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Molecular and Cellular Biology, November 1998, p. 6719-6728, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Inactivation of DNA-Dependent Protein Kinase by
Protein Kinase C
: Implications for Apoptosis
Ajit
Bharti,1
Stine-Kathrein
Kraeft,2
Mrinal
Gounder,1
Pramod
Pandey,1
Shengfang
Jin,3
Zhi-Min
Yuan,1
Susan P.
Lees-Miller,4
Ralph
Weichselbaum,5
David
Weaver,3
Lan Bo
Chen,2
Donald
Kufe,1 and
Surender
Kharbanda1,*
Cancer Pharmacology1
and
Cancer Biology,2 Dana-Farber Cancer
Institute, and
Department of Microbiology and Molecular
Genetics,3 Harvard Medical School, Boston,
Massachusetts 02115;
Department of Biological Sciences
University of Calgary, Calgary, Alberta, Canada T2N
IN44; and
Department of Radiation and
Cellular Oncology University of Chicago, Chicago, Illinois
606375
Received 6 April 1998/Returned for modification 2 June
1998/Accepted 28 July 1998
 |
ABSTRACT |
Protein kinase C
(PKC
) is proteolytically cleaved and
activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKC
in
apoptosis is supported by the finding that overexpression of the
catalytic fragment of PKC
(PKC
CF) in cells is associated with
the appearance of certain characteristics of apoptosis. However, the
functional relationship between PKC
cleavage and induction of
apoptosis is unknown. The present studies demonstrate that PKC
associates constitutively with the DNA-dependent protein kinase
catalytic subunit (DNA-PKcs). The results show that PKC
CF
phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKC
CF inhibits the function of DNA-PKcs to form complexes with DNA and to
phosphorylate its downstream target, p53. The results also demonstrate
that cells deficient in DNA-PK are resistant to apoptosis
induced by overexpressing PKC
CF. These findings support the
hypothesis that functional interactions between PKC
and DNA-PK
contribute to DNA damage-induced apoptosis.
 |
INTRODUCTION |
The cellular response to ionizing
radiation (IR) and other DNA-damaging agents includes cell cycle arrest
and activation of DNA repair. In the event of irreparable DNA damage,
cells respond with induction of apoptosis. Apoptosis is an
ultrastructurally and biochemically distinct form of cell death that
occurs in response to a variety of stimuli and is carried out by a
genetically determined cell suicide program (21, 23). Cells
undergoing apoptosis exhibit morphological and biochemical
characteristics that include blebbing of the cell membrane, a decrease
in cell volume, nuclear condensation, and internucleosomal cleavage of
DNA (26, 57). However, the intracellular signals that
control the induction of apoptosis are unclear.
The induction of apoptosis by a variety of stress inducers,
including DNA damage, is associated with activation of
aspartate-specific cysteine proteases (caspases) (1, 12, 41,
42). Direct involvement of caspases in the induction of
apoptosis is supported by studies with the cowpox virus protein
CrmA (48), the baculovirus protein p35 (6), and
peptide inhibitors (3, 46, 47). CrmA inhibits the induction
of apoptosis in cells treated with Fas ligand or tumor necrosis
factor (15, 39, 53). By contrast, IR-induced
apoptosis involves activation of a CrmA-insensitive pathway
(10). These findings have suggested that DNA damage-induced apoptosis is conferred by signals that are distinct from those activated by Fas and tumor necrosis factor (10). The
demonstration that IR induces the activation of caspase 3 and that this
event, like IR-induced apoptosis, is mediated by a
CrmA-insensitive, p35-sensitive pathway (10) has provided
support for caspase 3 as a key effector. IR-induced activation of
caspase 3 is associated with proteolytic cleavage of poly(ADP-ribose)
polymerase (25, 32, 43) and other proteins. Activation of
caspase 3 in irradiated cells is regulated by members of the
Bcl-2/Bcl-xL family (10, 14). Bcl-2 and
Bcl-xL block the release of cytochrome c from mitochondria of cells treated with IR and other agents (28, 31,
58). In this context, cytochrome c release activates
caspase 9, and this event is upstream to activation of caspase 3 (36).
The protein kinase C (PKC) family of serine/threonine kinases consists
of multiple isoforms that possess a conserved catalytic domain
(29). Studies have demonstrated that the calcium-independent
isoform is cleaved in cells induced to undergo apoptosis in response to DNA-damaging agents (13, 14). PKC
is cleaved by caspase 3 at the third variable region (V3) to a 40-kDa
catalytically active fragment (13, 14, 17). The finding that
overexpression of the PKC
catalytic fragment (PKC
CF) is
associated with chromatin condensation, nuclear fragmentation,
appearance of sub-G1 DNA, and lethality has supported a
role for PKC
cleavage in induction of apoptosis
(17). The ubiquitously expressed PKC
is unique among the
PKC isoforms as a substrate for tyrosine phosphorylation (36). Transformation by Ras (11) or v-Src
(60) results in tyrosine phosphorylation of PKC
. Other
studies have demonstrated that PKC
is phosphorylated and activated
by c-Abl during the response to DNA damage (59). PKC
has
been shown to activate the MEK-extracellular signal-regulated kinase
(ERK) pathway by a mechanism dependent on Raf and independent of Ras
(37). In concert with a potential tumor suppressor function
(40), PKC
has also been linked to induction of growth
arrest (16, 54) and apoptosis (17).
The DNA-dependent protein kinase (DNA-PK) is essential in the repair of
DNA double-strand breaks that form in irradiated cells and in V(D)J
recombination (20, 22, 55). DNA-PKcs is the 470-kDa
catalytic subunit of DNA-PK that contains a protein kinase homology
domain at the C terminus. DNA-PKcs activity is induced by binding to
the 70- and 80-kDa Ku heterodimer (2, 33). Ku binds to DNA
in double-strand break repair reactions and thereby recruits DNA-PKcs
to sites of DNA damage (5, 34, 50). Recent studies have
demonstrated that DNA-PKcs is a self-contained kinase that is activated
by direct interaction with double-stranded DNA and that the role of Ku
is to stabilize the binding of DNA-PKcs to DNA ends (9, 18).
DNA-PKcs, but not Ku, is cleaved by caspase-3 during apoptosis
(7, 19, 51). The available evidence indicates that DNA-PKcs
is cleaved into 240-, 150-, and 120-kDa fragments and that cleavage is
associated with loss of DNA-PK activity (51). The 240-kDa
fragment is derived from the N terminus, and the 150-kDa fragment,
which contains the kinase homology domain, is from the C terminus
(51). The 150-kDa fragment can also undergo further cleavage
to a 120-kDa protein that retains the kinase domain (51).
The present studies demonstrate that PKC
interacts with DNA-PKcs.
The results show that PKC
CF phosphorylates the cleaved fragment of
DNA-PKcs and inactivates it in vitro. Phosphorylation of DNA-PKcs by
PKC
also inhibits the binding of DNA-PKcs to DNA. We further show
that cells deficient in DNA-PK exhibit resistance to apoptosis
induced by overexpressing the catalytically active form of PKC
.
 |
MATERIALS AND METHODS |
Cell culture.
U-937 monoblastic leukemia cells (American
Type Culture Collection, Rockville, Md.) were grown in RPMI 1640 medium
containing 10% heat-inactivated fetal bovine serum (FBS), 100 U of
penicillin per ml, 100 mg of streptomycin per ml, and 2 mM
L-glutamine. The cell lines SCSV3, SCH8-1, CHO, and CHO/V-3
were cultured in Dulbecco's modified Eagle's medium containing 10%
heat-inactivated FBS. Irradiation was performed at room temperature
with a Gammacell-1000 (Atomic Energy of Canada, Ottawa, Ontario,
Canada) under aerobic conditions with a 137Cs source
emitting at a fixed-dose rate of 0.76 Gy/min as determined by
dosimetry.
Immunoprecipitation and immunoblot analysis.
Cell lysates
and immunoprecipitations were prepared as described previously
(45). Soluble proteins (150 µg) were incubated with
anti-PKC
(Santa Cruz Biotechnology, Santa Cruz, Calif.) or
anti-DNA-PK (Upstate Biotechnology, Inc., Upstate, N.Y.) for 1 h
and precipitated with protein A-Sepharose for an additional 1 h.
Preimmune rabbit serum (PIRS) was used as a negative control. The
resulting immune complexes were washed three times with lysis buffer,
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and transferred to nitrocellulose filters. Total-cell
lysate (30 µg) was used as a positive control. The residual binding
sites were blocked by incubating the nitrocellulose paper in 5% dry
milk in phosphate-buffered saline (PBS)-0.05% Tween 20 for 1 h
at room temperature and then with anti-PKC
or anti-DNA-PK antibodies
for 1 h. The antigen-antibody complexes were visualized by
enhanced chemiluminescence (ECL detection system; Amersham). Signal
intensities were determined by densitometric analysis (UltroScan; LKB,
Bromma, Sweden).
Far-Western analysis.
Purified DNA-PK protein (1 µg;
provided by S. P. Lees-Miller) was subjected to SDS-PAGE and
transferred to a nitrocellulose filter. Three identical filters were
made. The filters were then incubated with purified glutathione
S-transferase (GST)-full-length PKC
(PKC
FL),
GST-PKC
CF, or GST for 1 h at room temperature. The filters
were then analyzed by immunoblotting with anti-PKC
.
Generation of expression constructs.
FL, CF, and
kinase-inactive (CF K-R) PKC were prepared by cloning the appropriate
PCR product of human PKC
into pGEX-2T (Pharmacia Biotech, Uppsala,
Sweden). The resultant plasmids, pGEX-PKC
FL, pGEX-PKC
CF, and
pGEX-PKC
CF K-R contain a tac promoter controlling the expression of
a fusion protein consisting of GST linked to the N terminus of human
PKC
FL or CF. A similar strategy was used for green fluorescence
protein (GFP) fusion constructs by cloning the PCR product of PKC
into a eukaryotic expression vector, pEGFP-c1 (Clontech, Palo Alto,
Calif.). The resultant plasmids, pEGFP-PKC
CF and pEGFP-PKC
CF
K-R, contain the cytomegalovirus promoter controlling the expression of
a fusion protein consisting of GFP linked to the N terminus of PKC
CF.
Dissociation of DNA-PKcs from DNA by PKC
.
DNA-PK/Ku (1 µg; Promega) was incubated with double-stranded DNA-cellulose (15 µg; Sigma) in kinase buffer (25 mM HEPES [pH 7.4], 75 mM KCl, 10 mM
MgCl2, 1 mM dithiothreitol [DTT], 0.2 mM EGTA, 0.1 mM
EDTA) for 30 min at room temperature. The DNA-cellulose beads were then
washed and resuspended in kinase buffer. The kinase reaction mixtures
containing beads, 100 µM ATP, and GST-PKC
CF or GST-PKC
CF K-R
were incubated for 15 min at 30°C. To ensure that phosphorylation was
not due to DNA-PKcs, wortmannin was added to inhibit DNA-PKcs activity
(27). The supernatant fraction was obtained by sedimentation
of the beads. After the beads were washed with kinase buffer, they and
the supernatant fraction were boiled in SDS sample buffer. The proteins
were separated by SDS-PAGE (5% polyacrylamide) and analyzed by
immunoblotting with anti-DNA-PK.
In vitro phosphorylation of DNA-PKcs by PKC
.
The
recombinant GST-PKC
CF or GST-PKC
CF K-R linked to glutathione
beads was resuspended in kinase buffer II (KBII) (20 mM Tris-HCl [pH
7.4], [
-32P]ATP, 20 mM MgCl2, 4 mM DTT).
Purified DNA-PK (0.5 µg) in the absence of sonicated DNA was
incubated in kinase buffer containing [
-32P]ATP with
GST-PKC
CF or GST-PKC
CF K-R at 30°C for 20 min. The reaction
was terminated by the addition of SDS-PAGE sample buffer (100 mM
Tris-HCl [pH 7.0], 4% SDS, 720 nM 2-mercaptoethanol, 5 mg of
bromophenol blue per ml), and the reaction products were analyzed by
SDS-PAGE and autoradiography.
In vitro transcription-translation of DNA-PKcs fragments.
Specific DNA-PKcs polypeptides were formed by using a coupled in vitro
transcription-translation method (Promega) with templates generated
from the cDNA by PCR as described previously (24).
DNA-PKcs polypeptide binding assays.
PKC
CF binding to in
vitro-translated DNA-PKcs polypeptides was tested by incubating
GST-PKC
CF (5 µg) in 20 µl of MB (10 mM Tris [pH 7.4], 150 mM
NaCl, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl
fluoride, 1 µg of leupeptin per ml, 1 µg of pepstatin per ml, 1 µg of aprotinin per ml) with equal amounts of each
[35S]DNA-PKcs in vitro-translated product for 1 to 2 h at 4°C. A separate incubation of the in vitro-translated
[35S]DNA-PKcs product with GST was used as a negative
control. The beads were washed four times in 1 ml of MB at 4°C, and
the proteins were eluted by boiling in SDS sample buffer. The samples
were analyzed by SDS-PAGE and autoradiography. Signal intensities were determined by densitometric analysis.
Proteolysis of DNA-PKcs in vitro.
Purified DNA-PKcs (1 µg)
was incubated with 2.5 µg of purified recombinant caspase-3 per ml in
CB (50 mM HEPES [pH 7.5], 10% glycerol, 2.5 mM DTT, 0.24 mM EDTA) at
room temperature. The reaction products were analyzed by SDS-PAGE
(7.5% polyacrylamide), transferred to nitrocellulose membranes, and
immunoblotted with anti-DNA-PKcs.
Inactivation of the proteolytic fragment of DNA-PKcs by
phosphorylation with PKC
CF.
Purified DNA-PKcs was incubated
with or without 2.5 µg of purified recombinant caspase 3 per ml in CB
at room temperature for 30 min to 1 h as described above. An
aliquot was saved for immunoblotting with anti-DNA-PKcs. The cleaved
fragments of DNA-PKcs were mixed with purified GST-PKC
CF or
GST-PKC
CF K-R linked to GST-beads and further incubated for 15 min
at 30°C in KB containing [
32P]ATP. The GST-PKC
CF
and GST-PKC
CF K-R were then removed by sedimentation to avoid
substrate phosphorylation by PKC
CF in the next kinase reaction. The
supernatants containing the phosphorylated DNA-PKcs fragments were then
incubated for an additional 15 min at 30°C with GST-p53 in KBII
containing [
-32P]ATP. The kinase reactions were
stopped by boiling in 2× SDS sample buffer. The eluted proteins were
analyzed by SDS-PAGE and autoradiography.
Transient transfections.
The cells were transiently
transfected with pEGFP, pEGFP-PKC
CF, or pEGFP-PKC
CF K-R in the
presence of Lipofectamine (Life Technologies, Gaithersburg, Md.). After
12 to 18 h, the cells were harvested and sorted by FACscan
analysis.
Confocal microscopy.
The cells were grown on coverslips and
transfected with GFP-PKC
CF or GFP-PKC
CF K-R. After 48 h,
the cells on the coverslips were fixed in 4% paraformaldehyde in PBS
(pH 7.4) for 20 min at room temperature. They were permeabilized with
0.1% Triton X-100 and stained with 0.5 µg of
4',6-diamidino-2-phenylindole (DAPI) per ml for 10 min at room
temperature. The coverslips were mounted on slides with antibleach
mounting medium (Molecular Probes, Eugene, Oreg.) and viewed by
confocal microscopy with an LSM410 microscope (Zeiss) equipped with an
argon-krypton and a UV laser.
Cell sorting and propidium iodide staining for cells with
sub-G1 DNA.
At 18 h after transfection, the cells
were trypsinized and washed with Dulbecco's modified Eagle's medium.
GFP-positive cells were sorted in a Becton-Dickinson FACS Vantage. The
GFP-positive cells were replated in the culture medium-10%
heat-inactivated FBS for 40 h and then fixed with 40% ethyl
alcohol for 30 min. They were washed with PBS, resuspended in 0.5 µg
of propidium iodide per ml in PBS, and incubated with 50 µg of RNase
per ml at 37°C for 30 min. Numbers of cells with sub-G1
DNA were assessed by FACScan analysis.
 |
RESULTS |
DNA-PKcs associates with PKC
in vivo.
Whereas IR induces
the activation of PKC
(14), we asked if PKC
interacts
with proteins, such as DNA-PKcs, that are involved in DNA double-strand
break repair. Analysis of anti-PKC
immunoprecipitates with an
anti-DNA-PKcs antibody demonstrated reactivity with a protein of >350
kDa (Fig. 1A). PIRS was used as a
negative control of immunoprecipitation. As a positive control,
analysis of anti-DNA-PKcs immunoprecipitates by immunoblotting with
anti-DNA-PKcs demonstrated a similar pattern of reactivity (Fig. 1A).
To evaluate the stoichiometry of the interaction between DNA-PKcs and
PKC
, we subjected U-937 cell lysates to immunoprecipitation with
anti-PKC
and analyzed the supernatants and precipitates by
immunoblotting with anti-DNA-PKcs. Signal intensities from before and
after anti-PKC
immunoprecipitation were compared by laser
densitometric scanning. The results demonstrate that approximately 50%
of DNA-PKcs is associated with PKC
(Fig. 1B). When anti-DNA-PKcs
immunoprecipitates were analyzed by immunoblotting with anti-PKC
in
the reciprocal experiment, the results confirmed a constitutive
association of DNA-PKcs with PKC
(Fig. 1C). The interactions between
DNA-PKcs and PKC
are specific, since the anti-DNA-PKcs antibody does
not cross-react with PKC
and the anti-PKC
antibody does not
cross-react with DNA-PKcs (Fig. 1A and C). Activation of caspase 3 in
irradiated cells is associated with proteolytic cleavage of PKC
to
an active 40-kDa fragment (hereafter termed PKC
CF) (14).
To assess the interaction of DNA-PKcs with PKC
CF, U-937 cells were
irradiated and harvested at different time intervals. Lysates from
control and irradiated cells were subjected to immunoprecipitation with
anti-DNA-PKcs. Analysis of the precipitates with anti-PKC
demonstrated binding between DNA-PKcs and PKC
CF (Fig. 1D). The
finding that DNase has no effect on the coimmunoprecipitation of
DNA-PKcs and PKC
CF indicated that the association between these
proteins is not dependent on DNA binding (data not shown).

