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Carcinogenesis, Vol. 24, No. 2, 317-325, February 2003
© 2003 Oxford University Press

CARCINOGENESIS

PKC isozyme S-cysteinylation by cystine stimulates the pro-apoptotic isozyme PKC{delta} and inactivates the oncogenic isozyme PKC{varepsilon}

Feng Chu, Nancy E. Ward and Catherine A. O’Brian1

Department of Cancer Biology, University of Texas M.D.Anderson Cancer Center, Houston, TX 77030, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein kinase C (PKC) is a family of ten isozymes that play distinct and in some cases opposing roles in cell growth and survival. We recently reported that diamide, a diazene carbonyl derivative which oxidizes thiols to disulfides through addition/displacement reactions at the diazene bond, induces potent GSH-dependent inactivation of several PKC isozymes, including the oncogenic isozyme PKC{varepsilon}, via S-glutathiolation. PKC{delta}, a pro-apoptotic isozyme, was distinguished by its resistance to inactivation. In this report, we show that PKC-regulatory S-thiolation modifications produced by physiological disulfides elicit opposing effects on PKC{delta} and PKC{varepsilon} activity. We report that PKC{delta} is stimulated 2.0–2.5 fold by GSSG, (Cys–Gly)2 and cystine, under conditions where PKC{gamma} and PKC{varepsilon} are fully inactivated by cystine, and PKC{alpha} activity is affected marginally or not at all by the disulfides. Focusing on cystine, we show that DTT quenches cystine-induced PKC{delta} stimulation and PKC{gamma} and PKC{varepsilon} inactivation, indicative of oxidative regulation. By analyzing DTT-reversible isozyme radiolabeling by [35S]cystine, we demonstrate that PKC{gamma}, PKC{delta} and PKC{varepsilon} are each [35S] S-cysteinylated in association with the concentration-dependent regulation of isozyme activity by cystine. The restricted reactivity of cystine, together with the effects of DTT and thioredoxin on cystine-induced PKC isozyme regulation reported here, indicate that the cystine-induced PKC-regulatory effects entail isozyme S-cysteinylation. We recently hypothesized that antagonism of tumor promotion/progression by small cellular thiols may involve PKC regulation via oxidant-induced S-thiolation reactions with PKC isozymes. The findings of cystine-induced PKC isozyme regulation by S-cysteinylation reported here offer correlative support to the hypothetical model. Thus, PKC{delta}, a potent antagonist of DMBA–TPA-induced tumor promotion/progression in mouse skin, is stimulated by S-cysteinylation, PKC{varepsilon}, an important mediator of the tumor promotion/progression response, is inactivated by S-cysteinylation, and PKC{alpha}, which is not influential in DMBA–TPA-induced tumor promotion/progression, is not regulated by cystine. Furthermore, PKC{gamma} has oncogenic activity, and S-cysteinylation inactivated PKC{gamma} and PKC{varepsilon} similarly. These findings provide evidence that S-cysteinyl acceptor-sites in PKC isozymes may offer attractive targets for development of novel cancer preventive agents.

Abbreviations: (Cys–Gly)2, Cys–Gly disulfide; DAG, sn-1,2-dioleoylglycerol; DTT, dithiothreitol; GSH, glutathione; GSSG, glutathione disulfide; PKC, protein kinase C; [Ser25]PKC(19–31), RFARKGALRQKNV


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Protein kinase C (PKC) is a family of ten isozymes that play distinct and in some cases opposing roles in cell growth, survival and differentiation (1). The isozyme PKC{varepsilon} stimulates the growth of fibroblasts and renders the cells tumorigenic when it is overproduced by enforced expression (2,3). The oncogenic activity of PKC{varepsilon} in fibroblasts has been shown to result from its engagement of the mitogenic Erk1/2 activation pathway through direct interactions with Raf1 that stimulate Raf1 kinase activity (4,5). The importance of PKC{varepsilon} to cell growth regulation and transformation has also been demonstrated in epithelial tissues in vivo. Overexpression of PKC{varepsilon} in the epidermis of transgenic mice markedly enhances carcinoma formation in response to topical treatment with a carcinogen and a phorbol ester PKC activator and renders the tumor cells metastatic (6,7). In contrast, overexpression of PKC{delta} in the epidermis of the mice has the opposite effect of suppressing tumor formation in response to these agents (8). Consistent with this, PKC{delta} has been shown to play a pro-apoptotic role in mitochondrial-dependent apoptotic pathways in epithelial and other types of cells in culture (9–12). Because most human malignancies are of epithelial origin (7), these observations offer a rationale for cancer prevention strategies that enhance PKC{delta} or antagonize PKC{varepsilon} function.

The most well understood mechanism of PKC activation entails phosphatidylserine-dependent binding of the second-messenger sn-1,2-diacylglycerol to tandem Cys-rich binding regions in the kinase regulatory domain (1). In addition, pro-oxidant conditions support PKC regulation by redox signaling (13). For example, hydrogen peroxide treatment of COS7 cells activates PKC isozymes by a lipid-independent mechanism that entails stabilization of the phosphotyrosine content of the isozymes at conserved sites in the catalytic domain (14). In addition, reversible oxidative modifications of Cys residues in the PKC structure profoundly influence the kinase activity (13,15). Human PKC isozymes contain 16–28 Cys residues including one or two Cys-rich regions in the regulatory domain, with six Cys per region, and five to eight catalytic domain Cys (13). In an analysis of seven PKC isozymes ({alpha}, ß1, ß2, {gamma}, {delta}, {varepsilon}, {zeta}), we have shown that the thiol-specific oxidant diamide, which is a diazenecarbonyl derivative, supports S-glutathiolation of each isozyme, i.e. the disulfide linkage of glutathione (GSH) to kinase Cys residues (PKCS-SG), and that S-glutathiolation at 1–3 redox-sensitive Cys residues is sufficient to inactivate fully the kinase activity of most of the isozymes, including PKC{varepsilon} (16,17). PKC{delta} was unique among the isozymes in its resistance to inactivation. A several-fold higher diamide concentration was required to achieve PKC{delta} inactivation by S-glutathiolation, whereas the other isozymes examined were similarly sensitive to the GSH-dependent inactivation mechanism (17). The selectivity of the diamide-induced PKC S-thiolation reaction was indicated by the inability of cysteine to substitute for GSH as the S-thiolating ligand for any of the isozymes analyzed (17).

