeled with [32P]orthophosphate and were left untreated or were treated for 1 h with the indicated doses of rottlerin, then activated with ZOP as indicated. After activation, cells were lysed, and p47phox was immunoprecipitated using a goat anti-human polyclonal Ab. Immune complexes were resolved by SDS-PAGE and electrophoretically transferred to a PVDF membrane. Phosphorylation of p47phox was quantified by analysis on a phosphorimager. As shown in the lower panel, the same PVDF membrane was subjected to Western blot analysis with a p47phox mAb. The data are representative of three similar experiments. B, Rottlerin inhibits p47phox translocation. Monocytes were plated as previously described and then were left untreated or were treated for 1 h with rottlerin at the doses indicated. Cells were then not activated or were activated with ZOP. After activation, cells were fractionated as described in Materials and Methods, and the membrane fractions were resolved by SDS-PAGE and analyzed by Western blot analysis. The p47phox protein was detected using a rabbit polyclonal p47phox Ab. The data are representative of three similar experiments. C, Rottlerin inhibits NADPH oxidase activity. Monocytes were plated in 96-well plates (100 µl; 1 x 106/ml). Cells were left untreated or were treated with rottlerin for 1 h at the indicated doses. O2 production was measured as described in Materials and Methods. These data represent the mean ± SEM for three experiments.

To determine whether inhibiting the phosphorylation and translocation of p47^phox affected oxidase activity, we tested the effect of rottlerin on O

PKC antisense ODN inhibits phosphorylation and translocation of p47^phox

Our inhibitor studies suggested that PKC is required for p47^phox phosphorylation and translocation. To confirm these findings, because pharmacologic inhibitors are often limited in their specificity, and particularly because rottlerin has been reported to inhibit PKB, we chose to perform similar experiments using antisense ODN to PKC. We treated monocytes with an equimolar mixture of two different antisense ODN specific for PKC. Representative results of this treatment on the expression of PKC are shown in Fig. 4A (upper panel). Antisense ODN to PKC selectively inhibited PKC protein expression without inhibiting expression of PKC or tubulin (Fig. 4A, lower and middle panels). When normalized for sample loading, as determined by the tubulin blot (Fig. 4A, middle panel), the antisense ODN mixtures caused 21 ± 2% inhibition at 5 µM, whereas a 10 µM total concentration caused 66 ± 2% inhibition of PKC expression (p < 0.01) in Fig. 4A (upper panel). In similar experiments, PKC-specific antisense ODN also did not affect the protein level of p47^phox (data not shown). We then tested the effect of PKC antisense ODN on the phosphorylation and translocation of p47^phox. The data shown in Fig. 4B demonstrate that PKC antisense ODN inhibited the phosphorylation of p47^phox. This membrane was probed with Ab to p47^phox, and equal levels of p47^phox protein were present in each well (data not shown). As shown in the upper panel of Fig. 4C, translocation of p47^phox was inhibited by 23 and 83% in monocytes pretreated with 5 and 10 µM antisense ODN, respectively. This blot was stripped and reprobed with the membrane marker CD45 to assess loading (Fig. 4C, lower panel).

View larger version (26K): [in this window] [in a new window]   FIGURE 4. PKC, but not PKC, regulates p47phox phosphorylation and function. A, PKC antisense inhibits PKC protein expression. Monocytes were plated as previously described and then were left untreated or were treated with PKC antisense or sense ODNs for 48 h with two feedings at the doses indicated. Cells were lysed as described in Materials and Methods, resolved by SDS-PAGE, and analyzed by Western blot analysis. PKC protein was detected using a rabbit polyclonal PKC Ab. The blot was stripped and reprobed with anti--tubulin Ab to ensure equal loading. The same blot was reprobed with Ab to PKC to show that PKC antisense ODN do not inhibit PKC expression. B, PKC antisense ODN inhibits phosphorylation of p47phox. Monocytes were plated and treated as in A. After PKC antisense ODN treatment, monocytes were radiolabeled with [32P]orthophosphate for 12 h. The cells were then not activated or were activated with ZOP. After activation, cells were lysed, and the lysates were immunoprecipitated using a goat anti-human polyclonal p47phox Ab. Immune complexes were resolved by SDS-PAGE and electrophoretically transferred to a PVDF membrane. Phosphorylation of p47phox was quantified by analysis on a phosphorimager. A reprobe of this blot with anti-p47phox indicated equal loading of protein (data not shown). C, PKC antisense inhibits translocation of p47phox. Monocytes were plated and treated with antisense ODN as described in A. Cells were left unactivated or were activated with ZOP. After activation, cells were fractionated as described in Materials and Methods, and the membrane fractions were resolved by SDS-PAGE and analyzed by Western blot analysis. The p47phox protein was detected using a rabbit polyclonal p47phox Ab (upper panel). The blot was stripped and reprobed with the membrane marker CD45 (lower panel). D, PKC antisense inhibits NADPH oxidase activity. Monocytes were plated in 24-well plates (500 µl; 1 x 106/ml). Cells were left untreated or were treated with PKC antisense or sense ODN for 48 or 72 h at 10 µM. O2 production was measured as described in Materials and Methods. The data represent the mean ± SEM of four experiments.

