From the Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
The tripeptide glutathione (GSH) is
the predominant low molecular weight thiol reductant in mammalian
cells. In this report, we show that at concentrations at which GSH is
typically present in the intracellular milieu, GSH and the oxidized GSH
derivatives GSH disulfide (GSSG) and glutathione sulfonate each
irreversibly inactivate up to 100% of the activity of purified
Ca2+- and phosphatidylserine
(PS)-dependent protein kinase C (PKC) isozymes in a
concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC.
Our evidence for a nonredox mechanism of PKC inactivation can be
summarized as follows. GSSG antagonized the Ca2+- and
PS-dependent activity of purified rat brain PKC with the same efficacy (IC50 = 3 mM) whether or not the
reductant dithiothreitol was present. Glutathione sulfonate, which is
distinguished from GSSG and GSH by its inability to undergo
disulfide/thiol exchange reactions, was as effective as GSSG in
antagonizing Ca2+- and PS-dependent PKC
catalysis. The irreversibility of the inactivation mechanism was
indicated by the stability of the inactivated form of PKC to dilution
and extensive dialysis. The inactivation mechanism did not involve the
nonspecific phenomena of denaturation and aggregation of PKC because it
obeyed pseudo-first order kinetics and because the hinge region of
PKC-
remained a preferential target of tryptic attack following GSH
inactivation. The selectivity of GSH in the inactivation of PKC was
also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and
Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the
Ser/Thr kinase casein kinase 2 was by comparison weak (<25%).
Inactivation of PKC-
was not accompanied by covalent modification of
the isozyme by GSH or other irreversible binding interactions between
PKC-
and the tripeptide, but it was associated with an increase in the susceptibility of PKC-
to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with
N-acetylcysteine resulted in a substantial loss of
Ca2+- and PS- dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over
cellular PKC isozymes that may be lost when oxidative stress depletes
the cellular GSH pool.
 |
INTRODUCTION |
The phospholipid-dependent isozyme family protein
kinase C (PKC)1 plays an
important role in diverse physiological processes, including cell
growth and differentiation, muscle contraction, and neurotransmission, and pathological developments, e.g. tumor promotion and
multiple drug resistance in cancer (1-4). The PKC family consists of
Ca2+-dependent, phorbol ester-activated
isozymes (
,
1,
2, and
), Ca2+-independent, phorbol ester-activated isozymes (
,
,
,
, and µ), and Ca2+- and phorbol
ester-independent isozymes (
and
). Phosphatidylserine (PS)
dependence is universal among PKC isozymes (1, 5). Both stimulatory and
inhibitory endogenous regulators of PKC activity have been identified.
The second messenger, diacylglycerol, activates phorbol
ester-responsive PKC isozymes in a PS-dependent manner (5).
In addition to PS, arachidonic acid and other endogenously produced
fatty acids can support diacylglycerol activation of PKC (4-6).
Intracellular Ca2+ contributes to the activation of
PKC-
, PKC-
, and PKC-
(4, 5). Fully processed PKC isozymes are
phosphorylated at multiple sites (5, 7-9), and phosphatases can
inactivate PKC isozymes by dephosphorylation of critical residues (5,
10, 11). The sphingolipid metabolite ceramide has been implicated as a stimulator of PKC-inactivating phosphatase catalysis (12), and the
related metabolite sphingosine is a reversible inhibitor of PKC
(13).
PKC isozymes contain multiple Cys residues in their catalytic and
regulatory domains (14, 15), including Cys-rich sequences present in
the regulatory domain that are critical to the phorbol ester
responsiveness of the isozyme family (2, 16). A highly reactive Cys
residue of unknown function that is expressed in the catalytic domain
of each PKC isozyme subfamily is subject to S-thiolation by
the synthetic peptide-substrate analog
N-biotinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu, i.e.
an intermolecular disulfide bridge is formed between the Cys residue
and the synthetic peptide, and this modification inactivates the enzyme
(17, 18). The susceptibility of PKC isozymes to inactivation by an
S-thiolating peptide-substrate analog suggests that PKC
activity might also be subject to redox regulation by the endogenous
molecule glutathione (18). The tripeptide glutathione (GSH),
L-
-glutamyl-L-cysteinyl-glycine, is the
predominant low molecular weight thiol reductant in mammalian cells
(19). Protein S-glutathiolation, i.e. the
formation of a disulfide-linked protein-GSH complex, is a selective
protein modification that can be induced in cells by mild oxidative
stress (20). GSH disulfide (GSSG) has been shown to oxidatively
regulate the function of several purified enzymes, including carbonic
anhydrase III, aldose reductase, and HIV-1 protease by
S-glutathiolation, and in each case the effects of GSSG can
be fully reversed by the reducing agent dithiothreitol (DTT) (21-23).
Mammalian cells typically contain millimolar concentrations of GSH,
e.g. 0.5-10 mM, and oxidized GSH generally
amounts to less than 1% of the total cellular GSH content (19, 24). In this report, we show that at GSH concentrations typically present in
the intracellular milieu, GSH and oxidized GSH derivatives irreversibly
inactivate purified PKC by a novel nonredox mechanism that does not
involve glutathiolation of the enzyme. N-Acetylcysteine, a
precursor of cellular GSH (25, 26), likewise irreversibly inactivated
purified PKC in a nonredox manner. Treatment of cultured cells with
N-acetylcysteine induced a substantial decline in the level
of cellular PKC activity, providing evidence that the PKC inactivation
mechanism observed with N-acetylcysteine and GSH in the
purified enzyme system may also be operative in mammalian cells.
 |
MATERIALS AND METHODS |
Rat brain PKC was purified to near-homogeneity according to
silver-stained polyacrylamide gels by a previously described method (27). The histone kinase activity of the purified PKC preparation was
stimulated 10-15-fold by 0.2 mM Ca2+ and 30 µg/ml PS but was unaffected by either Ca2+ or PS alone.
The purified PKC preparation is a mixture of the isozymes
,
,
,
, and
(18). A fully active catalytic domain fragment of PKC
was generated from the purified PKC preparation by limited
trypsinolysis with a yield of >50%, as described previously (17, 28).
Where indicated, the catalytic domain fragment was purified from
regulatory domain fragment and residual intact PKC by DEAE ion-exchange
chromatography using a 0.0-0.4 M NaCl gradient (17, 28).
