Potent inactivation of representative members of each PKC isozyme subfamily and PKD via S-thiolation by the tumor-promotion/progression antagonist glutathione but not by its precursor cysteine

Feng Chu, Nancy E. Ward and Catherine A. O'Brian,1

Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 173, Houston, TX 77030, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We recently established that S-glutathiolation of cPKC{alpha} fully inactivates the isozyme, at a stoichiometry of ~1 mol GSH/mol cPKC{alpha}. In this report we demonstrate that, in addition to cPKC{alpha}, six other PKC isozymes that are representative of the three subfamilies within the PKC family (cPKCß1, cPKCß2 and cPKC{gamma}, nPKC{delta} and nPKC{varepsilon} and aPKC-{zeta}) are subject to inactivation by S-glutathiolation induced by the thiol-specific oxidant diamide, which induces disulfide bridge formation. Among PKD and the seven PKC isozymes examined in this report only nPKC{delta} has been directly implicated as an antagonist of tumor promotion/progression, while several of the kinases have been implicated in the mediation of tumor promotion/progression. We report that of the kinases examined nPKC{delta} was the most resistant to inactivation by diamide-induced S-glutathiolation. In the absence of GSH only nPKC{delta} activity exhibited a biphasic response to diamide, with low diamide concentrations oxidatively enhancing nPKC{delta} activity and higher concentrations inactivating the isozyme; the other seven kinases were subject to monophasic, concentration-dependent, oxidative inactivation by diamide to various extents. The results provide evidence that at least some pro-oxidant environments may support the potent inactivation of nPKC{varepsilon} and other PKC isozymes implicated in tumor promotion/progression by the mechanisms of S-glutathiolation and, in some cases, disulfide bridge formation among the isozyme thiols, without inducing substantial nPKC{delta} inactivation. The results also show that neither the seven PKC isozymes examined nor PKD are inactivated by S-cysteinylation under conditions that support potent inactivation by S-glutathiolation. This indicates that the protection that the tumor promotion/progression antagonist GSH may afford against oxidative tumor promotion/progression mechanisms by S-thiolating and inactivating PKC isozymes and PKD cannot be afforded by the metabolic GSH precursor cysteine. These observations support a role for PKC inactivation via S-glutathiolation in the mechanism of tumor promotion/progression antagonism by GSH in pro-oxidant environments.

Abbreviations: DAG, diacylglycerol; DMBA, 7,12-dimethylbenz[a]anthracene; DTT, dithiothreitol; GSH, glutathione; PKC, protein kinase C; PKD, protein kinase D; PS, phosphatidylserine; ROS, reactive oxygen species; TCA, trichloroacetic acid; TPA, 12-O-tetradecanoyl phorbol 13-acetate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The protein kinase C (PKC) isozyme family is divided into three subfamilies based on structural differences in isozyme regulatory domains that correspond to distinct modes of phospholipid-dependent regulation (1). The Ca2+-dependent cPKC ({alpha}, ß1, ß2 and {gamma}) and Ca2+-independent nPKC ({delta}, {varepsilon}, {theta} and {eta}) subfamilies are activated by phorbol esters, while the aPKC subfamily ({zeta} and {iota}) is Ca2+- and phorbol ester-independent (1). The phorbol ester-stimulated protein kinase D (PKD, also known as nPKCµ) is not included in the PKC family, due to its distinct catalytic domain structure (1).

The discovery that phorbol ester tumor promoters potently and selectively activate PKC (2) was the first indication that PKC may play an important role in tumor promotion, i.e. clonal expansion of initiated cells. A positive role for PKC activation in the proliferation of non-transformed cells and in the development of a transformed phenotype was later confirmed, primarily through studies of enforced PKC isozyme expression in immortalized fibroblasts (37). These studies also shed light on the potential roles of individual PKC isozymes in tumor promotion and progression. Thus, enforced expression of cPKCß1, cPKC{gamma} and nPKC{varepsilon} in immortalized fibroblasts produced tumorigenic cell lines that exhibited dysregulated growth in vitro (37) and enforced PKD expression enhanced the in vitro proliferation of immortalized fibroblasts (8), whereas enforced expression of cPKC{alpha} and nPKC{delta} in the cells had the opposite effect of producing cell lines that remained non-tumorigenic and exhibited growth inhibition in vitro (6,7). Consistent with a role for nPKC{delta} activation in tumor promotion antagonism (6), enforced nPKC{delta} expression induced keratinocyte growth arrest and differentiation in vitro; in contrast, enforced cPKC{alpha} expression did not affect keratinocyte growth properties or expression of the differentiation markers examined (9).

