PKCζ Regulates TNF-α–Induced Activation of NADPH Oxidase in Endothelial Cells
Although oxidant generation by NADPH oxidase is known to play an important role in signaling in endothelial cells, the basis of activation of NADPH oxidase is incompletely understood. The atypical isoform of protein kinase C, PKCζ, has been implicated in the mechanism of tumor necrosis factor-α (TNF-α)–induced oxidant generation in endothelial cells; thus, in the present study, we have addressed the role of PKCζ in regulating NADPH oxidase function. We showed by immunoblotting and confocal microscopy the presence of the major cytosolic NADPH oxidase subunits, p47phox and membrane-bound gp91phox in human pulmonary artery endothelial (HPAE) cells. TNF-α failed to activate oxidant generation in lung vascular endothelial cells derived from p47phox−/− and gp91phox−/− mice, indicating the requirement of NADPH oxidase in mediating the oxidant generation in endothelial cells. Stimulation of HPAE cells with TNF-α resulted in the phosphorylation of p47phox and its association with gp91phox. Inhibition of PKCζ by multiple pharmacological and genetic approaches prevented the TNF-α–induced phosphorylation of p47phox, and its translocation to the membrane. PKCζ was shown to colocalize with p47phox, and inhibition of PKCζ activation prevented the interaction of p47phox with gp91phox induced by TNF-α. Furthermore, inhibition of association of p47phox with gp91phox prevented the oxidant generation in endothelial cells. These data demonstrate a novel function of PKCζ in signaling oxidant generation in endothelial cells by the activation of NADPH oxidase, which may be important in mediating endothelial activation responses.
Oxidant generation in the endothelium may have an important role in signal transduction, gene expression, cell proliferation, apoptosis, and in the pathophysiology of various diseases such as acute respiratory distress syndrome (ARDS) and tissue injury from ischemia/reperfusion.1–7⇓⇓⇓⇓⇓⇓ Studies have shown that oxidant generation is involved in the signaling cascade mediating the tumor necrosis factor-α (TNF-α)–induced activation of nuclear factor (NF)–κB and expression of adhesion molecules such as ICAM-1 in endothelial cells.2–4⇓⇓ Despite the requirement of oxidant signaling in these responses, the upstream regulation of oxidant production in endothelial cells is not known.
NADPH oxidase is a highly regulated membrane-bound enzyme complex that catalyzes the 1-electron reduction of oxygen to superoxide anion with the simultaneous oxidation of cytosolic NADPH. Components of NADPH oxidase complex include the membrane-bound cytochromeb558, composed of 2 subunits, p22phox and gp91phox, and 4 cytosolic subunits, p47phox, p67phox, p40phox, and the small GTP-binding protein, Rac1/Rac2.8,9⇓ Assembly of the active NADPH oxidase complex requires the translocation of the cytosolic factors, p47phox, p67phox, and Rac1/Rac2 to the plasma membrane where these components interact with cytochrome b558.8–10⇓⇓ During complex assembly, p47phox first interacts with cytochrome b558.11 Translocation is initiated by signaling events, including the phosphorylation of p47phox, which contains a number of protein kinase C (PKC), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) phosphorylation sites (RXXS/TXRX, RRXS, and PXSP, respectively).12,13⇓ Although NADPH oxidase has been implicated in oxidant signaling in endothelial cells, there is little information on the regulation of NADPH oxidase activation and the generation of oxidants. In the present study, we addressed the role of PKC, a family of serine/threonine kinases,14–16⇓⇓ in mediating NADPH oxidase activation in endothelial cells. PKC isoforms are classified into three groups based on their structure and activation mechanisms: phosphatidylserine (PS)-, diacylglycerol (DAG)-, and Ca2+-dependent conventional PKC (cPKC; α, βΙ, βΙΙ, and γ), Ca2+-independent novel PKC (nPKC; δ, ε, μ, θ, and η) isoforms, and DAG-, and Ca2+-independent atypical PKC (aPKC; ζ, and λ/ι) isoforms. Tissue distribution of PKC-α, -δ, and -ζ is widespread, whereas the others are localized in a tissue- and cell type–specific manner. In addition to PKC-α, -δ, and -ζ, endothelial cells also express the PKC-β, -ε, -η, and -θ isoforms.17,18⇓ In a previous study, we showed that TNF-α–induced oxidant generation requires the activation of PKCζ,18 the atypical PKC isoform abundantly expressed in endothelial cells. In the present study, we investigated mechanisms by which PKCζ induces endothelial oxidant generation. We show that PKCζ is required for signaling oxidant generation in response to TNF-α and does so through the activation of NADPH oxidase. The mechanism of activation involves PKCζ-induced phosphorylation of p47phox and its targeting to the membrane where it associates with gp91phox to generate the active NADPH oxidase complex.
