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J. Biol. Chem., Vol. 275, Issue 31, 24136-24145, August 4, 2000

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Superoxide-induced Stimulation of Protein Kinase C via Thiol Modification and Modulation of Zinc Content^*

Lauren T. Knapp and Eric Klann^§¶

From the  Department of Neuroscience and the ^§ Center for the Neural Basis of Cognition, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Received for publication, March 10, 2000, and in revised form, May 19, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We investigated the effects of mild oxidation on protein kinase C (PKC) using the xanthine/xanthine oxidase system of generating superoxide. Exposure of various PKC preparations to superoxide stimulated the autonomous activity of PKC. Similarly, thiol oxidation increased autonomous PKC activity, consistent with the notion that superoxide stimulates PKC via thiol oxidation. The superoxide-induced stimulation of PKC activity was partially reversed by reducing agents, suggesting that disulfide bond formation contributed to the oxidative stimulation of PKC. In addition, superoxide increased the autonomous activity of the , _II, , and  PKC isoforms, all of which contain at least one cysteine-rich region. Taken together, our observations suggested that superoxide interacts with PKC at the cysteine-rich region, zinc finger motif of the enzyme. Therefore, we examined the effects of superoxide on this region by testing the hypothesis that superoxide stimulates PKC by promoting the release of zinc from PKC. We found that a zinc chelator stimulated the autonomous activity of PKC and that superoxide induced zinc release from an PKC-enriched enzyme preparation. In addition, oxidized PKC contained significantly less zinc than reduced PKC. Finally, we have isolated a persistent, autonomously active PKC by DEAE-cellulose column chromatography from hippocampal slices incubated with superoxide. Taken together, these data suggest that superoxide stimulates autonomous PKC activity via thiol oxidation and release of zinc from cysteine-rich region of PKC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein kinase C (PKC)¹ is an intracellular signaling enzyme that has been shown to contribute to many cellular processes including proliferation, differentiation, immune response, transcriptional regulation, synaptic transmission, and learning and memory (1, 2). Therefore, understanding the mechanisms involved in regulating PKC activity is necessary for an understanding of many cellular functions. Classically, PKC is defined by the dependence of its phosphotransferase activity on the cofactors phosphatidylserine, diacylglycerol, and Ca²⁺ (3). However, the regulation of PKC activity appears to be more complex in that the enzyme can become persistently active in the absence of cofactors. Mechanisms that can result in persistent stimulation of PKC include membrane insertion (4-6), proteolytic cleavage resulting in free catalytic domain (PKM) (7), or autophosphorylation (8). In addition, previous studies have suggested that PKC can be activated by exposure to oxidants (for an example, see Ref. 9). However, the type of oxidative modification that is responsible for the activation of PKC is not well understood. Therefore, we investigated the effects of superoxide on PKC activity in the absence and presence of cofactors using several different preparations as well as several isoforms of PKC. We found that superoxide increased the activity of PKC in a cofactor-independent manner. In addition, we present data that are consistent with the hypothesis that the superoxide-induced activation of PKC is via oxidation of thiols and release of zinc from the zinc finger motif of the cysteine-rich region.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Xanthine (X), superoxide dismutase (SOD), and catalase were purchased from Calbiochem; xanthine oxidase (XO), rat brain purified PKC, and dithiothreitol (DTT) from Roche Molecular Biochemicals; 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), mannitol, deferoxamine mesylate (Desferal), nitro blue tetrazolium, nitro blue diformazan, p-hydroxyphenylacetate, ZnCl₂, 4-(2-pyridylazo)resorcinol (PAR), Sephadex G-100, and DEAE-cellulose from Sigma; tris-(2-cyanoethyl)phosphine (Cyano) and tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) form Molecular Probes; and -mercaptoethanol (-ME) from Bio-Rad.

PKC Enzyme Preparations-- Hippocampi were dissected from 4-6-week-old male Harlan Sprague-Dawley rats (Hilltop, PA). For kinase activity assays, hippocampal homogenates were prepared in homogenization buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 100 ng/ml leupeptin, 100 ng/ml aprotinin, and 10 µM benzamidine). The protein concentration of the homogenates was 0.54 µg/µl. The soluble fraction of hippocampal homogenates was prepared by homogenization and fractionation by centrifugation at 45,000 × g at 4 °C for 45 min.

The soluble fraction was separated from the pellet and stored at 80 °C. Protein concentrations of the soluble fractions were 0.54 µg/µl. PKC from the soluble fraction of hippocampal homogenates was eluted (30-54 drops) from a 1 ml Sephadex G-100 column and samples were concentrated using Microcon-3 ultrafugation devices (Millipore) for 2.5 h at 4 °C. Protein concentration of the PKC-enriched fractions from the Sephadex G-100 was 1-2.5 µg/ml. Protein concentration was measured by the method of Bradford (10) using bovine serum albumin as the standard.

Purified PKC from rat brain was diluted 1:40 to 1:100 in enzyme buffer (20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, and 0.5 mM EGTA) immediately before use. PKC isoforms were provided by Dr. Alexandra C. Newton (University of California, San Diego, CA) in the form of cell lysates that overexpressed a baculovirus for either the , _II, , or  PKC isoforms; (11). Lysates were diluted 1:25 in enzyme buffer immediately before use.

The PKC  or  isoforms were immunoprecipitated from hippocampal homogenates using anti-PKC  or  antibodies (10 µg, Life Technologies, Inc.) and resolved by SDS-polyacrylamide gel electrophoresis on 10% gels as described previously (12). PKC was localized in the gel by comparison with molecular weight markers (Bio-Rad) and with samples transferred to polyvinylidene difluoride and probed by Western blot hybridization for the respective PKC isoform. PKC was eluted from the gel using 50 mM ammonium bicarbonate, pH 7.8, and 0.1% SDS. Samples from five immunoprecipitates were pooled and concentrated using Microcon-3 ultrafugation devices (Millipore) for 2.5 h at 4 °C. Protein concentration of the immunoprecipitated PKC was 1-7 µg/µl. The protein was acid hydrolyzed (6 N HCl; 16 h at 37 °C) and neutralized before measurement of zinc content by zinc assay with PAR.