Induction of disulfide bridge formation by diamide entails the addition of a thiol, e.g. GSH, to the diazene bond followed by its displacement by a second thiol species, e.g. a reactive protein thiol, yielding a mixed disulfide (18). Because the diazene-based chemistry of disulfide bridge formation by diamide is not shared by physiological or environmental agents, the demonstration of diamide-induced PKC regulation by S-glutathiolation (16,17) left open the question of whether PKC-regulatory S-thiolation modifications could be produced by reaction mechanisms that commonly occur in biological systems. In this report, we show that thiol–disulfide exchange with the physiological disulfide cystine produces opposing effects on the activity of the oncogenic isozyme PKC{varepsilon} and the pro-apoptotic isozyme PKC{delta} through S-cysteinylation of the isozymes. Cystine-induced isozyme S-cysteinylation stimulated PKC{delta} 2–3-fold and fully inactivated PKC{varepsilon}. These results suggest that the anticancer effects of small physiological thiols such as cysteine (19) may include PKC regulation by S-thiolation under pro-oxidant conditions. Furthermore, the S-cysteinylation sites in the isozyme structures may offer novel targets for the development of cancer preventive or therapeutic agents that stimulate the pro-apoptotic isozyme PKC{delta} or inactivate the oncogenic isozyme PKC{varepsilon}.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Purified human recombinant PKC isozymes ({alpha}, ß1, {gamma}, {delta}, {varepsilon}, {zeta}) were purchased from Pan Vera Corporation (Madison, WI). [{gamma}-32P]ATP, [35S]cystine, Hyperfilm MP and Amplify fluorographic reagent were from Amersham Pharmacia Biotech. [Ser25]PKC(19–31), a synthetic peptide substrate of PKC isozymes, was purchased from Bachem Bioscience (King of Prussia, PA), and sn-1,2-dioleoylglycerol (DAG) from Avanti Polar Lipids (Alabaster, AL). Slide-A-Lyzer® Dialysis Cassettes (0.5 ml capacity; 7000 MW cut-off) (Pierce Chem, Rockford, IL) were employed in dialysis experiments. Molecular weight markers and all other SDS–PAGE reagents were purchased from Bio-Rad (Hercules, CA); gels were run on the Bio-Rad Mini-Protean II System. Purified Escherichia coli thioredoxin reductase was purchased from American Diagnostica (Greenwich, CT), and recombinant E.coli thioredoxin and NADPH from Calbiochem-Novabiochem (La Jolla, CA). GSH, GSSG, Cys–Gly, (Cys–Gly)2, cysteine, cystine, cystamine, {gamma}-Glu-Cys, DTT, 2-mercaptoethanol, diamide, bovine brain PS (>98% pure), ATP, histone III-S, G-25 Sephadex, protease inhibitors, buffers, chelators and all other reagents were purchased from Sigma.

Analysis of the regulation of PKC isozyme activity by disulfides and other thiol-specific oxidants
To analyze effects of thiol-specific oxidants on PKC isozyme activity, PKC isozyme thiols were first refreshed by pretreatment of the kinase (5 µg) with 2 mM DTT for 30 min at 4°C (total volume = 500 µl), and then excess reducing agent was removed from the isozyme by gel filtration chromatography on a 2 ml G-25 Sephadex column at 4°C, as previously described (16,17). Next, the kinase (500 ng) was preincubated with the oxidant under analysis in the buffer employed for G-25 chromatography (total volume = 100 µl). For experiments with diamide, the kinase was incubated with diamide and small thiols (GSH, etc.) specified in the Results section in 20 mM Tris–HCl, pH 7.5, for 5 min at 30°C, placed on ice, and assayed. For analysis of the effects of disulfides such as GSSG and cystine on PKC isozyme activity, the isozyme was preincubated with the disulfide in 50 mM Tris–HCl, 200 mM NaCl, pH 8.0, for 20–30 min at 30°C, placed on ice, and assayed. All preincubation mixtures contained 1 mM EDTA and 1 mM EGTA. In some experiments, DTT reversal of the oxidant-induced changes in isozyme activity was measured. This was done by conducting a second preincubation of the kinase with/without 10 mM DTT (10 min, 30°C) immediately following kinase preincubation with disulfides, etc. In experiments that measured thioredoxin-mediated reversal of disulfide-induced PKC isozyme regulation, the first preincubation period with the disulfide was followed by a 1 h preincubation at 30°C with/without 10 µM thioredoxin–0.2 µM thioredoxin reductase–0.4 mM NADPH or, for comparison, 10 mM DTT.

The PKC activity of the preincubated isozyme was measured by an established assay procedure (16,17) immediately after placing the sample on ice at the end of the preincubation period. Kinase assay mixtures contained 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 DAG, 50 µM [Ser25]PKC(19–31), 6 µM [{gamma}-32P]ATP (5000–8000 c.p.m./pmol) and 40–100 ng of the PKC isozyme under analysis (16,17). CaCl2 was present only in assays of PKC{alpha}, PKCß1 and PKC{gamma}; DAG was omitted from assays of PKC{zeta} (17). PS and DAG were added to the reaction mixtures as sonicated dispersions. Where indicated, the peptide substrate was replaced by 50 µg/ml histone-III-S. Assays (10 min, 30°C) were initiated by the addition of [{gamma}-32P]ATP and terminated on phosphocellulose paper as previously described (16,17).

