Protein kinase C-dependent and -independent activation of the NADPH oxidase of human neutrophils

The protein kinase C inhibitor, staurosporine, inhibited NADPH oxidase activity of human neutrophils activated by phorbol myristate acetate. However, this inhibitor had no effect on either the initiation or the maximal rate of O2- secretion activated by the chemotactic peptide, fMet-Leu-Phe, but resulted in a more rapid termination of oxidant production. Similarly, staurosporine had no effect on the rapid (1 min) increase in luminol-dependent chemiluminescence activated by fMet-Leu-Phe, but the second (intracellular) phase of oxidant production was inhibited. The initial burst of oxidant production during phagocytosis was similarly protein kinase C-independent, but again the later phases of oxidase activity were staurosporine-sensitive. Neutrophils loaded with Quin-2 at concentrations sufficient to act as a Ca2+ buffer could not secrete O2- in response to fMet-Leu-Phe; although the initial (protein kinase C-independent) burst of luminol chemiluminescence was not observed in fMet-Leu-Phe-stimulated Ca2(+)-buffered cells, the second phase of (protein kinase C-dependent) oxidant production was largely unaffected. Hence, the initial burst of oxidant production activated by fMet-Leu-Phe, opsonized zymosan, and latex beads is independent of the activity of protein kinase C-dependent intracellular activation processes, but the activity of this kinase is required to extend or sustain the duration of oxidant production.     

Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is converted from an inactive to an active form upon stimulation of the neutrophil. This activation process was examined using a recently developed cell-free system in which dormant oxidase is activated by arachidonic acid in the presence of a soluble factor from the neutrophil (Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743). NADPH oxidase from unstimulated human neutrophils was detected only in the membrane fraction. The soluble activation factor was localized entirely to the cytosolic fraction and exhibited two peaks of activity when partially purified under nondenaturing conditions: a major peak with a molecular mass of approximately 250 kDa and a variable minor peak with a mass of approximately 40 kDa. Both forms activated NADPH oxidase in a similar manner and did not exhibit synergy when combined. The cytosolic factor is not protein kinase C (or another kinase) as both peaks of factor activity could be resolved from the protein kinase C peak and neither required calcium or ATP to activate the oxidase. Activation of NADPH oxidase did require the simultaneous presence of the membrane fraction, the cytosolic factor, arachidonic acid, and magnesium. Following activation, however, only the membrane fraction was then required for O2- production. Cytosolic factor levels were normal in five patients with either X-linked or autosomal recessive cytochrome b-negative chronic granulomatous disease. In contrast, the membrane fractions from each failed to generate O2-, indicating that the defects in these two genetic forms of chronic granulomatous disease reside either in the oxidase itself or in a membrane component required for activation.     

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O2*- generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme

Protein kinase C may be important in leukocyte function, because it is activated by phorbol myristate acetate (PMA), a potent stimulus of the respiratory burst in neutrophils. The localization of protein kinase C was compared in unstimulated and PMA-stimulated human neutrophils. Protein kinase C was primarily cytosolic in unstimulated cells but became associated with the particulate fraction after treatment of cells with PMA. The particulate-associated kinase activity did not require added calcium and lipids, but when extracted by Triton X-100 (greater than or equal to 0.2%), calcium and phospholipid dependence could be demonstrated. The EC50 of PMA for stimulating kinase redistribution and activation of NADPH oxidase, the respiratory burst enzyme, were similar (30 to 40 nM). Redistribution of protein kinase C occurred rapidly (no lag) and preceded NADPH oxidase activation (30 sec lag). These results suggest that redistribution of protein kinase C is linked to activation of the respiratory burst in human neutrophils.     

Fluoride can activate the respiratory burst independently of Ca2+, stimulation of phosphoinositide turnover and protein kinase C translocation in primed human neutrophils

Evidences have been provided in our laboratory that in neutrophils different signal transduction sequences for the activation of O2(-)-forming NADPH oxidase can be triggered by the same stimulus (Biochem. Biophys. Res. Commun. 1986, 135, 556-565; 1986, 135, 785-794; 1986, 140, 1-11). The results presented here show that the transduction sequence triggered by fluoride via dissociation of G-proteins and involving messengers produced by stimulation of phosphoinositide turnover, Ca2+ changes and translocation of protein kinase C from the cytosol to the plasmamembrane, can be bypassed when a primed state of neutrophils is previously induced. In fact: i) fluoride causes a pertussis toxin insensitive and H-7 sensitive respiratory burst in human neutrophils, which is linked to the activation of hydrolysis of PIP2, rise in [Ca2+]1 and translocation of PKC. In Ca2+-depleted neutrophils these responses to fluoride do not occur and are restored by addition of CaCl2. ii) The pretreatment of Ca2+-depleted unresponsive neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase by fluoride but not the turnover of phosphoinositides and PKC translocation. The nature of the alternative transduction sequence, the reactions different from phospholipase C activated by G-protein for the alternative sequence and the role of these discrete pathways for NADPH oxidase activation are discussed.     