The influence of cPKC{alpha}, nPKC{delta} and nPKC{varepsilon} on multistage skin carcinogenesis has been directly examined through the use of transgenic mice that express PKC isozyme transgenes in the epidermis. The results confirm key predictions made from the in vitro studies of enforced PKC isozyme expression. Thus, nPKC{varepsilon} overexpression enhanced carcinoma formation, which is an end-point of tumor progression, in response to sequential topical treatment with the initiator 7,12-dimethylbenz[a]anthracene (DMBA) and the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) (10). nPKC{delta} overexpression suppressed both papilloma and carcinoma formation, i.e. tumor promotion and progression, in response to DMBA+TPA (11). cPKC{alpha} overexpression did not affect the production of papillomas or carcinomas by DMBA+TPA, but did produce a marked increase in the inflammatory response of the epidermis to TPA treatment (12).

Based on the critical role played by PKC in phorbol ester-mediated tumor promotion and progression, we recently hypothesized that antagonism of tumor promotion and progression by the endogenous antioxidant glutathione (GSH) (1315) may involve regulatory effects on PKC isozymes. In support of the hypothesis we demonstrated that cPKC{alpha}, cPKCß1, cPKC{gamma} and nPKC{varepsilon} are inhibited to different extents via a non-redox mechanism by high concentrations of GSH (>3 mM), i.e. under conditions that model a highly reducing intracellular environment (16,17). Tumor promotion is associated with a pro-oxidant environment (18,19). Phorbol ester tumor promoters induce reactive oxygen species (ROS) production in keratinocytes (20,21) and inflammatory cells (22,23) and other tumor promoters, e.g. benzoyl peroxide, are themselves ROS (24). In addition, a pro-oxidant environment supports tumor progression (25,26). With this in mind we turned our focus to the potential influence of GSH on PKC isozymes in a pro-oxidant environment. We established that cPKC{alpha} is fully and reversibly inactivated by the oxidative mechanism of cPKC{alpha} S-glutathiolation (27). The potent inactivation of cPKC{alpha} by a GSH-dependent mechanism fits the hypothesis that GSH-mediated tumor promotion antagonism may involve regulatory effects on PKC isozymes, because PKC inactivation by a tumor promotion antagonist is logically consistent with the fact that selective PKC activators induce tumor promotion (2). In this report we investigate whether oxidant-induced S-glutathiolation modifies the function of representative members of each PKC isozyme subfamily and the novel phorbol ester-dependent kinase PKD.


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 Materials and methods
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Materials
Purified human recombinant PKC isozymes ({alpha}, ß1, ß2, {gamma}, {delta}, {varepsilon} and {zeta}) were purchased from Pan Vera Corporation (Madison, WI) and purified human recombinant PKD (nPKCµ) from Calbiochem-Novabiochem (San Diego, CA). [{gamma}-32P]ATP and [35S]cysteine were purchased from Amersham Pharmacia Biotech and [35S]GSH from New England Nuclear (Boston, MA). [Ser25]PKC(1931), a synthetic peptide substrate of PKC isozymes, and Syntide-2, a synthetic peptide substrate of PKD, were obtained from Bachem Bioscience (King of Prussia, PA) and Calbiochem-Novabiochem, respectively. Millipore GSWP nitrocellulose filter circles (25 mm diameter, 0.2 µm pore size) and P-81 phosphocellulose paper were from Fisher Scientific (Houston, TX). sn-1,2-Dioleoylglycerol was purchased from Avanti Polar Lipids. Diamide, GSH, dithiothreitol (DTT), bovine brain phosphatidylserine (PS) (>98% pure), ATP, buffers, chelators and all other reagents were from Sigma (St Louis, MO).

GSH-potentiated oxidative inactivation of PKC isozymes and PKD
To investigate GSH-potentiated, oxidative PKC isozyme/PKD inactivation the kinase thiols were first refreshed by pretreatment with 2 mM DTT for 30 min at 4°C, followed by removal of the reducing agent from the kinase by gel filtration chromatography on a 2 ml G-25 Sephadex column at 4°C, as previously described (27). Next, the kinases (500 ng/preincubation mixture) were preincubated in 20 mM Tris–HCl, pH 7.5, with or without the oxidant diamide, which was freshly prepared from the solid on the day of the experiment, and, where indicated, 100 µM GSH or cysteine (total volume 105 µl) in capped tubes for 5 min at 30°C. For the analysis of DTT reversal of GSH-potentiated kinase inactivation preincubation of the kinase with diamide/GSH was followed by a second preincubation in the presence of 30 mM DTT for 10 min at 30°C (27). At the end of the preincubation period(s) the kinase samples were placed on ice and then assayed at once, using previously described methods (27,28).