The histone kinase activity of the catalytic domain fragment
preparation was stimulated less than 1.5-fold by Ca2+ and
PS. Purified, baculovirus-produced recombinant human PKC-
was
purchased from Pan Vera Corp. (Madison, WI). Immunoblot analysis of
PKC-
was done as described previously (29) using an ECL detection
system (Amersham Pharmacia Biotech) and monoclonal anti-PKC-
(Transduction Laboratories, Lexington, KY). Dialysis of PKC-
was
done as described under "Results," using a 0.5-3 ml Slide-A-Lyzer unit with a molecular mass cut-off of 10,000 Da. (Pierce). Electrospray ionization mass spectrometric analysis of PKC-
was done at the University of Texas-Houston Analytical Chemistry Center. Samples of
purified recombinant control PKC-
and GSH-inactivated PKC-
(10 pmol/µl) were prepared for analysis by dialysis against 5% acetic
acid under bubbling nitrogen for 48 h. Horse skeletal muscle apomyoglobin (16951.5 Da) and bovine serum albumin (66430.3 Da) served
as standard proteins and were analyzed in parallel. The accuracy of
molecular masses determined from the spectra was within ±20 Da.
The synthetic peptide RKRTLRRL (>98% pure) was prepared at the
Synthetic Antigen Core Facility at the University of Texas M. D.
Anderson Cancer Center. The tripeptides
-Glu-Gly-Gly, Tyr-Gly-Gly, and Gly-Ala-Gly and the amino acids Gly, Glu, and
N-acetyl-Cys were purchased from Bachem Bioscience Inc.
(King of Prussia, PA), and [Ser25]PKC (19-31) was from
Peninsula Laboratories (Belmont, CA). [
-32P]ATP and
peroxidase-linked sheep anti-mouse Ig were purchased from Amersham
Pharmacia Biotech, and [3H]glutathione was from NEN Life
Science Products. Frozen rat brains were obtained from Harlan
Sprague-Dawley (Indianapolis, IN). Protein assay solutions and
SDS-polyacrylamide gel electrophoresis reagents, including molecular
mass markers, were obtained from Bio-Rad. Go6976 was purchased from
Calbiochem. MCF7-MDR cells and R6-PKC3 fibroblasts were kindly provided
by Dr. Kenneth Cowan (National Institutes of Health) and Dr. I. Bernard
Weinstein (Columbia University, NY), respectively, and tissue culture
reagents were purchased from Life Technologies, Inc. GSH, glutathione
disulfide (GSSG), glutathione sulfonic acid (GSO3), histone
IIIS, PS, ATP, DTT, L-1-tosylamido-2-phenylethyl
chloromethyl ketone-treated trypsin, phenylmethylsulfonyl fluoride,
DEAE-Sepharose, and all other reagents were purchased from Sigma.
Protein Kinase Assays--
The Ca2+- and
PS-dependent histone kinase activity of purified PKC was
measured as described previously (28). The histone kinase reaction
mixture (120 µl) contained 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.2 mM CaCl2,
30 µg/ml PS (or none), 6 µM [
-32P]ATP
(5000-8000 cpm/pmol), 0.67 mg/ml histone III-S, and 5 ng of purified
PKC. In indicated experiments, histone was replaced with the synthetic
peptide substrate RKRTLRRL (20 µM) (28). A 10-min
reaction period at 30 °C, which yields linear kinetics, was
initiated by the addition of [
-32P]ATP. The reaction
was terminated on phosphocellulose paper, and histone (or peptide)
phosphorylation was quantitated as described previously (28). GSX (GSH,
GSSG, or GSO3) was added to PKC assay mixtures as described
under "Results." In some experiments, PKC or its catalytic domain
fragment was preincubated with GSX alone for 5 min at 30 °C and then
briefly kept on ice prior to its addition to assay mixtures. The
preincubation mixtures were diluted 12-30-fold into assay mixtures as
specified under "Results." In other experiments, PKC and GSX were
added directly to PKC assay mixtures as separate components. All assays
were performed in triplicate and expressed as the mean value ± S.D. Purified casein kinase 2 and a casein kinase 2 assay kit were
purchased from Upstate Biotechnology (Lake Placid, NY). Casein kinase 2 was assayed by monitoring the phosphorylation of the synthetic peptide
substrate RRRDDDSDDD according to the instructions provided by the
manufacturer. Each reaction mixture contained 20 ng of casein kinase
2.
Measurement of Ca2+- and PS-dependent PKC
Activity in N-Acetylcysteine-treated Cultured Mammalian
Cells--
Human breast cancer MCF7-MDR cells and rat R6-PKC3
fibroblasts were chosen for analysis because they express levels of
Ca2+- and PS-dependent PKC activity that can be
measured accurately by assays of DEAE-extracted cell lysates. The
abundance of Ca2+- and PS-dependent PKC
activity is primarily due to enforced expression of
PKC-
1 in the R6-PKC3 cells (30) and to the increase in
PKC-
expression that occurred in association with the selection of the multidrug resistant line MCF7-MDR by doxorubicin (31). Prior to
treatment with N-acetylcysteine, cells were cultured as
previously reported, with MCF7-MDR cells in Eagle's minimum essential
medium containing 5% heat-inactivated fetal calf serum (32) and
R6-PKC3 cells in Dulbecco's modified Eagle medium containing 10%
heat-inactivated fetal calf serum and 50 µg/ml G418 (30). The culture
media also contained nonessential amino acids, vitamins, sodium
pyruvate, L-glutamine, and penicillin-streptomycin (30,
32). Near-confluent cells cultured in T150 flasks (approximately 5 × 107 R6-PKC3 cells and 4 × 107 MCF7-MDR
cells) were treated with N-acetylcysteine at the indicated concentration in culture medium (without serum) buffered with 80 (R6-PKC3) or 100 (MCF7-MDR) mM Tris-HCl, pH 7.5, for 30 min at 37 °C. The treatment conditions employed were determined to have
no effect on cell viability, as measured by trypan blue exclusion. At
the end of the treatment period, PKC was extracted from the cells by a
previously described procedure (33). Briefly, cells were washed with
phosphate-buffered saline, harvested from the flasks in ice-cold Buffer
A (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 20 µg/ml soybean trypsin inhibitor, 100 µg/ml
leupeptin, 0.25 mM phenylmethylsulfonyl fluoride)
containing 1% Triton X-100, 5 mM chelators (EDTA and
EGTA), and 15 mM 2-mercaptoethanol, and stirred for 1 h at 4 °C. All subsequent procedures were done at 4 °C. Cell
lysates were centrifuged for 15 min at 13,800 × g to remove debris and then loaded onto 0.5 ml DEAE-Sepharose columns equilibrated in Buffer A. After washing each column with 3 ml Buffer A,
Ca2+- and PS-dependent PKC was eluted with 2 ml
of Buffer A containing 0.2 M NaCl, and the protein
concentration of the eluted sample was determined (33). Part of the
sample was mixed with 2× SDS-polyacrylamide gel electrophoresis sample
buffer for immunoblot analysis, and the remainder was reserved for PKC
assays.