Materials and Methods
Human recombinant TNF-α with a specific activity of 2.3×107 was purchased from Promega (Madison, Wis). Monoclonal and polyclonal antibodies against p47phox or gp91phox subunit of NADPH oxidase were kindly provided by Ulla G. Knaus and Bernard M. Babior (The Scripps Research Institute, La Jolla, Calif), Michio Nakamura (Nagasaki University, Nagasaki, Japan), and Mark T. Quinn (Montana State University, Bozeman, Mont). The synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, was a kind gift from Chris Ross (Kansas State University College of Veterinary Medicine, Manhattan, Kans). The following items were purchased: monoclonal antibody to PKCζ (Alexis Biochemicals), secondary Western blotting antibodies (Santa Cruz Biotechnology), nitrocellulose membrane from Bio-Rad Laboratories; phorbol esters (PMA) and calphostin C from Sigma Chemical; chelerythrine from Calbiochem-Novabiochem Corp; bicinchoninic acid (BCA) protein assay reagents were from Pierce. Myristoylated membrane-permeable peptide inhibitor of PKCζ and PKCθ were obtained from BioSource International. All other materials were from Sigma.
Endothelial Cell Cultures
Human pulmonary artery endothelial (HPAE; Clonetics, La Jolla, Calif) cells were cultured as described18 in gelatin-coated flasks using endothelial basal medium 2 (EBM2) with bullet kit additives. Mouse lung vascular endothelial cell cultures were obtained from wild-type and p47phox−/− and gp91phox−/− mice. The knockout mice19,20⇓ were provided by Drs Mary C. Dinauer (University of Indiana School of Medicine, Indianapolis, Ind) and Steven M. Holland (NIH, Bethesda, Md). Mice were housed in the University of Illinois Animal Care Facilty in specific pathogen-free conditions with free access to food and water. Studies were performed in accordance with institutional and NIH guidelines and after approval from the Institutional Review Board. Mouse endothelial cells were cultured as described by us.21
Immunoprecipitation and Immunoblotting
HPAE cells were washed with ice-cold TBS and lysed in 10 mmol/L Tris-HCl (pH 7.5), 5 mmol/L EDTA, 10 mmol/L EGTA, 50 μg/mL PMSF, and a mixture of protease inhibitors. Lysates were sonicated for 10 seconds and then ultracentrifuged at 100 000g for 1 hour at 4°C, and the supernatants were collected and designated cytosolic fraction. The remaining pellets were resuspended in the above lysis buffer containing 1% Triton X-100, sonicated, and incubated for 30 minutes at 4°C. These lysates were microfuged at 4°C, and the supernatants were designated membrane fraction. For study of association of p47phox with gp91phox, 300 μg precleared lysates were incubated with 10 μL monoclonal anti-gp91phox IgG for 1 hour at 4°C. Immunocomplexes were Western blotted as described.18
Phosphorylation of p47phox
HPAE cells grown to confluence were washed and incubated with [32P]orthophosphate (100 μCi/dish) in phosphate-free medium overnight. Cells were lysed with 1 mL of lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.5% NP40, 1 mmol/L Na3VO4, 150 mmol/L NaCl, 50 μg/mL PMSF, 1% Triton X-100, 50 mmol/L NaF, 30 mmol/L NaPPi, and protease inhibitors). p47phox was immunoprecipitated from the lysates as described above. Immunocomplexes were washed and resolved by 4% to 15% SDS/PAGE. Gels were transferred to nitrocellulose, and the phosphorylated form of p47phox was detected by autoradiography.