Hippocampal Slice Preparation-- Hippocampi from male Harlan Sprague-Dawley rats (100-150 g) were removed, and 400-µm slices were prepared with a McIlwain tissue chopper. The slices were perfused for 1 h with a standard saline solution (124 mM NaCl, 4.4 mM KCl, 26 mM NaHCO₃, 10 mM D-glucose, 2 mM CaCl₂, 2 mM MgCl₂, gassed with 95% O₂, 5% CO₂, pH 7.4) in an interface tissue slice chamber at 32 °C. After 1 h, slices were incubated with either X/boiled XO or X/XO at the concentrations and times described in Fig. 12 legend.

Oxidative Treatment of PKC-- Sixty µl of either hippocampal homogenates, soluble fraction, or PKC isoforms were incubated with X/XO (20 µg/ml X and 2 µg/ml XO) for 2 min at 37 °C. Thirty µl of purified PKC from rat brain was incubated with X/XO (20 µg/ml X and 2 µg/ml XO) for 30 s at 25 °C. At the end of the incubation, the samples were placed on ice and immediately assayed for PKC phosphotransferase activity.

Generation of Superoxide and Hydrogen Peroxide-- To estimate the extent of oxidant exposure of the various PKC preparations, we performed two assays to measure concentrations of superoxide and hydrogen peroxide (H₂O₂) generated by X/XO. Superoxide and H₂O₂ were generated by X/XO at 37 °C in a total reaction volume of 2 ml containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 2 mM sodium pyrophosphate, 100 ng/ml leupeptin, 100 ng/ml aprotinin, 10 µM benzamidine, 1.3 mM xanthine, and various concentrations of XO (0-20 milliunits/ml). The initial rates of superoxide generation were determined spectrophotometrically (570 ) by measuring the superoxide dismutase-inhibitable conversion of nitro blue tretrazolium to diformazan (13). The production of superoxide ranged from 2.5 to 10 nmol/min (2.5-10 µM) at 37 °C. H₂O₂ production by X/XO at 37 °C was determined by measuring the catalase-inhibitable conversion of p-hydroxyphenylacetic acid to 2.2'-dihyroxybuphenyl-5,5'-diacetate (14). The production of H₂O₂ ranged from 2 to 20 nmol/min (2-20 µM) at 37 °C.

DEAE Column Chromatography of PKC-- The soluble fraction of hippocampal homogenates was applied to a 1-ml DEAE-cellulose column. The column was washed with 3 ml of column buffer (20 mM Tris-HCl, 1 mM EDTA, leupeptin (100 ng/ml), aprotinin (100 ng/ml), and benzamindine (10 µg/ml)). PKC was eluted in 500-µl fractions by stepwise addition of column buffer supplemented with 0.1 M NaCl, followed by column buffer supplemented with 0.25 M NaCl. The fractions were concentrated and desalted using Microcon-3 ultrafugation devices (Millipore) for 2.5 h at 4 °C. The activity of PKC in each fraction was determined as described below.

Protein Kinase Activity Assays-- Autonomous and cofactor-dependent PKC activity in the various preparations were determined by measuring the transfer of [-³²P]ATP (ICN) to the selective PKC substrate NG-(28-43) in reaction mixtures, as described previously (15, 16). Autonomous PKC activity was defined as transferase activity assayed in the presence of 2 mM EGTA. Cofactor-dependent PKC activity was defined as activity assayed in the presence of 100 µM CaCl₂, 320 µg/ml phosphatidylserine, and 30 µg/ml dioctanoylglycerol. Reactions were initiated by the addition of 5 µl of the enzyme preparation and incubated for 2 min at 37 °C in the reaction mixture. Reactions were terminated by the addition of 25 µl of ice-cold stop buffer containing 100 mM ATP and 100 mM EDTA. Duplicate 25-µl aliquots were spotted onto P-81 phosphocellulose filter papers. The papers were washed with 150 mM H₃PO₄, dried, and immersed in 2.5 ml of ScintiSafe (Fisher), and the radioactivity was measured by scintillation counting. For each experimental condition, values for control reactions lacking the substrate peptide were subtracted as blanks. In addition, autonomous PKC activity values were subtracted from values obtained for cofactor-dependent PKC activity.

Zinc Assay-- Soluble zinc was measured spectrophotometrically (500 ) (17) by observing an increase in absorbance that indicates the formation of the PAR-zinc complex at a PAR concentration of 100 µM in 20 mM Tris, pH 7.5, 1 mM EDTA, 100 ng/ml leupeptin, 100 ng/ml aprotinin, and 10 µM benzamidine.

Western Blot Analyses-- Tissue samples were homogenized and protein concentrations determined as described above. Equivalent amounts of protein for each sample were resolved in 10% SDS-polyacrylamide gels, blotted electrophoretically to Immobilon membranes, and probed with a rabbit polyclonal antibody to either phospho-S657-PKC (1 µg/ml; Upstate Biotechnology, Inc.), phosphotyrosine (1 µg/ml; Upstate Biotechnology, Inc.), or PKC (Life Technologies, Inc.). The blots then were exposed to a goat anti-rabbit IgG-peroxidase linked antibody, and developed using enhanced chemiluminescence reagent (Amersham Pharmacia Biotech).

Data Analysis-- Student's t tests were used for comparisons between two treatments, and one-way ANOVA for comparisons between three or more treatments. The Bonferroni correction was used for post hoc comparisons between treatments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Oxidative Activation of PKC-- We investigated whether or not the X/XO system of generating superoxide could regulate the activity of PKC. As depicted in Fig. 1, exposure of various PKC preparations to X/XO increased autonomous PKC activity. In hippocampal homogenates, X/XO increased both autonomous PKC activity (control = 0.19 ± 0.03 pmol/min/µg; X/XO = 1.62 ± 0.13 pmol/min/µg) and cofactor-dependent PKC activity (control = 4.83 ± 0.25 pmol/min/µg; X/XO = 6.34 ± 0.37 pmol/min/µg). Incubation of the soluble fraction of hippocampal homogenates with X/XO resulted in increased autonomous PKC activity with a magnitude similar to that observed in the homogenate preparation (control = 0.65 ± 0.06 pmol/min/µg; X/XO = 4.91 ± 0.71 pmol/min/µg); however, no X/XO-induced increase cofactor-dependent PKC activity was observed in this preparation (control = 7.58 ± 0.74 pmol/min/µg; X/XO = 6.92 ± 0.73 pmol/min/µg). Incubation of purified PKC with X/XO similarly resulted in an increase in autonomous PKC activity (control = 1.89 ± 0.23 pmol/min; X/XO = 8.41 ± 1.44 pmol/min), but not in cofactor-dependent PKC activity (control = 14.76 ± 2.52 pmol/min; X/XO = 15.10 ± 3.78 pmol/min). These data indicate that X/XO is capable of activating PKC in a number of different preparations.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effects of X/XO treatment on PKC activity in various enzyme preparations. Hippocampal homogenates (n = 90), the soluble fraction from hippocampal homogenates (n = 28), and rat brain purified PKC (n = 18) were incubated with X/XO (20 µg/ml/2 µg/ml) for either 2 min at 37 °C (homogenates and soluble fraction of hippocampal homogenates) or 30 s at 25 °C (purified PKC). Autonomous and cofactor-dependent activity were measured as described under "Experimental Procedures." Values are mean ± S.E. and are expressed as a percentage of control samples. *, p < 0.05 using Student's t test for independent groups (control versus X/XO treatment).