Non-reducing SDS–PAGE analysis of PKC isozyme [35S] S-cysteinylation
PKC isozyme thiols were refreshed and the purified isozymes gel filtered. The isozymes were then incubated with 0.1–2.5 mM [35S]cystine (40–250 mCi/mmol) in 80 mM Tris–HCl, pH 8.2, 1 mM EDTA, 1 mM EGTA for 20 min at 30°C (1 µg PKC in a total volume of 200 µl). The incubation period was terminated by the addition of non-reducing SDS–PAGE sample buffer, and samples were boiled and then run on 10% SDS–PAGE (300 ng PKC isozyme/lane). Gels were stained with Coomassie dye to visualize MW markers, rinsed with deionized water, soaked in Amplify fluorographic reagent for 30 min, and exposed to Hyperfilm MP with an intensifying screen. Band intensities in autoradiograms were quantitated by densitometric analysis.

Measurement of the stoichiometry of PKC isozyme [35S] S-cysteinylation
To measure the stoichiometry of PKC isozyme [35S] S-cysteinylation by [35S]cystine, PKC isozymes were covalently modified and then dialyzed extensively for removal of unbound radiolabel. In these experiments, PKC isozyme thiols were first refreshed and the purified isozymes gel-filtered. The isozymes were incubated with 2.5 mM [35S]cystine in 80 mM Tris–HCl, pH 8.2, 1 mM EDTA, 1 mM EGTA, 8% glycerol for 20 min at 30°C followed by continued incubation (10 min) with/without 10 mM DTT. Incubation mixtures were then pipetted into Pierce 0.5 ml Slide-A-Lyzer® Dialysis Cassettes (15–18 pmol isozyme in a sample volume of 205 µl per cassette), and dialyzed under bubbling nitrogen against 80 mM Tris–HCl, pH 8.2, 1 mM EDTA, 1 mM EGTA, 8% glycerol (5.8 l) for a total of 26 h at 4°C (three buffer changes); this procedure produced minimal post-dialysis changes in sample volume (<5%). The stoichiometry of PKC [35S] S-cysteinylation was calculated as the c.p.m. recovered post-dialysis from the isozyme sample that lacked DTT minus the c.p.m. recovered from the corresponding DTT-containing sample, divided by the pmol PKC in the sample (pmol [35S]cysteine equivalents per pmol PKC).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
We recently reported that diamide inactivates several PKC isozymes by inducing S-glutathiolation of ~1–3 redox-sensitive Cys residues within the isozyme structures (16,17,20). In this report, as an initial approach to dissect the structural features within GSH ({gamma}-Glu–Cys–Gly) that are involved in the PKC-inactivating S-thiolation mechanism, we investigated whether dipeptides corresponding to the NH2 terminus ({gamma}-Glu–Cys) or COOH terminus (Cys–Gly) of the tripeptide could potentiate diamide-induced PKC isozyme inactivation. We focused on PKC{alpha}, because it has the most fully characterized diamide-induced S-glutathiolation inactivation mechanism (16), and on PKC{delta} and PKC{varepsilon}, in light of their prominent, opposing roles in phorbol ester-induced tumor promotion/progression (6–8). Our previous studies established that PKC{alpha} and PKC{varepsilon} are equally sensitive to inactivation by diamide-induced S-glutathiolation, whereas PKC{delta} is resistant to the inactivation mechanism and responds biphasically to diamide, whether administered alone or in combination with GSH (17).

Figure 1Go (panel C) shows that Cys–Gly (100 µM) (open circles) is nearly as effective as GSH (open triangles) in potentiating diamide-induced PKC{varepsilon} inactivation (filled circles). The lack of effect of Cys–Gly (open circles) on PKC{varepsilon} activity at the lowest diamide concentration examined (20 µM diamide) indicates that the PKC{varepsilon}-regulatory effects of Cys–Gly are diamide-dependent (Figure 1Go, panel C). In contrast with Cys–Gly, 100 µM {gamma}-Glu–Cys (filled triangles) failed to potentiate diamide-induced PKC{varepsilon} inactivation (Figure 1Go, panel C). Similarly, diamide-induced PKC{alpha} inactivation (filled circles) was potentiated by 100 µM GSH (open triangles) and Cys–Gly (open circles) but not by 100 µM {gamma}-Glu–Cys (filled triangles) (Figure 1Go, panel A). The results of the PKC{delta} analysis appear complicated due to the biphasic responses of the isozyme to diamide with/without thiol peptides (Figure 1Go, panel B). However, by comparing the curves it can be discerned that the responses of PKC{delta} to diamide alone (filled circles) and diamide plus {gamma}-Glu–Cys (filled triangles) are similar, indicating that the effects of {gamma}-Glu–Cys were negligible in this system. Furthermore, the curves corresponding to diamide plus Cys–Gly (open circles) and diamide plus GSH (open triangles) are distinct from the diamide alone curve (filled circles) but resemble each other, consistent with analogous diamide-potentiating effects of Cys–Gly and GSH on PKC{delta} activity. Thus, Cys–Gly contains structural features of GSH that are necessary and sufficient for potentiation of diamide-induced regulatory effects on PKC{alpha}, PKC{delta} and PKC{varepsilon}.



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  		Fig. 1. Potentiation of diamide-induced PKC isozyme regulation by GSH and GSH-related dipeptides. PKC isozymes were preincubated with diamide (0.02–5 mM) alone or in the presence of thiol peptide (100 µM Cys–Gly, {gamma}-Glu–Cys, or GSH) in 20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA for 5 min at 30°C, and then assayed. Each assay mixture contained 45 ng of the PKC isozyme under analysis. Other experimental details are provided in Materials and methods. 100% activity values shown correspond to 28 pmol 32P/min (cPKC{alpha}), 1.5 pmol 32P/min (nPKC{delta}), and 10 pmol 32P/min (nPKC{varepsilon}). Each data point is the average ± SD of assays done in triplicate. The results shown were reproduced in an independent analysis.