HPAE cells grown on gelatinized cover slips were treated as indicated, washed with HBSS and fixed in 4% paraformaldehyde, and blocked with 5% goat serum containing 0.2% BSA, 0.01% NaN3, and 0.1% Triton X-100. Thereafter, cells were incubated for 1 hour at room temperature with 1 μg of the indicated primary antibody. After 3 washes in HBSS, 4 μg/mL secondary Ab conjugated with rhodamine or fluorescene (Molecular Probes) was added for an additional 2 hours at room temperature. Cells were extensively washed in HBSS, mounted on glass slides with ProLong Antifade mounting media (Molecular Probes), and images were acquired with the Zeiss LSM 510 confocal microscope.
Oxidant generation in HPAE cells was measured as described18 with slight modifications. After treatment, cells were washed ×2 with HBSS and fixed in 4% paraformaldehyde for 20 minutes at room temperature. Cultures were then viewed with fluorescence microscopy.
Transfection of HPAE Cells
Phosphorothioate oligonucleotides to PKCζ sense (ATGCCCAGCAGGACC) and antisense (GGTCCTGCTGGGCAT) have been previously described.22,23⇓ Both are targeted to the translation initiation codon of PKCζ mRNA. Phosphorothioate antisense oligonucleotide to PKCα (GTTCTCGCTGGTGAGTTTCA) is directed to the 3′-untranslated region of PKCα mRNA. Oligonucleotides were transfected using Lipofectin (GIBCO-BRL) as described.3,18⇓ The expression vector pcDNA3 containing HA-tagged kinase defective PKC-ζ, -α, and -ε isoforms and catalytically active PKCζ mutant24 were gifts from Dr Jae-Won Soh (Columbia University, New York, NY) and were transfected as described.22
TNF-α Induces Phosphorylation and Translocation of p47phox in Endothelial Cells
We determined the phosphorylation status of p47phox, a requirement for activation NADPH oxidase.13,25,26⇓⇓ TNF-α challenge of HPAE cells induced p47phox phosphorylation in a time-dependent manner. Phosphorylated form of p47phox increased as early as 1 minute, peaked at 5 minutes after TNF-α challenge, and declined after 15 minutes (Figure 1A). We next determined whether p47phox phosphorylation was accompanied by its translocation to the membrane. Time course of membrane translocation of p47phox was shown to parallel phosphorylation of p47phox, except that remained membrane-associated even at 15 minutes after TNF-α challenge (Figure 1B) and returned to the basal level by 30 minutes (data not shown).
TNF-α Induces Association of p47phox With gp91phox
To determine if membrane translocation of p47phox resulted in the association with gp91phox, membrane fractions from control and TNF-α–treated HPAE cells were immunoprecipitated with an antibody against gp91phox and immunoblotted with an antibody against p47phox. Results showed that TNF-α promoted the association of p47phox with gp91phox (Figure 2A). Confocal immunofluorescence confirmed the association of p47phox with gp91phox induced by TNF-α (Figure 2B). In resting cells, p47phox was localized in cytosol (Figure 2B). TNF-α stimulation of HPAE cells increased the staining of p47phox throughout the cytosol, especially in the perinuclear region (Figure 2B). Double labeling with antibodies against p47phox (red) and cytochrome b558 (green) yielded strong yellow-orange staining in TNF-α–stimulated HPAE cells (Figure 2B) indicative of colocalization.
Inhibition of PKCζ Prevents TNF-α–Induced Phosphorylation and Targeting of p47phox
We used general and isoform-specific inhibitors to delineate the involvement of PKCζ in mediating the translocation of p47phox. Confocal imaging and Western blotting showed that pretreatment of HPAE cells with PKC inhibitors, calphostin C and chelerythrine, prevented TNF-α–induced translocation of p47phox (Figures 3A and 3B). In another experiment, we observed that depletion of cPKC and nPKC by prolonged exposure of HPAE cells to phorbol esters (500 nmol/L; 20 hours)18 failed to prevent membrane targeting of p47phox induced by TNF-α (Figure 3C), thus excluding the involvement of cPKC and nPKC in the mechanism of the response.