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.     

Oxidation-induced persistent activation of protein kinase C in hippocampal homogenates.

Incubation of purified protein kinase C with H2O2 results in the generation of a persistently activated form of the enzyme which is no longer dependent on Ca2+ or lipid cofactors. This oxidative activation of purified protein kinase C requires added Fe2+ in the incubation medium. Treatment of the soluble fraction of hippocampal homogenates with H2O2 also leads to persistent activation of protein kinase C; however, oxidative activation of protein kinase C under these conditions does not require the addition of Fe2+. The persistently activated form of protein kinase C appears as a novel peak of activity on DE52 anion exchange columns, suggesting a modification of the charge character of the enzyme. Thus, oxidative modification of protein kinase C can result in its persistent activation, and this mechanism may constitute a pathway for physiological activation of the enzyme in the hippocampus.     

Activation of the respiratory burst oxidase in a fully soluble system from human neutrophils

The O2(-)-forming respiratory burst oxidase is present in a dormant state in a fully soluble system containing both cytosol and a deoxycholate extract of membranes from resting human neutrophils. Sodium dodecyl sulfate at low concentrations converts this soluble dormant oxidase into its catalytically active form. The Vmax for the activated oxidase was 2.1 mumol of O2-/min/mg of membrane protein. Michaelis constants for NADPH and NADH (38 microM and 1.7 mM, respectively) were similar to those measured previously in other systems. Oxidase activity was not detected after sodium dodecyl sulfate treatment of systems containing solubilized neutrophil membranes obtained from patients with X-linked chronic granulomatous disease. These results suggest that the deoxycholate extract contains both the resting oxidase and those membrane-associated components needed for its activation, all in functioning states.     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

Redox regulation of platelet-derived-growth-factor-receptor: role of NADPH-oxidase and c-Src tyrosine kinase

This study identifies some early events contributing to the redox regulation of platelet-derived growth factor receptor (PDGFr) activation and its signalling in NIH3T3 fibroblasts. We demonstrate for the first time that the redox regulation of PDGFr tyrosine autophosphorylation and its signalling are related to NADPH oxidase activity through protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K) activation and H2O2 production. This event is also essential for complete PDGF-induced activation of c-Src kinase by Tyr416 phosphorylation, and the involvement of c-Src kinase on H2O2-induced PDGFr tyrosine phosphorylation is demonstrated, suggesting a role of this kinase on the redox regulation of PDGFr activation. Finally, it has been determined that not only PI3K activity, but also PKC activity, are related to NADPH oxidase activation due to PDGF stimulation in NIH3T3 cells, as it occurs in non-phagocyte cells. Therefore, we suggest a redox circuit whereby, upon PDGF stimulation, PKC, PI3K and NADPH oxidase activity contribute to complete c-Src kinase activation, thus promoting maximal phosphorylation and activation of PDGFr tyrosine phosphorylation.     

Ectopic expression of an Arabidopsis calmodulin-like domain protein kinase-enhanced NADPH oxidase activity and oxidative burst in tomato protoplasts

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.     

Reconstitution of the partially purified membrane component of the superoxide-generating NADPH oxidase of pig neutrophils with phospholipid

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.     

Phorbol 12, myristate 13, acetate potentiates the respiratory burst while inhibits phosphoinositide hydrolysis and calcium mobilization by formyl-methionyl-leucyl-phenylalanine in human neutrophils

It is widely believed that the transduction pathway in the activation of the NADPH oxidase by formyl-methionyl-leucyl-phenylalanine (FMLP) in neutrophils involves the stimulation of phosphoinositide hydrolysis, the increase in [Ca2+]i and the activity of the Ca2+ and phospholipid dependent protein kinase C. The results presented here show that the activation of the respiratory burst by FMLP can be dissociated by the stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate and Ca2+ changes. In fact, in neutrophils pretreated (primed) with non stimulatory doses of phorbol myristate acetate the respiratory burst by chemotactic peptide is greatly potentiated while the increase in [3H] inositol phosphates formation and in [Ca2+]i are depressed due to the inhibition of phospholipase C. This finding indicates that FMLP can trigger also a sequence of transduction reactions for the activation of the NADPH oxidase different from that involving the formation of the second messengers diacylglycerol and inositol phosphates and the increase in free Ca2+ concentration.     