Exposure of the various preparations to either allopurinol or oxypurinol (both XO inhibitors) alone had nonspecific effects on PKC activity (data not shown). Therefore, the use of XO inhibitors was not suitable to determine whether or not the enzymatic activity of XO was required for the X/XO-induced increase in autonomous PKC activity. However, exposure of homogenates, soluble fraction, or purified PKC to either xanthine alone (20 µg/ml) or xanthine with heat-inactivated XO (20 µg/ml/2 µg/ml) had no effect on either autonomous or cofactor-dependent PKC activity (data not shown). These results demonstrate that the catalytic action of xanthine oxidase on xanthine is necessary for the increase in PKC activity induced by X/XO.

To characterize further the effect of X/XO on autonomous PKC activity, we performed extended incubations of hippocampal homogenates with X/XO. The X/XO-induced increase in PKC activity occurred rapidly. For example, hippocampal homogenates treated with X/XO exhibited maximal activation within 30 s (data not shown). Maximum increases in autonomous PKC activity were observed whether X/XO treatment lasted on the order of several minutes or hours, which demonstrates that prolonged incubations with X/XO do not result in the inactivation of PKC (data not shown). Thus, the X/XO-induced increase in autonomous PKC activity was rapid in onset and persistent in duration.

In agreement with previous observations, PKC from hippocampal homogenates phosphorylates NG-(28-43) in a manner that is consistent with Michaelis-Menten kinetics (Fig. 2A). Lineweaver-Burke analysis of the phosphorylation data revealed a linear double-reciprocal plot, using NG-(28-43) concentrations from 0.1 to 100 µM (Fig. 2B). From these data the apparent K_m for both control and X/XO-treated PKC in the presence of calcium and lipid cofactors was 27 µM, whereas the apparent K_m values for control and X/XO-treated PKC in the presence of excess EGTA were 15 and 17 µM, respectively. The calculated V_max values for control and X/XO-treated PKC in the presence of calcium and lipid cofactors were 12.05 and 11.93 pmol/µg/min, respectively; whereas the calculated V_max values for control and X/XO-treated PKC in the presence of excess EGTA were 0.24 and 2.62 pmol/µg/min, respectively. No inhibition of PKC activity was observed even at NG-(28-43) concentrations of 1 mM, which indicates that there is not a substrate inhibition effect. Hill plot analysis revealed Hill coefficients of 0.24 for control and 0.91 for X/XO-treated PKC in the presence of excess EGTA. The Hill coefficients for control and X/XO-treated PKC in the presence of calcium and lipid cofactors were 0.94 and 0.92, respectively. Taken together, these data suggest that there is noncooperative binding of the substrate peptide, which is consistent with previous reports (15, 18).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Concentration curve for phosphorylation of NG-(28-43) by X/XO-stimulated PKC in the soluble fraction of hippocampal homogenates. A, NG-(28-43) (0.1-500 µM) was phosphorylated in the absence (Auto.) or in the presence of enzyme cofactors (Cofactor) as described under "Experimental Procedures." Values are mean ± S.E. for four determinations assayed in duplicate. B, double-reciprocal plot of the data in A.

The X/XO system generates both superoxide and hydrogen peroxide. Therefore, we tested whether either superoxide and/or hydrogen peroxide was responsible for the X/XO-induced increase in autonomous PKC activity. In experiments in which either hippocampal homogenates or purified PKC were exposed to X/XO, we added either SOD or catalase to remove superoxide and H₂O₂, respectively. As illustrated in Fig. 3, SOD significantly reduced the X/XO-induced increase in autonomous PKC activity in both homogenates and purified PKC. In contrast, catalase had no significant effect on the X/XO-induced increase in autonomous PKC activity. Taken together, these results demonstrate that superoxide is responsible for the X/XO-induced increase in autonomous PKC activity.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Identification of reactive species responsible for the X/XO-induced stimulation of autonomous PKC activity. Hippocampal homogenates (n = 9-15) and rat brain purified PKC (n = 16) were incubated for either 2 min at 37 °C or 30 s at 25 °C, respectively, with X/XO (20 µg/ml/2 µg/ml) in the presence of SOD, catalase (Cat.), mannitol, or Desferal. Values are mean ± S.E. and are expressed as a percentage of X/XO-induced increase in autonomous PKC activity. *, p < 0.05 using a one-way ANOVA with Bonferroni correction for multiple comparisons within each of the two preparations.

In the presence of transition metals, superoxide can drive the production of hydroxyl radical. Therefore, we investigated whether or not hydroxyl radical was responsible for the X/XO-induced increase in autonomous PKC activity. Mannitol, a hydroxyl radical scavenger, and Desferal, an iron chelator, were added immediately prior to X/XO treatment of the various PKC preparations. As depicted in Fig. 3, neither mannitol nor Desferal had an effect on X/XO-induced increase of autonomous PKC activity in either hippocampal homogenates or soluble fraction. Incubation of the various preparations with either mannitol or Desferal alone had no effect on autonomous or cofactor-dependent PKC activity (data not shown). These results demonstrate that the superoxide-driven production of hydroxyl radical is not responsible for the X/XO-induced increase in autonomous PKC activity.