 
Diamide-induced protein S-glutathiolation entails the formation of diamide–GSH adducts that react with protein thiols to form S-glutathiolated protein species (18). The potentiation of diamide-induced PKC isozyme regulation achieved by 100 µM GSH and Cys–Gly in Figure 1Go is most likely reflective of the abilities of diamide–GSH and diamide–Cys–Gly adducts to react with regulatory PKC-thiols, although it may also involve formation of adducts between PKC-thiols and diamide that react with GSH and Cys–Gly. Given that diamide is neither a physiological nor an environmental agent in biological systems (18), this raised the critical question of whether PKC could be S-thiolated by the thiol peptides through delivery mechanisms other than adduct formation with diamide. Therefore, as a second approach to evaluate structural features of GSH that may be involved in PKC isozyme regulation by S-thiolation, we compared the PKC regulatory effects of the physiological disulfides GSSG, (Cys–Gly)2, and cystine (21,22).

Figure 2Go shows that the lipid cofactor-stimulated activity of PKC{delta} (filled triangles) was enhanced up to ~2.5-fold by incubating the isozyme with GSSG (panel A), (Cys–Gly)2 (panel B), and cystine (panel C) for 20–30 min at 30°C. Concentrations required to enhance PKC{delta} activity 2.0-fold were 1.8 ± 0.1 mM GSSG, 1.9 ± 0.1 mM (Cys–Gly)2, and 0.93 ± 0.01 mM cystine (averaged values ± SE from graphical analysis of Figure 2Go and an independent analysis). In addition to PKC{delta}, several other PKC isozymes were surveyed for effects of GSSG (0.1–2.5 mM) on lipid cofactor-stimulated isozyme activity. GSSG effects were either modestly inactivating (PKC{gamma}, PKC{zeta}) or negligible (PKC{alpha}, PKCß1, PKC{varepsilon}) (Figure 2Go, panel A).



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  		Fig. 2. Regulatory effects of physiological disulfides on PKC isozyme activity. PKC isozymes were preincubated with GSSG (30 min) (A), (Cys–Gly)2 (20 min) (B), or cystine (20 min) (C) in 50 mM Tris–HCl, 200 mM NaCl, pH 8.0, 1 mM EDTA, 1 mM EGTA at 30°C, and then assayed. Each assay mixture contained 60 ng of the PKC isozyme under analysis. For other experimental details, see Materials and methods. In (A), 100% activity values are 28 pmol 32P/min (cPKC{alpha}), 1.4 pmol 32P/min (cPKCß1), 3.6 pmol 32P/min (cPKC{gamma}), 1.4 pmol 32P/min (nPKC{delta}), 8.6 pmol 32P/min (nPKC{varepsilon}) and 3.3 pmol 32P/min (aPKC{zeta}). Similar 100% activity levels were achieved when the corresponding isozymes were analyzed in (B) and (C). Each data point is the average ± SD of assays done in triplicate. The results shown were reproduced in an independent analysis.

 
To compare PKC isozyme responses to (Cys–Gly)2, we returned to the original focus on PKC{alpha}, PKC{delta} and PKC{varepsilon} but widened it to include PKC{gamma}, because it was the most sensitive of the phorbol ester-responsive isozymes surveyed to inactivation by GSSG (Figure 2Go, panel A). As was the case for GSSG, the effects of (Cys–Gly)2 on the isozymes examined other than PKC{delta} were only partially inactivating (PKC{gamma}, PKC{varepsilon}) or negligible (PKC{alpha}) (Figure 2Go, panel B). In contrast, cystine fully inactivated PKC{gamma} (open circles) and PKC{varepsilon} (open triangles), while it had modest inactivating effects against PKC{alpha} (filled circles) (Figure 2Go, panel C). Cystine inactivated PKC{gamma} and PKC{varepsilon} with IC50s of 350 ± 50 µM and 370 ± 20 µM respectively (averaged values ± SE of the IC50 obtained from the graph in Figure 2Go and an independent analysis). Thus, cystine was the most potent PKC-regulatory disulfide in the analysis, and it produced opposing effects on the activity of the pro-apoptotic isozyme PKC{delta} (9–12) versus the growth-stimulatory isozymes PKC{gamma} (23) and PKC{varepsilon} (2,3) (Figure 2Go). Intriguingly, from the perspective of structure–activity relationships, the results in Figure 2CGo are in sharp contrast with the complete lack of effect of oxidized cysteine on PKC isozyme activity, when delivered as a diamide adduct (17) rather than a disulfide.

To determine whether PKC{delta}-stimulatory effects of GSSG, (Cys–Gly)2 and cystine were selective for the lipid cofactor-stimulated form of the isozyme, we utilized a histone kinase assay of PKC{delta} activity, because the lipid dependence of purified PKC isozyme activity is especially pronounced with histone as substrate (Chu and O’Brian, unpublished observations). Figure 3Go (panels A–E, first versus third bar) shows that the histone kinase activities of PKC{gamma}, PKC{delta} and PKC{varepsilon} were stimulated 9–50-fold by lipid cofactors. In panels A–C, comparison of the basal PKC{delta} activity recovered after preincubation alone (first bar) versus with GSSG, (Cys–Gly)2, or cystine (second bar) indicates that the disulfides did not stimulate basal PKC{delta} activity. In contrast, each disulfide enhanced the lipid cofactor-stimulated histone–kinase activity of PKC{delta} ~2.0-fold (Figure 3Go, panels A–C, third versus fourth bar), i.e. to about the extent observed for the peptide kinase activity of PKC{delta} in Figure 2Go. Thus, the PKC{delta}-stimulatory effects of GSSG, (Cys–Gly)2 and cystine are selective for the lipid cofactor-activated form of the isozyme. Figure 3Go also shows that 2 mM cystine inactivated the lipid cofactor-stimulated histone kinase activities of PKC{gamma} (panel D) and PKC{varepsilon} (panel E) with about the same efficacy as it achieved against the peptide kinase activities of the isozymes in Figure 2Go.