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FIG. 1.
Association of DNA-PKcs and PKC . (A) Lysates from
U-937 cells were subjected to immunoprecipitation with anti-DNA-PK
( DNA-PK), PIRS or anti-PKC ( PKC ). Immunoprecipitates were
analyzed by immunoblotting with anti-DNA-PKcs. Whole-cell lysate
(Lysate) was used as a positive control for the immunoblot analysis.
(B) Soluble proteins from U-937 cells were subjected to
immunoprecipitation with anti-PKC . Lysates before and after
immunoprecipitation were analyzed by immunoblotting with anti-DNA-PKcs
(top). The results are expressed as the mean ± standard deviation
(SD) of three independent experiments (bottom). (C) U-937 cell lysates
were immunoprecipitated with PIRS, anti-PKC , or anti-DNA-PKcs.
Immunoprecipitates were analyzed by immunoblotting with anti-PKC .
Lysate was used as a positive control for the immunoblotting. (D) U-937
cells were treated with 20 Gy of IR and harvested at the indicated
times. Lysates were immunoprecipitated with anti-DNA-PKcs, and the
precipitates were analyzed by immunoblotting with anti-PKC .
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|
DNA-PKcs binds directly to PKC
in vitro.
To assess the
interaction of DNA-PKcs with PKC
FL and PKC
CF in vitro, we
incubated GST fusion proteins prepared from PKC
FL and PKC
CF
with U-937 cell lysates. Analysis of the adsorbates with anti-DNA-PKcs
demonstrated that whereas both PKC
FL and PKC
CF bind to
DNA-PKcs, the apparent affinity of the interaction with PKC
CF is
greater than that with PKC
FL (Fig.
2A). To determine whether the interaction
between DNA-PKcs and PKC
is direct, purified DNA-PKcs was incubated
separately with GST-PKC
FL, GST-PKC
CF, or GST. After being
washed, the bound proteins were separated by SDS-PAGE (5%
polyacrylamide) and analyzed by immunoblotting with anti-DNA-PKcs.
Reactivity with anti-DNA-PKcs in the GST-PKC
CF and GST-PKC
FL,
but not GST, adsorbates supported a direct interaction of DNA-PKcs with
PKC
FL and PKC
CF (Fig. 2B). To further demonstrate direct
interaction between DNA-PKcs and PKC
, purified DNA-PKcs was resolved
by SDS-PAGE, transferred to a nitrocellulose filter, and renatured
in aquaous buffer. After incubation with GST-PKC
FL, GST-PKC
CF,
or GST, the filters were washed and probed with anti-PKC
. Reactivity
with anti-PKC
at the position corresponding to DNA-PKcs confirmed
the direct interaction of DNA-PKcs with PKC
(Fig. 2C). The absence
of reactivity when the filters were incubated with GST indicated that
binding of DNA-PKcs with PKC
is specific (Fig. 2B).