Requirement of cysteine modification for superoxide-induced activation of PKC-- We investigated whether thiol oxidation enhanced PKC activity by using the thiol-oxidizing agent DTNB. As depicted in Fig. 4, treatment of purified PKC with DTNB increased autonomous PKC activity (control = 2.47 ± 0.35 pmol/min; DTNB = 4.94 ± 0.70 pmol/min). The increase in autonomous PKC activity was reversed by treatment with the reducing agent DTT (2.54 ± 0.32 pmol/min). These results demonstrate that thiol oxidation with DTNB is able to increase autonomous PKC activity. However, as also depicted in Fig. 4, DTNB reduced cofactor-dependent PKC activity (control (7.94 ± 0.40 pmol/min); DTNB (4.56 ± 0.80 pmol/min)). The DTNB-induced inhibition of cofactor-dependent activity was reversed partially by DTT (6.06 ± 0.28 pmol/min). These results demonstrate that thiol oxidation with DTNB inhibits the stimulation of PKC activity by the cofactors, which suggests that the thiol oxidation occurs within the cysteine-rich, cofactor-binding region of PKC.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of thiol oxidation on autonomous and cofactor-dependent PKC activity. Rat brain purified PKC (n = 8) was either incubated with DTNB for 30 s at 25 °C or preincubated with DTNB for 30 s at 25 °C and then incubated with DTT (50 mM) for an additional 1 min at 25 °C. Autonomous and cofactor-dependent PKC activity were measured as described under "Experimental Procedures." Values are mean ± S.E. and are expressed as percentage of control samples, which were treated with H2O. *, p < 0.05 using a one-way ANOVA with Bonferroni correction for multiple comparisons for each of the two types of PKC activity.

A well characterized oxidative modification of thiols is the formation of the disulfide bond. Therefore, we investigated whether disulfide bond formation was responsible for the superoxide-induced increase in autonomous PKC activity. As illustrated in Fig. 5, we tested the effects of several reducing agents on the superoxide-induced increase in PKC activity. Specifically, all of the reducing agents tested, DTT, -ME, and Cyano, partially reversed the superoxide-induced increase in autonomous PKC activity. The partial reversal by the reducing agents commenced at 30 min of incubation and reached the maximum reversal (~40% inhibition) at 60 min of incubation with reducing agent. We observed no additional reversal of the superoxide-induced stimulation of PKC at times greater than 60 min up to 12 h of incubation with reducing agents. These results suggest that superoxide induced the formation of very stable disulfide bond that increases autonomous PKC activity.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Effect of reducing agents on superoxide-induced increase in autonomous PKC activity. Rat brain purified PKC was incubated with X/XO (20 µg/ml X to 2 µg/ml XO) in the presence of DTT (100 mM; n = 8), -ME (100 mM; n = 8), or Cyano (250 µM; n = 8) for 60 min at 4 °C. Values are mean ± S.E. and are expressed as percentage of the X/XO-induced increase in autonomous PKC activity. *, p < 0.05 using Student's t test for independent groups (control versus X/XO treatment).

Requirement of the Regulatory Domain for the Oxidative Activation of PKC-- We investigated the region of PKC that was targeted by superoxide to increase the autonomous activity of PKC. First, we determined whether superoxide increased the activity of the free catalytic domain of PKC (PKM). We found that superoxide did not increase PKM activity. In fact, superoxide tended to inhibit the activity of PKM (control = 0.67 ± 0.23 pmol/min; X/XO = 0.36 ± 0.1 pmol/min). This finding is consistent with previous reports indicating that oxidation inhibits PKM activity (9) and suggests that the regulatory domain is required for superoxide to increase autonomous PKC activity.

To determine the region of the regulatory domain that was necessary for the superoxide-induced increase of autonomous PKC activity, we studied the effects of superoxide on the activity of various PKC isoforms. The isoforms studied represented the three classes of PKC: (a) the  and _II isoforms of the classical PKC subfamily, (b) the  isoform of the novel PKC subfamily, and (c) the  isoform of the atypical PKC subfamily (19). As illustrated in Fig. 6, superoxide increased the autonomous activity of the classical PKC subfamily as represented by the  and _II isoforms. For the  isoform, superoxide increased autonomous PKC activity (control = 1.22 ± 0.08 pmol/min; X/XO = 2.22 ± 0.31 pmol/min) but had no effect on cofactor-dependent PKC activity (control = 32.64 ± 2.13 pmol/min; X/XO = 26.84 ± 2.92 pmol/min). Likewise, for the _II isoform, superoxide increased autonomous PKC activity (control = 0.40 ± 0.08 pmol/min; X/XO = 1.15 ± 0.08 pmol/min), but had no effect on cofactor-dependent PKC activity (control = 8.81 ± 1.56 pmol/min; X/XO = 10.09 ± 1.42 pmol/min). Similar to the results we observed with isoforms in the classical PKC subfamily, superoxide increased the autonomous activity of the novel and atypical PKC subfamilies, as represented by the  and  isoforms (for the  isoform: control = 4.26 ± 1.11 pmol/min; X/XO = 6.32 ± 1.11 pmol/min; for the  isoform: control = 24.54 ± 1.21 pmol/min; X/XO = 32.56 ± 1.22 pmol/min). Each of the PKC subfamilies contains a common cysteine-rich region (C1). Therefore, the observation that superoxide increased the activity of PKC from each of the PKC subfamilies is consistent with notion that superoxide interacts with the thiol group of cysteine to increase the autonomous activity of PKC.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   The effects of superoxide on autonomous PKC activity of various PKC isoforms. Lysates (n = 4) from cells overexpressing either the , II, , or  isoforms were incubated with X/XO (20 µg/ml/2 µg/ml) for 30 s at 25 °C. Values are mean ± S.E. and are expressed as a percentage of control samples, which were treated with H2O. *, p < 0.05 using Student's t test for independent groups (control versus X/XO treatment).

Modulation of Zinc Status by Superoxide-- The C1 region of PKC is characterized by a zinc finger motif that renders the thiol functional groups of the cysteines of the zinc finger in close apposition, separated by the incorporated zinc molecule. Thus, release of zinc from the zinc finger and formation of a disulfide within the zinc finger region would render a stable modification. Interestingly, a number of proteins with zinc-thiolate moieties are regulated by oxidation at the zinc binding site in a manner that results in the loss of zinc and disulfide formation within the metal binding site (for an example, see Ref. 20). Therefore, we investigated the hypothesis that superoxide could stimulate the autonomous activity of PKC via oxidation of the zinc finger region of the enzyme by promoting the loss of zinc from the zinc finger motif.