The assay employed for the Ca+- and
PS-dependent PKC activity of the eluted samples (10 µg of
sample protein/assay) was modified from the assay used with purified
PKC (see above, under "Protein Kinase Assays") as follows. The
pseudosubstrate-based synthetic peptide substrate 10 µM
[Ser25]PKC-(19-31) (34) was employed as the
phosphoacceptor substrate, the cofactor Ca2+ was present at
1 mM, and the Ca2+- and
PS-dependent PKC-selective inhibitor Go6976 (35) was
employed at 100 nM to distinguish Ca2+- and
PS-dependent PKC activity from background kinase activity. Ca2+- and PS-dependent PKC activity was
calculated by subtracting the cpm obtained from assay mixtures
containing Ca2+,, PS, and Go6976 from the cpm obtained from
assay mixtures containing Ca2+ and PS.
 |
RESULTS |
As an initial test of whether PKC is subject to regulation by
glutathiolation, we preincubated a purified rat brain PKC preparation with GSSG at the concentrations shown in Fig.
1, diluted each preincubation mixture
30-fold into a PKC assay mixture, and measured the Ca2+-
and PS-dependent histone kinase activity of the enzyme.
Preincubation with GSSG inhibited PKC in a
concentration-dependent manner, achieving 50% inhibition
at about 2.6 mM GSSG and complete inhibition at
5.0
mM GSSG (Fig. 1, closed circles). The loss of
100% of the activity indicated that each Ca2+- and
PS-dependent isozymic component of the PKC preparation was subject to inhibition by GSSG. In a control experiment, the
preincubation step was omitted, and GSSG was added directly to PKC
assay mixtures at the 30-fold diluted final concentration corresponding
to each preincubation mixture. Under these conditions, inhibition was negligible (Fig. 1, open circles). This indicates that the
inhibition of PKC achieved by GSSG in Fig. 1 (closed
circles) was a result of the preincubation step and was stable
when the preincubation mixture was diluted. These results were
consistent with an irreversible inactivation mechanism that could
involve glutathiolation of PKC.

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Fig. 1.
Irreversible inactivation of PKC by
GSSG. Purified rat brain PKC was preincubated with GSSG at the
concentration shown for 5 min at 30 °C, briefly placed on ice, and
then diluted 30-fold into histone kinase assay mixtures. The
Ca2+- and PS-dependent histone kinase activity
of PKC was measured; it represents the total activity of PKC- ,
PKC- , and PKC- (18). Shown is the % inactivation of PKC achieved
by preincubation with GSSG alone ( ) and in the presence of 100 mM DTT ( ) and 250 mM DTT ( ). Also shown
is the % inhibition of PKC achieved by omitting the preincubation step
and directly adding GSSG and PKC to assay mixtures as separate
components to achieve the 30-fold diluted final concentration ( ),
i.e. the GSSG concentration in the assay mixtures was
30-fold less than the GSSG concentration indicated on the x
axis. 100% activity was 3.77 ± 0.32 pmol of 32P
transferred per min. For other experimental details, see "Materials
and Methods."
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|
Glutathiolation of proteins is readily reversed by reducing agents,
such as DTT (21, 23, 36). To test whether GSSG-induced inactivation of
Ca2+- and PS-dependent PKC (Fig. 1,
closed circles) involved PKC glutathiolation, DTT was
included in the PKC/GSSG preincubation mixtures. The efficacy of GSSG
in inactivating Ca2+- and PS-dependent PKC was
virtually unaffected by the presence of either 100 mM DTT
(Fig. 1, open triangles) or 250 mM DTT (Fig. 1,
open squares) in the preincubation mixtures. We also
determined that removal of the
-mercaptoethanol (5 mM)
in the PKC preparation by gel filtration (17, 18) prior to
preincubation of the enzyme with GSSG had no effect on the
concentration dependence of GSSG inactivation of PKC (data not shown).
These results ruled out glutathiolation as the mechanism of GSSG
inactivation of Ca2+- and PS-dependent PKC.
We next examined whether GSH and GSO3, a GSH analog that
cannot participate in thiol/disulfide exchange reactions, could
inactivate Ca2+- and PS-dependent PKC. When PKC
was preincubated with GSH and then diluted 30-fold into reaction
mixtures, GSH inactivated PKC with roughly half the potency of GSSG,
achieving 50% inactivation at about 5.1 mM GSH (Fig.
2, closed triangles). No
inhibition was observed if the preincubation step was omitted, and GSH
was added directly to reaction mixtures at the corresponding final (30-fold diluted) concentrations (Fig. 2, open triangles),
providing evidence for an irreversible inactivation mechanism. The
observed potency of GSH in the inactivation of Ca2+- and
PS-dependent PKC was consistent with the equivalence of 2 GSH with DTT-reduced GSSG (Figs. 1 and 2). The capacity of GSH to
inactivate PKC was not merely a nonspecific effect of the free sulfhydryl of the tripeptide, because the reducing agent DTT did not
inactivate PKC at concentrations as high as 250 mM (Fig.
1). Furthermore, the inactivation potency of GSH could not be
attributed to trace amounts of GSSG, because it was unaffected by the
presence of 100 mM DTT in PKC/GSH preincubation mixtures
(data not shown). We also examined the ability of GSH to inactivate a
purified preparation of the Ser/Thr kinase casein kinase 2 (37). In
these experiments, casein kinase 2 was preincubated with GSH under the
conditions employed in Fig. 2, and the preincubation mixture was
diluted 12-fold into casein kinase assay mixtures. Preincubation with 5 and 10 mM GSH, which effected approximately 40 and 90%
inactivation of PKC (Fig. 2), resulted in a loss of only 8 ± 9%
and 23 ± 2% of casein kinase 2 activity, respectively (values
are averages obtained from three independent experiments done in
triplicate). These results indicate that the potent inactivation of PKC
achieved by GSH at physiological concentrations (Fig. 2) cannot be
attributed to broad and nonspecific effects of the tripeptide against
isolated protein kinases.