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  		Fig. 3. Regulatory effects of cystine and other physiological disulfides on lipid cofactor-stimulated versus basal PKC isozyme activity. nPKC{delta} was preincubated with 2 mM GSSG (30 min) (A), 5 mM (Cys–Gly)2 (20 min) (B), or 2 mM cystine (20 min) (C). cPKC{gamma} (D) and nPKC{varepsilon} (E) were preincubated for 20 min with 2 mM cystine. All preincubations were done in 50 mM Tris–HCl, 200 mM NaCl, pH 8.0, 1 mM EDTA, 1 mM EGTA at 30°C. The histone kinase activities of the preincubated isozymes were assayed, as shown, in the absence or presence of the PKC-stimulatory cofactors 30 µg/ml PS, 30 µg/ml DAG and 0.2 mM Ca2+. Each assay mixture contained 95 ng of the PKC isozyme under analysis. Other experimental details are provided in Materials and methods. 100% activity is the isozyme activity observed under control conditions in the presence of stimulatory cofactors. 100% activity values shown correspond to 6.3 pmol 32P/min (nPKC{delta}) (A), 1.5 pmol 32P/min (nPKC{delta}) (B), 1.5 pmol 32P/min (nPKC{delta}) (C), 0.67 pmol 32P/min (cPKC{gamma}) (D) and 3.9 pmol 32P/min (nPKC{varepsilon}) (E). Each data point is the mean ± SD of triplicate determinations; the results shown were reproduced in an independent analysis.

 
To establish whether the regulation of PKC isozyme activity by the physiological disulfides involves an oxidative mechanism, we first evaluated whether DTT could quench the observed PKC-regulatory effects. We previously reported that >2 mM GSSG and >4 mM GSH each antagonized the activity of purified PKC isozymes in a DTT-insensitive manner, indicative of a non-oxidative/reductive (non-redox) inhibitory mechanism (24). Those studies were done by preincubating purified PKC with GSH/GSSG in 20 mM Tris–HCl, pH 7.5 (5 min, 30°C) (24), whereas the analyses in Figures 2 and 3GoGo utilize a higher ionic strength (50 mM Tris–HCl, 200 mM NaCl, pH 8.0), to dampen potentially inactivating ionic interactions between PKC and GSH/GSSG, and a longer preincubation period (20–30 min, 30°C), to provide more time for thiol–disulfide exchange reactions to occur.

Figure 4Go (panels A–C) shows that, under conditions where preincubation with GSSG, (Cys–Gly)2, and cystine each stimulated PKC{delta} activity 2.0–2.5-fold in Figure 2Go, co-incubation with 10 mM DTT fully quenched the stimulatory response, while 10 mM DTT alone had no effect on PKC{delta} activity. In addition, replacement of the disulfide agent with the corresponding reduced thiol (GSH, Cys–Gly, cysteine) at twice the disulfide concentration, e.g. 2.0 mM GSSG versus 4.0 mM GSH, had no effect on PKC{delta} activity (Figure 4Go, panels A–C). These results provide evidence that the disulfide bridge in GSSG, (Cys–Gly)2, and cystine plays a critical role in the stimulation of PKC{delta} activity by these agents. Because cystine was the only disulfide agent in Figure 2Go to markedly inactivate PKC isozymes, we confined the analysis of disulfide agent-mediated PKC isozyme inactivation to cystine. Figure 4Go (panels D and E) shows that 10 mM DTT quenched cystine-induced PKC{gamma} and PKC{varepsilon} inactivation and produced only modest or negligible effects on the isozyme activity in the absence of cystine. In addition, cysteine affected neither PKC{gamma} nor PKC{varepsilon} activity, when administered at twice the concentration of cystine. Taken together, the results in Figure 4Go provide evidence that cystine stimulates the activity of PKC{delta} and inactivates PKC{gamma} and PKC{varepsilon} by oxidative mechanisms.



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  		Fig. 4. Cystine and other physiological disulfides regulate PKC isozyme activity by an oxidative mechanism. nPKC{delta} was pre-incubated with/without 2.0 mM GSSG (A), 5.0 mM (Cys–Gly)2 (B), or 2.5 mM cystine (C). cPKC{gamma} (D) and nPKC{varepsilon} (E) were pre-incubated with 2 mM cystine. All preincubations were done in 50 mM Tris–HCl, 200 mM NaCl, pH 8.0, 1 mM EDTA, 1 mM EGTA at 30°C; preincubation periods were 30 min (A) and 20 min (B–E). Where indicated, the preincubation mixtures contained 10 mM DTT or the reduced amino-acid (cysteine) or peptide (Cys–Gly or GSH) at twice the concentration of the disulfide reagent in the analysis. The samples were assayed for PKC isozyme activity immediately following the preincubation period. Each assay mixture contained 45 ng of the PKC isozyme under analysis. 100% activity is defined as the PKC activity observed in the absence of disulfide and thiol reagents; 100% activity values shown correspond to 0.48 pmol 32P/min (nPKC{delta}) (A), 0.33 pmol 32P/min (nPKC{delta}) (B), 0.68 pmol 32P/min (nPKC{delta}) (C), 5.3 pmol 32P/min (cPKC{gamma}) (D) and 12 pmol 32P/min (nPKC{varepsilon}) (E). Each data point represents the mean ± SD of triplicate determinations; the results shown were reproduced in an independent analysis.