We next determined the possibility that aPKC isoforms are involved in the mechanism of TNF-α–induced membrane translocation of p47phox and NADPH oxidase activation. Myristoylated membrane-permeable peptide antagonist or antisense oligonucleotide (which specifically inhibit the function or synthesis of PKCζ,18,27⇓ an abundantly expressed aPKC isoform in endothelial cells) was used to address the role of this PKC isoform in mediating the response. Preincubation of cells with PKCζ peptide inhibitor prevented TNF-α–induced membrane translocation of p47phox (Figures 4A and 4B). The antisense oligonucleotide to PKCζ, which inhibits the expression of PKCζ,18 also prevented TNF-α–induced membrane targeting of p47phox (Figure 4C). In contrast, antisense oligonucleotide to PKCα failed to modify the TNF-α response (Figure 4C). We also determined the effects of kinase-defective mutant of PKCζ (PKCζK281R) on the membrane translocation of p47phox. Expression of PKCζK281R prevented the TNF-α–induced membrane targeting of p47phox, whereas in control experiments, inhibition of PKCα by the kinase-defective mutant (PKCαK368R) failed to prevent the response (Figure 4D).
Expression of kinase-defective mutant of PKCε (PKCεK437R) also failed to inhibit the response (data not shown). In another experiment, we showed that expression of constitutively active PKCζ mutant induced membrane targeting of p47phox in the absence of TNF-α challenge (Figure 4E). Thus, these data indicate that PKCζ is required and sufficient to mediate the TNF-α–induced membrane translocation of p47phox.
As p47phox phosphorylation is required for NADPH oxidase activation,13,25,26⇓⇓ we evaluated whether PKCζ is involved in mediating the phosphorylation of p47phox. Inhibition of PKCζ by the specific peptide antagonist27 prevented TNF-α– induced p47phox phosphorylation (Figure 5A). In contrast, inhibition of PKCθ had no effect on this response (Figure 5A). We determined the ability of PKCζ to associate with p47phox after TNF-α challenge because this may be required for phosphorylation. Analysis by confocal microscopy showed ubiquitous staining of PKCζ (green) and general cytosolic and perinuclear staining of p47phox (red), without apparent colocalization (Figure 5B). However, TNF-α stimulation of HPAE cells altered the cellular distribution of both PKCζ and p47phox, resulting in colocalization of PKCζ with p47phox (Figure 5B). In contrast, phorbol esters (PMA) failed to induce the same pattern of colocalization of PKCζ with p47phox (data not shown). In another control experiment, we determined that TNF-α failed to induce the colocalization of PKCα with p47phox (data not shown). Thus, activation of PKCζ is involved in the phosphorylation p47phox and its targeting to the membrane in response to TNF-α challenge of endothelial cells.
Inhibition of PKCζ Prevents TNF-α–Induced Association of p47phox With gp91phox and Oxidant Generation
We evaluated the effects of inhibition of PKCζ on TNF-α–induced association of p47phox with gp91phox and resultant oxidant generation in HPAE cells. Coimmunoprecipitation studies showed that pretreatment of cells with the peptide antagonist of PKCζ inhibited the TNF-α–induced association of p47phox with gp91phox (Figure 6A). This finding is consistent with the role of PKCζ in phosphorylating and membrane targeting of p47phox as described in Figure 4. We used the fluorescent redox-sensitive dye carboxy-H2DCFDA to determine if the effects of PKCζ inhibition in TNF-α–induced NADPH oxidase assembly prevented the oxidant generation. Cells were challenged with TNF-α for 1 hour to allow maximum oxidant accumulation during this period. Control cells showed little fluorescence. In contrast, TNF-α induced marked oxidant generation (Figure 6B), which was evident as early as 5 minutes after TNF-α exposure of HPAE cells (data not shown). Inhibition of PKCζ by the peptide antagonist prevented TNF-α–induced oxidant generation (Figure 6B). In another experiment in lung vascular endothelial cells obtained from p47phox−/− and gp91phox−/− mice, we showed that TNF-α failed to activate oxidant generation (Figure 7). These data indicate that PKCζ is crucial in signaling NADPH oxidase activation and oxidant generation in endothelial cells.
We investigated the basis of NADPH oxidase activation in endothelial cells because oxidant signaling triggered by this complex has an important function in mediating TNF-α–induced activation of NF-κB and resultant expression of adhesion molecules such as ICAM-1.2–4⇓⇓ The present results show that the atypical PKC isoform PKCζ plays a critical role in signaling NADPH oxidase activation and the generation of oxidants in TNF-α–exposed endothelial cells. The mechanism of NADPH oxidase activation involved PKCζ-induced phosphorylation of p47phox, thus targeting this subunit to endothelial membranes where it associated with gp91phox.