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Fig. 2.
Irreversible inactivation of PKC by GSH and
GSO3. The % inactivation of purified rat brain PKC by
preincubation with GSH and GSO3 was measured. The
Ca2+- and PS-dependent histone kinase activity
of PKC was assayed subsequent to preincubation of the enzyme with GSH
( ) and GSO3 ( ) at the concentrations shown, or the
preincubation step was omitted, and GSH ( ) and GSO3
( ) were added to assay mixtures independently of PKC. In every case,
the GSH/GSO3 concentration in the assay mixture was 30-fold
less than the concentration indicated on the abscissa. 100%
activity was 2.87 ± 0.14 pmol of 32P transferred per
min. For other experimental details, see the legend to Fig.
1 and "Materials and Methods."
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|
Potent, irreversible inactivation of Ca2+- and
PS-dependent PKC was also achieved by preincubation of the
enzyme with GSO3 (Fig. 2, closed circles). We
next tested whether preincubation with GSH and the oxidized derivatives
would inactivate PKC in assays employing the synthetic
peptide-substrate RKRTLRRL (28) in lieu of histone. GSH, GSSG, and
GSO3 were each equally effective in inactivating the
histone and synthetic peptide phosphorylation reactions of PKC (data
not shown). The ability of GSO3 to inactivate PKC, taken
together with the DTT insensitivity of GSH- and GSSG-mediated PKC
inactivation, definitively demonstrates that the inactivation mechanism
does not require reduction, formation, or isomerization of disulfide
bridges within PKC.
To determine whether the catalytic domain of PKC was sufficient for GSH
inactivation of the enzyme, we generated a Ca2+- and
PS-independent catalytic domain fragment of PKC by limited trypsinolysis of the purified rat brain PKC preparation (17, 28) (see
"Materials and Methods"). The catalytic domain fragment was
preincubated with GSH and oxidized GSH derivatives as described under
"Materials and Methods" and then diluted 12-fold into assay mixtures. Fig. 3 shows that GSH, GSSG,
and GSO3 each effected concentration-dependent
inactivation of the Ca2+- and PS-independent histone kinase
activity of the catalytic domain fragment. No inactivation was observed
when the preincubation step was omitted and GSH, GSSG, or
GSO3 was diluted 12-fold into reaction mixtures as a
component separate from the catalytic domain fragment of PKC. Catalytic
domain fragment that was purified from residual intact PKC and the
regulatory domain fragment by DEAE chromatography (28) was likewise
subject to inactivation by GSH, GSSG, and GSO3 (data not
shown). Although GSH, GSSG, and GSO3 were each about
1.5-fold more potent in the inactivation of the catalytic domain
fragment compared with inactivation of Ca2+- and
PS-dependent PKC, the relative potencies of GSH, GSSG, and GSO3 against the catalytic domain fragment of PKC and
intact PKC were similar, with GSH being about half as potent as GSSG
and GSO3 (Figs. 1-3). These results indicate that the
mechanism of GSH inactivation of PKC is catalytic domain-directed.

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Fig. 3.
Irreversible inactivation of the catalytic
domain of PKC by glutathione. A catalytic domain fragment of PKC
generated by limited trypsinolysis of the purified rat brain PKC
preparation was preincubated with GSH ( ), GSSG ( ), or
GSO3 ( ) for 5 min at 30 °C, briefly placed on ice,
and then diluted 12-fold into histone kinase assay mixtures. The % inactivation of the Ca2+- and PS-independent histone kinase
activity of the catalytic domain fragment achieved by preincubation
with GSH, GSSG, and GSO3 is shown. 100% activity was
1.52 ± 0.2 pmol of 32P transferred per min. For other
details, see "Materials and Methods."
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In the above experiments, PKC or its catalytic domain fragment was
preincubated with GSH or oxidized GSH derivatives at inactivating concentrations in the absence of PKC substrates and cofactors. To test
whether PKC substrates and cofactors might protect PKC from
inactivation by GSX (GSH, GSSG, or GSO3), we next added
inactivating concentrations of GSX directly to the PKC assay mixtures,
which contained 0.2 mM CaCl2, 30 µg/ml PS, 10 mM MgCl2, 6 µM
[
-32P]ATP, 0.67 mg/ml histone III-S, and 5 ng of
purified PKC. Under these conditions, the IC50 values of
GSSG, GSH and GSO3 against Ca2+- and
PS-dependent PKC activity estimated by graphical analysis were 2.2, 4.2, and 1.7 mM (Fig.
4). These IC50 values are
actually somewhat smaller than those obtained when PKC was preincubated in the absence of cofactors and substrates with GSSG (IC50 = 2.6 mM), GSH (IC50 = 5.1 mM), and
GSO3 (IC50 = 2.4 mM) prior to its addition to assay mixtures (Figs. 1 and 2). Thus, the presence of PKC
substrates and cofactors in PKC assays at concentrations sufficient to
optimally support Ca2+- and PS-dependent PKC
activity does not afford protection against GSH inactivation of
PKC.

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Fig. 4.
PKC inhibition by GSX added directly to assay
mixtures. The % inhibition of the Ca2+- and
PS-dependent histone kinase activity of purified rat brain
PKC by the presence of GSH ( ), GSSG ( ), and GSO3
( ) in assay mixtures at the concentrations shown was measured. 100%
activity was 9.51 ± 0.78 pmol of 32P transferred per
min. For other experimental details, see the legend to Fig. 1 and
"Materials and Methods."
