Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 173, Houston, TX 77030, USA
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Abstract |
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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.
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Introduction |
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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 and nPKC
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
and nPKC
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
activation in tumor promotion antagonism (6), enforced nPKC
expression induced keratinocyte growth arrest and differentiation in vitro; in contrast, enforced cPKC
expression did not affect keratinocyte growth properties or expression of the differentiation markers examined (9).
The influence of cPKC, nPKC
and nPKC
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
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
overexpression suppressed both papilloma and carcinoma formation, i.e. tumor promotion and progression, in response to DMBA+TPA (11). cPKC
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, cPKCß1, cPKC
and nPKC
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
is fully and reversibly inactivated by the oxidative mechanism of cPKC
S-glutathiolation (27). The potent inactivation of cPKC
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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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 TrisHCl, 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 TrisHCl, 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 [-32P]ATP (50008000 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 [
-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 (711 pmol) was preincubated under the conditions employed in the kinase inactivation analysis in the presence of 100 µM [35S]GSH (250300 c.p.m./pmol) or [35S]cysteine (450500 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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Results |
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We first analyzed whether GSH could potentiate diamide-induced inactivation of the kinases under conditions where marked GSH-potentiated inactivation of cPKC was shown to be caused by cPKC
S-glutathiolation in our previous study (27). In these experiments cPKC
(which served as a positive control), cPKCß1, cPKCß2 and cPKC
, nPKC
and nPKC
, aPKC
and PKD were preincubated with diamide (0.025.0 mM) ± 100 µM GSH for 5 min at 30°C and then assayed. Figure 1
(closed circles) shows concentration-dependent inactivation of the PKC isozymes and PKD by diamide in the absence of GSH (non-potentiated inactivation). Diamide (0.025.0 mM) induced monophasic inactivation of all of the kinases tested with variable potencies, with the exception of nPKC
, which is the sole kinase in the study that has been implicated in the antagonism of tumor promotion/progression (6,9,11; Figure 1
, closed circles). Table I
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
was the most resistant to diamide treatment (IC50 = 4.45 ± 0.55 mM) and cPKC
was the most sensitive (IC50 = 0.06 ± 0.01 mM) (Table I
). Comparing the IC50 values that correspond to the non-potentiated inactivation mechanism in Table I
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
> cPKCß2 > aPKC
> cPKCß1 > nPKC
> PKD > cPKC
(Table I
). nPKC
alone showed a biphasic response to diamide. nPKC
activity was enhanced by low diamide concentrations and this effect peaked at 1.0 mM diamide, where 1.8-fold activation was observed (Figure 1
, closed circles). Higher diamide concentrations produced a decline in nPKC
activity, but 50% inactivation was not reached in the analysis. A maximal effect of 27 ± 1% nPKC
inactivation was achieved at 5.0 mM diamide, which was the highest diamide concentration tested (Figure 1
, closed circles).
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Among the kinases that were monophasically inactivated by diamide alone (all except nPKC) (Figure 1
, 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
) to 100 ± 10 µM (PKD) (Table I
). 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 (0.25.0 mM diamide) and full inactivation was attained by the GSH-potentiated mechanism at 1 mM diamide (Figure 1
, open circles). In contrast, PKD was incompletely inactivated by the GSH-potentiated inactivation mechanism across the entire diamide concentration range (Figure 1
, 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 was also inactivated by the GSH-potentiated mechanism (Figure 1
, open circles). However, the IC50 value observed for nPKC
inactivation by the GSH-potentiated mechanism (IC50 = 1.25 ± 0.46 mM diamide) was ~1020 times greater than the IC50 values observed for PKD and the other PKC isozymes (51100 µM diamide) (Table I
). In addition, GSH produced a modest enhancement of diamide-induced nPKC
activation at low concentrations of the oxidant (75100 µM diamide) (Figure 1
, open circles); this enhancement of diamide-induced nPKC
activation by GSH was reproduced in a second, independent analysis (data not shown). Thus, nPKC
proved to be the most resistant of the kinases to the GSH-potentiated inactivation mechanism and the results in Figure 1
and Table I
show that several phorbol ester-responsive kinases that have been implicated in the mediation of tumor promotion/progression by phorbol esters (cPKCß1, cPKC
, nPKC
and PKD) (38,10) are more readily inactivated by the GSH-potentiated inactivation mechanism than nPKC
. 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 1, open circles), and in some cases by preincubation of the kinases with diamide alone (Figure 1
, 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 2
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 2
, first versus fourth bar per set) indicated that preincubation with 3 mM DTT negligibly affected the kinase activity of cPKC
, cPKCß2, cPKC
and PKD and produced a modest loss of the activity of the other kinases in the study. Thus, a 1535% enhancement of kinase activity was observed for cPKCß1, nPKC
, nPKC
and aPKC
when DTT was omitted from the preincubation mixtures (Figure 2
, first versus fourth bar per set). The relatively minor effects of DTT on kinase activity in Figure 2
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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We next investigated whether the GSH-dependent, oxidative inactivation of PKC isozymes and PKD in Figure 1 (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 1
. 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 TrisHCl, 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 1623 Cys residues (31). We previously reported that a diamide concentration sufficient for full cPKC
inactivation by S-glutathiolation (100 µM diamide) induced binding of 0.95 ± 0.03 mol [35S]GSH/mol cPKC
(27). Table II
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 1
is mediated by select S-glutathiolation of 13 Cys residues in the kinase structures.
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Preincubation with 100 µM cysteine in the absence of diamide affected the activity of the kinases only modestly, i.e. 15% (Figure 3
, compare the first and third bars in the histograms). Consistent with the results shown in Figure 1, 2
.5 mM diamide enhanced the activity of nPKC
and 500 µM diamide inactivated the other kinases to variable extents (Figure 3
, compare the first and fourth bars in the histograms). Also consistent with Figure 1
, GSH potentiated the diamide-induced inactivation to >90% for all of the kinases except nPKC
and PKD and each of the latter kinases exhibited a major GSH-dependent component of inactivation under the conditions employed (Figure 3
, 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 1
(all but nPKC
) co-preincubation with cysteine produced a 520% 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
pre-incubated with diamide (Figure 3
, 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 II
. 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 (Figure 3
), 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 3
, 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
and nPKC
to ~60% of the activity of the untreated positive controls. In addition, DTT restored S-glutathiolated aPKC
activity to ~30% of the positive control and produced a recovery of activity of S-glutathiolated nPKC
that was 2.5-fold greater than the activity of the positive control. In contrast, the GSH-potentiated inactivation of cPKCß1, cPKCß2 and cPKC
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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Discussion |
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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 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
is highly resistant to oxidative regulation by either intramolecular disulfide bridge formation or by formation of disulfide linkages between cPKC
monomers. In contrast, cPKC
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 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
, 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
, 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
but not that of any other kinase in the analysis. In addition to nPKC
, other kinases implicated in the mediation of tumor promotion/progression, e.g. cPKCß1, cPKC
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
.
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. 13 residues out of a total of 1623.
The results in this report clearly demonstrate differential responses of closely related PKC isozymes, e.g. cPKC versus cPKC
and nPKC
versus nPKC
(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.
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Notes |
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Acknowledgments |
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References |
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