Cleavage of
PKC but Not
/
PKC by Caspase-3 during
UV-induced Apoptosis*
Sonia
Frutos,
Jorge
Moscat, and
María T.
Diaz-Meco
From the Laboratorio Glaxo Wellcome-CSIC de Biología
Molecular y Celular, Centro de Biología Molecular "Severo
Ochoa" (Consejo Superior de Investigaciones
Científicas-Universidad Autónoma de Madrid), Universidad
Autónoma, Canto Blanco, 28049 Madrid, Spain
 |
ABSTRACT |
The stimulation of caspases is a critical event
in apoptotic cell death. Several kinases critically involved in
cell proliferation pathways have been shown to be cleaved by
caspase-mediated mechanisms. Thus, the degradation of
protein
kinase C (PKC) and MEKK-1 by caspase-3 generates activated fragments
corresponding to their catalytic domains, consistent with the
observations that both enzymes are important for apoptosis. In
contrast, other kinases reported to have anti-apoptotic properties,
such as Raf-1 and Akt, are inactivated by proteolytic degradation by
the caspase system. Since the atypical PKCs have been shown to play
critical roles in cell survival, in the study reported here we have
addressed the potential degradation of these PKCs by the caspase system in UV-irradiated HeLa cells. Herein we show that although
PKC and
/
PKC are both inhibited in UV-treated cells, only
PKC but not
/
PKC is cleaved by a caspase-mediated process. This cleavage generates a fragment that corresponds to its catalytic domain that is
enzymatically inactive. The sequence where caspase-3 cleaves
PKC was
mapped, and a mutant resistant to degradation was shown to protect
cells from apoptosis more efficiently than the wild-type enzyme.
 |
INTRODUCTION |
The two members of the atypical protein kinase C
(PKC)1 subfamily of isozymes
(aPKCs), namely
PKC and
/
PKC, have recently been shown to be
involved in a number of important cellular functions including cell
proliferation and survival (1-5). The mechanisms whereby the aPKCs
control these functions most probably involve their ability to regulate
ERK-AP1 (6-11) and NF-
B (10, 12-18) signaling pathways, both
important intermediaries in the cascades controlling cell growth and
apoptosis (2, 19-26). Contrary to the other PKCs, the atypicals are
insensitive to diacylglycerol and
12-O-tetradecanoylphorbol-13-acetate but have been proposed to be regulated by ceramide (18, 27, 28), phosphatidylinositol 3-kinase
(10, 29-34), and Ras (8, 35, 36). In addition, the aPKCs selectively
bind to, and are regulated, by two novel proteins (LIP and Par-4) which
seem to be critical components of pathways controlling cell growth and
survival. Thus, LIP (for
/
PKC-interacting protein) potently
induces NF-
B in a
/
PKC-dependent manner (14),
whereas Par-4 is a potent inhibitor of the atypical PKC enzymatic
activity and, interestingly, was initially identified by differential
screening in cells that were undergoing growth arrest and apoptosis (3,
37). Consistently, the ectopic expression of Par-4 in NIH-3T3 cells
induces apoptosis in a manner that is dependent on its ability to block
the atypical PKCs (2, 3). Recent studies demonstrate that the
expression of Par-4 sensitizes prostate cancer and melanoma cells to
apoptotic stimuli (38), as well as that it may be a mediator of
neuronal apoptosis (39). Overexpression of the atypical PKCs inhibits
UV- and drug-induced apoptosis in NIH-3T3 (2, 3) and human leukemia
cells (5), respectively. Taken together, these results indicate that
the aPKCs are potent anti-apoptotic kinases that can be inactivated by
pro-apoptotic regulators such as Par-4.
The activation of the caspase system is a critical event in apoptosis
(40-42), and recent studies demonstrate that a number of signal
transduction kinases are subjected to a caspase-mediated breakdown
(43). Early work reported that the degradation of
PKC by caspase-3
generates an active fragment corresponding to its catalytic domain
(44), suggesting that
PKC activity may be important during apoptotic
cell death (45). Consistently, inhibition of
PKC impairs UV-induced
apoptosis in keratinocytes (46). MEKK-1 is also cleaved during
apoptosis by anoikis (47), Fas ligation (48), and genotoxic stress
(49). In all three cases, the caspase-mediated degradation of MEKK-1
produced a catalytically active fragment, in keeping with the
pro-apoptotic properties of MEKK-1 (47-49). PKN is another example of
a kinase degraded by the caspase system producing a catalytically
active fragment (50). However, other kinases, such as Raf-1 and Akt are
inactivated by proteolytic degradation by the caspase system during
apoptosis (43). This is particularly relevant because both Raf-1 and
Akt are anti-apoptotic kinases (50-56). Therefore, a model is emerging whereby the activation of caspases in apoptotic cells leads to the
cleavage of different kinases with opposite outcomes, depending on
whether the given kinase plays a pro- or an anti-apoptotic role.