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We also tested for protective effects of PKC substrates and cofactors
against GSX inactivation of Ca2+- and
PS-dependent PKC by including substrates/cofactors in the PKC-GSX preincubation mixtures prior to dilution into histone kinase
assay mixtures. Inclusion of the activating cofactors 1 mM
CaCl2 and 30 µg/ml PS in PKC/GSSG preincubation mixtures
did not afford protection against GSSG inactivation of
Ca2+- and PS-dependent PKC but instead reduced
the IC50 of GSSG estimated by graphical analysis to 1 mM (Fig. 5). Similarly, the
presence of 1 mM CaCl2 and 30 µg/ml PS in
PKC/GSH preincubation mixtures reduced the IC50 of GSH to
about 3.8 mM (data not shown). Histone also failed to
afford substantial protection against GSH inactivation of PKC. The
percentage of inactivation of Ca2+- and
PS-dependent PKC achieved by preincubation with 10 mM GSH in the absence of histone (100 ± 1%) was
negligibly affected by including histone III-S in the preincubation
mixtures at concentrations of 0.5 mg/ml (100 ± 1% inactivation),
1.0 mg/ml (93 ± 4% inactivation), and 1.5 mg/ml (89 ± 5%
inactivation). As seen in Table III, a comparison of the inactivation
of recombinant PKC-
achieved by 10 mM GSH when 3 mM MgATP was present (87 ± 11% inactivation) or
absent (97 ± 4% inactivation) in PKC-
/GSH preincubation
mixtures (which were extensively dialyzed to remove excess nucleotide
prior to dilution into histone kinase assay mixtures) indicates that
MgATP affords little protection against GSH inactivation of PKC.

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Fig. 5.
Irreversible inactivation of Ca2+
and phosphatidylserine (CaPS)-activated PKC by GSSG.
Shown is the % inactivation of purified rat brain PKC achieved by
preincubation of the enzyme with GSSG at the concentrations indicated
in the presence of the activating cofactors 1 mM
CaCl2 and 30 µg/ml PS. Preincubation mixtures were
prepared and diluted into histone kinase assay mixtures as described in
the legend to Fig. 1. 100% activity was 1.13 ± 0.05 pmol of
32P transferred per min.
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Having determined that preincubation of PKC with glutathiones (GSH,
GSSG, and GSO3) and the direct addition of the glutathiones to PKC assay mixtures at physiological GSH concentrations (5-10 mM) both potently antagonize the enzyme (Figs. 1, 2, and
4), we next asked the question of whether PKC antagonism by
glutathiones reflected selective interactions between PKC and the
tripeptides or whether, on the other hand, any tripeptide at a
concentration of 10 mM could nonspecifically antagonize PKC
activity under the same conditions. Of the three tripeptides that we
analyzed,
-Glu-Gly-Gly bore the most structural resemblance to GSH,
and it effected >90% antagonism of PKC activity at a concentration of
10 mM, whether the peptide was preincubated with PKC or
directly added to PKC assays (Table I).
In contrast, the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly were related
to GSH only by virtue of a C-terminal Gly residue. These peptides had
negligible effects on PKC activity (<10% antagonism), whether
preincubated with the enzyme or directly added to PKC assay mixtures at
a concentration of 10 mM (Table I). These results clearly
show that GSH antagonism of PKC cannot be ascribed to nonspecific
effects of millimolar concentrations of a small peptide on the purified
enzyme. We also analyzed the ability of amino acid constituents of GSH
to antagonize PKC. At a concentration of 10 mM, Glu and Gly
had little or no effect on PKC activity, whereas
N-acetyl-Cys resembled GSH in that it potently inactivated
PKC in a DTT-insensitive manner (Table I). The >90% inactivation of
PKC by 10 mM
-Glu-Gly-Gly and 10 mM N-acetyl-Cys (± 100 mM DTT) provides evidence
that distinct structural features of GSH may contribute to its ability
to antagonize PKC.
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Table I
Effects of glutathione-related tripeptides and amino acid constituents
of glutathione on the Ca2+- and PS-dependent
histone kinase activity of rat brain PKC
Results represent an average of two independent experiments done in
triplicate.
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The results shown in Table II demonstrate
that purified recombinant human PKC-
(38) is subject to inactivation
by GSH and oxidized GSH derivatives. PKC-
was inactivated by
preincubation with GSSG, GSSG + DTT, GSH, and GSO3, and in
each case, the inactivated form of PKC-
was stable following a
12-fold dilution (Table II), providing evidence for an irreversible
inactivation mechanism. Comparable inactivation of PKC-
was achieved
by GSH and the oxidized GSH derivatives whether histone III-S (Table
II) or the synthetic peptide RKRTLRRL (data not shown) was employed as
the phosphoacceptor substrate. Kinetic analysis of the inactivation of
PKC-
by 5 mM GSH indicated that the inactivation
mechanism was time-dependent and obeyed pseudo-first order
kinetics (Fig. 6). The pseudo-first order
rate constant kobs, which is the slope of the
linear plot (Fig. 6) (39), was determined to be 0.43 ± 0.02 min
1 by averaging the values obtained from two kinetic
analyses.
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Table II
Inactivation of PKC- by glutathione and oxidized glutathione
derivatives
PKC- was preincubated under the conditions indicated for 5 min at
30 °C and then diluted 12-fold into histone kinase assay mixtures.
Each assay mixture contained 100 ng of PKC- . Each % inactivation
value shown is the mean value from two experiments done in triplicate.
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Fig. 6.
Kinetics of inactivation. The kinetics
of GSH inactivation of PKC- were analyzed by preincubating PKC-
with 5 mM GSH at 30 °C for the time periods indicated.
Preincubation mixtures with and without GSH were analyzed in parallel.
Preincubated mixtures were immediately diluted 12-fold into histone
kinase reaction mixtures on ice, and PKC- activity was measured as
described under "Materials and Methods." Control values ( ) show
the PKC- activity remaining after preincubation in the absence of
GSH for the indicated time period expressed as a percentage of the
activity observed prior to preincubation (time = 0 min). shows
the time-dependent loss of PKC- activity induced by GSH;
each closed circle denotes the PKC- activity remaining
after preincubation with GSH for the indicated time period expressed as
a percentage of the control value observed at the same time period
(linear correlation coefficient = 0.9945).
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As a more rigorous test of the irreversibility of GSX (GSH, GSSG, or
GSO3)-mediated inactivation of PKC-
, we determined the stability of the GSX-inactivated form of PKC-
to dialysis. We compared the postdialysis recovery of PKC-
activity and PKC-
protein from samples of GSX-inactivated PKC-
and mock-inactivated (control) PKC-
subjected to dialysis for 8-10 h against Tris-HCl buffer, pH 7.5, in the presence of 10 or 25 mM DTT. Based
on Western analysis with a PKC-
mAb, the postdialysis recovery of
PKC-
protein from GSX-inactivated PKC-
and control PKC-
was
virtually identical, and the electrophoretic mobility of the isozyme
was unchanged whether PKC-
was inactivated by GSSG, GSH, or
GSO3 (Fig. 7). Comparison of
the PKC-
activity recovered postdialysis from GSX-inactivated
PKC-
versus control PKC-
indicated that each of the
GSX-inactivated PKC-
species (GSSG-, GSH-, or
GSO3-inactivated PKC-
) remained fully inactivated
following dialysis (Table III). These
results firmly establish the irreversibility of the inactivation of
PKC-
by GSH and oxidized GSH derivatives.