Briefly, PKC isozymes and PKD were assayed using the peptide substrates 50 µM [Ser25]PKC(1931) and 500 µM Syntide-2, respectively. Other components of the kinase assay mixtures (total volume 120 µl) were 20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 0.2 mM CaCl2 (or 1 mM EGTA), 30 µg/ml PS, 30 µg/ml sn-1,2-dioleoylglycerol (or none), 6 µM [{gamma}-32P]ATP (5000–8000 c.p.m./pmol) and ~50 ng preincubated kinase. PS and diacylglycerol (DAG) were added to the reaction mixtures in the form of sonicated dispersions. Ca2+ was included in cPKC assays and DAG was included in both cPKC and nPKC assays. Kinase assays (10 min, 30°C) were initiated by addition of [{gamma}-32P]ATP to the reaction mixtures and terminated by pipetting aliquots of the assay mixtures onto phosphocellulose paper, which binds the [32P]phosphopeptide product, as previously described (27,28).

Stoichiometry of PKC isozyme/PKD 35S S-thiolation
To measure the stoichiometry of diamide-induced kinase S-thiolation each kinase (7–11 pmol) was preincubated under the conditions employed in the kinase inactivation analysis in the presence of 100 µM [35S]GSH (250–300 c.p.m./pmol) or [35S]cysteine (450–500 c.p.m./pmol) ± 500 µM diamide. At the end of the preincubation period (5 min, 30°C) the kinase was trichloroacetic acid (TCA) precipitated by addition of an equal volume (125 µl) of ice-cold TCA/PPi solution (20% TCA, 1% pyrophosphate) and the sample placed on ice. The radiolabeled kinase samples were pipetted onto 25 mm nitrocellulose membrane circles, which were washed with the TCA/PPi solution by vacuum filtration (3x5 ml) and then counted, as previously described (27). Covalent 35S-labeling of the kinases was calculated by subtracting the c.p.m. obtained from kinase samples that contained [35S]GSH or [35S]cysteine alone (background c.p.m.) from the c.p.m. obtained from kinase samples containing [35S]GSH or [35S]cysteine plus 500 µM diamide (total c.p.m.). 35S kinase labeling assays were done in duplicate and the results were reproduced in independent experiments.


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 Materials and methods
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Protein S-thiolation is an oxidative post-translational modification that is produced non-enzymatically and involves disulfide linkage of GSH or other low molecular weight thiol species to selected protein thiols (29). We recently established that cPKC{alpha} is fully inactivated by S-glutathiolation (27). This was accomplished by demonstrating that potent GSH-dependent oxidative inactivation of cPKC{alpha} was induced by the thiol-specific oxidant diamide in association with cPKC{alpha} S-glutathiolation (~1 mol GSH/mol cPKC{alpha}) (27). Other potential inactivation mechanisms were ruled out by the restricted oxidant activity of diamide, which is limited to disulfide bridge formation (30), by the DTT reversibility of the inactivation (27), which is characteristic of disulfide-based mechanisms (30), and by the GSH-dependent nature of the inactivation (27). In this report we have investigated whether PKD and representative members of the PKC isozyme subfamilies cPKC (ß1, ß2 and {gamma}), nPKC ({delta} and {varepsilon}) and aPKC ({zeta}) (1) are subject to inactivation by S-glutathiolation. Among these kinases nPKC{delta} and nPKC{varepsilon}, respectively, have been directly implicated in antagonism of tumor promotion/progression and mediation of tumor promotion/progression by phorbol esters in the mouse skin carcinogenesis model (10,11).

We first analyzed whether GSH could potentiate diamide-induced inactivation of the kinases under conditions where marked GSH-potentiated inactivation of cPKC{alpha} was shown to be caused by cPKC{alpha} S-glutathiolation in our previous study (27). In these experiments cPKC{alpha} (which served as a positive control), cPKCß1, cPKCß2 and cPKC{gamma}, nPKC{delta} and nPKC{varepsilon}, aPKC{zeta} and PKD were preincubated with diamide (0.02–5.0 mM) ± 100 µM GSH for 5 min at 30°C and then assayed. Figure 1Go (closed circles) shows concentration-dependent inactivation of the PKC isozymes and PKD by diamide in the absence of GSH (non-potentiated inactivation). Diamide (0.02–5.0 mM) induced monophasic inactivation of all of the kinases tested with variable potencies, with the exception of nPKC{delta}, which is the sole kinase in the study that has been implicated in the antagonism of tumor promotion/progression (6,9,11; Figure 1Go, closed circles). Table IGo shows the IC50 values for non-potentiated, diamide-induced inactivation of the PKC isozymes and PKD, i.e. the diamide concentrations required to achieve 50% kinase inactivation in the absence of GSH. Of the kinases monophasically inactivated by diamide cPKC{alpha} was the most resistant to diamide treatment (IC50 = 4.45 ± 0.55 mM) and cPKC{gamma} was the most sensitive (IC50 = 0.06 ± 0.01 mM) (Table IGo). Comparing the IC50 values that correspond to the non-potentiated inactivation mechanism in Table IGo it is evident that the relative sensitivities of the monophasically inactivated PKC isozymes and PKD to the inactivating effects of diamide were, starting with the most sensitive kinase, cPKC{gamma} > cPKCß2 > aPKC{zeta} > cPKCß1 > nPKC{varepsilon} > PKD > cPKC{alpha} (Table IGo). nPKC{delta} alone showed a biphasic response to diamide. nPKC{delta} activity was enhanced by low diamide concentrations and this effect peaked at 1.0 mM diamide, where 1.8-fold activation was observed (Figure 1Go, closed circles). Higher diamide concentrations produced a decline in nPKC{delta} activity, but 50% inactivation was not reached in the analysis. A maximal effect of 27 ± 1% nPKC{delta} inactivation was achieved at 5.0 mM diamide, which was the highest diamide concentration tested (Figure 1Go, closed circles).