We have used several independent pharmacological and genetic approaches to define the role of the PKCζ isoform in mediating NADPH oxidase activation. First, we showed that chelerythrine and calphostin C, broad spectrum inhibitors of PKC,18 prevented the TNF-α–induced membrane translocation of p47phox. To exclude the involvement of cPKC and nPKC isoforms in the mechanism, we depleted both cPKC and nPKC (but not the aPKC isoforms) by prolonged exposure of endothelial cells to PMA.18 Depletion of cPKC and nPKC failed to prevent membrane translocation of p47phox in response to TNF-α challenge. These results pointed to the involvement of aPKC isoform such as PKCζ in the membrane targeting of p47phox. Although the present studies show an important role of PKCζ in the TNF-α–induced membrane translocation of p47phox and NADPH oxidase activation, it is possible that cPKC and nPKC isoforms signal activation of NADPH oxidase in response to other agonists, such as thrombin, which activate 7 transmembrane G protein–coupled receptors.28 PKCα, PKCβ, and PKCδ have been shown to contribute to the assembly and activation of NADPH oxidase in a stimulus- and cell-specific manner.29–31⇓⇓
To delineate the specific role of PKCζ signaling as a requirement for the TNF-α–induced activation of NADPH oxidase in endothelial cells, we used 3 different approaches in which PKCζ activation was inhibited. First, pretreatment of HPAE cells with a myristoylated membrane-permeable peptide antagonist corresponding to the pseudosubstrate region of PKCζ, known to inhibit protein kinase activity,32 markedly decreased TNF-α–induced membrane translocation of p47phox. Second, inhibition of PKCζ synthesis by antisense oligonucleotide18,23⇓ also blocked the membrane translocation of p47phox induced by TNF-α. In contrast, inhibition of PKCθ or PKCα failed to prevent the TNF-α response. Third, expression of kinase-defective mutant of PKCζ prevented TNF-α–induced p47phox translocation, whereas expression of the mutants of PKCα and PKCε isoforms had no effect. Finally, in a gain of function experiment, we showed that expression of the constitutively active PKCζ increased p47phox translocation. Taken together, these results show the critical role of PKCζ in signaling TNF-α–induced p47phox activation.
We next addressed whether the role of PKCζ-induced phosphorylation of p47phox is requirement for NADPH oxidase activation. Studies have shown that phosphorylation causes a conformational change in p47phox, which releases the complexed p47phox and allows its translocation and association to membrane-bound cytochrome b558.33 We observed that TNF-α induced time-dependent phosphorylation of p47phox, paralleling the membrane translocation of p47phox and activation of NADPH oxidase. These data are in accord with the finding of Dang et al34 showing that p47phox is an in vitro substrate for PKCζ. We also showed that inhibition of PKCζ activation prevented the TNF-α–induced phosphorylation of p47phox, indicating the causal role of PKCζ in the response.
The finding that PKCζ is required for phosphorylation of p47phox led us to examine if PKCζ directly phosphorylates p47phox or an intermediate kinase is activated that in turn is responsible for phosphorylation. Our results showed that TNF-α induced the colocalization of PKCζ with p47phox, whereas in control studies PMA failed to produce the same pattern of PKCζ and p47phox association. In another control experiment, TNF-α failed to cause the colocalization of PKCα with p47phox. Thus, these data show that activation of PKCζ is involved in the phosphorylation of p47phox and its targeting to the membrane in response to TNF-α challenge of endothelial cells. The present findings show that p47phox is a substrate of PKCζ; however, it is possible that PKCζ may also phosphorylate p47phox through MAP kinases (ERK1/2 and p38), which are known to signal downstream of PKCζ.35–37⇓⇓
To address the functional effects of phosphorylation of p47phox and its translocation to the membrane, we assessed the assembly of NADPH oxidase complex and its effect on oxidant generation. The data showed that inhibition of phosphorylation and membrane translocation of p47phox induced by blocking PKCζ activation prevented the association of p47phox with gp91phox as well as oxidant generation. Results also showed that pretreatment of HPAE cells with an endogenous proline-arginine (PR)–rich antibacterial peptide, PR-39, known to inhibit NADPH oxidase assembly through interaction with Src homology 3 domains of p47phox, 38 prevented the TNF-α–induced assembly of NADPH oxidase complex and oxidant generation (data not shown). We further demonstrated that TNF-α failed to activate oxidant generation in lung vascular endothelial cells derived from p47phox−/− and gp91phox−/− mice. It should be noted that there is a controversy concerning the role of gp91phox in nonphagocytic cells, and on the relative importance of Nox-1, a homologue of gp91phox39, (or other members of Nox family)39 in these cells. However, the present data are consistent with an important role of gp91phox in the mechanism of oxidant generation in endothelial cells.40–42⇓⇓
The site of oxidant generation activated by NADPH oxidase in endothelial cells is unclear. The finding that p47phox fluorescence after TNF-α challenge did not appear as a ring associated with the plasma membrane suggests an intracellular source of oxidants. The previous finding that endothelial NADPH oxidase components (gp91phox and p22phox) are localized in the endoplasmic reticulum42 supports an intracellular source of oxidants,40,41⇓ whereas other studies have shown extracellular production of superoxide anion (O2·−) in endothelial cells.43,44⇓ Thus in the present study, we cannot exclude that there is also extracellular oxidant generation induced by the NADPH oxidase complex in these cells.