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Fig. 7.
Postdialysis immunoblot analysis of
GSX-inactivated PKC- . Recombinant PKC- (10 µg) was
inactivated by incubation with 10 mM GSH, 5 mM
GSSG, or 10 mM GSO3 for 5 min at 30 °C in 20 mM Tris-HCl, pH 7.5, 10% glycerol; control PKC- was
mock-inactivated by incubation as described above but in the absence of
GSX. PKC- incubation mixtures (1 ml) were dialyzed for 8-10 h at
4 °C against 500 ml of 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.25 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin,
plus either 10 or 25 mM DTT as shown. Western analysis of
dialyzed GSX-inactivated PKC- samples (+) and dialyzed control
PKC- samples ( ) is shown. The samples employed in the Western
analysis shown here were also employed in the analysis of catalytic
activity shown in Table III. Sample volumes were first equalized by
adjusting for slight discrepancies (<5%) with the addition of buffer.
For Western analysis, a 20-µl aliquot of each adjusted sample was
prepared in SDS-polyacrylamide gel electrophoresis sample buffer (1:1)
and then loaded onto the gel. Anti-PKC- mAb (0.05 µg/ml) was
employed as the primary Ab, horseradish peroxidase-linked sheep
anti-mouse Ig (1:300 dilution) was the secondary Ab, and immunoreactive
bands were detected by enhanced chemiluminescence. The region of the
blots spanning 45-116 kDa is shown for each GSX +/ paired sample;
PKC- is indicated by an arrow at 82 kDa.
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Table III
The stability of the GSSG/GSH/GSO3-inactivated form of PKC-
under dialysis
GSX-inactivated PKC- and mock-inactivated control PKC- were
dialyzed for 8-10 h under identical conditions against Tris-HCl buffer
containing either 10 or 25 mM DTT. For dialysis buffer
components and other experimental details, see the legend to Fig. 7.
The postdialysis % inactivation was calculated by expressing the
activity recovered from the GSX-inactivated PKC- sample after
dialysis as a percentage of the activity recovered postdialysis from
mock-inactivated PKC- . The recovery of PKC- protein was
determined to be equivalent for GSX-inactivated and
mock-inactivated PKC- by Western analysis (see Fig. 7).
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To determine whether GSH-inactivated PKC-
was covalently modified by
GSH, we incubated PKC-
with [3H]GSH under conditions
that achieve full inactivation (>95%) of the isozyme (Table III).
After a 5-min incubation of PKC-
(10 µg) and 10 mM
[3H]GSH (30.0 cpm/pmol) at 30 °C, the incubation
mixture (320 µl) was dialyzed extensively against 20 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10%
glycerol, 50 mM DTT, 10 µg/ml leupeptin, and 0.25 mM phenylmethylsulfonyl fluoride (500 ml/cycle; 3 cycles of > 8 h each). Incubation mixtures containing PKC-
alone
(320 µl) and [3H]GSH alone (320 µl) were dialyzed in
parallel in the same vessel. The results reported below are averaged
values based on two independent experiments. Western analysis and
densitometry of PKC-
-containing samples postdialysis indicated that
the recovery of PKC-
was >95% in each case (data not shown). We
found that the dialyzed [3H]GSH-inactivated PKC-
sample retained only 0.16 ± 0.06 pmol of
[3H]GSH/pmol of PKC-
. The sample containing PKC-
alone did not contain the radiolabel (0.00 ± 0.01 pmol of
[3H]GSH/pmol of PKC-
). The sample containing
[3H]GSH alone retained residual amounts of
[3H]GSH postdialysis (0.032 ± 0.006 pmol of
[3H]GSH/unit volume equivalent to 1 pmol of PKC-
).
Thus, limited exchangeability of [3H]GSH in this system
could account for about 20% of the radiolabel retained by the
[3H]-GSH-inactivated PKC-
sample. However, even if the
contribution of limited exchangeability is not factored in, the
stoichiometry of [3H]GSH associated with fully
inactivated PKC-
postdialysis is less than 0.2 GSH/PKC-
. This
clearly indicates that neither covalent modification of PKC-
by 10 mM GSH nor other irreversible binding mechanisms can
account for the fully inactivated state of the isozyme. The conclusion
that GSH inactivation of PKC-
does not entail glutathiolation of the
isozyme was confirmed by mass spectrometric analysis of control and
GSH-inactivated PKC-
. Monoglutathiolation of a protein increases the
MW by 305 Da, and phosphorylation produces an increase of 80 Da.
Control and GSH-inactivated PKC-
each contained a major species with
a molecular mass that corresponded (± 20 Da) to monophosphorylated
PKC-
(predicted molecular mass, 76,843 Da (40); control PKC-
molecular mass, 76,832 Da; GSH-inactivated PKC-
molecular mass,
76,838 Da) and a minor species with a molecular mass that corresponded
to diphosphorylated PKC-
(predicted molecular mass, 76, 923 Da (40);
control PKC-
molecular mass, 76,922 Da; GSH-inactivated PKC-
molecular mass, 76,924 Da). These results confirm that GSH-inactivated
PKC-
is not glutathiolated, and they provide evidence that
GSH-inactivated PKC-
does not differ from control PKC-
in its
phosphorylation state.
Having established that GSH inactivation of PKC-
was stable
following extensive dialysis and that inactivated PKC-
did not contain covalently attached or tightly bound GSH, we next addressed the
question of whether the inactivation was associated with a conformational change in PKC-
. The susceptibility of PKC isozymes to
limited trypsinolysis serves as an indicator of conformational changes
that involve alterations in the exposure of the hinge region (8, 41,
42). Fig. 8 shows a comparison of the
sensitivity of control PKC-
(A) and GSH-inactivated
PKC-
(B) to limited trypsinolysis. GSH-inactivated and
control PKC-
(15 µg each) were prepared and then dialyzed for
removal of GSH from the inactivated PKC-
sample, as described in the
legend to Fig. 7. Following dialysis, the GSH-inactivated PKC-
sample was determined to be fully inactivated (percentage of
inactivation = 98 ± 2%). The dialyzed samples were
subjected to limited trypsinolysis under identical conditions.