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Fig. 1. GSH potentiates the oxidative inactivation of PKC isozymes and PKD. Kinases were preincubated for 5 min at 30°C in the presence of diamide (0.02–5.0 mM) with (open circles) or without (closed circles) 100 µM GSH, as described in Materials and methods. 100% activity values, which represent the kinase activity recovered after preincubation in the absence of diamide and GSH, were 25.2 ± 0.2 (cPKC{alpha}), 5.3 ± 0.4 (cPKCß1), 9.0 ± 0.3 (cPKCß2), 4.3 ± 0.3 (cPKC{gamma}), 1.1 ± 0.1 (nPKC{delta}), 11.8 ± 0.5 (nPKC{varepsilon}), 2.3 ± 0.1 (aPKC{zeta}) and 1.0 ± 0.1 (PKD) pmol 32P/min. Each experimental value is the mean ± SD of assays done in triplicate. The experimental results shown were reproduced in a second, independent analysis.

 

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Table I. Diamide concentrations that achieve 50% inactivation of PKC isozymes and PKD by GSH-potentiated and non-potentiated inactivation mechanisms
 
In the absence of diamide, preincubation of the seven PKC isozymes and PKD with 100 µM GSH (5 min, 30°C) affected the kinase activities by <10% (Figure 3Go, compare first and second bars in each histogram). This indicates that any substantial enhancement of the diamide-induced kinase inactivation (Figure 1Go, closed circles) that is produced by including 100 µM GSH in the preincubation mixtures (Figure 1Go, open circles) can be attributed to GSH potentiation of diamide-induced kinase inactivation.



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Fig. 3. Cysteine cannot substitute for GSH in the potentiation of diamide-induced kinase inactivation. Kinases were preincubated in the presence or absence of 500 µM diamide with or without 100 µM GSH or 100 µM cysteine, as indicated, under the conditions employed in Figure 1Go and then assayed. The alternative diamide concentration of 2.5 mM was employed for nPKC{delta}. For further experimental details see Materials and methods. 100% activity values, which represent the kinase activity recovered after preincubation in the absence of diamide and GSH/cysteine, were 17.2 (cPKC{alpha}), 2.6 (cPKCß1), 6.6 (cPKCß2), 2.2 (cPKC{gamma}), 0.4 (nPKC{delta}), 7.4 (nPKC{varepsilon}), 0.9 (aPKC{zeta}) and 4.9 (PKD) pmol 32P/min (experimental error <5%). Each experimental value is the mean ± SD of assays done in triplicate; the experimental results shown were reproduced in a second, independent analysis.

 
Figure 1Go (open circles) shows that 100 µM GSH markedly potentiated diamide-induced inactivation of cPKC isozymes {alpha}, ß1 and ß2, the nPKC isozymes {delta} and {varepsilon}, the aPKC isozyme {zeta} and PKD. In each case the GSH-dependent component of the total GSH-potentiated inactivation represented 30% or more of the initial activity at the optimal diamide concentration, e.g. the GSH-dependent component of nPKC{varepsilon} inactivation was 74 ± 1% of the initial kinase activity at 200 µM diamide. In addition, although cPKC{gamma} was very sensitive to inactivation by diamide alone, GSH-potentiated inactivation was also detected for this isozyme, e.g. 200 µM diamide achieved 82 ± 1% and 100 ± 3% cPKC{gamma} inactivation, respectively, in the absence and presence of 100 µM GSH (Figure 1Go).