Redistribution of protein kinase C activity in human monocytes: correlation with activation of the respiratory burst

Protein kinase C (PKC) was found to be present in purified human monocytes and lymphocytes isolated by countercurrent centrifugal elutriation. In unstimulated monocytes and lymphocytes, approximately 90% of the PKC activity was cytosolic when the cells were disrupted in the presence of EGTA. The role of this kinase in the stimulation of the respiratory burst in monocytes was investigated. Phorbol esters capable of triggering the release of O2- caused a loss of PKC activity from the cytosol and the appearance of the kinase activity in the particulate cell fraction. Kinase activity was partially extractable from the particulate fraction by 0.1% Triton X-100, whereupon it demonstrated calcium and lipid dependence. The EC50 for the phorbols in initiating the respiratory burst correlated well with their EC50 for stimulating the appearance of PKC activity in the particulate fraction (R = 0.998). Redistribution of PKC activity in monocytes by phorbol myristate acetate (PMA) was rapid and appeared to precede the release of O2-. PMA also shifted PKC activity from the cytosol to the extractable particulate fraction of lymphocytes. We conclude that redistribution of PKC activity by active phorbols or other cell stimulants could provide substrate specificity for phosphorylation reactions. By shifting PKC activity to the monocyte particulate fraction, active phorbols may initiate the phosphorylation of a substrate required for stimulation of the respiratory burst.     

Photolabeling of a O2-. generating protein in bovine polymorphonuclear neutrophils by an arylazido NADP+ analog

A plasma membrane fraction of bovine polymorphonuclear neutrophils enriched in NADPH-dependent, O2-. generating oxidase activity, and a number of fractions solubilized in detergent, recovered during the course of the purification of this oxidase have been tested for their ability to react with radiolabeled N-4-azido-2-nitrophenyl aminobutyryl NADP+ (arylazido NADP+ or NAP4-NADP+). In the dark, NAP4-NADP+ and its reduced form NAP4-NADPH, were found to inhibit competitively the NADPH-dependent O2-. generating oxidase activity of the plasma membrane fraction of bovine neutrophils activated by phorbol myristate acetate. The nitrene derivative formed upon photoirradiation of NAP4-NADP(H) bound covalently to different proteins of the plasma membrane. Photolabeling of these proteins was prevented by preincubation with an excess of NADPH. Photolabeling of a protein of 65,000 Mr was decreased by omission of phorbol myristate acetate as activating agent of the respiratory burst in neutrophils or by addition of micromolar amounts of Cibacron Blue and mersalyl which are known to inhibit the production of O2-. by the plasma membrane fraction. During the course of the purification procedure, the 65000 Mr protein emerged as the preferentially photolabeled protein. These data, in agreement with previous findings concerning the purification of an NADPH-dependent, O2-. generating oxidase protein of Mr 65000 from bovine neutrophils (Doussière, J. and Vignais, P.V. (1985) Biochemistry 24, 7231-7239), strongly suggest that a single protein of Mr 65000, located in the plasma membrane fraction of bovine neutrophils, is able to act both as an NADPH deshydrogenase and as an oxygen reductase to generate O2-.     

A phosphatidic acid-activated protein kinase and conventional protein kinase C isoforms phosphorylate p22(phox), an NADPH oxidase component

Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935), we previously showed that p47(phox), a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22(phox), a subunit of the NADPH oxidase component flavocytochrome b(558), also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22(phox). We identified two protein kinase families capable of phosphorylating p22(phox): 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47(phox) and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47(phox). In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22(phox). These findings suggest that phosphorylation of p22(phox) by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase.     

The respiratory burst oxidase of neutrophils. Separation of an FAD enzyme and its characterization

Pig blood neutrophils were briefly activated by various fatty acids and then fractionated into membrane vesicles with different NADPH oxidase activities. Treatment of these membranes with a detergent, octyl glucoside, resulted in a high yield of solubilized oxidase, which was subjected to isoelectric focusing on gels (pI 4.0-8.0). 1) A distinct band staining with NADPH-nitroblue tetrazolium focused at pI 5.0. The enzyme (pI 5.0) showed high specificity for NADPH and similar characteristics to the oxidase involved in the respiratory burst. 2) The enzyme was extracted from gel slices and analyzed. When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutase-insensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. However, it could produce superoxide anion (O2-) under some micelle conditions. 3) Therefore, the formation of the enzyme-substrate complex of yeast cytochrome c peroxidase was employed for the detection of H2O2. A fresh extract of stimulated cells catalyzed equimolar NADPH oxidation and H2O2 production of 306 and 300 nmol min-1 (mg protein)-1, respectively. The Km value of the enzyme for NADPH was 30 +/- 13 (S.D.) microM. The recovery of the extract (pI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg of protein, giving a turnover number of 300-600 min-1 in terms of O2- generation/FAD. No heme protein was found in the enzyme. The enzyme was mainly of 67-kDa molecular mass.     