Trypsinolysis was measured by Western analysis of the samples (18) with
a monoclonal antibody that recognizes PKC-
and its catalytic domain
fragment (Upstate Biotechnology, Inc.). Fig. 8A shows that
in the control PKC-
sample, the 82-kDa intact PKC-
species
(lane 1) persisted following incubation with 1× (lane
2), 2× (lane 3), and 4× (lane 4) units of
trypsin/ml. In contrast, Fig. 8B shows that under identical
conditions, the GSH-inactivated 82-kDa PKC-
species virtually
disappeared after limited proteolysis by 2× (lane 3) and
4× (lane 4) units of trypsin/ml. The results shown in Fig.
8 were reproduced in two additional experiments (data not shown). The
fact that limited trypsinolysis generated a 50-kDa catalytic domain
fragment (18) from both control (Fig. 8A) and
GSH-inactivated (Fig. 8B) PKC-
indicates that the hinge
region remains a major target of trypsinolysis of PKC-
following GSH
inactivation. The results also indicate that GSH-inactivated PKC-
is
more sensitive than control PKC-
to trypsinolysis, providing
evidence that GSH inactivation of PKC-
is accompanied by either a
stable conformational change in the isozyme or a destabilization of the
isozyme structure that persists even after removal of GSH by prolonged
dialysis.

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Fig. 8.
Limited trypsinolysis of GSH-inactivated
PKC- . Control (A) and GSH-inactivated (B)
PKC- were prepared and dialyzed as described in the legend to Fig.
7. 10 mM GSH was employed for inactivation of PKC-
(98 ± 1% and 98 ± 2% inactivation were observed pre- and
postdialysis, respectively), and the dialysis buffer contained 10 mM DTT (for other buffer components, see the legend to Fig.
7). Trypsinolysis was done by incubating 1.5 µg of PKC- (control
or GSH-inactivated) with 0 (lane 1), 325 (lane
2), 650 (lane 3), or 1300 (lane 4) units/ml
trypsin for 30 min on ice, and the reaction was terminated with an
equal volume of 2× SDS-polyacrylamide gel electrophoresis sample
buffer. PKC- (the band indicated at 82 kDa) and the catalytic domain
fragment of PKC- (the band indicated at 50 kDa) were detected by
Western analysis of the samples (100 ng of sample protein/lane) with a
PKC- mAb (Upstate Biotechnology, Inc.) using enhanced
chemiluminescence.
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N-Acetylcysteine is a precursor of cellular GSH that is
readily taken up by mammalian cells and incorporated into GSH (25, 26).
Because GSH and N-acetylcysteine inactivated purified
Ca2+- and PS-dependent PKC isozymes similarly,
treatment of cells with N-acetylcysteine afforded a
convenient approach to assess whether
GSH/N-acetylcysteine-mediated PKC inactivation could also occur in mammalian cells. Fig. 9 shows
that when cultured rat R6-PKC3 fibroblasts were treated with 40 mM N-acetylcysteine for 30 min at 37 °C, the
level of extractable Ca2+- and PS-dependent PKC
activity in the cells was reduced by 53 ± 3%. Similarly, the
level of Ca2+- and PS-dependent PKC activity in
cultured human breast cancer MCF7-MDR cells declined 64 ± 3% in
response to a 30 min treatment with 50 mM
N-acetylcysteine at 37 °C (Fig. 9). We determined by Western analysis that the N-acetylcysteine treatment of
R6-PKC3 and MCF7-MDR cells shown in Fig. 9 did not change the level of expression of the Ca2+- and PS-dependent PKC
isozymes (
,
, and
) in the cells (experimental error,
10%)
(data not shown). We also measured the intracellular level of
GSH/N-acetylcysteine in each cell line prior to and at the
end of the treatment period indicated in Fig. 9, by an established method that subjects deproteinized cell lysates to a
5,5'-dithiobis-(2-nitrobenzoic acid) colorimetric assay (43). In
R6-PKC3 fibroblasts, prior to treatment, the GSH level was 4.03 ± 0.23 nmol/106 cells, and subsequent to treatment, the
GSH/N-acetylcysteine level was 10.60 ± 0.33 nmol/106 cells. In MCF7-MDR cells, the initial GSH level
was 4.24 ± 0.37 nmol/106 cells, and the
GSH/N-acetylcysteine level measured at the end of the
treatment period was 12.24 ± 0.04 nmol/106 cells.
These results provide evidence that a 3-fold increase in the
intracellular level of GSH may be sufficient to induce a marked
inactivation of Ca2+- and PS-dependent PKC
isozymes in mammalian cells.

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Fig. 9.
Effect of N-acetylcysteine
treatment on cellular PKC activity. MCF7-MDR and R6-PKC3 cells
were treated with N-acetylcysteine for 30 min at 37 °C,
and PKC activity was extracted from the cells and assayed with the
PKC- pseudosubstrate-based synthetic peptide substrate
[Ser25]PKC (19-31) as described under "Materials and
Methods." Ca2+- and PS-dependent PKC
activity is shown as filled bars, and the basal activity of
the PKC preparation observed in the absence of stimulatory cofactors is
shown as hatched bars. pmol/min indicates pmol of
32P transferred to [Ser25]PKC (19-31) per
min by 10 µg of DEAE-extracted protein (see "Materials and
Methods" for assay conditions). Each bar represents the
average value ± SD obtained from assays done in triplicate. The
results shown were reproduced in several independent experiments.
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DISCUSSION |
The oxidative modification of select proteins by oxidized GSH,
i.e. protein S-glutathiolation, has been shown to
serve as a reversible mechanism of regulation of several enzymes,
including carbonic anhydrase III and aldose reductase (21, 22). Liu and
Hannun (24) recently reported that at physiological concentrations, GSH
reversibly inhibits isolated neutral magnesium-dependent
sphingomyelinase by a nonredox mechanism but does not antagonize acidic
sphingomyelinase. We report here that Ca2+- and
PS-dependent PKC activity is also subject to antagonism by
GSH by a nonredox mechanism, but in the case of PKC, the antagonism is
irreversible. Incubation with GSH at physiological concentrations fully
inactivated PKC, whereas it achieved less than 25% loss of casein
kinase 2 activity, indicating that potent inactivation by GSH is not a
general property of purified Ser/Thr protein kinases. Our evidence for
a non-oxidative/reductive irreversible mechanism of PKC inactivation by
GSH at physiological concentrations can be summarized as follows. GSSG
antagonized the Ca2+- and PS-dependent activity
of purified rat brain PKC with the same efficacy (IC50 = 3 mM) whether or not the reductant DTT (
100 mM)
was present, and 2 equivalents of GSH antagonized PKC to the same
extent as 1 equivalent of GSSG in the presence of DTT.
GSO3, which is distinguished from GSSG and GSH by its
inability to undergo disulfide/thiol exchange reactions, was as
effective as GSSG in antagonizing Ca2+- and
PS-dependent rat brain PKC catalysis. Purified recombinant PKC-
activity was likewise subject to antagonism by GSH, GSSG, and
GSO3. An irreversible mechanism of PKC inactivation by GSX (GSH, GSSG, or GSO3) was demonstrated by the stability of
the inactivated form of PKC to dilution into PKC assay mixtures and to
extensive dialysis.
The fact that GSH inactivation of PKC-
obeyed pseudo-first order
kinetics indicates that the observed inactivation cannot be attributed
to GSH-induced random unfolding and/or aggregation of PKC-
, because
random protein unfolding (denaturation) and aggregation do not obey
first-order kinetics (44). In addition, the production of a 50-kDa
catalytic domain fragment (18) from fully inactivated PKC-
by
limited trypsinolysis corroborates the kinetic evidence against an
inactivation mechanism involving random unfolding (denaturation) of
PKC, because it shows that the hinge region of PKC-
continues to
serve as a preferential target of trypsinolysis even after irreversible
inactivation of the isozyme by GSH. An independent line of evidence
that GSH inactivation of PKC involves specific interactions between the
enzyme and the tripeptide is provided by the observation that PKC is
also potently inactivated by the closely related tripeptide
-Glu-Gly-Gly but not by the amino acids Glu and Gly or the distantly
related tripeptides Tyr-Gly-Gly and Gly-Ala-Gly. GSH and
N-acetyl-Cys both induced DTT-insensitive PKC inactivation.
The potent inactivation of PKC by
-Glu-Gly-Gly and N-acetyl-Cys
suggests that at least two structural features of GSH represented by
-Glu-X-Gly and X-Cys-X contribute to its ability to inactivate PKC.
We determined that GSH-inactivated PKC-
did not contain covalently
bound GSH by two experimental approaches, mass spectrometric analysis
of the inactivated isozyme and quantitation of radiolabel irreversibly
bound to [3H]GSH-inactivated PKC-
. The latter approach
also demonstrated that the inactivated form of PKC-
does not contain
tightly associated, reversibly bound GSH. In other words, the
GSH-inactivated state of PKC-
persists in the absence of bound GSH.
Proteolytic sensitivity of PKC can be used as a probe of the
conformation of the enzyme (8, 41, 42). Our observation that the
GSH-inactivated form of PKC-
is much more sensitive than control
PKC-
to the proteolytic action of trypsin provides evidence that the
inactivation mechanism involves either the induction of a stable
conformational change in the isozyme that exposes the hinge region or
the conversion of the isozyme structure to a destabilized state with
increased conformational flexibility at the hinge region. Physical
studies of the solution structures of native versus
GSH-inactivated PKC-
will be required to distinguish between these
two possibilities.
Comparable inactivation of intact PKC and a proteolytically derived
catalytic domain fragment of the enzyme by GSH, GSSG, and
GSO3 indicated that the inactivation mechanism was
catalytic domain-directed. The protection against GSX inactivation of
PKC offered by the substrates MgATP and histone was negligible, arguing against an active site-directed mechanism. In addition, the activating cofactors Ca2+ and PS failed to protect PKC against
GSX-mediated inactivation, providing evidence that both resting and
activated conformations of PKC are susceptible to GSX inactivation.
Although the close homology among the catalytic domains of PKC isozymes
(15) would suggest that all PKC isozymes may be subject to GSH
inactivation, the observations reported here are confined to GSH
inactivation of isozymes in the Ca2+- and
PS-dependent PKC subfamily, and the question of whether isozymes in the Ca2+-independent PKC subfamilies are
subject to GSX inactivation remains to be addressed.
Because N-acetylcysteine is a GSH precursor that is readily
taken up by cells, N-acetylcysteine treatment is commonly
used as a means of increasing the intracellular GSH pool (25, 26). N-Acetylcysteine itself also functions effectively in cells
as a free radical scavenger and reducing agent (25, 26). In this report, we show that GSH and N-acetylcysteine irreversibly
inactivate purified PKC isozymes by a nonredox mechanism with similar
efficacy, and we report that brief treatment of cultured rat
fibroblasts and human cancer cells with N-acetylcysteine
results in a sharp decline in the level of Ca2+- and
PS-dependent PKC activity in the cells, according to
assays of extracts prepared from the cells. These results provide
evidence that the mechanism of PKC inactivation by
GSH/N-acetylcysteine in a purified enzyme system may
also be operative in mammalian cells. Physiological stimuli,
e.g. transforming growth factor
1, tumor
necrosis factor, and growth factor withdrawal, have been reported to
increase the GSH level in mammalian cells by about 1.5-3.5-fold
(45-47). We observed a marked inactivation of Ca2+- and
PS-dependent PKC isozymes when cells were exposed to
N-acetylcysteine under conditions that induced a GSH + N-acetylcysteine level that exceeded the original GSH level
by about 3-fold. Thus, our results suggest that the induction of
increased GSH levels in mammalian cells by physiological stimuli may in
some cases be associated with GSH-induced inactivation of PKC isozymes.
Just how GSH-mediated PKC inactivation may regulate the enzymatic
activity in cells is currently under investigation.
The importance of PKC as a molecular target in phorbol ester-mediated
tumor promotion is well documented (1), but it is not yet clear whether
PKC also plays an important role in oxidant-mediated tumor promotion
(48). Based on the evidence reported here that GSH can antagonize PKC,
we hypothesize that depletion of the intracellular GSH pool and the
consequential loss of a negative regulatory mechanism over PKC isozymes
may contribute to the tumor-promoting action of oxidants in
nontransformed cells.