Among the kinases that were monophasically inactivated by diamide alone (all except nPKC{delta}) (Figure 1Go, closed circles) the diamide concentration required to induce 50% inactivation by the GSH-potentiated mechanism, i.e. the IC50 value, ranged from 51 ± 11 (cPKC{gamma}) to 100 ± 10 µM (PKD) (Table IGo). The similarity of the IC50 values provides evidence that a conserved GSH-potentiated oxidative inactivation mechanism may be shared by the kinases. However, another feature of the inactivation curves distinguished PKD from the PKC isozymes in the analysis. For the monophasically inactivated PKC isozymes GSH-potentiated inactivation was >90% across a broad diamide concentration range (0.2–5.0 mM diamide) and full inactivation was attained by the GSH-potentiated mechanism at 1 mM diamide (Figure 1Go, open circles). In contrast, PKD was incompletely inactivated by the GSH-potentiated inactivation mechanism across the entire diamide concentration range (Figure 1Go, open circles). This suggests that PKC isozymes may share a common GSH-dependent inactivation mechanism that abolishes kinase activity and PKD, which is a member of a separate kinase family (1), may be subject to a related but distinct mechanism in which residual kinase activity resists inactivation.

nPKC{delta} was also inactivated by the GSH-potentiated mechanism (Figure 1Go, open circles). However, the IC50 value observed for nPKC{delta} inactivation by the GSH-potentiated mechanism (IC50 = 1.25 ± 0.46 mM diamide) was ~10–20 times greater than the IC50 values observed for PKD and the other PKC isozymes (51–100 µM diamide) (Table IGo). In addition, GSH produced a modest enhancement of diamide-induced nPKC{delta} activation at low concentrations of the oxidant (75–100 µM diamide) (Figure 1Go, open circles); this enhancement of diamide-induced nPKC{delta} activation by GSH was reproduced in a second, independent analysis (data not shown). Thus, nPKC{delta} proved to be the most resistant of the kinases to the GSH-potentiated inactivation mechanism and the results in Figure 1Go and Table IGo show that several phorbol ester-responsive kinases that have been implicated in the mediation of tumor promotion/progression by phorbol esters (cPKCß1, cPKC{gamma}, nPKC{varepsilon} and PKD) (38,10) are more readily inactivated by the GSH-potentiated inactivation mechanism than nPKC{delta}. These results are consistent with a role for the GSH-potentiated kinase inactivation mechanism in antagonism of tumor promotion/progression by GSH.

Next, experiments were done to verify that the marked loss of PKC isozyme and PKD activity that was produced by the GSH-potentiated inactivation mechanism (Figure 1Go, open circles), and in some cases by preincubation of the kinases with diamide alone (Figure 1Go, closed circles), was indeed due to an oxidative inactivation mechanism. In these experiments we included 3 mM DTT in the kinase preincubation mixtures to quench the oxidant activity of diamide (30). The preincubation mixtures were prepared by mixing DTT with diamide ± GSH prior to addition of the kinase. Following a 5 min preincubation period at 30°C the kinase activity was measured. Figure 2Go compares the kinase activity recovered after PKD and each of the PKC isozymes under investigation was preincubated with: (i) 3 mM DTT alone (defined as 100% activity); (ii) 3 mM DTT in the presence of 500 µM diamide; (iii) 3 mM DTT in the presence of 500 µM diamide plus 100 µM GSH; (iv) without DTT. A comparison of the activity recovered after preincubation of the kinases with versus without 3 mM DTT (Figure 2Go, first versus fourth bar per set) indicated that preincubation with 3 mM DTT negligibly affected the kinase activity of cPKC{alpha}, cPKCß2, cPKC{gamma} and PKD and produced a modest loss of the activity of the other kinases in the study. Thus, a 15–35% enhancement of kinase activity was observed for cPKCß1, nPKC{delta}, nPKC{varepsilon} and aPKC{zeta} when DTT was omitted from the preincubation mixtures (Figure 2Go, first versus fourth bar per set). The relatively minor effects of DTT on kinase activity in Figure 2Go indicated that 3 mM DTT could be employed to ascertain the effects of abrogating the oxidant activity of diamide on the non-potentiated and GSH-potentiated, diamide-induced PKC/PKD inactivation mechanisms.



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Fig. 2. DTT quenches GSH-potentiated, diamide-induced PKC isozyme/PKD inactivation. Kinases were preincubated with 3 mM DTT alone (first bar per set), 3 mM DTT in the presence of 500 µM diamide (second bar), 3 mM DTT in the presence of 500 µM diamide + 100 µM GSH (third bar) or without DTT (fourth bar) for 5 min at 30°C and then assayed, as described in Materials and methods and Results. 100% activity values, which represent the kinase activity recovered after preincubation with 3 mM DTT alone, were 24.4 (cPKC{alpha}), 2.0 (cPKCß1), 5.5 (cPKCß2), 1.7 (cPKC{gamma}), 1.5 (nPKC{delta}), 8.5 (nPKC{varepsilon}), 1.4 (aPKC{zeta}) and 7.2 (PKD) pmol 32P/min (experimental error < 5%). Each experimental value is the mean ± SD of assays done in triplicate; the experimental results shown were reproduced in a second, independent analysis.