Because the aPKCs control pro-survival signals, in the study reported
here we have addressed the potential degradation of the atypical PKCs
by the caspase system in HeLa cells exposed to UV irradiation.
 |
MATERIALS AND METHODS |
Plasmids--
pCDNA3-
PKCmyc containing the
full-length rat
PKC with the Myc tag at the COOH terminus was
obtained by polymerase chain reaction. pCDNA3-Myc-
/
PKC and
pCDNA3-GST-
PKCDEL (residues 240-592) constructs
were tagged with the Myc epitope or with the GST protein at the amino
terminus, respectively. The corresponding fragments excised from
pCDNA3-HA-
/
PKC and pCDNA3-HA-
PKCDEL,
respectively (6), were subcloned into pCDNA3-Myc or pCDNA3-GST. The
PKC caspase mutants were obtained by site-directed mutagenesis (Quick change, Stratagene):
PKC1, D200G;
PKC2, D210G;
PKC3, D239G;
PKC1/2, DD200/210GG;
PKC2/3, DD210/239GG;
PKC1/2/3, DDD200/210/239GGG.
Cell Culture, Transfections, and Reagents--
HeLa cells were
maintained in high glucose Dulbecco's modified Eagle's medium
containing 10% fetal calf serum, 100 µg/ml penicillin G, and 100 µg/ml streptomycin (FLOW). Subconfluent cells were transfected by the
calcium phosphate method (CLONTECH, Inc.). Some
experiments were performed in the presence of the ICE inhibitors YVAD-CHO, DEVD-CHO, or z-VAD-fmk (Bachem, United Kingdom). For UV
treatment, culture medium was removed, dishes were washed once with
phosphate-buffered saline, UVC irradiated (180 J/m2), and
fresh medium was added to the cells. The monoclonal anti-
/
PKC was
from Transduction Laboratories. Anti-Myc epitope (9E10) antibody was
from UBI.
aPKC Activity--
HeLa cells incubated at different times after
UV light exposure were extracted with lysis buffer (50 mM
Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM EGTA, and protease inhibitors) and immunoprecipitated with a monoclonal anti-
/
PKC.
Immunoprecipitates were washed seven times with lysis buffer containing
0.5 M NaCl. For in vitro kinase assay,
immunocomplexes were incubated with 1 µg of myelin basic protein and
5-10 µCi (100 µM) of [
-32P]ATP in
kinase buffer (35 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 0.5 mM EGTA, 0.1 mM
CaCl2, 1 mM phenyl phosphate) for 30 min at
30 °C in a final volume of 20 µl. Reactions were stopped by the
addition of concentrated sample buffer. Samples were boiled for 3 min
and separated by SDS-polyacrylamide gel electrophoresis (PAGE) followed by exposure and quantitation in an InstantImager (Packard). The activity of transfected Myc-
PKC was determined in immunoprecipitates with an anti-Myc antibody (9E10, UBI) as described above.
In Vitro Protease Assays--
Wild-type
PKC and the different
mutants were translated in vitro using a coupled
transcription and translation system with T7 polymerase (Promega,
Madison, WI). Apoptotic cells extract was prepared as follows. HeLa
cells were UV-irradiated for 10 h, washed once in ice-cold
phosphate-buffered saline, and resuspended at 2 × 108/ml in lysis buffer (25 mM HEPES, pH 7.5, 5 mM EDTA, 2 mM dithiothreitol, 0.1% CHAPS, 10%
sucrose, and 1 mM phenylmethylsulfonyl fluoride). The
lysate was then subjected to four freeze-thaw cycles prior to
centrifugation at 10,000 × g for 10 min. Recombinant
caspase-3 was from Pharmingen. Cell extracts, in the absence or
presence of caspase inhibitors (10 nM; YVAD-CHO or
DEVD-CHO), or purified caspase-3 were incubated for 1 h at
37 °C with 5 µl of 35S-labeled in vitro
translated wild-type or mutant
PKC. The reaction was stopped by
addition of Laemmli sample buffer and subjected to SDS-PAGE prior to
drying and exposure in an InstantImager (Packard).