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FIG. 2.
Direct interaction of DNA-PKcs with PKC . (A) U-937
cell lysate was incubated with GST, GST-PKC FL, or GST-PKC CF.
The protein adsorbates were analyzed by immunoblotting with
anti-DNA-PKcs. (B) Purified DNA-PK (1 µg) was incubated with GST,
GST-PKC FL, or GST-PKC CF. After extensive washing, the bound
proteins were eluted by boiling in SDS sample buffer and analyzed by
immunoblotting with anti-DNA-PK. (C) Purified DNA-PK (1 µg) was
resolved by SDS-PAGE and transferred to three nitrocellulose filters.
The filters were incubated with GST, GST-PKC FL, or GST-PKC CF
for 1 h at room temperature and then analyzed by immunoblotting
with anti-PKC antibody.
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PKC
CF-mediated phosphorylation of DNA-PKcs inhibits DNA-PK
binding to DNA in vitro.
To determine whether PKC
phosphorylates DNA-PK, we incubated GST-PKC
FL or GST-PKC
CF with
purified DNA-PKcs in the presence of [
-32P]ATP. The
phosphorylation reactions were carried out in the absence of DNA to
inhibit DNA-PKcs autophosphorylation. Analysis of the products by
autoradiography indicated that DNA-PKcs is a substrate for PKC
(Fig.
3A, top) PKC
CF is at least 40 times
more active than PKC
FL, as determined by their phosphorylation of
myelin basic protein (Fig. 3A, bottom). The present results also
demonstrate approximately 10-fold more phosphorylation of DNA-PKcs by
PKC
CF (Fig. 3A, top).

