A direct redox regulation of protein kinase C isoenzymes mediates oxidant-induced neuritogenesis in PC12 cells

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl(2) was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl(2) and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCepsilon plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCepsilon and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser(41) was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser(133)) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both L and D-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCepsilon.     

Protein kinase C contains two phorbol ester binding domains

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.     

Independent functioning of cytosolic phospholipase A2 and phospholipase D1 in Trp-Lys-Tyr-Met-Val-D-Met-induced superoxide generation in human monocytes

Recently, a novel peptide (Trp-Lys-Tyr-Met-Val-D-Met, WKYMVm) has been shown to induce superoxide generation in human monocytes. The peptide stimulated phospholipase A2 (PLA2) activity in a concentration- and time-dependent manner. Superoxide generation as well as arachidonic acid (AA) release evoked by treatment with WKYMVm could be almost completely blocked by pretreatment of the cells with cytosolic PLA2 (cPLA2)-specific inhibitors. The involvement of cPLA2 in the peptide-induced AA release was further supported by translocation of cPLA2 to the nuclear membrane of monocytes incubated with WKYMVm. WKYMVm-induced phosphatidylbutanol formation was completely abolished by pretreatment with PKC inhibitors. Immunoblot showed that monocytes express phospholipase D1 (PLD1), but not PLD2. GF109203X as well as butan-1-ol inhibited peptide-induced superoxide generation in monocytes. Furthermore, the interrelationship between the two phospholipases, cPLA2 and PLD1, and upstream signaling molecules involved in WKYMVm-dependent activation was investigated. The inhibition of cPLA2 did not blunt peptide-stimulated PLD1 activation or vice versa. Intracellular Ca2+ mobilization was indispensable for the activation of PLD1 as well as cPLA2. The WKYMVm-dependent stimulation of cPLA2 activity was partially dependent on the activation of PKC and mitogen-activated protein kinase, while PKC activation, but not mitogen-activated protein kinase activation, was an essential prerequisite for stimulation of PLD1. Taken together, activation of the two phospholipases, which are absolutely required for superoxide generation, takes place through independent signaling pathways that diverge from a common pathway at a point downstream of Ca2+.     

Flavin-linked Erv-family sulfhydryl oxidases release superoxide anion during catalytic turnover

Typically, simple flavoprotein oxidases couple the oxidation of their substrates with the formation of hydrogen peroxide without release of significant levels of the superoxide ion. However, two evolutionarily related single-domain sulfhydryl oxidases (Erv2p; a yeast endoplasmic reticulum resident protein and augmenter of liver regeneration, ALR, an enzyme predominantly found in the mitochondrial intermembrane) release up to ~30% of the oxygen they reduce as the superoxide ion. Both enzymes oxidize dithiol substrates via a redox-active disulfide adjacent to the flavin cofactor within the helix-rich Erv domain. Subsequent reduction of the flavin is followed by transfer of reducing equivalents to molecular oxygen. Superoxide release was initially detected using tris(3-hydroxypropyl)phosphine (THP) as an alternative reducing substrate to dithiothreitol (DTT). THP, and other phosphines, showed anomalously high turnover numbers with Erv2p and ALR in the oxygen electrode, but oxygen consumption was drastically suppressed upon the addition of superoxide dismutase. The superoxide ion initiates a radical chain reaction promoting the aerobic oxidation of phosphines with the formation of hydrogen peroxide. Use of a known flux of superoxide generated by the xanthine/xanthine oxidase system showed that one superoxide ion stimulates the reduction of 27 and 4.5 molecules of oxygen using THP and tris(2-carboxyethyl)phosphine (TCEP), respectively. This superoxide-dependent amplification of oxygen consumption by phosphines provides a new kinetic method for the detection of superoxide. Superoxide release was also observed by a standard chemiluminescence method using a luciferin analogue (MCLA) when 2 mM DTT was employed as a substrate of Erv2p and ALR. The percentage of superoxide released from Erv2p increased to ~65% when monomeric mutants of the normally homodimeric enzyme were used. In contrast, monomeric multidomain quiescin sulfhydryl oxidase enzymes that also contain an Erv FAD-binding fold release only 1-5% of their total reduced oxygen species as the superoxide ion. Aspects of the mechanism and possible physiological significance of superoxide release from these Erv-domain flavoproteins are discussed.     