Apoptosis Assays--
-Galactosidase co-transfection assays
for determination of cell death were performed as described (2, 3).
TUNEL analysis was performed using the "in situ cell death
detection kit" (Boehringer Mannheim).
 |
RESULTS |
We initially determined the effect of UV irradiation on the
activity and potential cleavage of
/
PKC and
PKC during
apoptosis. HeLa cells were UV-irradiated for different times, after
which endogenous
/
PKC was immunoprecipitated and its activity was determined. Parallel extracts were analyzed by immunoblot. Results of
Fig. 1A demonstrate that short
exposures (5-20 min) to UV irradiation provokes a measurable
activation of
/
PKC activity. However, longer treatments leads to
a robust reduction of
/
PKC activity to below basal levels, that
is detectable at 2 h, and maximal at 4-20 h. Immunoblot analysis
reveals that the protein levels of
/
PKC remain unchanged at all
the time points measured. Identical results were obtained when this
experiment was performed in HeLa cells that have been transfected with
a Myc-tagged
/
PKC construct and in which the activity and levels
of the enzyme were determined with an anti-Myc antibody (not shown).
Because there are not reliable antibodies selective for the
PKC
isoform, we next transfected HeLa cells with a Myc-tagged version of
PKC after which cells were UV-irradiated as above. Results of Fig.
1B show that, in contrast to
/
PKC, short-term
irradiation does not affect
PKC activity. However, longer exposures
to UV light produces a dramatic reduction on
PKC activity that
precedes its proteolytic degradation that produces a fragment of
approximately 50 kDa. Because this
PKC construct was Myc-tagged at
the COOH terminus, this fragment must include the catalytic domain.
Therefore, the anti-Myc immunoprecipitates contained not only the
full-length protein but also the fragments generated by UV irradiation
(not shown). The fact that the enzymatic activity is reduced in
UV-irradiated cells indicates that the
PKC 50-kDa fragment generated
by UV irradiation is catalytically inactive. TUNEL analysis of parallel
cultures indicated that apoptosis becomes detectable at 10 h. At
this time point, 12 ± 2% of the cells showed signs of DNA
fragmentation. At 20 h, the number of apoptotic cells increased to
30 ± 10%, whereas at 40 h, virtually all cells were
apoptotic.

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Fig. 1.
Activity and levels of the atypical PKC
isotypes in UV-irradiated cells. A, HeLa cell extracts were
prepared at different times following UV-irradiation, after which the
levels of / PKC were determined by immunoblot with a monoclonal
/ PKC-specific antibody (upper panel). This is a
representative experiment of three with identical results. Parallel
extracts were immunoprecipitated with the same antibody, and the
activity of / PKC was determined as described under "Materials
and Methods" (lower panel). Results are the mean ± S.D. of four independent experiments including the one shown in the
upper panel. B, extracts from HeLa cells transfected with a
COOH-terminal Myc-tagged version of PKC were prepared at different
times following UV irradiation, after which the levels of PKC were
determined by immunoblot with a monoclonal anti-Myc antibody
(upper panel). This is a representative experiment of three
with very similar results. Parallel extracts were immunoprecipitated
with the same antibody, and the activity of PKC was determined as
described under "Materials and Methods" (lower panel).
Results are the mean ± S.D. of four independent experiments
including the one shown in the upper panel.
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|
In the next series of experiments the involvement of the caspase system
in the degradation of
PKC was determined. Thus, HeLa cells
transfected with Myc-tagged
PKC were UV-irradiated either in the
absence or presence of z-VAD, a broad-specific inhibitor of caspases,
and the activity and degradation of
PKC was determined. Results of
Fig. 2A demonstrate that the
presence of z-VAD completely abrogated the UV-induced cleavage of
PKC with little or no effect on its inhibition. Of note, the
presence of z-VAD did not affect the changes in activity of
/
PKC
produced by UV irradiation (Fig. 2B). Collectively these
results indicate that
PKC is degraded by a caspase-mediated process
during UV irradiation but that its inhibition is
caspase-independent.

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Fig. 2.
Effect of z-VAD on the inhibition of the
atypical PKC activity by UV irradiation. A, HeLa cells
transfected with the COOH-terminal Myc-tagged version of PKC were
incubated or not with z-VAD prior to UV irradiation. Afterward cell
extracts were prepared at different times following UV irradiation and
the levels of PKC were determined by immunoblot with the monoclonal
anti-Myc antibody (upper panel). This is a representative
experiment of three with very similar results. Parallel extracts were
immunoprecipitated with the same antibody, and the activity of PKC
was determined as described under "Materials and Methods"
(lower panel). Results are the mean ± S.D. of four
independent experiments including the one shown in the upper
panel. HeLa cells were incubated or not with z-VAD prior to UV
irradiation. Afterward cell extracts were prepared at different times
following UV irradiation and the levels of / PKC were determined
by immunoblot with the monoclonal anti- / PKC antibody (upper
panel). This is a representative experiment of three with very
similar results. Parallel extracts were immunoprecipitated with the
same antibody, and the activity of / PKC was determined as
described under "Materials and Methods" (lower panel).
Results are the mean ± S.D. of four independent experiments
including the one shown in the upper panel.
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|
To further explore the possible breakdown of
PKC by caspases,
35S-labeled in vitro translated
PKC or
/
PKC were incubated in vitro with extracts from cell
cultures that were either untreated or exposed to UV light for 10 h. Interestingly, a fragment of identical size to that produced
in vivo and most probably corresponding to the catalytic
domain of the enzyme was observed when
PKC but not
/
PKC was
incubated with extracts from UV-irradiated cells (Fig.
3A). In addition, another
fragment of about 20-30 kDa was also detected which most probably
corresponds to the regulatory domain of
PKC (Fig. 3A).
The cleavage of
PKC is prevented by the incubation with z-VAD or
DEVD, but not by YVAD (Fig. 3B). Because DEVD is relatively
specific for caspase-3 whereas YVAD is for caspase-1, these results
strongly suggest that
PKC may be a substrate of caspase-3. This is
confirmed in the results of Fig. 3C demonstrating that
recombinant caspase-3 efficiently cleaves
PKC but not
/
PKC
in vitro.

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Fig. 3.
Caspase-mediated breakdown of
PKC in vitro.
35S-Labeled in vitro translated PKC
(A-C) or / PKC (A and C) were
incubated with extracts from either control or UV-irradiated cells
(A and B), either in the absence (A
and B) or presence of different caspase inhibitors
(B), or with recombinant caspase-3 (C). Afterward
the reaction was fractionated by SDS-PAGE followed by autoradiography
in an InstantImager. Essentially identical results were obtained in
another two experiments.
|
|
The size of the
PKC fragments generated by the caspase action
suggests that the cleavage must occur at the hinge region linking the
regulatory and the catalytic domains. In addition, because
/
PKC
is resistant to caspase-mediated breakdown, the caspase cleavage
site(s) should be present in a region of
PKC with no sequence
homology with
/
PKC. The hinge domain in the atypical PKCs is,
together with the V1 region, where the major differences exist between
PKC and
/
PKC (57). Interestingly, the alignment of the hinge
regions of
PKC and
/
PKC reveals the existence of three
potential caspase sites in
PKC that are absent in
/
PKC. These
sites are underlined in Fig. 4 and named
as 1, 2, and 3. In order to determine which of these potential sites
are important for
PKC breakdown, different
PKC mutants that
abrogate the caspase consensus sequence were constructed, translated
in vitro, and incubated with extracts from UV-irradiated
cells. The individual disruption of sites 1, 2, and 3, produced no
effect on the degradation of
PKC (Fig.
5). However, although the double 1/2
mutant was also cleaved, the 2/3 and 1/2/3 mutants were completely
resistant to degradation (Fig. 5). This indicates that sites 2 and 3 are the targets of the caspase-mediated breakdown of
PKC. In
order to explore the role of sites 2 and 3 in vivo,
Myc-tagged
PKC2/3 mutant (myc-
PKC2/3) was
transfected into HeLa cells, after which they were UV-irradiated for
different times and the activity and levels of this
PKC mutant were
determined. Results of Fig. 6 demonstrate
that although myc-
PKC2/3 is completely resistant to
degradation, its enzymatic activity is dramatically inhibited by UV
irradiation.

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Fig. 4.
Alignment of the hinge region of
PKC and
/ PKC. The putative
caspase sites in the PKC sequence are underlined and
named 1, 2, and 3.
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Fig. 5.
Effect of mutations of the caspase sites on
the cleavage of PKC.
35S-Labeled in vitro translated PKC, wild
type (WT), or mutated at the different caspase sites as
described under "Materials and Methods," was incubated with
extracts from either control or UV-irradiated cells. Afterward the
reaction was fractionated by SDS-PAGE followed by autoradiography in an
InstantImager. Essentially identical results were obtained in another
two experiments.
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Fig. 6.
Mutation of the caspase sites does not affect
the inhibition of PKC activity by UV
irradiation. Extracts from HeLa cells transfected with a
Myc-tagged PKC2/3 mutant were prepared at different
times following UV irradiation, after which the levels of
PKC2/3 were determined by immunoblot with a monoclonal
anti-Myc antibody (upper panel). This is a representative
experiment of three with very similar results. Parallel extracts were
immunoprecipitated with the same antibody, and the activity of
PKC2/3 was determined as described under "Materials
and Methods" (lower panel). Results are the mean ± S.D. of four independent experiments including the one shown in the
upper panel.
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|
Collectively these findings indicate that UV irradiation inhibits both
/
PKC and
PKC activities. This is consistent with previous data
from this laboratory and may be due to the interaction of these PKCs
with the selective protein inhibitor Par-4 (2, 3). A previously
unreported observation is the degradation of
PKC mediated by
caspase-3. This generates a fragment that is identical to the catalytic
domain of
PKC, that should be permanently active because it lacks
the regulatory region which keeps the enzyme blocked through the
binding of the catalytic domain to the pseudosubstrate sequence (57).
However, the data presented in this study (Fig. 1B), suggest
that the catalytic fragment generated by UV irradiation is inactive,
since at 20 h its enzymatic activity still is inhibited despite
the fact that all the
PKC has been broken-down to its catalytic
fragment. To more firmly demonstrate this possibility, HeLa cells were
transfected with a GST-tagged version of the catalytic domain of
PKC
(GST-
PKCCAT) encompassing residues from site 3 to the
end of the protein. Afterward, cells transfected with the catalytic
PKC were UV irradiated for different times, and the levels and
activity of the GST-
PKCCAT construct were determined.
Results of Fig. 7 demonstrate that UV
irradiation produces a robust inhibition of the catalytic construct, indicating that some signal is generated by this stress treatment that
not only inhibits the full-length protein, as previously reported (2)
and shown in Fig. 1B, but also the catalytic fragment that
is generated during the UV-irradiation process.

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Fig. 7.
UV irradiation inhibits the activity of
catalytic PKC. Extracts from HeLa cells
transfected with a GST-tagged version of the PKCCAT
construct were prepared at different times following UV irradiation,
after which the levels of PKCCAT were determined by
immunoblot with a monoclonal anti-GST antibody (upper
panel). This is a representative experiment of three with very
similar results. Parallel extracts were immunoprecipitated with the
same antibody, and the activity of PKCCAT was determined
as described under "Materials and Methods" (lower
panel). Results are the mean ± S.D. of four independent
experiments including the one shown in the upper
panel.
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The results of Table I demonstrate that
the cleavage of
PKC by caspase-3 is a critical event for apoptosis.
To address this, we used a
-galactosidase cell viability assay. HeLa
cells were transfected with either a control expression vector or
plasmids for wild-type or caspase-resistant
PKC, after which the
cultures were UV-irradiated and the number of
-galactosidase-positive (blue) cells were scored 36 h
thereafter. Interestingly, the expression of caspase-resistant
PKC
protected HeLa cells from UV-induced apoptosis more efficiently than
the wild-type enzyme (Table I). These results suggest that the
caspase-mediated cleavage of
PKC significantly contributes to
apoptotic cell death.
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Table I
Enhanced protection from UV-induced apoptosis by the caspase-resistant
PKC mutant
HeLa cells were transfected with pCMV- gal (2.5 µg) and either 2 or
10 µg of either plasmid control pCDNA3 (control) or expression
vectors for wild-type ( PKC) or caspase-resistant ( PKC2/3)
PKC. Twenty-four h post-transfection cells were induced to undergo
apoptosis by UV irradiation and 36 h later they were fixed and
stained with 5-bromo-4-chloro-3-indoyl -D-galactoside.
Results (number of blue cells per 35-mm dish) are the mean ± S.D.
of three independent experiments with incubations in duplicate.
Statistical differences from control untreated cell cultures.
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 |
DISCUSSION |
Caspase activation is a critical and ultimate step in apoptotic
cell death. A number of substrates have been reported to be the target
of these proteolytic enzymes (40-42). Among those, considerable attention has recently been focused on the caspase-mediated degradation of enzymes involved in cell growth and survival. Thus, the
caspase-mediated cleavage seems to trigger the activation of
pro-apoptotic kinases, such as MEKK-1 or PKN (46-49), and the
inactivation of anti-apoptotic or proliferative kinases, such as Raf-1
or Akt (43). Regarding PKC, the
isoform has been shown to be
cleaved by caspases, which results in the generation of a catalytically
active fragment (44, 45). This suggests that
PKC activation by
proteolytic degradation may be considered a critical step in apoptosis.