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FIG. 3.
PKC CF phosphorylates DNA-PKcs and releases DNA-PKcs
from Ku-DNA beads. (A) GST-PKC FL or GST-PKC CF was incubated
with purified DNA-PK-Ku complex (top) or myelin basic protein (MBP)
(bottom) in the presence of [ -32P]ATP for 15 min at
30°C. In vitro kinase reactions were analyzed by SDS-PAGE and
autoradiography. (B) Purified DNA-PK/Ku was incubated with DNA beads,
and the beads were washed and suspended in kinase buffer. Kinase
reaction mixtures containing beads, 20 µM wortmannin, ATP, and
GST-PKC CF or kinase-inactive GST-PKC CF K-R were incubated for
15 min at 30°C. The supernatant fraction was obtained by
sedimentation of the beads. The beads and supernatant fractions were
boiled in SDS sample buffer. Proteins were separated by SDS-PAGE (5%
polyacrylamide) and analyzed by immunoblotting with anti-DNA-PKcs.
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|
Recent studies have demonstrated that c-Abl-mediated phosphorylation of
DNA-PK on tyrosine inhibits DNA-PKcs activity (
27).
Other
studies have shown that autophosphorylation of DNA-PKcs
inhibits its
activity by inducing the dissociation of DNA-PKcs
from DNA
(
8). To further assess the functional significance
of the
interaction between DNA-PKcs and PKC