The thiol proteinase inhibitors improve the abnormal rapid down-regulation of protein kinase C and the impaired natural killer cell activity in (Chediak-Higashi syndrome) beige mouse

Protein kinase C (PKC) is essential in intracellular signal transduction for various cell functions including natural killer (NK) cell activity. This enzyme is hydrolysed by calpain, which is Ca2+-dependent thiol proteinase. We showed here that in NK activity-deficient beige (bg/bg) mouse, the model of Chediak-Higashi syndrome, the translocated membrane-bound PKC activity declined rapidly in NK cell-enriched lymphocytes after TPA stimulation. However, the rapid decline was abolished by the pretreatment of cells with leupeptin (a thiol and serine proteinase inhibitor) or E64 (a thiol proteinase inhibitor). Furthermore, these reagents improved the impaired NK cell activity in beige mouse whereas they did not affect NK cell activity in C57BL/6 (+/+) and the heterozygous (+/bg) mice. Meanwhile, TPA stimulation induced only low levels in NK cytotoxic factors (NKCF) release from beige NK cells, but these reagents augmented the lowered NKCF release. These results suggest that the improvement of impaired NK cell activity in beige mouse by the thiol proteinase inhibitors may be due to the elimination of abnormal rapid down-regulation of PKC, resulting in the augmentation of the lowered PKC activity.     

Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.     

Xanthine oxidase-dependent ROS production mediates vitamin A pro-oxidant effects in cultured Sertoli cells

Several studies have suggested that vitamin A (retinol, ROH) presents pro-oxidant properties in biological systems. Recent studies point out that xantine oxidase, a ROS-generating enzyme, catalyses ROH oxidation to RA in vitro. These works stimulated the authors to investigate whether xanthine oxidase could be involved on the ROH pro-oxidative effects reported in cultured Sertoli cells. In vitro, it was demonstrate that xanthine oxidase generates superoxide in the presence of ROH as assessed by superoxide mediated-NBT reduction. Superoxide production is potentiated in the presence of NADH and inhibited by allopurinol. In Sertoli cells, ROH treatment increased xanthine oxidase activity and inhibition of the enzyme with allopurinol attenuated ROH-induced ROS production, protein damage and cytotoxicity. Moreover, inhibition of ROH oxidation to RA by retinaldehyde dehydrogenase inhibitor potentiated both xanthine oxidase-dependent ROS production and cell damage in ROH-treated cells. The data show that xanthine oxidase may play a role on vitamin A pro-oxidant effects.     

Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.     

Xanthine oxidase-dependent ROS production mediates vitamin A pro-oxidant effects in cultured Sertoli cells

Several studies have suggested that vitamin A (retinol, ROH) presents pro-oxidant properties in biological systems. Recent studies point out that xantine oxidase, a ROS-generating enzyme, catalyses ROH oxidation to RA in vitro. These works stimulated the authors to investigate whether xanthine oxidase could be involved on the ROH pro-oxidative effects reported in cultured Sertoli cells. In vitro, it was demonstrate that xanthine oxidase generates superoxide in the presence of ROH as assessed by superoxide mediated-NBT reduction. Superoxide production is potentiated in the presence of NADH and inhibited by allopurinol. In Sertoli cells, ROH treatment increased xanthine oxidase activity and inhibition of the enzyme with allopurinol attenuated ROH-induced ROS production, protein damage and cytotoxicity. Moreover, inhibition of ROH oxidation to RA by retinaldehyde dehydrogenase inhibitor potentiated both xanthine oxidase-dependent ROS production and cell damage in ROH-treated cells. The data show that xanthine oxidase may play a role on vitamin A pro-oxidant effects.     

Endothelin-1 induces vascular endothelial growth factor synthesis in osteoblasts: involvement of p38 mitogen-activated protein kinase

We previously reported that endothelin-1 (ET-1) stimulates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of ET-1 on the synthesis of vascular endothelial growth factor (VEGF) in these cells. ET-1 significantly stimulated VEGF secretion time-dependently 18 hours after the stimulation. The stimulatory effect was dose-dependent in the range between 0.1 nM and 0.1 micro;M. BQ123, an antagonist of endothelin(A) (ET(A)) receptor, inhibited the ET-1-induced VEGF secretion. The ET-1-induced VEGF secretion was suppressed by SB203580 and PD169316, inhibitors of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, stimulated VEGF secretion. Calphostin C, a specific PKC inhibitor, suppressed the VEGF secretion by ET-1. TPA-induced VEGF secretion was suppressed by SB203580. Taken together, our results strongly suggest that ET-1 stimulates VEGF synthesis via ET(A) receptor in osteoblasts and that p38 MAP kinase is involved at a point downstream from PKC in the VEGF synthesis.     