PKC does not regulate p47^phox phosphorylation or translocation

Our previous studies have shown that PKC is required for ZOP-activated, monocyte-mediated O

View larger version (41K): [in this window] [in a new window]   FIGURE 5. PKC antisense ODN inhibits PKC protein expression, but does not inhibit p47phox phosphorylation or translocation. Monocytes were treated identically to those in Fig. 4, A–C, except that PKC antisense or sense ODNs (10 µM) were used to treat the cells for 24 h. A, PKC protein was detected using a PKC mAb (A, upper panel). The same membrane was then reprobed with a mAb to tubulin to examine protein loading (lower panel). B and C, p47phox phosphorylation and translocation were examined in monocytes treated with two doses of antisense and sense ODN. Results are from a representative experiment of three performed.

ZOP induces phosphorylation and activation of PKC in human monocytes

Our studies of p47^phox phosphorylation and translocation (Figs. 3 and 4) suggest that PKC may be one of the upstream kinases regulating p47^phox phosphorylation in ZOP-activated human monocytes. To determine whether PKC is phosphorylated/activated after ZOP treatment, we used Abs specifically recognizing phosphotyrosine (PY99) or the phosphoserine at position 643 of PKC. Monocytes were treated with ZOP for different times, then lysed, and whole cell lysates were immunoprecipitated with anti-PKC Abs, followed by Western blot analysis. As shown in Fig. 6, ZOP induced both tyrosine and serine phosphorylation of PKC in a time-dependent fashion (Fig. 6, A and B). The phosphorylated form of PKC was detected as early as 5 min, suggesting that activation of PKC in ZOP-treated cells is one of the early signaling events. The phosphorylation signals continued to increase thereafter and were sustained up to 60 min after ZOP treatment. To determine whether phosphorylation of PKC resulted in enhanced kinase activity, we performed an in vitro PKC kinase assay in which monocyte PKC was immunoprecipitated with anti-PKC Ab, and histone H1 was used as a PKC substrate. ZOP treatment resulted in a strong induction of PKC kinase activity, as indicated by the phosphorylation of histone H1 (Fig. 6C). Pretreatment of cells with rottlerin for 30 min before ZOP activation markedly inhibited ZOP-induced PKC kinase activity, whereas pretreatment with GF109203X failed to block the kinase activation (Fig. 6D).

View larger version (52K): [in this window] [in a new window]   FIGURE 6. PKC is phosphorylated and activated in ZOP-stimulated human monocytes. A and B, Monocytes (5 x 106/2 ml culture medium) were plated in each well of six-well plates and treated with ZOP for various times as indicated. Cells were lysed, and 100 µg of total protein was immunoprecipitated with anti-PKC Abs and blotted with either anti-PY99 Ab (A) or anti-phospho-PKC (Ser643) Ab (B). The blots were stripped and reprobed with anti-PKC Ab to assess loading (lower panels). C, Monocytes were plated as described in A and B and were treated with ZOP for the indicated times. D, Monocytes were either left untreated or were treated with rottlerin (5 µM) or GF109203X (1 µM) for 30 min before ZOP treatment for an additional 30 min. Cells were lysed, and 100 µg of total protein was immunoprecipitated with anti-PKC Abs. PKC activity was measured by the immune complex protein kinase assay using histone H1 as the substrate, as described in Materials and Methods. Upper panels, Phosphorimages; lower panels, Western blot results using anti-PKC Ab. The data are from a representative experiment of at least three repeat experiments that gave similar results.