First, we studied whether or not stabilizing the zinc finger and preventing the release of zinc from the motif with excess zinc would block the superoxide-induced stimulation of PKC. As demonstrated in Fig. 7, incubation of the soluble fraction of hippocampal homogenates with either zinc salt or sodium chloride (ionic control) had no effect on either autonomous or cofactor-dependent PKC activity. However, addition of zinc blocked the superoxide-induced stimulation of PKC, which suggests that the excess zinc stabilized the finger motif. It is important to note that zinc did not prevent the generation of reactive oxygen species by X/XO (data not shown). These results are consistent with the hypothesis that superoxide stimulates the autonomous activity of PKC by acting on the cysteine-rich zinc finger of PKC.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Inhibition of superoxide-induced stimulation of autonomous PKC activity by zinc. The soluble fraction from hippocampal homogenates (n = 12) was incubated with X/XO (20 µg/ml X to 2 µg/ml XO) in the presence or absence of either ZnCl2 (1 mM) or NaCl (2 mM) for 2 min at 37 °C. Autonomous and cofactor-dependent activity were measured as described under "Experimental Procedures." Values are mean ± S.E. and are expressed as a percentage of control PKC activity. *, p < 0.05 compared with control samples using one-way ANOVA with Bonferroni post hoc comparisons between treatments.

Next, we investigated whether removal of zinc from the finger using a selective zinc chelator would stimulate autonomous PKC activity. As shown in Fig. 8, chelating zinc resulted in a time-dependent stimulation of autonomous PKC activity. The increase in autonomous PKC activity induced by the chelator was blocked by including zinc at a 1:1 molar ratio of zinc:chelator. In a manner to that observed for superoxide, chelating zinc did not increase the cofactor-dependent activity of PKC. Therefore, these observations support the hypothesis that superoxide-induced stimulation of autonomous PKC activity involves the removal of zinc from the enzyme.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Stimulation of autonomous PKC activity with a zinc chelator. PKC-enriched fractions were eluted from a Sephadex G-100 column as described under "Experimental Procedures." Fractions (n = 6-12) were incubated either with or without TPEN (1 mM) in the presence or absence of ZnCl2 (1 mM) for the indicated times at 4 °C. Autonomous and cofactor-dependent activity were measured as described under "Experimental Procedures." Values are mean ± S.E. and are expressed as a percentage of control PKC activity. *, p < 0.05 compared with control samples using one-way ANOVA with Bonferroni post hoc comparisons between treatments.

If superoxide stimulates PKC by promoting the release of zinc from the zinc finger motif, then exposure to superoxide should result in the release of zinc from the protein. We tested this hypothesis by measuring the amount of zinc in a PKC-enriched column fraction before and after exposure to superoxide. As shown in Fig. 9A, incubation of the PKC-enriched column fraction with catalytically competent X/XO (PKC Fract. + X/XO) resulted in an increase in absorbance, reflecting the release of zinc and binding to PAR, a zinc-binding dye. In contrast, we observed no change in absorbance when the PKC-enriched solution was incubated with either xanthine alone or boiled X/XO. These data are consistent with the hypothesis that superoxide results in release of zinc from PKC.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Effect of superoxide on the zinc content of PKC. A, PAR (100 µM) was incubated with X (20 µg/ml), X/XO (20 µg/ml X to 2 µg/ml XO), X/boiled XO (X/bXO; 20 µg/ml X to 2 µg/ml bXO), the PKC-enriched fraction from a Sephadex G-100 column (PKC fraction; 100-150 µg), PKC fraction and X, PKC fraction and X/XO, or PKC fraction and X/bXO for 30 min at 37 °C. The formation of the PAR-Zn complex was determined by measuring a change in absorbance at 500 . Values are mean ± S.E. *, p < 0.05 using one-way ANOVA with Bonferroni post hoc comparisons between treatments. B, after treatment with X/XO (20 µg/ml X to 2 µg/ml XO) for 2 min at 37 °C, either the PKC  or  isoform was isolated from hippocampal homogenates by immunoprecipitation and subjected to acid hydrolysis as described under "Experimental Procedures." The zinc content of the PKC isoforms was determined by measuring the change in absorbance of PAR (100 µM) at 500  after a 30-min incubation at 37 °C. Values are mean ± S.E. *, p < 0.05 using Student's t test for independent groups (control versus X/XO treatment).

The observations that the superoxide-induced stimulation of PKC was blocked by excess zinc, was mimicked by chelating zinc, and was correlated with a release of zinc from a PKC-enriched column fraction support the hypothesis that superoxide stimulates PKC in a manner that promotes the loss of zinc from the enzyme. Therefore, we investigated whether we could measure a decrease in zinc from PKC that had been treated with superoxide. As described under "Experimental Procedures," PKC was treated with or without superoxide, immunoprecipitated, isolated, acid-hydrolyzed, and incubated with PAR. An increase in absorbance would represent an increase in soluble zinc, which in turn would represent the zinc originating from the immunoprecipitated PKC. As demonstrated in Fig. 9B, we observed a decrease in the content of zinc after treatment of either PKC or PKC with superoxide. These results provide direct evidence that oxidation of PKC results in the loss of zinc from the enzyme.

Isolation of Superoxide-stimulated, Zinc-depleted PKC Activity-- In order to investigate the mechanism of the superoxide-induced stimulation of PKC activity via the release of zinc in an intact cellular preparation, we isolated superoxide-stimulated, zinc-depleted PKC using ion exchange column chromatography, which facilitated the direct measurement of oxidatively activated PKC from tissue. As has been reported previously, we observed that two fractions of cofactor-dependent PKC activity eluted from a DEAE-cellulose column with 0.1 M NaCl and 0.25 M NaCl salt washes after application of the soluble fraction of hippocampal homogenates to the column (Fig. 10; Refs. 9 and 21). We also observed a peak of autonomous activity that eluted from the column with the 0.25 M NaCl wash in samples that were treated with X/XO prior to application to the column. The 0.25 M NaCl peak of autonomous PKC activity was blocked when the X/XO-treated samples were incubated with either SOD (25 µg/ml; 1.11 pmol/min) or SOD/catalase (25 µg/ml, 25 µg/ml; 0.87 pmol/min) prior to application to the column. However, the 0.25 M NaCl peak of autonomous PKC activity was not blocked when the X/XO-treated samples were incubated by treatment with catalase alone (25 µg/ml; 9.54 pmol/min). Neither X/XO nor the antioxidants altered the elution profile of cofactor-dependent PKC from the DEAE-cellulose column (data not shown). These results demonstrate that superoxide is the reactive oxygen species responsible for the autonomous activity eluted with 0.25 M NaCl. Next, we tested whether the peak of superoxide-stimulated PKC activity eluted with 0.25 M NaCl was also indicative of zinc-depleted PKC. As shown in Fig. 11, treatment of the soluble fraction of hippocampal homogenates with the zinc chelator TPEN resulted in the elution of autonomously active PKC with 0.25 M NaCl. TPEN did not alter the elution profile of cofactor-dependent PKC. Taken together, these results demonstrate that DEAE-cellulose column chromatography is a suitable technique to isolate and measure the activity of superoxide-stimulated, zinc-depleted PKC.