Protein kinase C (PKC) isoforms translocate to Triton-insoluble fractions in stimulated human neutrophils: correlation of conventional PKC with activation of NADPH oxidase.

The responses of human neutrophils (PMN) involve reorganization and phosphorylation of cytoskeletal components. We investigated the translocation of protein kinase C (PKC) isoforms to PMN cytoskeletal (Triton-insoluble) fractions, in conjunction with activation of the respiratory burst enzyme NADPH oxidase. In resting PMN, PKC-delta (29%) and small amounts of PKC-alpha (0.6%), but not PKC-betaII, were present in cytoskeletal fractions. Upon stimulation with the PKC agonist PMA, the levels of PKC-alpha, PKC-betaII, and PKC-delta increased in the cytoskeletal fraction, concomitant with a decrease in the noncytoskeletal (Triton-soluble) fractions. PKC-delta maximally associated with cytoskeletal fractions at 160 nM PMA and then declined, while PKC-alpha and PKC-betaII plateaued at 300 nM PMA. Translocation of PKC-delta was maximal by 2 min and sustained for at least 10 min. Translocation of PKC-alpha and PKC-betaII was biphasic, plateauing at 2-3 min and then increasing up to 10 min. Under maximal stimulation conditions, PKC isoforms were entirely cytoskeletal associated. Translocation of the NADPH oxidase component p47phox to the cytoskeletal fraction correlated with translocation of PKC-alpha and PKC-betaII, but not with translocation of PKC-delta. Oxidase activity in cytoskeletal fractions paralleled translocation of PKC-alpha, PKC-betaII, and p47phox. Stimulation with 1,2-dioctanoylglycerol resulted in little translocation of PKC isoforms or p47phox, and in minimal oxidase activity. We conclude that conventional PKC isoforms (PKC-alpha and/or PKC-betaII) may regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase. PKC-delta may modulate other PMN responses that involve cytoskeletal components.     

Divalent cation requirements for mounting a respiratory burst in response to phorbol diesters by macrophages from SENCAR and C57BL/6 mice

Oxygen radicals are thought to play an important role in the promotion phase of carcinogenesis and the action of phorbol esters. Inflammatory cells are an abundant source of reactive oxygen intermediates (ROI) in the body and release large quantities of ROI when exposed to phorbol esters. Both protein kinase C (PKC), the receptor for phorbol esters, and the NADPH oxidase which generates ROI are Ca2+- and Mg2+-dependent. We investigated the requirements for Ca2+ and Mg2+ of macrophages from strains of mice sensitive and resistant to the promotion of tumors by phorbol esters. Macrophages from SENCAR mice, which are sensitive to phorbol ester promotion, required much lower levels of Ca2+ or Mg2+ to mount a full respiratory burst, as measured by the release of H2O2 in response to phorbol ester stimulation, than macrophages from C57BL/6 mice, which are resistant to promotion by phorbol esters. Conversely, when the particulate stimulus zymosan was used, there was little difference between macrophages from the two strains in requirements for Ca2+ and Mg2+ to release H2O2. Lowering the concentration of either cation in the absence of the other was more inhibitory than in the presence of the other cation. The studies demonstrate that differences in sensitivity to divalent cations by macrophages from these two strains is selective for phorbol ester stimulation and that lower requirements for Ca2+ and Mg2+ for ROI release correlates with sensitivity to the promotion of tumors by phorbol esters.     

Protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca2+ and reactive oxygen species

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in signalling and development. Given the high toxicity of ROS, their production is tightly regulated. In Arabidopsis, respiratory burst oxidase homologue F (AtrbohF) encodes NADPH oxidase. Here we characterised the activation of AtRbohF using a heterologous expression system. AtRbohF exhibited ROS-producing activity that was synergistically activated by protein phosphorylation and Ca2+. The two EF-hand motifs of AtRbohF in the N-terminal cytosolic region were crucial for its Ca2+-dependent activation. AtrbohD and AtrbohF are involved in stress responses. Although the activation mechanisms for AtRbohD and AtRbohF were similar, AtRbohD had significantly greater ROS-producing activity than AtRbohF, which may reflect their functional diversity, at least in part. We further characterised the interrelationship between Ca2+ and phosphorylation regarding activation and found that protein phosphorylation-induced activation was independent of Ca2+. In contrast, K-252a, a protein kinase inhibitor, inhibited the Ca2+-dependent ROS-producing activity of AtRbohD and AtRbohF in a dose-dependent manner, suggesting that protein phosphorylation is a prerequisite for the Ca2+-dependent activation of Rboh. Positive feedback regulation of Ca2+ and ROS through AtRbohC has been proposed to play a critical role in root hair tip growth. Our findings suggest that Rboh phosphorylation is the initial trigger for the plant Ca2+-ROS signalling network.