 
Figure 2Go shows that inclusion of 500 µM diamide plus 100 µM GSH in kinase preincubation mixtures that contained 3 mM DTT altered the amount of kinase activity recovered for each of the kinases under investigation by <=20%. In comparison, inactivation of the kinases induced by 500 µM diamide plus 100 µM GSH in the absence of DTT in Figure 1Go was 43 ± 3% for nPKC{delta}, 88 ± 2% for PKD and >90% for the rest of the kinases examined. Thus, the GSH-potentiated, diamide-induced inactivation that was observed for all of the kinases in Figure 1Go was indeed due to an oxidative mechanism, because inactivation was quenched by DTT (Figure 2Go). The results in Figure 2Go also show that preincubation of the kinases with 500 µM diamide in the absence of GSH produced minor losses of activity (<25%) for all of the kinases surveyed when preincubation mixtures contained 3 mM DTT. Comparing these results with Figure 1Go it is evident that the enhancement of nPKC{delta} activity and substantial inactivation of cPKCß2, cPKC{gamma}, nPKC{varepsilon} and aPKC{zeta} achieved by 500 µM diamide (Figure 1Go, closed circles) were quenched by DTT (Figure 2Go) and therefore involved oxidative mechanisms.

We next investigated whether the GSH-dependent, oxidative inactivation of PKC isozymes and PKD in Figure 1Go (open circles) was associated with S-glutathiolation of the kinases. Because of the restricted oxidant activity of diamide the only type of covalent linkage that diamide can induce between the kinases and GSH is a disulfide bond (30), i.e. kinase S-glutathiolation. Diamide-induced covalent labeling of each kinase with [35S]GSH was measured under conditions where diamide induced substantial GSH-dependent inactivation of every kinase examined in Figure 1Go. This was done by preincubating the kinases with 500 µM diamide and 100 µM [35S]GSH for 5 min at 30°C in 20 mM Tris–HCl, pH 7.5. [35S]GSH covalent labeling of the kinases was quantitated by capturing TCA-precipitated, radiolabeled kinases on nitrocellulose membranes by vacuum filtration, as described in Materials and methods. Human PKC isozymes and PKD contain 16–23 Cys residues (31). We previously reported that a diamide concentration sufficient for full cPKC{alpha} inactivation by S-glutathiolation (100 µM diamide) induced binding of 0.95 ± 0.03 mol [35S]GSH/mol cPKC{alpha} (27). Table IIGo shows that the stoichiometries of kinase 35S S-glutathiolation induced by 500 µM diamide ranged from 0.7 to 3.2 mol [35S]GSH/mol kinase. Thus, GSH-dependent oxidative inactivation of the kinases was associated with 35S S-glutathiolation of a small subset of Cys residues in the kinase structures. These results provide strong correlative evidence that the GSH-dependent, oxidative inactivation of PKC isozymes and PKD shown in Figure 1Go is mediated by select S-glutathiolation of 1–3 Cys residues in the kinase structures.


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Table II. Stoichiometry of kinase 35S S-glutathiolation induced by diamide under conditions that produce potent GSH-dependent oxidative kinase inactivation
 
In addition to GSH, the amino acid cysteine is a major endogenous S-thiolating species (32), but it is considerably less effective than GSH in the antagonism of tumor promotion/progression (13,15) (see Discussion). To assess the importance of GSH availability to inactivation of PKC isozymes and PKD by S-thiolation we investigated whether cysteine, a metabolic precursor of GSH (33), could substitute for GSH in the oxidative inactivation mechanism. PKC isozymes and PKD were preincubated in the presence of 500 µM diamide ± 100 µM GSH under the conditions employed in Figure 1Go; an alternative diamide concentration of 2.5 mM was employed for nPKC{delta} to ensure robust GSH-dependent inactivation of that isozyme. Where indicated, 100 µM cysteine was employed in place of GSH, to allow a direct comparison of the effects of cysteine and GSH on diamide-induced kinase inactivation.