View larger version (30K): [in this window] [in a new window]   Fig. 10.   Effect of superoxide on the elution profile of PKC from DEAE column. The soluble fraction from hippocampal homogenates (n = 8) was incubated with X/XO (20 µg/ml/2 µg/ml) for 2 min at 37 °C. The samples were applied and eluted from a DEAE column as described under "Experimental Procedures." Autonomous and cofactor-dependent activity were measured as described under "Experimental Procedures." Inset, representative Western blots for phosphorylated serine 657 on PKC (P657), phosphotyrosine (PY), and PKC of the DEAE column fractions indicated.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   Effect of a zinc chelator on the elution profile of PKC from DEAE column. The soluble fraction from hippocampal homogenates (n = 4) was incubated with TPEN (1 mM) for 7 days at 4 °C. The samples were applied and eluted from a DEAE column as described under "Experimental Procedures." Autonomous and cofactor-dependent activities were measured as described under "Experimental Procedures."

We investigated the possibility that superoxide-induced stimulation of PKC might also involve alteration of the phosphorylation status of the enzyme. Thus, we examined the migration pattern of PKC, the phosphorylation of serine 657 on PKC, and the tyrosine phosphorylation PKC isolated from DEAE column eluants of hippocampal homogenates treated with and without superoxide. As shown in the inset to Fig. 10, the superoxide-induced stimulation PKC did not alter the mobility of PKC on SDS-polyacrylamide gel electrophoresis, which suggests that there is no alteration in the phosphorylation of PKC. We also observed no alteration in the phosphorylation of serine 657 on PKC and no alteration in PKC tyrosine phosphorylation (inset of Fig. 10). Therefore, we can reject the hypothesis that changes in phosphorylation contribute to superoxide-induced stimulation of PKC in our preparation.

We tested the hypothesis that superoxide can persistently increase the autonomous activity of PKC in an intact cellular preparation, specifically the hippocampal slice preparation. Hippocampal slices were incubated for 10 min with X/XO; 45 min after washout of X/XO, the slices were homogenized and subjected to DEAE-cellulose chromatography. Similar to homogenate preparation experiments in Figs. 10 and 11, we were able to isolate a superoxide-stimulated, zinc-depleted peak of autonomous activity of PKC from hippocampal slices that were incubated with X/XO (Fig. 12). In slices treated with X/XO, the peak of autonomous PKC activity was blocked by SOD (25 µg/ml; 0.13 pmol/min), but not by catalase (25 µg/ml; 3.15 pmol/min), a result similar to the homogenate experiments in Figs. 10 and 11. Finally, neither SOD nor catalase had an effect on the elution profile of cofactor-dependent PKC activity in slices incubated with X/XO (data not shown). Taken together, these date demonstrate that a brief incubation of superoxide with hippocampal slices results in a persistent increase in autonomous PKC activity.

View larger version (21K): [in this window] [in a new window]   Fig. 12.   Effect of superoxide on PKC from hippocampal slices. Hippocampal slices (n = 5) were prepared as described under "Experimental Procedures." Slices were incubated with either X/boiled XO or X/XO (20 µg/ml X to 2 µg/ml bXO or XO) for 10 min at 30 °C in an interface perfusion chamber. The slices were incubated with perfusate in the absence of X/boiled XO or X/XO for an additional 45 min at 30 °C, at which point the slices were frozen on dry ice, homogenized, the soluble fraction collected, and applied to DEAE column. Autonomous and cofactor-dependent activities were measured as described under "Experimental Procedures."

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

These studies demonstrate that superoxide regulates PKC by increasing the autonomous activity of PKC. To the best of our knowledge, the work reported here is the first to determine the stringency of X/XO-induced oxidation of PKC by measuring the amount of superoxide and H₂O₂ generated by X/XO and to characterize the kinetics of oxidation on the activity of PKC. The work reported here also is the first to investigate the effects of oxidation on PKC activity in different PKC preparations and the effects of oxidation of various PKC isoforms. Finally, we have provided direct evidence demonstrating that cysteine oxidation and release of zinc are required for the superoxide-induced activation of PKC: the first such demonstration not only for PKC, but of any kinase.

Previous studies on the effects of reactive oxygen species (ROS) on PKC have indicated that the redox regulation of PKC is complex. The effect of oxidation on PKC is likely to be a function of a number of factors that contribute to the stringency of oxidation. Various ROS have different oxidative strengths. For instance, hydroxyl radical is a stronger oxidant than H₂O₂, which, in turn, is stronger than superoxide (22). In previous studies using mild oxidizing conditions, either superoxide (Fig. 1; Refs. 23 and 24) or H₂O₂ (25) were found to increase PKC activity, whereas oxidizing conditions that promote the production of the strong oxidant hydroxyl radical were found to inactivate PKC (26). The stringency of oxidation can be influenced by the length of oxidant exposure. For example, brief exposure of PKC to H₂O₂ was found to increase activity, whereas prolonged exposure of PKC to H₂O₂ was reported to result in proteolysis and inactivation of PKC (9). In addition, the type PKC preparation studied can influence the effects of oxidation on PKC. For example, products of lipid oxidation have been shown to increase PKC activity (27-29). Therefore, in either cellular or homogenate preparations of PKC, ROS may activate PKC in an indirect manner via the generation of oxidized lipids. However, studies using purified PKC preparations have demonstrated direct effects of oxidation on PKC (23, 24). In these types of studies, shorter times of incubation with the oxidant were employed to limit the stringency of oxidation.