 
The ability of cystine, but not cysteine, to stimulate PKC{delta} and inactivate PKC{gamma} and PKC{varepsilon} (Figure 4Go) suggested PKC isozyme S-cysteinylation as the mechanism responsible for the observed PKC-regulatory effects. Protein S-thiolation is readily reversed by DTT via reduction of the disulfide linkage (25,26). In Figure 5Go, PKC{gamma}, PKC{delta} and PKC{varepsilon} were each preincubated with cystine for 20 min at 30°C followed by a second preincubation with/without the addition of 10 mM DTT or 2.5 mM 2-mercaptoethanol (10 min, 30°C), and then assayed. Cystine-induced inactivation of PKC{varepsilon} (panel A) and PKC{gamma} (panel B) was fully reversed by DTT, while DTT had little or no effect on the isozyme activity in the absence of cystine (Figure 5Go, panels A and B). The DTT reversibility of cystine-induced PKC{gamma} and PKC{varepsilon} inactivation supports isozyme S-cysteinylation as the inactivation mechanism.



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  		Fig. 5. DTT reverses PKC{varepsilon} and PKC{gamma} inactivation while potentiating PKC{delta} stimulation by cystine. PKC isozymes were subjected to two successive preincubations in 50 mM Tris–HCl, 200 mM NaCl, pH 8.0, 1 mM EDTA, 1 mM EGTA at 30°C. In the first preincubation (20 min), cystine was present, where indicated, at 2.0 mM (A, B, D) or 2.5 mM (C). At the initiation of the second preincubation period (10 min), 10 mM DTT (A, B, C) or 2.5 mM 2-mercaptoethanol (D) was added, where indicated. Isozymes were assayed immediately after the second preincubation period. Each assay mixture contained 50 ng of the isozyme under analysis. 100% activity values shown correspond to 5.7 pmol 32P/min (nPKC{varepsilon}) (A), 2.4 pmol 32P/min (cPKC{gamma}) (B), 0.69 pmol 32P/min (nPKC{delta}) (C) and 0.85 pmol 32P/min (nPKC{delta}) (D). Each data point is the mean ± SD of triplicate determinations; the results shown were reproduced in an independent analysis.

 
Cystine-induced PKC{delta} stimulation was further enhanced by subsequent incubation of the isozyme with 10 mM DTT (Figure 5Go, panel C). This phenomenon was also produced when 2.5 mM 2-mercaptoethanol was substituted for 10 mM DTT (Figure 5Go, panel D). Each reducing agent enhanced cystine-induced PKC{delta} stimulation to an extent that was ~4-fold over the original activity level. Neither DTT nor 2-mercaptoethanol modified PKC{delta} activity in the absence of cystine treatment (Figure 5Go, panels C and D), eliminating a trivial explanation for the observed enhancement of isozyme activity. These results provide evidence that, upon exposure to DTT, cystine-induced structural changes in PKC{delta} yield to additional changes in the isozyme structure that further enhance activity. However, the results left open the question of whether cystine-induced PKC{delta} stimulation is associated with isozyme S-cysteinylation and, if so, whether the effects of DTT include reversal of that modification.

To establish whether PKC{gamma}, PKC{delta} and PKC{varepsilon} were S-cysteinylated (PKC–S–S–Cys) in association with isozyme regulation by cystine, the isozymes were incubated with [35S]cystine (0.1–2.5 mM) for 20 min at 30°C and analyzed by non-reducing 10% SDS–PAGE/autoradiography. Figure 6Go shows concentration-dependent labeling of PKC{gamma} (panel A), PKC{delta} (panel B) and PKC{varepsilon} (panel C) by [35S]cystine at 0.1 mM (lane 1), 0.5 mM (lane 2), 1.0 mM (lane 3), and 2.5 mM (lane 4). When DTT was included in the SDS–PAGE sample buffer that was added to isozyme samples incubated with 2.5 mM [35S]cystine, isozyme labeling was abrogated (panels A–C, lane 5). The DTT reversibility of [35S]cystine labeling of the isozymes (panels A–C, lanes 4 versus 5) is indicative of [35S] S-cysteinylation (25,26). The concentration dependence of PKC{gamma}, PKC{delta} and PKC{varepsilon} [35S] S-cysteinylation by [35S]cystine (Figure 6Go) was in general agreement with the concentration-dependence of the cystine-induced regulatory effects observed for each isozyme (Figure 2Go, panel C). Thus, cystine-induced PKC{gamma} and PKC{varepsilon} inactivation (Figure 2Go, panel C) is associated with isozyme S-cysteinylation, as predicted from the DTT reversibility of the inactivation (Figure 5Go, panels A and B). Furthermore, the DTT-reversible labeling of PKC{delta} by [35S]cystine in Figure 6Go (panel B) indicates that cystine-induced PKC{delta} stimulation (Figure 2Go, panel C) is associated with isozyme S-cysteinylation, and that the structural changes in PKC{delta} associated with DTT-induced enhancement of S-cysteinylated PKC{delta} activity (Figure 5Go, panel C) include reversal of S-cysteinylation.



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  		Fig. 6. [35S] S-cysteinylation of PKC isozymes by [35S]cystine. PKC{gamma} (A), PKC{delta} (B), and PKC{varepsilon} (C) were incubated for 20 min at 30°C in 80 mM Tris–HCl, pH 8.2, 1 mM EDTA, 1mM EGTA with [35S]cystine at 0.1 mM (lane 1), 0.5 mM (lane 2), 1.0 mM (lane 3), and 2.5 mM (lanes 4 and 5). After the incubation period was terminated by adding non-reducing SDS–PAGE sample buffer to each sample, DTT was added to samples corresponding to lane 5 (A and C, 5 mM DTT; B, 10 mM DTT). Samples were boiled and analyzed by 10% SDS–PAGE/autoradiography (300 ng PKC isozyme/lane). (The lane designated as 5 is from the same gel as lanes 1–4 but was separated from the heavily labeled lane 4 by skipping one lane). In each panel, the bar graph depicts averaged densitometric values ± SD obtained from the autoradiogram shown and a second, independent analysis.