Monocyte-derived, PKC immune complexes phosphorylate p47^phox

Our data indicate that PKC regulates p47^phox phosphorylation, but do not prove that PKC directly phosphorylates p47^phox. To explore whether this might be the case, we performed an immune complex kinase assay in which the digested GST-p47^phox fusion protein was used as a substrate for lysate-derived PKC. We found that PKC immunoprecipitated from both ZOP- and PMA-treated cells can phosphorylate p47^phox (Fig. 7A). In contrast, PKC immune complexes from unactivated monocytes mediated substantially lower, but detectable, p47^phox phosphorylation. Activation induced a >3-fold increase in p47^phox phosphorylation by 15 min. The ZOP-induced phosphorylation of p47^phox by PKC was inhibited by pretreatment of the monocytes with rottlerin, but not by pretreatment with GF109203X (Fig. 7B). These data demonstrate for the first time that PKC is activated in ZOP-treated monocytes, and that PKC is one of the crucial kinases regulating p47^phox phosphorylation.

View larger version (35K): [in this window] [in a new window]   FIGURE 7. Phosphorylation of recombinant p47phox fusion protein by PKC using monocyte-derived PKC and recombinant PKC. A and B, Phosphorylation by PKC immune complexes. Human monocytes were treated with ZOP for 15 or 60 min or with 50 ng/ml PMA for 15 min. Inhibitors (5 µM rottlerin or 10 µM GF109203X) were added to the culture 30 min before ZOP treatment. Human monocytes were lysed, and 100 µg of total protein was immunoprecipitated with anti-PKC Abs. The immune complexes were incubated with GST-p47phox digested with thrombin. Phosphorimages are shown in the top panels, and lower panels show the Western blot results of the respective membranes shown in the top panels. C, In vitro phosphorylation of p47phox. The recombinant GST-p47phox fusion protein (20 pmol) was exposed to 2 pmol of PKC human recombinant. Controls, using 20 pmol of PKC substrate peptide or GST (at the same concentration as in the p47phox-GST fusion protein), were treated similarly. The samples were resolved by SDS-PAGE, transferred to a PVDF membrane, and exposed to a phosphorimage screen. The data shown in each part of this figure are representative of the results obtained in three similar experiments.

PKC directly phosphorylates p47^phox in vitro

To assess whether PKC is able to directly phosphorylate p47^phox, we conducted in vitro kinase assays using recombinant active PKC and the GST-p47^phox fusion protein. As shown in Fig. 7C, recombinant PKC was active in the assay, causing substantial autophosphorylation (shown in all lanes) as well as phosphorylation of the PKC substrate peptide used as a positive control (lane 2). Recombinant active PKC caused strong phosphorylation of p47^phox (lane 4) compared with the GST control (lane 1).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The phosphorylation and assembly of the components of the NADPH oxidase have been extensively studied in human neutrophils. In contrast, similar studies involving regulation of this enzyme complex in human monocytes have been limited. One key element in neutrophil NADPH oxidase assembly and activation is believed to be stimulus-induced phosphorylation of the essential components of the NADPH oxidase and their translocation from the cytosol to the plasma membrane (2, 7). p47^phox is the most well studied of the phosphorylated NADPH oxidase components. Previous studies with PMA-stimulated neutrophils or B lymphocytes have indicated that activation of NADPH oxidase requires phosphorylation of p47^phox, which occurs before its translocation (19, 40, 41, 42).

Phosphorylation of components of the NADPH oxidase has been investigated primarily through the use of broad-spectrum protein kinase inhibitors as well as some moderately selective PKC inhibitors. Through these investigations it has been found that PKCs, stimulated by PMA, play a significant role in regulating the phosphorylation of oxidase components in human neutrophils (21, 24, 43). Our data presented in this study suggest an important role for PKC in ZOP-stimulated human monocytes, because both general and moderately selective PKC inhibitors inhibited phosphorylation and translocation of p47^phox. Our present studies also indicate, as predicted, that inhibition of phosphorylation prevents translocation and blocks O

Previous studies using cell-free systems have implicated PKC involvement in p47^phox phosphorylation and NADPH oxidase activity in neutrophils (44). A recent study using in vitro systems with fractionated neutrophil components and cell-free assays reported that PKC, -, -II, and - can phosphorylate p47^phox (45). These investigators found that phosphorylation of p47^phox by the aforementioned isoforms induced translocation of p47^phox and stimulated oxidase activity. It should be noted, however, that results obtained using cell-free, fractionated neutrophil components may not reflect the physiological role of PKCs in intact cells.