, we asked if PKC
affects the
association of DNA-PKcs with DNA. To address this
issue, DNA-PKcs was
bound to DNA-beads and incubated in the presence
of wortmannin to
inhibit DNA-PKcs autophosphorylation and hence
its autodissociation
from DNA. After being washed to remove unbound
DNA-PKcs, the beads were
incubated with GST-PKC

CF or GST-PKC
CF K-R in the presence of
[
32P]ATP. The bound and supernatant fractions were then
analyzed
by immunoblotting with anti-DNA-PKcs. The results demonstrate
that addition of GST-PKC

CF K-R has no detectable effect on the
release of DNA-PKcs from DNA (Fig.
3B). By contrast, incubation
with
GST-PKC

CF resulted in the release of DNA-PKcs from the
beads (Fig.
3B). Whereas DNA-PK requires DNA for activity, these
results suggest
that PKC

CF inhibits DNA-PK activity by abrogating
the ability of
DNA-PK to associate with DNA.
PKC
CF binds to DNA-PKcs at its catalytic domain.
To
determine the regions of DNA-PKcs responsible for the association with
PKC
CF, fragments of the DNA-PKcs polypeptide were generated from
mouse DNA-PKcs cDNAs. Fourteen different DNA-PKcs fragments,
representing the entire open reading frame, were synthesized by in
vitro transcription-translation (Fig. 4A)
(24). Purified GST-PKC
CF was incubated separately with
the in vitro-translated fragments of DNA-PK, washed, and analyzed by
autoradiography. The signal intensities of GST-PKC
CF-bound DNA-PKcs
fragments were compared to the total amount of product in the reaction
mixture by densitometeric scanning. The results demonstrate that
DNA-PKcs-6 (amino acids 2333 to 2774; 10 to 15% bound to PKC
CF),
DNA-PKcs-8 (amino acids 3414 to 3850; 12 to 18% bound), DNA-PKcs-9
(amino acids 3757 to 4124; 22 to 25% bound), and DNA-PKcs-15 (amino
acids 3414 to 4123; 18 to 25% bound) associate with GST-PKC
CF but not with GST (Figs. 4B and C and data not shown). Thus, PKC
CF binds
to DNA-PKcs through a 1,790-amino-acid region that in part includes the
PK homology domain.

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FIG. 4.
Association of PKC CF with specific DNA-PKcs protein
fragments. (A) Positions of the DNA-PKcs protein fragments. Protein
fragments from various regions of the DNA-PKcs gene were prepared by
PCR and in vitro transcription-translation as described in the text.
(B) GST-PKC CF bound to glutathione-Sepharose was mixed with in
vitro-translated products from DNA-PKcs regions to allow binding. After
being washed, samples were separated by SDS-PAGE (10% polyacrylamide)
and analyzed by autoradiography. (C) GST-PKC CF or GST bound to
glutathione-Sepharose was mixed with DNA-PKcs fragment 6, 8, or 9. After being washed, the bound proteins were analyzed by SDS-PAGE and
autoradiography.
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Cleavage of DNA-PKcs by caspase 3 and inhibition of DNA-PKcs
activity by phosphorylation with PKC
CF.
Recent studies have
shown that DNA-PKcs kinase activity is reduced in apoptotic
cells and that the inhibition correlates with proteolytic cleavage of
DNA-PKcs (51). To investigate which protease is responsible
for cleaving DNA-PKcs in vitro, we treated purified DNA-PKcs with
caspase 3 or interleukin-converting enzyme (ICE). In contrast to ICE,
caspase 3 induced the cleavage of purified DNA-PKcs to 240- and 150-kDa
fragments (Fig. 5A). Overexposed gels
demonstrate a minor cleaved fragment of 120 kDa (data not shown). To
determine whether cleavage of DNA-PKcs by caspase 3 inhibits DNA-PKcs
activity in vitro, we incubated purified DNA-PK-Ku-DNA complexes with
caspase 3 for 30 min to 1 h and used autoradiography to analyze
the products of a kinase reaction performed in the presence of
[
-32P]ATP. As assessed by autophosphorylation and/or
phosphorylation of the cleaved fragments, the results demonstrate that
the 240-kDa cleavage fragment of DNA-PKcs (DNA-PK CL1) is
phosphorylated (Fig. 5B). In vitro, DNA-PKcs phosphorylates p53, and
this event requires binding of DNA-PKcs to DNA (35).
Therefore, to further assess the effect of caspase 3-mediated cleavage
on DNA-PK activity, we incubated purified DNA-PK-Ku-DNA complexes
with caspase 3 and assessed DNA-PK activity with GST-p53 as a
substrate. The results demonstrate that cleavage of DNA-PKcs by caspase
3 inhibits in part the DNA-PKcs activity as assessed by its
phosphorylation of p53 (Fig. 5C).