 
Having established an association between cystine-induced regulation of PKC{gamma}, PKC{delta} and PKC{varepsilon} activity with isozyme S-cysteinylation, we next employed a selective mode of disulfide reduction to address whether isozyme S-cysteinylation was causally linked to the observed effects on isozyme activity. For this purpose, we employed thioredoxin, which is a dithiol–disulfide oxidoreductase that catalyzes the reduction of disulfide linkages when coupled with thioredoxin reductase (27). PKC{gamma}, PKC{delta} and PKC{varepsilon} were incubated with 2 mM cystine for 20 min at 30°C and then gel-filtered in order to remove excess cystine, which would otherwise interfere with thioredoxin catalysis. The gel-filtered, S-cysteinylated isozymes were incubated for 1 h at 30°C with/without thioredoxin–thioredoxin reductase or DTT, and then assayed. In these experiments, the use of isozymes that were gel-filtered after cystine treatment precluded utilization of isozymes that had not been exposed to cystine as internal positive controls. Therefore, as an alternative, activity values observed for the DTT-treated isozymes were normalized to 100%. Table IGo shows that DTT treatment of gel-filtered, S-cysteinylated PKC{gamma} and PKC{varepsilon} induced marked increases in isozyme activity (>=5-fold) that were in close agreement with the effects of DTT on S-cysteinylated PKC{gamma} and PKC{varepsilon} in Figure 5Go. The 30% enhancement of the activity of gel-filtered, S-cysteinylated PKC{delta} by DTT (Table IGo) is consistent with the stimulatory effect of the reducing agent on S-cysteinylated PKC{delta} in Figure 5Go. Thus, the gel-filtered, S-cysteinylated forms of the isozymes in Table IGo (designated as untreated control), correspond to cystine-inactivated PKC{gamma} and PKC{varepsilon} and cystine-stimulated PKC{delta}.


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  		Table I. Thioredoxin-mediated effects on PKC isozyme regulation by S-cysteinylation
 
Table IGo shows that thioredoxin was as effective as DTT in restoring the activity of cystine-inactivated PKC{gamma}, indicating that PKC{gamma} inactivation by cystine entailed isozyme S-cysteinylation. Thioredoxin was also effective in restoring the activity of cystine-inactivated PKC{varepsilon}, indicating that PKC{varepsilon} inactivation likewise entailed isozyme S-cysteinylation. The activity of cystine-inactivated PKC{varepsilon} was increased ~3-fold by thioredoxin and 5-fold by DTT (Table IGo). Interestingly, thioredoxin shared with DTT the property of further enhancing, rather than reversing, cystine-induced PKC{delta} stimulation (Table IGo). In fact, thioredoxin stimulated PKC{delta} activity to twice the extent achieved by DTT (Table IGo). Thus, two distinct modalities of disulfide reduction, thioredoxin catalysis and exposure to reducing agents, further enhanced the activity of S-cysteinylated PKC{delta} (Table IGo; Figure 5Go, panels C and D).

Superoxide treatment has been shown to stimulate the activity of several PKC isozymes by inducing release of Zn2+, which is bound to Cys-rich Zn2+ finger structures in the PKC regulatory domain (15). A facile and effective mode of reversing the Zn2+ release mechanism of PKC stimulation is incubation of the enzyme with millimolar ZnCl2 (15). To test for involvement of Zn2+ release in the mechanism of PKC{delta} stimulation by S-cysteinylation, we treated PKC{delta} with/without 2 mM cystine for 20 min at 30°C, under the conditions employed in Figure 2Go, followed by further incubation with/without 2 mM ZnCl2 for 10 min at 30°C. Incubation with ZnCl2 abrogated PKC{delta} stimulation by S-cysteinylation and had only minor effects on the activity of unmodified PKC{delta}. In two independent experiments, a >2.5-fold stimulation of PKC{delta} by cystine was abrogated to 70 ± 3% of the original activity level by further incubation with ZnCl2. In the absence of cystine treatment, 84 ± 6% of the original activity was recovered after incubation with ZnCl2. These results provide strong evidence that S-cysteinylation stimulates PKC{delta} activity by provoking Zn2+ release from the regulatory domain.

To measure the stoichiometries of isozyme [35S] S-cysteinylation associated with PKC{gamma} and PKC{varepsilon} inactivation and PKC{delta} stimulation, the isozymes were incubated with 2 mM [35S]cystine for 20 min at 30°C followed by a further 10 min incubation with/without 10 mM DTT. The isozymes (15–18 pmol/sample) were dialyzed extensively at 4°C (26 h with three changes), and then analyzed for DTT-sensitive incorporation of the radiolabel (c.p.m. in DTT-free isozyme samples minus c.p.m. in DTT-containing samples). For each isozyme, the stoichiometry of [35S] S-cysteinylation was close to one; the values were 0.41 pmol [35S]cysteine/pmol PKC{gamma}, 0.60 pmol [35S]cysteine/pmol PKC{delta} and 0.82 pmol [35S]cysteine/pmol PKC{varepsilon} (Table IIGo). These results provide evidence that selective S-cysteinylation of one or at most a few redox-sensitive Cys in PKC isozymes containing 21–23 Cys residues (13) is sufficient to inactivate PKC{gamma} and PKC{varepsilon} and to stimulate PKC{delta}.


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  		Table II. Stoichiometry of PKC isozyme [35S] S-cysteinylation
 
Finally, we extended the analysis of PKC isozyme regulation by physiological disulfides to the cystine analog cystamine, reasoning that elimination of anionic character in cystine might improve reactivity with PKC-regulatory thiols. Cystamine proved to be several-fold more effective than cystine in the oxidative regulation of PKC{gamma}, PKC{delta} and PKC{varepsilon}, inducing DTT-reversible inactivation of PKC{gamma} and PKC{varepsilon} with IC50s of 55 ± 5 µM and 95 ± 5 µM and stimulating PKC{delta} 2-fold in a DTT-sensitive manner at 120 ± 20 µM.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this report, we demonstrate differential responses of several PKC isozymes to cystine and other physiological disulfides. We report that PKC{delta} is stimulated 2.0–2.5-fold by GSSG, (Cys–Gly)2, and cystine, under conditions where PKC{gamma} and PKC{varepsilon} are fully inactivated by cystine, and PKC{alpha} activity is affected marginally or not at all by the disulfides. Focusing on cystine, we show that DTT quenches cystine-induced PKC{delta} stimulation and PKC{gamma}, {varepsilon} inactivation, indicative of oxidative regulation. By analyzing DTT-reversible [35S]cystine-induced labeling of the isozymes by non-reducing SDS–PAGE, we demonstrate that PKC{gamma}, PKC{delta} and PKC{varepsilon} are each [35S] S-cysteinylated with a concentration dependence which indicates an association between the cystine-induced regulatory effects on the isozymes and isozyme S-cysteinylation.