Effects of vanadate on the oxidation of NADH by xanthine oxidase

Vanadate (V(V)) stimulates the oxidation of NADH by xanthine oxidase and superoxide dismutase eliminates the effect of V(V). Paraquat stimulates both the oxidation of NADH by xanthine oxidase and the V(V) enhancement of that oxidation. Xanthine, which is a better substrate for xanthine oxidase than is NADH, causes a V(V)-dependent co-oxidation of NADH which is transient and eliminated by SOD. Urate inhibits the V(V)-stimulated oxidation of NADH by xanthine oxidase or by Rose Bengal plus light. Measurement of rates of both O2- production and V(V)-stimulated NADH oxidation showed that many molecules of NADH were oxidized per O2-. These chain lengths were an inverse function of overall reaction rate. Minimum chain lengths, calculated on the basis of 100% univalent reduction of O2 to O2-, were smaller than measured average chain lengths by a factor of five. All of these results are in accord with the view that V(V) does not directly affect the activity of the enzyme, but rather catalyzes the free radical chain oxidation of NADH by O2-. It was further shown that phosphate was not involved and that the active form of V(V) was orthovanadate, rather than decavanadate.     

Activation of protein kinase C is not required for glyceraldehyde-stimulated insulin secretion from rat islets

Glyceraldehyde-induced insulin release from rat islets of Langerhans was not affected following down-regulation of protein kinase C (PKC) by prolonged exposure to the tumour-promoting phorbol ester, 4 beta-phorbol myristate acetate (PMA). Glyceraldehyde did not cause translocation of islet PKC under conditions in which PMA stimulated redistribution of enzyme activity. These results indicate that activation of PKC is not required for glyceraldehyde stimulation of insulin secretion from normal rat islets.     

Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C.

A large Mg2+ cell uptake against concentration gradients is stimulated in collagenase-dispersed rat myocytes by carbachol and in hepatocytes by carbachol or vasopressin. The signalling pathway(s) responsible for this stimulation of Mg2+ uptake was investigated by using various activators or inhibitors of protein kinase C (PKC) and by correlating Mg2+ uptake with cell PKC activity and cAMP content. In both cell preparations, the direct stimulation of PKC by diacylglycerol analogs or phorbol esters reproduce the same pattern of Mg2+ uptake as that induced by carbachol or vasopressin. These data indicate that the activation of PKC is responsible for a stimulation of Mg2+ uptake by myocytes or hepatocytes, whereas increase in cAMP in these cells stimulates Mg2+ release.     

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.     

Oxidative processes in human promonocytic cells (THP-1) after differentiation into macrophages by incubation with Chlamydia pneumoniae extracts

Human monocytes differentiated into macrophages by Chlamydia pneumoniae were able to oxidize blood lipoproteins, as discovered by Kalayoglu et al. (1998). Using a model of human promonocytic cells (THP-1), the cells were differentiated into macrophages by preincubation with C. pneumoniae extract, and further stimulated by phorbol myristate acetate. In these conditions, the differentiated cells oxidized a thiol compound and released superoxide anion as demonstrated respectively by gas liquid chromatography and electron spin resonance. The thiol oxidation and superoxide anion release were inhibited by diphenyliodonium, a NADPH oxidase and NOsynthase inhibitor, proving that the respiratory burst and the NOsynthase were involved in the oxidation processes occurring in the differentiated THP-1. The role of H(2)O(2) (derived from superoxide anion) was indicated by the enhancing effect of a peroxidase on the thiol oxidation. The presence of alpha-tocopherol in the surrounding medium strongly diminished the oxidation of the thiol target.     

EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol

Resveratrol (3,4',5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes, and red wine, has received considerable attention because of its apparent protective effects against various degenerative diseases due to its potential antioxidant activities. However, direct evidence for the superoxide-scavenging capacity of resveratrol is lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5-(diethoxyphosphoryl)-5-methylpyrroline-N-oxide (DEPMPO)-spin trapping technique was utilized to determine the ability of resveratrol in scavenging superoxide anions generated from both potassium superoxide and the xanthine oxidase/xanthine system. We have demonstrated here for the first time that the presence of resveratrol resulted in decreased formation of DEPMPO-superoxide adduct (DEPMPO-OOH) in both the potassium superoxide and xanthine oxidase/xanthine systems, indicating that resveratrol could directly scavenge superoxide anions. The inhibition of DEPMPO-OOH in the xanthine oxidase/xanthine system, however, was found to be much potent as compared to that observed in potassium superoxide system. It was further shown that resveratrol could also directly inhibit xanthine oxidase activity as assessed by oxygen consumption and formation of uric acid. Taken together, the dual role of resveratrol in directly scavenging superoxide and inhibiting its generation via xanthine oxidase reported in this study may explain, at least in part, the protective role of this compound against oxidative injury in various disease processes.     

Phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in MDCK cells

We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the polymeric immunoglobulin receptor (pIgR) in MDCK cells. Apical release of pre-endocytosed ligand (dimeric IgA) bound to the pIgR can be stimulated twofold within 7 min of addition of PMA while recycling of the ligand from the basal surface is not affected. In addition, apical surface delivery of pIgR and cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA. The recycling of apically internalized ligand back to the apical surface is similarly stimulated. These results suggest that the stimulation of apical delivery is from an apical recycling compartment. The effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 h and observed that transcytosis could no longer be stimulated by PMA. Western blots show that the PKC isozymes alpha and to a lesser extent epsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16 h and that treatment of MDCK cells with PMA for 5 min causes a dramatic translocation of the PKC alpha isozyme and a partial translocation of the epsilon isozyme from the cytosol to the membrane fraction of cell homogenates. This translocation suggests that the alpha and/or epsilon isozymes may be involved in PMA mediated stimulation of transcytosis. A mutant pIgR in which serines 664 and 726, the major sites of phosphorylation, are replaced by alanine is stimulated to transcytose by PMA, suggesting that phosphorylation of pIgR at these sites is not required for the effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosis may involve the activation of PKC alpha and/or epsilon, and that this stimulation occurs independently of the major phosphorylation sites on the pIgR. Finally, PMA stimulates transcytosis of basolaterally internalized transferrin, suggesting that PMA acts to generally stimulate delivery of endocytosed proteins to the apical surface.     

Effects of oxidized low density lipoprotein on nitric oxide synthetase and protein kinase C activities in bovine endothelial cells.

Oxidized low-density lipoprotein (Ox-LDL) is an atherogenic lipoprotein. It has been suggested that Ox-LDL causes endothelial dysfunction by decreasing the release of endothelium-derived factors (EDRF-NO) or increasing the inactivation of EDRF-NO. The mechanism by which Ox-LDL causes dysfunctional NO during early stages of atherosclerosis is not clear. The purpose of this study was to examine the role of Ox-LDL on nitric oxide synthetase (eNOS), protein kinase C (PKC) activities and cAMP production in bovine aortic endothelial cells (BAEC). Ox-LDL stimulated PKC activity of BAEC but it inhibited both eNOS activity and cAMP production. Ox-LDL partially inhibited the forskolin stimulated cAMP production. Furthermore, we observed that 8Br-cAMP treatment decreased the activity of eNOS in a concentration dependent manner. Serotonin which has a profound inhibitory effect on cAMP production also stimulated eNOS activity. Pertusis toxin treatment blocked the stimulatory action of serotonin on the stimulation of eNOS activity. Our results thus suggest that Ox-LDL inhibit the endothelium-dependent relaxation. One possible mechanism is that Ox-LDL stimulates PKC activity, which in turn increases the phosphorylation of the Gi-protein. Inhibition of Gi-protein then leads to reduced release of NO from endothelial cells and thus causes endothelial dysfunction.     

Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification. Modification of beta-PKC or the minor PKC isozyme by calpain yielded Ca2+/phospholipid-independent forms (PKM) that retained only 50% of the original activities. In contrast, PKM formed from alpha-PKC retained full catalytic activity. For each native isozyme the rate of conversion by calpain was accelerated in the presence of Ca2+ and the lipid effectors, and the PKM form generated in each case was resistant to further digestion by calpain. All three PKC isozymes were also modified by a neutral serine proteinase isolated from human neutrophils, with this proteinase the major effect being loss of kinase activity, via a transient production of a Ca2+/phospholipid-independent form. This neutral serine proteinase appears to be localized at sites of interaction of cytoskeletal proteins with the cell membrane. Following stimulation of intact neutrophils with phorbol 12-myristate 13-acetate complete loss of native cytosolic kinase activity was observed, with recovery of approximately 30% of the original activity as a cytosolic Ca+/phospholipid independent form, presumably PKM. Loss of native PKC activity was greatest for the beta-isozyme. In cells stimulated by fMet-Leu-Phe approximately 60% of the original PKC activity was recovered as native cytosolic PKC and 30% as cytosolic PKM. Inhibitors of calpain reduced the extent of down-regulation of PKC, increased the proportion of PKC that remained associated with the plasma membrane and significantly reduced the proteolytically generated fully active PKM. Taken together, the in vitro and in vivo results suggest that calpain is involved primarily in the conversion of the PKC isozymes to the irreversibly activated PKM forms, and that the neutral serine proteinase may be the enzyme responsible for down-regulation, possibly via PKM as an intermediate.