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FIG. 5.
Caspase 3 cleaves DNA-PKcs and partially inhibits
DNA-PKcs activity. (A) Purified DNA-PK was incubated with recombinant
caspase 3 (2.5 µg/ml) (left) or with recombinant ICE (right) at room
temperature for 1 h or 30 min, respectively. The reaction products
were subjected to SDS-PAGE and analyzed by immunoblotting with
anti-DNA-PKcs. (B and C) Purified DNA-PK/Ku in the presence of
DNA-beads was incubated with recombinant caspase 3 for 1 h at room
temperature. In vitro kinase reactions containing
[ -32P]ATP were performed in the absence (B) or
presence (C) of GST-p53 as the substrate. The reactions were stopped by
the addition of SDS sample buffer, and the products were analyzed by
SDS-PAGE and autoradiography. DNA-PK CL1 and CL2, DNA-PK-cleaved
fragments 1 and 2.
|
|
Whereas the present findings demonstrate that cleavage of DNA-PKcs by
caspase 3 is associated with partial inhibition of DNA-PKcs
activity,
we asked whether the cleaved DNA-PKcs fragment is affected
by PKC

CF
phosphorylation. To address this issue, we first incubated
purified
DNA-PK-Ku complexes with in vitro-translated caspase
3 to generate its
fragments. The cleaved fragments of DNA-PKcs
were then incubated with
GST-PKC

CF, GST-PKC

CF K-R, or buffer
in the presence of
[

-
32P]ATP. The DNA-PKcs activity in kinase reaction
mixtures was assessed
by autophosphorylation or phosphorylation of the
DNA-PK CL1 or
by using GST-p53 as the substrate. The results
demonstrate that
autophosphorylation of DNA-PKcs, phosphorylation of
the DNA-PKcs
CL1, or phosphorylation of p53 by PKC

CF is associated
with inhibition
of DNA-PKcs activity (Fig.
6). PKC

CF binds to DNA-PKcs at the
catalytic domain (fragments 8, 9, and 15 [Fig.
4]), and this
interaction
contributes in part to the inhibition of DNA-PKcs (Fig.
6C). Similarly,
cleavage of DNA-PKcs by caspase 3 also partially
inhibits DNA-PKcs
activity (Fig.
5). However, more pronounced
inhibition of DNA-PKcs
activity was observed upon PKC

CF-mediated
phosphorylation of
DNA-PKcs (Fig.
6C). To assess whether cleavage of
DNA-PKcs facilitates
the inhibition of DNA-PKcs activity by PKC

CF-mediated phosphorylation,
we performed experiments in which
uncleaved DNA-PKcs was incubated
with PKC

CF or PKC

CF K-R and
then analyzed for DNA-PKcs-dependent
p53 phosphorylation. The results
demonstrate that phosphorylation
of uncleaved DNA-PKcs by PKC

CF
inhibits DNA-PKcs activity but
not to the extent observed when cleaved
DNA-PKcs is phosphorylated
by PKC

CF (Fig.
6C and data not shown).
Taken together, these
findings indicate that the interaction of PKC

CF with DNA-PKcs
and caspase 3-mediated cleavage of DNA-PKcs
independently contribute
in part to inhibition of DNA-PKcs activity.
However, nearly complete
inhibition of DNA-PKcs activity was observed
when PKC

CF phosphorylated
the cleaved fragment of DNA-PKcs (Fig.
5
and
6).

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|
FIG. 6.
Phosphorylation and inactivation of DNA-PKcs by PKC
CF. (A and B) Purified DNA-PK-Ku in the presence of DNA-beads was
incubated with recombinant caspase 3 for 1 h at room temperature.
In vitro phosphorylation of the cleaved fragments of DNA-PK was then
performed in the presence of [ -32P]ATP and GST-PKC
CF or GST-PKC CF K-R for 15 min at 30°C. After phosphorylation of
DNA-PKcs and removal of PKC -CF or PKC CF K-R by sedimentation,
kinase reactions were performed in the absence (A) or presence (B) of
GST-p53 for an additional 15 min at 30°C. The reactions were stopped
by the addition of SDS sample buffer, and the products were analyzed by
SDS-PAGE and autoradiography. Lanes: 1, DNA-PK-Ku with DNA; 2, DNA-PK-Ku without DNA; 3, DNA-PK-Ku with DNA and caspase 3; 4, DNA-PK-Ku with DNA caspase 3, and GST-PKC CF; 5, DNA-PK-Ku with
DNA, caspase 3, and GST-PKC CF K-R; 6, DNA-PK-Ku with DNA and
GST-PKC CF; 7, GST-p53; 8, buffer with [ -32P]ATP.
(C) The percent inhibition of DNA-PKcs-mediated GST-p53 phosphorylation
is expressed as the mean ± SD of four independent experiments.
|
|
Functional role of DNA-PK-PKC
complexes in
apoptosis.
Both PKC
and DNA-PKcs are cleaved after
apoptotic stimuli of cells (14, 51). If the cleavage
and regulation of these proteins is associated with functions in
apoptosis, mutant cells of DNA-PKcs may show alterations in
apoptotic pathways that are dependent on PKC
.
DNA-PK-deficient (ScSV3, scid) and DNA-PK+ (SCH8-1,
scid + human DNA-PKcs) cells (5) were transiently transfected with GFP vectors expressing PKC
CF. After 48 h, the morphology of GFP-positive cells was analyzed. PKC
was found to
stimulate the disintegration or shrinkage of nuclei of
DNA-PK+ cells, indicative of apoptosis (Fig.
7). In striking contrast, DNA-PK-deficient cells failed to demonstrate nuclear shrinkage in
response to PKC
CF. To confirm whether the differential response was
due to DNA-PK mutations, we examined a second set of DNA-PK mutant and
control Chinese hamster ovary cell lines in the same way.
DNA-PK-deficient CHO V-3 cells (52) failed to form PKC
CF-dependent apoptosis, unlike the DNA-PK+ parental
CHO cells (Fig. 8). Thus, DNA-PK is
linked to apoptotic mechanisms via PKC
.