Cystine-induced inactivation of PKC{gamma} and PKC{varepsilon} was reversed by DTT as well as by thioredoxin, indicating that inactivation entailed disulfide bridge formation. The stable S-cysteinylation of PKC{gamma} and PKC{varepsilon} in association with inactivation strongly supports an inactivation mechanism where PKC S-cysteinylation serves as a redox switch that turns off PKC{gamma},{varepsilon} activity in a thioredoxin-reversible manner. Furthermore, the stoichiometries of PKC{gamma} and PKC{varepsilon} [35S] S-cysteinylation are consistent with a single redox-sensitive Cys in each isozyme as the switch, although direct identification of the involved Cys residue(s) by mass spectroscopic or site-specific mutagenesis approaches will be required to ascertain whether this is the case. On the other hand, the data do not exclude a more complicated mechanism in which S-cysteinylation of a critical Cys residue(s) in PKC{gamma},{varepsilon} is an intermediate step in the inactivation mechanism that, while not in itself inactivating, leads to concerted displacement of the S-cysteinyl modification by another PKC-thiol and formation of an inactivating intramolecular disulfide bridge in the kinase.

The analysis of PKC{delta} produced the surprising result that DTT treatment not only failed to reverse cystine-induced PKC{delta} stimulation but actually enhanced the activity of S-cysteinylated PKC{delta} while having no effect on the activity of native (unmodified) PKC{delta}. Thioredoxin likewise further enhanced the activity of S-cysteinylated PKC{delta}. These results are intriguing, as they suggest that although PKC{delta} S-cysteinylation is a reversible modification, it may serve as an irreversible mode of kinase stimulation, given that a physiological mechanism of dethiolation, thioredoxin catalysis (26,27), produces structural changes in S-cysteinylated PKC{delta} that further stimulate the kinase activity. A recently described oxidative mode of PKC stimulation entails release of Zn2+ from the PKC regulatory domain, where it is bound to Cys-rich Zn2+ finger structures (15). In this report, we present evidence that S-cysteinylation-mediated stimulation of PKC{delta} entails induction of Zn2+ release from the regulatory domain. These findings offer a logical explanation for the persistence of S-cysteinylation-mediated PKC{delta} stimulation after reversal of the post-translational modification. Furthermore, the stoichiometry of PKC{delta} S-cysteinylation suggests that a single redox-sensitive Cys may mediate kinase stimulation.

The concentrations of cystine required to stimulate PKC{delta} and inactivate PKC{gamma},{varepsilon} in vitro are substantially higher than the physiological spectrum of intracellular cystine concentrations (22,28). That said, the robust PKC-regulatory effects achieved by cystine at low stoichiometries of isozyme S-cysteinylation suggest the existence of oxidative post-translational modifications that are physiological counterparts to the PKC-regulatory S-cysteinyl modifications produced by in vitro exposure to cystine. This is also supported by our findings that the physiological disulfide cystamine exerts potent oxidative regulatory effects on PKC{gamma}, PKC{delta} and PKC{varepsilon} in vitro at concentrations <200 µM. The potencies reported here for cystine-induced regulation of PKC isozymes by S-cysteinylation are in line with the potencies reported for S-thiolation-mediated regulation of other enzymes by physiological disulfides in vitro. For example, GSSG inactivates creatine kinase by S-glutathiolation with an IC50 of 0.5 mM (29), and aldose reductase is inactivated ~80% by S-thiolation reactions with 1.5 mM GSSG and 0.4 mM cystine (21). Other studies have utilized 50 mM GSSG to drive S-glutathiolation-mediated inactivation of protein tyrosine phosphatase 1B (30) and HIV protease (31).

We have hypothesized that the antagonism of tumor promotion/progression by GSH and other small cellular thiols may involve regulatory effects on PKC through S-thiolation reactions with PKC isozymes under pro-oxidant conditions (17). Our observations of cystine-induced PKC isozyme regulation by S-cysteinylation offer strong support to the hypothetical model. Thus, an isozyme that potently antagonizes DMBA–TPA-induced tumor promotion/progression when expressed as a transgene in mouse skin (PKC{delta}) (8) is stimulated by S-cysteinylation, an isozyme that has the opposite effect of markedly increasing carcinoma formation when expressed as a transgene in the skin (PKC{varepsilon}) (6) is inactivated by S-cysteinylation, and an isozyme that does not influence DMBA–TPA-induced tumor promotion/progression when expressed as a transgene in the skin (PKC{alpha}) (32) is not regulated by cystine. Furthermore, PKC{gamma}, while not widely expressed in mammalian tissues (33), is implicated as an oncogene through studies of transfected fibroblasts (23), and S-cysteinylation inactivated PKC{gamma} and PKC{varepsilon} similarly. Therefore, S-cysteinyl acceptor-sites in PKC isozymes may offer attractive targets for the development of drugs with isozyme-selective PKC-regulatory action for preventive intervention and treatment of proliferative disorders such as cancer.


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


   Acknowledgments
 
Supported by NIH grant R01-CA74831 and Robert A.Welch Foundation Grant G-1141.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 13, 2002; accepted October 16, 2002.


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