In summary, the present study implicates PKCζ as the critical kinase that signals TNF-α–induced oxidant generation in endothelial cells through the activation of NADPH oxidase. The activation of NADPH oxidase was dependent on PKCζ-induced phosphorylation of p47phox. Inhibition of PKCζ activity or its expression prevented the TNF-α–induced NADPH oxidase assembly and oxidant production. Thus, strategies aimed at preventing TNF-α–induced PKCζ activation and oxidant signaling may be useful in controlling the inflammatory components of diseases such as ARDS and ischemia/reperfusion tissue injury.
This work was supported by National Institutes of Health grants HL27016, HL46350, HL45638, and HL67424.
↵*Both authors contributed equally to this work.
Original received December 12, 2001; revision received March 5, 2002; accepted March 27, 2002.
- ↵Rahman A, Bando M, Kefer J, Anwar KN, Malik AB. Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene transcription. Mol Pharm. 1999; 55: 575–583.
- ↵Griendling KK, Alexander RW. Endothelial control of the cardiovascular system: recent advances. FASEB J. 1996; 10: 283–292.
- ↵Al-Mehdi AB, Zhao G, Dodia C, Tozawa K, Costa K, Muzykantov V, Ross C, Blecha F, Dinauer M, Fisher AB. Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+. Circ Res. 1998; 83: 730–707.
- ↵Abe JI, Baines CP, Berk BC. Role of mitogen-activated protein kinases in ischemia and reperfusion injury: the good and the bad. Circ Res. 2000; 86: 692–699.
- ↵Rotrosen D, Yeung CL, Leto TL, Malech HL, Kwong CH. Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. Science. 1992; 256: 1459–1462.
- ↵Heyworth PG, Curnutte JT, Nauseef WM, Volpp BD, Pearson DW, Rosen H, Clark RA. Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly: translocation of p47phox and p67phox requires interaction between p47phox and cytochrome b558. J Clin Invest. 1991; 87: 352–356.
- ↵Kleinberg ME, Malech HL, Rotrosen D. The phagocyte 47-kilodalton cytosolic oxidase protein is an early reactant in activation of the respiratory burst. J Biol Chem. 1990; 265: 15577–15583.
- ↵El Benna J, Faust LP, Babior BM. The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation: phosphorylation of sites recognized by protein kinase C and by proline-directed kinases. J Biol Chem. 1994; 269: 23431–23436.
- ↵El Benna J, Faust RP, Johnson JL, Babior BM. Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping: phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase. J Biol Chem. 1996; 271: 6374–6378.
- ↵Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992; 258: 607–614.
- ↵Moscat J, Diaz-Meco MT. The atypical protein kinase Cs: functional specificity mediated by specific protein adaptors. EMBO Rep. 2000; 1: 399–403.
- ↵Rahman A, Anwar KN, Malik AB. Protein kinase Cζ mediates TNF-α–induced ICAM-1 gene transcription in endothelial cells. Am J Physiol Cell Physiol. 2000; 279: C906–C914.
- ↵Jackson SH, Gallin JI, Holland SM. The p47phox mouse knock-out model of chronic granulomatous disease. J Exp Med. 1995; 182: 751–758.
- ↵Fan J, Frey RS, Rahman A, Malik AB. Role of neutrophil NADPH oxidase in the mechanism of tumor necrosis factor-α–induced NF-κB activation and intercellular adhesion molecule-1 expression in endothelial cells. J Biol Chem. 2002; 277: 3404–3411.
- ↵Rahman A, Anwar KN, Uddin S, Xu N, Ye RD, Platanias LC, Malik AB. Protein kinase Cδ regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase. Mol Cell Biol. 2001; 21: 5554–5565.
- ↵Dominguez I, Diaz-Meco MT, Municio MM, Berra E, Garcia de Herreros A, Cornet ME, Sanz L, Moscat J. Evidence for a role of protein kinase Cζ subspecies in maturation of Xenopus laevis oocytes. Mol Cell Biol. 1992; 12: 3776–3783.
- ↵Soh JW, Lee EH, Prywes R, Weinstein IB. Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol Cell Biol. 1999; 19: 1313–1324.
- ↵Rotrosen D, Leto TL. Phosphorylation of neutrophil 47-kDa cytosolic oxidase factor: translocation to membrane is associated with distinct phosphorylation events. J Biol Chem. 1990; 265: 19910–19915.
- ↵Inanami O, Johnson JL, McAdara JK, El Benna J, Faust LR, Newburger PE, Babior BM. Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47phox on serine 303 and 304. J Biol Chem. 1998; 273: 9539–9543.
- ↵Hofmann J. The potential for isoenzyme-selective modulation of protein kinase C. FASEB J. 1997; 11: 649–669.
- ↵Li Q, Subbulakshmi V, Fields AP, Murray NR, Cathcart MK. Protein kinase Cα regulates human monocyte O−2 production and low density lipoprotein lipid oxidation. J Biol Chem. 1999; 274: 3764–3771.
- ↵Korchak HM, Rossi MW, Kilpatrick LE. Selective role for β-protein kinase C in signaling for O2− generation but not degranulation or adherence in differentiated HL60 cells. J Biol Chem. 1998; 273: 27292–27299.
- ↵Seargeant S, McPhail LC. Opsonized zymosan stimulates the redistribution of protein kinase C isoforms in human neutrophils. J Immunol. 1997; 159: 2877–2885.
- ↵House C, Kemp BE. Protein kinase C contains a pseudosubstrate prototype in its regulatory domain. Science. 1987; 238: 1726–1728.
- ↵Sumimoto H, Kage Y, Nunoi H, Sasaki H, Nose T, Fukumaki Y, Ohno M, Minakami S, Takeshige K. Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. Proc Natl Acad Sci U S A. 1994; 91: 5345–5349.
- ↵Dang PMC, Fontayne A, Hakim J, El Benna J, Perianin A. Protein kinase Cζ phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst. J Immunol. 2001; 166: 1206–1213.
- ↵Kampfer S, Hellbert K, Villunger A, Doppler W, Baier G, Grunicke HH, Uberall F. Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKCλ, ε and ζ. EMBO J. 1998; 17: 4046–4055.
- ↵Mizukami Y, Kobayashi S, Uberall F, Hellbert K, Kobayashi N, Yoshida KI. Nuclear mitogen-activated protein kinase activation by protein kinase Cζ during reoxygenation after ischemic hypoxia. J Biol Chem. 2000; 275: 19921–19927.
- ↵Rahman A, Anwar KN, Uddin S, Xu N, Ye RD, Platanias LC, Malik AB. Protein kinase Cδ regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase. Mol Cell Biol. 2001; 21: 5554–5565.
- ↵Shi J, Ross CR, Leto TL, Blecha F. PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47phox. Proc Natl Acad Sci U S A. 1996; 93: 6014–6018.
- ↵Wagner AH, Schroeter MR, Hecker M. 17β-Estradiol inhibition of NADPH oxidase in human endothelial cells. FASEB J. 2001; 15: 2121–2130.
- ↵Gorlach A, Brandes RP, Nguyen K, Amidi M, Dehghani F, Busse R. A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall. Circ Res. 2000; 87: 26–32.
- ↵Bayraktutan U, Blayney L, Shah AM. Molecular characterization and localization of the NAD(P)H oxidase components gp91phox and p22phox in endothelial cells. Arterioscler Thromb Vasc Biol. 2000; 20: 1903–1911.