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|
FIG. 7.
Transient overexpression of PKC CF in SCH8-1
(DNA-PK+/+) and ScSV3 (DNA-PK / ) cells.
GFP-tagged PKC CF was transiently transfected in SCH8-1
(DNA-PK+/+) and ScSV3 (DNA-PK / ) cells. The
cells were stained with DAPI, and the GFP-positive cells were analyzed
by confocal microscopy. The results are shown as overlay photographs of
DAPI and GFP staining. Arrows indicate apoptotic cells. Bar, 10 µm.
|
|

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|
FIG. 8.
Transient overexpression of PKC CF in
DNA-PK / and DNA-PK+/+ CHO cells. GFP-tagged
PKC CF was transiently transfected in CHO (DNA-PK+/+)
and CHO V-3 (DNA-PK / ) cells. The cells were stained
with DAPI, and the GFP-positive cells were analyzed by confocal
microscopy. The results are shown as overlay photographs of DAPI and
GFP staining. Arrows indicate apoptotic cells. Bar, 10 µm.
|
|
If phosphorylation by PKC

is required in apoptosis, a PKC

mutant protein inactivated in kinase activity would be expected
to
differ in its properties in the above assay. To test this,
SCH8-1
and ScSV3 cells were transiently transfected with GFP vectors
expressing PKC

CF or the kinase-inactive mutant PKC

CF K-R.
After
48 h, the cells were sorted for GFP positivity and analyzed
for
sub-G
1 DNA content. Approximately 60% of the cells
were apoptotic
when PKC

CF was overexpressed in
DNA-PK
+/+ cells, in contrast to 20% in
DNA-PK
/
cells (Fig.
9). By contrast, only 10% cells were
apoptotic when
PKC

CF K-R was transfected into
DNA-PK
+/+ cells (Fig.
9). Taken together, these findings
demonstrate that
interaction of DNA-PKcs with PKC

CF contributes to
apoptosis.

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|
FIG. 9.
Transient overexpression of PKC CF and not PKC CF
K-R in DNA-PK+/+ cells is associated with induction of
apoptosis. GFP-tagged PKC CF or PKC CF K-R mutant was
transiently transfected in SCH8-1 (DNA-PK+/+) (lane 1, GFP;
lane 2, GFP-PKC CF; lane 3, GFP-PKC CF K-R) or ScSV3
(DNA-PK / ) (lane 4, GFP; lane 5, GFP-PKC CF; lane 6, GFP-PKC CF K-R) cells. As controls, cells were transfected with
GFP-expressing empty vector. The GFP-positive cells were sorted by
FACScan analysis and analyzed for DNA content by flow cytometry. The
results are expressed as the percentage (mean ± SD of three
independent experiments, each performed in duplicate) of cells with
sub-G1 DNA content.
|
|
 |
DISCUSSION |
Caspase-mediated proteolysis in apoptosis.
In addition
to PKC
, substrates that are activated by caspase-mediated cleavage
in apoptosis include the p21-activated kinase 2 (PAK2)
(49), cytosolic phospholipase A2 (56), sterol
regulatory binding proteins (44), the 45-kDa subunit of DNA
fragmentation factor (38), and PITSLRE kinase
2-1
(4). Expression of the cleaved fragments of PKC
or PAK2
in cells induces certain characteristics of apoptosis (17,
49). However, the precise role of these cleaved proteins in
apoptosis is unclear. By contrast, other proteins are
inactivated by caspases. Previous studies have demonstrated that
DNA-PKcs is a substrate of caspase 3 and that cleavage is accompanied
by loss of DNA-PKcs activity (7, 19, 51). The functional
role of DNA-PK cleavage in the induction of apoptosis, like
that for many of the other substrates of caspases, is unknown.
DNA-PK
cs is cleaved by caspase 3 at a
DEVD/N
2713 site into N-terminal 240-kDa and C-terminal
150-kDa fragments. The 150-kDa
fragment contains another DWVD/G site
that predicts the generation
of an additional fragment of 120 kDa
(
51). In the present studies,
cleavage of DNA-PKcs to the
240- and 150-kDa fragments by caspase
3 resulted in partial loss of
catalytic activity in the presence
of Ku and DNA. Moreover, cleavage of
DNA-PKcs also partially inhibited
the DNA-PKcs-mediated phosphorylation
of p53. These findings suggest
that mechanisms other than cleavage of
DNA-PKcs by caspase 3 are
responsible for the loss of DNA-PKcs activity
that has been observed
in cells induced to undergo apoptosis
(
51).
Regulation of DNA-PKcs activity.
DNA-PKcs autophosphorylation
inactivates DNA-PKcs by a mechanism in which DNA-PKcs disassociates
from Ku (8). Other studies have demonstrated that the c-Abl
tyrosine kinase negatively regulates DNA-PKcs activity in the response
to DNA damage (27). c-Abl phosphorylates the C-terminal
region of DNA-PKcs and induces the disassociation of DNA-PKcs from the
DNA-PKcs-Ku complex (24, 27). c-Abl and Ku both bind to
the C terminus of DNA-PKcs near the kinase domain, and c-Abl
phosphorylates the region of DNA-PKcs to which Ku binds
(24). Thus, autophosphorylation and c-Abl-mediated phosphorylation regulate DNA-PKcs activity by mechanisms that oppose
the activation of DNA-PKcs through binding to the Ku-DNA complex.
The present studies demonstrate that DNA-PKcs is also regulated by
PKC

. DNA-PKcs constitutively associates with the full-length
form of
PKC

in nonapoptotic cells and with PKC

CF in cells
induced
to undergo apoptosis. The results indicate that PKC

,
like c-Abl,
binds directly to the C terminus of DNA-PKcs at the
catalytic
domain. PKC

CF phosphorylates DNA-PKcs and results in the
disassociation
of DNA-PKcs from DNA. These findings are in concert with
the demonstration
that interaction of DNA-PKcs with PKC

inhibits
DNA-PKcs activity.
Taken together with the finding that cleavage of
DNA-PKcs by caspase
3 is partially sufficient to inhibit DNA-PKcs
activity, the results
support a model in which cleavage of PKC

to
the catalytically
active fragment results in association and
phosphorylation of
DNA-PKcs and thereby complete inhibition of DNA-PKcs
activity.
Thus, on the basis of these findings, we would propose that
in
addition to autophosphorylation (
8) and c-Abl-mediated
phosphorylation
(
24,
27), DNA-PKcs is downregulated by
PKC

. To our knowledge,
DNA-PKcs may be the first substrate found to
be regulated by PKC
CF-mediated phosphorylation.
Recent studies have shown that PKC

associates constitutively with
c-Abl (
59). Activation of c-Abl by DNA damage results
in
c-Abl-dependent phosphorylation of PKC

. Also, c-Abl-mediated
phosphorylation of PKC

results in activation of PKC

in vitro
and
in irradiated cells (
59). Whereas c-Abl functions in the
downregulation of DNA-PKcs by mediating direct phosphorylation
of
DNA-PKcs (
27), it may also contribute to the interaction
of
PKC

and DNA-PKcs by activating PKC

. In this context, c-Abl
associates with PKC

in a complex that includes DNA-PKcs (
27,
59). The activation of PKC

by c-Abl in the response to DNA
damage (
59) thus provides a mechanism by which both c-Abl
and
PKC

can downregulate DNA-PKcs activity. Subsequent cleavage of
PKC

to the catalytic fragment by caspase 3 would preclude further
activation by a c-Abl-dependent mechanism. The constitutive activation
of PKC

CF and thereby the phosphorylation of DNA-PKcs and/or
the
cleaved CL1 fragment is sufficient to inhibit disassociation
of
DNA-PKcs from the Ku-DNA complex.
Functional interaction of DNA-PKcs and PKC
in
apoptosis.
Overexpression of PKC
CF in cells is
associated with chromatin condensation, nuclear fragmentation,
induction of sub-G1 DNA, and lethality (17).
These findings have indicated that cleavage of PKC
to a
kinase-active fragment by caspase 3 contributes to multiple changes
that are characteristic of apoptosis. Whereas the mechanistic
basis for the proapoptotic effects of PKC
CF are unclear,
the demonstration that PKC
CF interacts with DNA-PKcs prompted
studies on the functional significance of this interaction. Cells
deficient in DNA-PKcs were transfected to express PKC
CF, and the
findings were compared to those obtained with cells that express
DNA-PK. The results obtained with ScSv3 (DNA-PK
/
)
and SCH8-1 (DNA-PK+/+) cells demonstrate that
PKC
CF-induced apoptosis is abrogated in part in
DNA-PK-deficient cells. Similar results were obtained with CHO V-3
(DNA-PK
/
) and CHO (DNA-PK+/+) cells
transfected to express PKC
CF. These findings suggest that PKC
CF
induces apoptosis, at least in part by a DNA-PK-dependent mechanism.
The demonstration that cells deficient in DNA-PK are hypersensitive to
DNA-damaging agents has supported a direct role for
DNA-PKcs in DNA
repair (
5,
30,
34). The inhibition of DNA-PKcs
by PKC

CF
would be expected to inhibit DNA repair and thereby
facilitate the DNA
fragmentation that is induced in apoptosis.
Thus, given that
PKC

inhibits DNA-PKcs activity, it is not evident
why
DNA-PK
/
cells would be less sensitive to PKC

CF-induced apoptosis. One
potential explanation is that the
direct binding of PKC

CF to
DNA-PKcs provides access for DNA-PKcs
substrates to phosphorylation
by PKC

CF. In this context, DNA-PKcs
associates with and phosphorylates
the p53 tumor suppressor and other
proteins. The present experiments
have not addressed whether the pools
of DNA-PKcs that associate
with PKC

are the same as those that form
complexes with other
proteins. However, it is conceivable that in the
absence of DNA-PKcs,
PKC

would not be positioned to interact with a
protein that is
central to the apoptotic response. Insights
into this potential
mechanism could be obtained from an analysis of
other proteins
associated with the PKC

-DNA-PKcs complex.
 |
ACKNOWLEDGMENTS |
We thank Stephen Jackson for critical reading of the manuscript
and for invaluable suggestions.
This investigation was supported by PHS grants CA75216 (S.K.) and
CA55241 (D.K.) awarded by the National Cancer Institute, DHHS.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Cancer
Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney St., Boston, MA 02115. Phone: (617) 632-2938. Fax: (617)
632-2934. E-mail:
Surender_Kharbanda{at}MacMailGW.dfci.harvard.edu.
 |
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Molecular and Cellular Biology, November 1998, p. 6719-6728, Vol. 18, No. 11
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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