Our data presented in this manuscript address the physiological role of PKC in regulating the activity of NADPH oxidase in intact primary human monocytes. In our studies we used ZOP as a physiological activator of oxidase activity. In contrast, cell-free systems do not require physiological stimuli to achieve activation of the oxidase. Thus, nonphysiological stimulation of the oxidase in cell-free systems may fail to address the relevant roles of specific PKC isoforms in intact human cells.

PKC was found to be involved in PMA-stimulated oxidase activity in human neutrophils (46). In other studies, PKC has been reported to mediate p47^phox phosphorylation in fMLP-activated, but not PMA-activated, neutrophils (47). The serine/threonine kinase Akt has also been reported to regulate p47^phox phosphorylation using in vitro neutrophil assays and in fMLP-activated, but not PMA-activated, intact neutrophils (48, 49). The roles of PKC and AKT in ZOP-activated monocytes remain to be determined. To date, no studies have shown that a specific kinase or isoform of PKC regulates phosphorylation of any of the key components of the NADPH oxidase in intact monocytes. As outlined above, we previously demonstrated that PKC is required for O

In this study we present data demonstrating that the PKC inhibitor, rottlerin, inhibits p47^phox phosphorylation and translocation and also regulates NADPH oxidase activity. This functional regulation of NADPH oxidase activity by PKC is corroborated by our results showing that antisense ODN to PKC also blocked monocyte O

This is the first report that a specific PKC isoform regulates the function of the monocyte NADPH oxidase by regulating phosphorylation and translocation of p47^phox. In addition to our data indicating isoform-specific regulation of monocyte NADPH oxidase activity, we report that recombinant p47^phox is a substrate for in vitro phosphorylation by PKC. We also show that rottlerin inhibits in vitro phosphorylation of p47^phox as well as ZOP-induced PKC activity. Our laboratory is currently investigating the amino acid residues on p47^phox that are phosphorylated by PKC. Our finding that PKC is an upstream kinase regulating p47^phox phosphorylation and NADPH oxidase activity in intact monocytes does not address whether other components of this enzyme complex can serve as substrates for PKC and does not rule out a role for other PKC isoforms as regulatory kinases in the pathways leading to NADPH oxidase activity.

In summary, we show for the first time that PKC specifically regulates the function of NADPH oxidase in ZOP-induced primary human monocytes. The PKC inhibitor rottlerin inhibited phosphorylation and translocation of p47^phox, an essential regulatory component of NADPH oxidase activity. In addition, we corroborated these inhibitor findings using antisense ODN specific to PKC. Finally, we show that recombinant p47^phox is a substrate for phosphorylation by monocyte-derived or recombinant PKC and that rottlerin inhibited this phosphorylation. Our results suggest that PKC is an important regulator of p47^phox phosphorylation and translocation. Future studies are needed to address whether PKC is an immediate upstream kinase of p47^phox and to define the role of PKC in regulating NADPH oxidase phosphorylation and activity in other cells expressing this oxidase, such as neutrophils, lymphocytes, and vascular smooth muscle cells.

	Footnotes

 
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.

¹ This work was supported by National Institutes of Health Grants HL51068 and HL61971 (to M.K.C.).

² Address correspondence and reprint requests to Dr. Martha K. Cathcart, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: cathcam{at}ccf.org

³ Abbreviations used in this paper: PKC, protein kinase C; BCS, bovine calf serum; cPKC, conventional PKC (PKC, -, and -); nPKC, novel PKC (PKC, -, -, and -); ODN, oligodeoxyribonucleotide; PBST, PBS/Tween; PVDF, polyvinylidene difluoride; SOD, superoxide dismutase; ZOP, opsonized zymosan.

Received for publication January 16, 2004. Accepted for publication August 19, 2004.

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