Preincubation with 100 µM cysteine in the absence of diamide affected the activity of the kinases only modestly, i.e. <=15% (Figure 3Go, compare the first and third bars in the histograms). Consistent with the results shown in Figure 1, 2GoGo.5 mM diamide enhanced the activity of nPKC{delta} and 500 µM diamide inactivated the other kinases to variable extents (Figure 3Go, compare the first and fourth bars in the histograms). Also consistent with Figure 1Go, GSH potentiated the diamide-induced inactivation to >90% for all of the kinases except nPKC{delta} and PKD and each of the latter kinases exhibited a major GSH-dependent component of inactivation under the conditions employed (Figure 3Go, compare the fourth and fifth bars in the histograms). In contrast, cysteine did not potentiate the diamide-induced inactivation of any of the kinases, but instead produced a modest increase in the activity recovered from kinases that were co-preincubated with 500 µM diamide. For the kinases monophasically inactivated by diamide in Figure 1Go (all but nPKC{delta}) co-preincubation with cysteine produced a 5–20% enhancement of the activity recovered when the kinases were preincubated with diamide and co-preincubation with cysteine robustly enhanced the recovery of activity of nPKC{delta} pre-incubated with diamide (Figure 3Go, compare the fourth and sixth bars in the histograms). These results demonstrate that cysteine cannot substitute for GSH in oxidative inactivation of the kinases by diamide-induced kinase S-thiolation. A question remaining was whether cysteine failed to S-thiolate the kinases or if it S-thiolated but did not thereby inactivate the kinases. To address this we measured induction of [35S]cysteine covalent labeling of the kinases by diamide, using the conditions and methods employed in the analysis of [35S]GSH covalent labeling of the kinases in Table IIGo. We found that none of the kinases was 35S S-cysteinylated after preincubation with 100 µM [35S]cysteine and 500 µM diamide (<0.05 mol [35S]cysteine/mol kinase).

The enhancing effects of cysteine on the recovery of kinase activity after co-preincubation with diamide, which were minor for every kinase except nPKC{delta} (Figure 3Go), may stem from the reductant activity of cysteine. In fact, when the kinases were subjected to a second preincubation period in the presence of the strong reductant DTT (30 mM DTT, 10 min at 30°C) after the initial preincubation with diamide and GSH shown in Figure 3Go, substantial reversal of the GSH-potentiated inactivation was observed for several of the kinases. Thus, DTT restored the kinase activity of S-glutathiolated PKD to ~100% and that of S-glutathiolated cPKC{alpha} and nPKC{varepsilon} to ~60% of the activity of the untreated positive controls. In addition, DTT restored S-glutathiolated aPKC{zeta} activity to ~30% of the positive control and produced a recovery of activity of S-glutathiolated nPKC{delta} that was 2.5-fold greater than the activity of the positive control. In contrast, the GSH-potentiated inactivation of cPKCß1, cPKCß2 and cPKC{gamma} was quite resistant to DTT reversal, with the extent of DTT reversal of inactivation ranging from 10 to 15% of the value of the positive control. These results provide evidence that the stability of the inactivating S-glutathiolating linkages to reducing conditions varied widely for the kinases examined.


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 References
 
ROS have been shown to regulate PKC activity through diverse oxidative mechanisms, in some cases producing PKC activation and in others PKC inactivation (34). Mechanisms of oxidative regulation of PKC activity (34) can be dichotomized into direct mechanisms that involve oxidative modifications of the PKC structure and indirect mechanisms that involve oxidative regulation of PKC regulatory proteins. Several direct mechanisms of oxidative regulation of PKC activity have been reported and one important indirect mechanism has been elucidated (34). The indirect mechanism involves oxidative regulatory effects on protein tyrosine phosphatases and protein tyrosine kinases that enrich the phosphotyrosine content of PKC isozymes at specific sites in the catalytic domain, with consequential lipid-independent PKC activation (35). Direct oxidative regulatory mechanisms can be subdivided into mechanisms that either activate or inactivate PKC. Whether kinase activation or inactivation is produced by the oxidative modifications introduced into PKC depends on the chemical nature of the ROS and, in some cases, its concentration (34). Thus, superoxide activates PKC by a mechanism that entails thiol oxidation of autoinhibitory zinc fingers in the regulatory domain of PKC and zinc release from those structures (36), nitric oxide and various S-nitrosylating species, e.g. S-nitrosocysteine, inactivate PKC by a mechanism involving thiol oxidation (37) and the tumor promoter periodate produces biphasic effects on PKC activity, with oxidative PKC activation at low periodate concentrations (<100 µM) and oxidative PKC inactivation at higher concentrations (38).

We recently investigated the effect of the thiol-specific oxidant diamide, which exclusively produces a single type of oxidative modification in proteins, i.e. disulfide bridges (30), on cPKC{alpha} activity. Our objective was to define the influence of intra/intermolecular disulfide bridge formation and S-glutathiolation on activity of the isozyme. Our analysis demonstrated that cPKC{alpha} is highly resistant to oxidative regulation by either intramolecular disulfide bridge formation or by formation of disulfide linkages between cPKC{alpha} monomers. In contrast, cPKC{alpha} was potently inactivated by S-glutathiolation, both in its purified form and in diamide-treated fibroblasts (27).