Taking all of these factors into consideration, we propose a continuum model for the effects of oxidation on PKC activity. The continuum of oxidative effects on PKC is dependent on the stringency of oxidation. At one end of the continuum, low stringency of oxidation results in a persistent increase in PKC activity. Moving along the continuum, more stringent oxidation can produce a transient increase in PKC activity, generation of PKM, and eventually the inactivation of PKC. The studies presented herein represent the redox regulation of PKC at the low stringency end of the continuum model. In support of the conclusion that our studies represent the low stringency conditions, we measured both superoxide and H₂O₂ production from X/XO and observed that low micromolar concentrations of each are produced, ROS concentrations that are likely to be produced in intact tissue preparations such as the hippocampal slice preparation (30). In addition, we demonstrated that the effects of X/XO on PKC were due to the weak oxidant superoxide and not to the stronger oxidants H₂O₂ or hydroxyl radical. Finally, our use of brief exposures of the various PKC preparations to superoxide supports the conclusion that our studies represent low stringency oxidizing conditions.

In this study we also characterized the kinetic behavior of oxidized PKC and the autonomous activity of the enzyme. We observed a difference between previously reported values of K_m and V_max for substrate NG-(28-43) that are likely to be due to differences of enzyme behavior in different preparations, as our studies are the first to characterize the behavior of PKC from hippocampal homogenates. Whereas previous studies have investigated the kinetics of the cofactor-dependent activity of the enzyme, we characterized the kinetic behavior of autonomous activity as well. As discussed above there have been a number of published studies of the role of oxidation in the regulation of PKC activity; however, we directed our attention to how oxidation affects the kinetics of PKC. The kinetic analysis in Fig. 2 shows that there are differences between the superoxide-stimulated autonomous activity and cofactor-dependent activity. The superoxide-stimulated PKC has a higher affinity for the substrate than cofactor-stimulated PKC, but the maximal activity of the superoxide-stimulated PKC is approximately 4.5-fold lower than that for cofactor-stimulated PKC. However, Hill plot analysis revealed Hill coefficients for superoxide-stimulated autonomous activity and cofactor-dependent activity that are virtually identical, which suggests that the interaction with the substrate is very similar for the different types of activity. It is important to note that superoxide exposure had no effect on the kinetics of cofactor-dependent activity. The most parsimonious interpretation of these findings is that the modification induced by superoxide is subtle rather than dramatic in nature, thus preserving the ability of the cofactors to activate the superoxide-modified enzyme. Finally, these data support the notion that superoxide acts as a novel, positive regulator of PKC.

Our studies are the first to examine the effects of oxidation on various PKC isoforms in an effort to identify regions of the regulatory domain that are necessary for oxidative activation of PKC. We demonstrated that all the isoforms tested (, II, , and ) resulted in increased autonomous PKC activity in response to superoxide exposure (Fig. 6), which indicates that each of the isoforms has the necessary elements required for oxidative activation of PKC. However, the increases in autonomous PKC activity observed with the  and the _II isoforms were more robust than those observed with the  and the  isoforms. Because neither the  nor the  isoforms contain the Ca²⁺-binding domain, our findings suggest that this domain is necessary for the maximum activation of PKC by superoxide. A common region of the , _II, , and  isoforms is the first cysteine-rich region in the conserved C1 domain that serves as a cofactor-binding region of PKC. Therefore, we postulate that the first cysteine-rich region of the C1 domain is an important site of superoxide interaction with PKC.

The susceptibility of cysteine residues to oxidation prompted us to examine the effects of thiol oxidation on PKC activity. We observed that treatment with DTNB increased autonomous PKC activity in a manner that was reversible by DTT (Fig. 4). The most likely interpretation of this finding is that thiol oxidation increases autonomous PKC activity. However, there are several differences between the effects of superoxide and DTNB on PKC activity. First, the DTNB-induced increase in autonomous PKC activity was of a lesser magnitude (Fig. 4; 200 ± 57% of control) than that induced by for superoxide (Fig. 1; 676 ± 50% of control). Second, DTNB inhibited cofactor-dependent PKC activity (Fig. 4), whereas superoxide had no effect on cofactor-dependent activity (Fig. 1). The inhibition of cofactor-dependent PKC activity by DTNB suggests that DTNB acts on thiols within the cysteine-rich cofactor-binding domain, which is consistent with a finding reported previously (31). The observation that superoxide did not affect cofactor-dependent PKC activity suggests that superoxide does not modify cysteines within the cofactor-binding region of PKC in the same manner as does DTNB. Therefore, the most likely explanation for the difference between the effects of superoxide and DTNB on cofactor-dependent activity is that DTNB oxidation of the cysteine-rich region results not only in disulfide formation but also the formation of mixed disulfides between the thiols of PKC and DTNB. Such mixed disulfides in the C1 region would be expected to inhibit the binding of cofactors in this region and therefore decrease cofactor-dependent activity, similar to what is shown in Fig. 4.

Oxidation of vicinal thiols can result in the formation of disulfide bonds. Because PKC is enriched in vicinal thiols, especially in the cysteine-rich region, we investigated whether disulfide bond formation was the modification induced by superoxide. In support of this hypothesis, we observed that incubation of purified PKC with DTT, -ME, or Cyano partially reversed the superoxide-induced increase in PKC activity (Fig. 5). There are several possible interpretations of these observations. One interpretation is that disulfide bond formation contributes to the increase in autonomous PKC activity induced by superoxide. It is possible that another type of thiol oxidation that is not reversible by reducing agents, such as sulfonic acid, may contribute to the superoxide-induced activation of PKC. However, such a thiol modification would require extremely stringent oxidizing conditions (32) that are not likely to be present in our experimental conditions. In addition, formation of sulfonic acid would alter the charge of the enzyme (32), and likely alter both autonomous and cofactor-dependent activity, which we did not observe. Alternatively, superoxide may induce disulfide bond formation that is resistant to reducing agents. For example, disulfide bond formation could result in a conformational change of PKC that renders the disulfide bond(s) inaccessible to reducing agents. However, this possibility is not likely to occur because we observed that Cyano, a hydrophobic reducing agent that should penetrate the hydrophobic interior of the protein, was no more effective in reversing the superoxide-induced stimulation of PKC than the hydrophilic reducing agents DTT and -ME (Fig. 5).