Actually, expression of the catalytic fragment of
PKC is sufficient
to induce programmed cell death (44, 45).
In this study we demonstrate a differential regulation of
PKC and
/
PKC during UV-induced apoptosis. Thus,
/
PKC is stimulated at early times after UV-irradiation whereas
PKC is not (Fig. 1).
Recent studies from this laboratory show that
/
PKC can be selectively activated by the novel regulator LIP (14). It is possible
that an increased interaction with this protein after UV irradiation
may account for the selective activation of
/
PKC by this stress
stimulus.2 Perhaps more
interesting from the point of view of the role of the atypical PKCs in
apoptosis, is our observation that both aPKCs are inhibited in
UV-irradiated cells, but that only
PKC is proteolytically cleaved.
This cleavage seems to be produced by the direct action of caspase-3 on
two sites in the hinge region of
PKC. However, although the
degradation of
PKC generates a fragment that under normal conditions
is more active than the full-length protein, in UV-irradiated cells
this fragment is completely inactive. This is in marked contrast to
what has been reported for
PKC and is in keeping with the notion
that
PKC is a pro-survival enzyme that needs to be blocked for
apoptosis to proceed (1-3, 44, 45). The observation that a
caspase-resistant mutant of
PKC protects cells from apoptosis more
efficiently than the wild-type enzyme, indicates that the proteolytic
degradation of
PKC is an additional and critical way to modulate
UV-induced apoptosis in HeLa cells contributing to the inevitability of
the apoptotic cell death. We have previously shown that Par-4 induction
during UV irradiation is a mechanism for the inactivation of the
atypical PKCs (2, 3). However, Par-4 cannot interact with the catalytic domain of those kinases (3); therefore, the inhibition of the enzymatic
activity of the catalytic fragment in the UV-irradiated cells, detected
in this study, indicates that mechanisms in addition to the binding of
Par-4 to the regulatory domain must operate for the blockage of
PKC
during apoptosis. These mechanisms will probably target the catalytic
domain of the kinase, but its precise nature remains to be determined.
However, we are tempted to speculate that the activation of
phosphatases may promote the de-phosphorylation of critical residues in
the activation loop of
PKC that are required for its basal activity
(58,59). These residues have been reported to be phosphorylated by
PDK-1, a phosphatidylinositol 3-kinase-dependent enzyme (30, 31).
Although there is no published evidence that phosphatidylinositol
3-kinase is inactivated in UV-irradiated HeLa cells, it is blocked in
other forms of apoptosis, such as in anoikis (56), in ceramide-treated
cells (60), or following genotoxic stress (61). Therefore, it is
possible that the inactivation of phosphatidylinositol 3-kinase by
stress leads to the de-phosphorylation of residues important in the
activation loop of
PKC which, together with the binding of Par-4,
may be the critical events on its inhibition.
 |
ACKNOWLEDGEMENTS |
We are indebted to Esther Garcia, Carmen
Ibañez, and Beatriz Ranera for technical assistance and Gonzalo
Paris and Isabel Perez for help and enthusiasm.
 |
FOOTNOTES |
*
This work was supported by Grants SAF96-0216 from
Comisión Interministerial de Ciencia y Tecnología, Spain,
PM96-0002-C02 from Dirección General de Investigación
Científica y Técnica (DGICYT), and BIO4-CT97-2071 from the
European Union, and by funds from Glaxo Wellcome Spain, and an
institutional grant from Fundación Ramón Areces to the
Centro de Biologia Molecular.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Centro de
Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad
Autónoma, Canto Blanco, 28049 Madrid, Spain. Tel.: 34-913978039;
Fax: 34-629690055; E-mail: tdiazmeco{at}cbm.uam.es.
2
L. Sanz, M. T. Diaz-Meco, and J. Moscat,
unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
PKC, protein kinase
C;
aPKC, atypical protein kinase C;
ERK, extracellular signal-regulated
kinase;
GST, glutathione S-transferase;
LIP,
/
PKC
interacting protein;
MAPK, mitogen-activated protein kinase;
MEKK, MAPK
kinase kinase;
NF-
B, nuclear factor
B;
PAGE, polyacrylamide gel
electrophoresis;
Par-4, prostate apoptosis response 4;
PDK-1, phosphoinositide-dependent protein kinase-1;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
 |
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