In view of the presence of conserved Cys residues in the PKC family (31), the potent inactivation of cPKC{alpha} by S-glutathiolation (27) suggested that inactivation by S-glutathiolation might be an oxidative regulatory mechanism common to additional members of the PKC family. This could potentially support a role for PKC isozyme S-glutathiolation in the antagonism of tumor promotion/progression by GSH in pro-oxidant enviroments. In the present report we have examined both independent effects of the oxidant diamide and the effects of diamide-induced S-glutathiolation on the kinase activity of PKD and representative members of each PKC isozyme subfamily. In support of the hypothesis that PKC S-glutathiolation may play a role in tumor promotion/progression antagonism by GSH we found that nPKC{varepsilon}, which is an important mediator of tumor promotion/progression (10), exhibited markedly GSH-dependent inactivation by diamide as a result of isozyme S-glutathiolation and that nPKC{delta}, which antagonizes tumor promotion/progression (11), was an order of magnitude less sensitive to inactivation via diamide-induced S-glutathiolation than the other six PKC isozymes analyzed and PKD. Furthermore, diamide oxidatively enhanced the activity of nPKC{delta} but not that of any other kinase in the analysis. In addition to nPKC{varepsilon}, other kinases implicated in the mediation of tumor promotion/progression, e.g. cPKCß1, cPKC{gamma} and PKD (3,4,8), were potently inactivated by the GSH-potentiated mechanism, although in some cases the contribution of S-glutathiolation to the inactivation mechanism was minimized by the sensitivity of the kinase to inactivation by diamide; this was especially true of cPKC{gamma}.

In addition to differential PKC isozyme sensitivity to S-glutathiolation, a second correlation between PKC inactivation by S-thiolation and tumor promotion antagonism is provided in this report by the observation that cysteine could not substitute for the tumor promotion/progression antagonist GSH (1315) in the PKC/PKD inactivation mechanism. In mouse skin carcinogenesis models cysteine is significantly less effective than GSH in antagonizing tumor promotion/progression and the limited efficacy of cysteine in this regard is primarily due to its incorporation into GSH (13,15). We found that the eight kinases analyzed were uniformly resistant to diamide-induced S-cysteinylation. This observation offers an indication of the selectivity of the S-thiolating mode of PKC/PKD inactivation for the endogenous S-thiolating species GSH and suggests dependence of the inactivation mechanism on regulation of cellular GSH levels. In addition, selectivity is also evident from the limited number of PKC/PKD cysteine residues that are potentially involved in this mode of inactivation, based on measurements of the stoichiometry of kinase S-glutathiolation under inactivating conditions, i.e. 1–3 residues out of a total of 16–23.

The results in this report clearly demonstrate differential responses of closely related PKC isozymes, e.g. cPKC{alpha} versus cPKC{gamma} and nPKC{delta} versus nPKC{varepsilon} (1), to a strictly delimited form of oxidative stress that produces disulfide bridges within proteins and protein S-glutathiolation. The results provide a solid foundation for investigations directed, first, at identifying physiological, pathophysiological and environmental oxidants, e.g. oxidant tumor promoters/progressors (18,19), that may induce PKC inactivation by S-glutathiolation and, second, characterizing the isozyme selectivity of the inactivation using purified isozymes and enforced isozyme expression in non-transformed cells (38), as a test of the potential significance of PKC S-glutathiolation to tumor promotion/progression antagonism.

Exogenous oxidative stimuli produce mitogenic responses in non-transformed cells by inducing activation of Src family kinases, Ras and the MAP kinases Erk1/2 (39) and Ras-mediated superoxide production induces a mitogenic response in Ras-transformed cells (40). We hypothesize that concomitant oxidative induction of PKC inactivation via S-glutathiolation may oppose these mitogenic responses. We further hypothesize that this mode of PKC/PKD inactivation may thus serve as a novel mechanism of tumor promotion/progression antagonism in pro-oxidant environments that complements other mechanisms that involve the quenching of free radicals and other ROS by GSH (15,33). Under pro-oxidant conditions that do not support the PKC S-glutathiolating inactivation mechanism oxidative stimuli may have the opposite effect of supporting PKC activation through other modes of thiol oxidation (36) or phosphotyrosine stabilization in the PKC structure (35), and this could potentially contribute to the mitogenic response of the cells and to tumor promotion/progression, depending in part on the isozymes involved. Studies underway to identify the cysteine residues(s) in PKC isozymes that mediate the S-glutathiolating inactivation mechanism may illuminate the role of S-glutathiolation and other types of thiol oxidation on PKC isozyme regulation and may allow the development of transgenic models (10,11) to directly test the importance of the residues to the oxidative regulation of PKC isozymes in vivo.


    Notes
 
1 To whom correspondence should be addressed Email obrian{at}mdacc.tmc.edu Back


    Acknowledgments
 
This work was supported by grants from the National Institutes of Health (CA 74831) and the Robert A. Welch Foundation (G-1141).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 4, 2001; revised March 22, 2001; accepted March 28, 2001.