The superoxide-induced increase in autonomous PKC activity results in the persistent activation of PKC. Other mechanisms that influence persistent stimulation of PKC include membrane insertion, proteolytic cleavage in the hinge region, and autophosphorylation (3). In addition, it recently has been reported that the addition of oxidizing agents to cell cultures can result in tyrosine phosphorylation and activation of PKC (12). We have addressed the possibility that the superoxide-induced activation of PKC results in either proteolysis or phosphorylation of PKC. First, we observed no generation of PKM after treatment of either hippocampal homogenates or slices with superoxide (data not shown), which excludes the possibility that superoxide exposure results in increased proteolysis of PKC. In addition, we observed that the autophosphorylation of PKC at serine 657 (Fig. 10, inset) and phosphorylation of PKCII at threonine 634/641 (data not shown) did not increase in response to exposure of hippocampal homogenates to superoxide. Finally, we did not observe any tyrosine phosphorylation of PKC when either hippocampal homogenates (inset of Fig. 10) or slices (data not shown) were treated with either superoxide or H₂O₂. Taken together, these results do not support the notion that the stimulation of PKC by superoxide involves mechanisms that have been previously described to be involved in the persistent activation of the enzyme.

The hypothesis that oxidation acts at zinc finger regions to modulate the function of a protein is not an idea that is peculiar to PKC. In fact, the kinds of modifications discussed here (promoting the release of zinc from zinc fingers and forming disulfide bonds) have been demonstrated for other zinc-containing proteins such as metallothionein (20, 33-35), the glucocorticoid receptor (36-38), the retinoic acid receptor (39), aspartate carbamoyltransferase (17), alcohol dehydrogenase (40), and replication protein A (41). Moreover, there has been speculation that that ROS and acidic conditions may alter the zinc content of PKC (23, 42-44). However, herein we present the first evidence that the C1 region is the likely target for superoxide, and that the mechanism involves both the release of zinc from this region in addition to disulfide bond formation. There are several lines of evidence consistent with this idea. First, we found that the superoxide-induced stimulation of autonomous PKC activity was blocked by excess zinc. In addition, we tested the hypothesis that stimulation of PKC by superoxide involves a loss of zinc from the zinc finger motif. Consistent with this hypothesis, we observed that a zinc chelator increased autonomous PKC activity. Furthermore, we observed that zinc was released when PKC was exposed to superoxide. Finally, we isolated and measured the zinc content of PKC after exposure to superoxide and found that superoxide decreased the zinc content of PKC. Because there is more than one zinc finger motif on the majority of the PKC isoforms, it would be interesting to determine whether there is a preferential susceptibility of one zinc finger over another for oxidation by superoxide. Such an observation would prove useful in developing a model of a redox site and aid in the identification of such sites in other proteins.

We have shown that a superoxide-stimulated, zinc-depleted form of PKC can be isolated via DEAE column chromatography (Figs. 10 and 11). In addition, we have shown that a persistent, autonomously active PKC can be isolated in the same manner from an intact cellular preparation, specifically the hippocampal slice preparation, after brief exposures to physiological concentrations of superoxide (Fig. 12). Thus, it will be useful to use this type of procedure to determine whether superoxide-stimulated, zinc-depleted PKC can be isolated from either physiological or pathological stimulation of intact cellular preparations.

There is a variety of evidence for the redox regulation of receptors, signal transduction cascades, and transcription factors (45, 46). However, the impact of redox regulation in various cellular functions, especially neuronal function, has not been well described. There is evidence that ROS contribute to many pathological conditions, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease (47-51). Therefore, it is tempting to speculate that redox regulation of intracellular signaling molecules may contribute to these diseases. In addition, ROS may be necessary for physiological cellular functions. For example, we recently reported that superoxide is necessary for the expression of a cellular model of learning and memory, N-methyl-D-aspartate (NMDA) receptor-dependent long term potentiation (LTP) (52, 53). In these studies, scavenging of superoxide prevented LTP and attenuated the LTP-associated increases in PKC activity (52), which suggests that superoxide contributes to the activation of PKC that is required for LTP. In addition, it has been demonstrated that NMDA receptor activation increases the production of superoxide in the rat hippocampal slice preparation (30). In this study, NMDA receptor activation was found to increase in superoxide to levels roughly comparable to the concentrations of superoxide we found to increase autonomous PKC activity in our experiments. Finally, deficient LTP and learning have been observed in animals in which the normal metabolism of superoxide has been perturbed by transgenic or deletion mutations (54, 55). Collectively, these observations suggest that superoxide may contribute to both physiological and pathological conditions via redox regulation, and that redox regulation of PKC by superoxide may contribute to these conditions.

	ACKNOWLEDGEMENTS

We thank Dr. Edda Thiels and Beatriz I. Kanterewicz for critically reading this manuscript and helpful suggestions. We also thank Dr. Joanne Johnson, Amelia Edwards, and Dr. Alexandra Newton for generously providing PKC isoforms, as well as Karen Hartnett for technical advice regarding the nitro blue tetrazolium assay.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants NS08950 and NS34007 (both to E. K.), National Institute of Mental Health Training Grant MH18273, National Institute of Mental Health National Research Service Award Fellowship MH1198301 (to L. T. K.), a University of Pittsburgh Central Research Development Fund award (to E. K.), and a grant from the Winters Foundation (to E. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260. Tel.: 412-624-4610; Fax: 412-624-9198; E-mail: klann@brain.bns.pitt.edu.

Published, JBC Papers in Press, May 22, 2000, DOI 10.1074/jbc.M002043200

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; X, xanthine; XO, xanthine oxidase; bXO, boiled xanthine oxidase; SOD, superoxide dismutase; DTT, dithiothreitol; -ME, -mercaptoethanol; ANOVA, analysis of variance; LTP, long term potentiation; NMDA, N-methyl-D-aspartate; PKM, free catalytic domain of PKC; Cyano, tris-(2-cyanoethyl)phosphine; TPEN, tetrakis-(2-pyridylmethyl)ethylenediamine; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid); ROS, reactive oxygen species; PAR, 4-(2-pyridylazo)resorcinol; Auto, autonomous PKC activity; Cofactor, cofactor-dependent PKC activity.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES