From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Received for publication, December 26, 2000, and in revised form, March 12, 2001
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ABSTRACT |
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DNA-dependent protein kinase
(DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the
DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA
double strand break repair by the process of nonhomologous end
joining. Nonhomologous end joining is a major mechanism for the
repair of DNA double strand breaks in mammalian cells. As such, DNA-PK
plays essential roles in the cellular response to ionizing radiation
and in V(D)J recombination. In vitro, DNA-PK undergoes
phosphorylation of all three protein subunits (DNA-PK catalytic
subunit, Ku70 and Ku80) and phosphorylation correlates with
inactivation of the serine/threonine protein kinase activity of DNA-PK.
Here we show that phosphorylation-induced loss of the protein kinase
activity of DNA-PK is restored by the addition of the purified
catalytic subunit of either protein phosphatase 1 or protein
phosphatase 2A (PP2A) and that this reactivation is blocked by the
potent protein phosphatase inhibitor, microcystin. We also show that
treating human lymphoblastoid cells with either okadaic acid or
fostriecin, at PP2A-selective concentrations, causes a 50-60%
decrease in DNA-PK protein kinase activity, although the protein
phosphatase 1 activity in these cells was unaffected. In
vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was
observed when cells were labeled with [32P]inorganic
phosphate in the presence of the protein phosphatase inhibitor, okadaic
acid. Together, our data suggest that reversible protein
phosphorylation is an important mechanism for the regulation of DNA-PK
protein kinase activity and that the protein phosphatase responsible
for reactivation in vivo is a PP2A-like enzyme.
The reversible phosphorylation of proteins, catalyzed by
protein kinases and protein phosphatases, is a major mechanism for the
regulation of many eukaryotic cellular processes, including metabolism, muscle contraction, pre-mRNA splicing, and cell cycle control (1). The eukaryotic protein phosphatases comprise several families of enzymes that catalyze the dephosphorylation of
intracellular phosphoproteins. The protein phosphatases responsible for
dephosphorylation of serine and threonine residues in the cytoplasmic
and nuclear compartments of eukaryotic cells are encoded by the
PPP and PPM gene families, which are
defined by distinct amino acid sequences and tertiary structures (2).
The type 1, 2A, and 2B protein phosphatases (PP1, PP2A, and PP2B,
respectively)1 belong to the
PPP family. This family also includes a growing list of novel protein
phosphatases (e.g. protein phosphatase 4, 5, 6, and 7) (2,
3).
Inhibitors of protein (serine/threonine) phosphatases include
endogenous proteins that regulate protein phosphatases in eukaryotic cells, such as inhibitor-1, DARPP 32, and inhibitor-2, which
specifically inhibit PP1 (4, 5). Several toxins, drugs, and tumor
promotors, including okadaic acid, microcystins, tautomycin, nodularin,
cantharidin, endothall, calyculin A, and fostriecin, are also highly
specific inhibitors of members belonging to the PPP family of
serine/threonine protein phosphatases. These toxins bind to the same
site on the enzymes but with different relative affinities (6, 7) and have been fundamental to understanding the role of protein
phosphorylation in various physiological processes and deciphering
which type of protein phosphatase is responsible for a given cellular
event (6).
The differential sensitivities to these inhibitors have provided
methods to identify and quantitate the levels of PP1 and PP2A in cell
and tissue extracts (8). Several of these inhibitors, including okadaic
acid and fostriecin, are membrane-permeable and potently inhibit
phosphatase activity in intact cells. Due to their differential
affinities to PP1 and PP2A and their distinct permeation properties,
these two inhibitors can inhibit PP1 and PP2A in a highly selective
manner (9). We have used these inhibitors at PP2A-selective
concentrations to investigate the regulation of
DNA-dependent protein kinase (DNA-PK) by protein
phosphorylation in vitro and in vivo.
DNA-PK is composed of a catalytic subunit (DNA-PKcs) and a
heterodimeric DNA end-binding protein, Ku70/Ku80. DNA-PKcs and Ku are
required for the repair of ionizing radiation induced DNA damage via
the process of nonhomologous end joining (reviewed in Ref. 10).
Although the exact role of DNA-PKcs and Ku in nonhomologous end joining
has yet to be determined, in vitro, DNA-PK acts as a
serine/threonine protein kinase and phosphorylates a variety of protein
substrates including p53 and the 32-kDa subunit of DNA replication
protein A (reviewed in Refs. 10 and 11). Several in vitro
DNA-PK phosphorylation sites (e.g. serine 15 of human p53
and serines and threonines in the amino-terminal 30 amino acids of the
replication protein A 32-kDa subunit) are phosphorylated in
vivo in response to DNA damage (12-14); however, it is not clear whether DNA-PK plays a direct role in these processes in
vivo. DNA-PK also undergoes phosphorylation of all three of its
protein components in vitro, and phosphorylation of the
DNA-PK complex correlates with loss of protein kinase activity and
disruption of DNA-PKcs from the Ku-DNA complex (15). The DNA-PK
phosphorylation sites on DNA-PKcs are presently unknown; however, Ku70
is phosphorylated by DNA-PK in vitro predominantly at serine
6, while Ku80 is phosphorylated on at least three carboxyl-terminal
sites, including serines 577 and 580 and threonine 715 (16). Although
the physiological substrates of DNA-PK are unknown, a conserved
aspartic acid residue in the kinase domain of DNA-PKcs is required to
rescue the radiosensitive phenotype associated with deficiency of
DNA-PKcs in rodent cells (17), strongly suggesting that DNA-PK acts as
a protein kinase in vivo. Ku is phosphorylated in
vivo (18), although the sites of in vivo
phosphorylation have yet to be identified.
Here we have examined the effects of protein dephosphorylation on the
activity of in vitro DNA-PK-mediated phosphorylation of DNA-PKcs and Ku. We show that dephosphorylation of
self-phosphorylated DNA-PK results in reactivation of DNA-PK protein
kinase activity. Moreover, we show that DNA-PK protein kinase
activity is significantly reduced in cells that have been treated with
the protein phosphatase inhibitors okadaic acid and fostriecin and that
inhibition of protein phosphatase activity by okadaic acid
significantly increases the in vivo phosphorylation state of
DNA-PKcs, Ku70, and Ku80. Together, our data show that DNA-PK protein
kinase activity can be regulated by reversible protein phosphorylation
in vitro and that DNA-PK is a target of protein kinases and
protein phosphatases in vivo.
Reagents--
Bovine serum albumin, phenylmethylsulfonyl
fluoride, Tris base, EGTA, leupeptin, pepstatin, and wortmannin were
from Sigma. A Mono Q (HR5/5) column and 32P-labeled
inorganic phosphate were from Amersham Pharmacia Biotech. [ Cell Culture and Inhibitor Treatment--
Lymphoblastoid cells
(BT) and MO59J cells were maintained in RPMI or Dulbecco's modified
Eagle's medium, respectively (Life Technologies, Inc.) supplemented
with 10% fetal calf serum in an atmosphere of 5% CO2. Log
phase cells (1 × 106 cells/10-ml dish) were incubated
in media containing OA or fostriecin. OA was prepared
by dissolving in dimethyl sulfoxide, and fostriecin was dissolved in
phosphate-buffered saline containing 0.1 mM ascorbic acid.
Control incubations included that same amount of vehicle alone.
Preparation of S10 and P10 Extracts--
Cells were harvested
either by centrifugation (BT) or trypsinization (MO59J), washed twice
in phosphate-buffered saline, and lysed by a single freeze thaw cycle
as described previously (19). Cytoplasmic (S10) and nuclear (P10)
extracts were prepared as described (20). Extracts were snap frozen in
liquid nitrogen and stored at Preparation of Recombinant PP1 and Purification of
PP2A--
Human PP1
Cells were grown in LB medium plus 1 mM
MnCl2, and, after reaching an
A600 of 0.3, cells were induced with 0.1 mM IPTG for 16 h and then pelleted by centrifugation
at 4000 × g for 30 min. Cells from 0.75 liters of
suspension were resuspended in 15 ml of 50 mM Hepes-KOH, pH
7.5, 100 mM KCl, 1 mM EDTA, 2 mM
MnCl2, 5% glycerol, 0.1% Preparation of 32P-Labeled Glycogen Phosphorylase a
and Protein Phosphatase Assays--
32P-Labeled glycogen
phosphorylase a containing 1.0 mol of phosphate/mol of
subunit was prepared using phosphorylase b (22) and
phosphorylase kinase (23) purified from rabbit skeletal muscle by the
methods described. Details of the assay can be found in Ref. 4.
Briefly, assays were performed at 30 °C in a total volume of 30 µl
that included 10 µl of protein phosphatase diluted as required into
buffer B (50 mM Tris-HCl, pH 7.5 (20 °C), 0.1 mM EGTA, 0.1% (v/v) Protein Purification and DNA-PK Activity Assays--
The
DNA-PKcs and Ku subunits of DNA-PK were purified from human placenta as
described previously (25). Kinase assays using purified proteins or
cell extracts from MO59J (3 µg) or lymphoblastoid cells (3 µg) were
as described (19) except that the synthetic peptide substrate used was
PESQEAFADLWKK (26). The peptide, PESEQAFADLWKK, which is not
phosphorylated by DNA-PK, was used as a control (26). Reactions
contained 25 mM Hepes-NaOH, pH 7.5, 0.25 mM
synthetic peptide, 100 mM KCl, 10 mM
MgCl2, 1 mM dithiothreitol, 0.2 mM
EGTA, 0.1 mM EDTA plus 10 µg/ml sonicated calf thymus
DNA, and 0.25 mM ATP containing stabilized
[ Autophosphorylation of DNA-PK--
Purified DNA-PKcs and Ku
proteins were preincubated at 30 °C as described for activity
assays, except that synthetic peptide was not present. Radiolabeled ATP
was present where indicated at 0.25 mM. After 0-10 min,
aliquots were removed and analyzed by SDS-PAGE followed by
autoradiography. In order to reassay samples for remaining protein
kinase activity, aliquots corresponding to 5-10% (v/v) or the
percentage indicated of the preincubation reaction were removed
and reassayed under standard kinase assay conditions with a full
complement of synthetic peptide, DNA, and radiolabeled ATP as described
above. For "add-back" experiments, after the indicated times,
either the free catalytic subunit of PP1 (50 milliunits/ml final
concentration), the free catalytic subunit of PP2A (50 milliunits/ml
final concentration) or protein extracts from lymphoblastoid or MO59J
cells (treated with Me2SO, OA, or fostriecin) were
added to aliquots from preincubation reactions containing
phosphorylated DNA-PK. At timed intervals, aliquots corresponding to
5-10% (v/v) of this reaction were removed and reassayed under
standard kinase assay conditions with a full complement of synthetic
peptide, DNA, and radiolabeled ATP. In identical experiments, aliquots
were removed and analyzed by SDS-PAGE, followed by autoradiography.
The stoichiometry of phosphorylation of in vitro
phosphorylated DNA-PKcs was estimated by excising the phosphorylated
bands from the dried Coomassie-stained gels and measuring
32P incorporation by Cerenkov counting in a scintillation
counter. The specific activity of the radioactive ATP was calculated in cpm/pmol, and the amount of DNA-PKcs and Ku was estimated from the
predicted molecular masses (~470, 80, and 70 kDa, respectively) and
the protein concentration as determined by the Bio-Rad protein assay.
In Vivo Labeling and Immunoprecipitation of DNA-PKcs and
Ku--
BT cells were grown to approximately midlog phase, and then
cells were transferred to 15-ml conical tubes and pelleted by centrifugation at 1500 × g for 5 min. Cells were then
rinsed twice with phosphate-free RPMI medium. For each
experiment, ~2 × 106 cells were resuspended in 1 ml
of phosphate-free medium containing 10% fetal calf serum and
incubated with okadaic acid (1 µM) or an equivalent
volume of Me2SO for 1 h in a humidified
CO2 incubator at 37 °C under 5% CO2. Cells
were allowed to incorporate 32P-labeled inorganic
phosphate (0.75 millicuries/ml) for a further 1 h, and then the
label was removed and cells were washed twice in ice-cold
phosphate-buffered saline and lysed in immunoprecipitation buffer
containing 20 mM Tris-HCl, pH 7.4, 250 mM NaCl,
1 mM EDTA, 1% (v/v) Nonidet P-40, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 10 mM
NaF. DNA-PKcs and Ku were immunoprecipitated as described previously (15) and transferred to nitrocellulose membrane. The membrane was
exposed to x-ray film overnight at We have previously shown that incubation of purified DNA-PKcs and
Ku heterodimer with DNA and Mg-ATP results in phosphorylation of
all three subunits of the DNA-PK holoenzyme and
time-dependent loss of protein kinase activity (15). Loss
of DNA-PK protein kinase activity did not occur when the purified
protein was incubated with Mg-ATP in the absence of DNA or with DNA,
magnesium, and the nonhydrolyzable ATP analogue AMP-PNP (15),
suggesting that loss of DNA-PK protein kinase activity correlates with
phosphorylation of one or more of the DNA-PK subunits.
An important prediction of these results is that treatment with a
protein phosphatase would reverse the phosphorylation-induced loss of
DNA-PK protein kinase activity. In order to test this hypothesis,
purified DNA-PKcs and Ku were first incubated with DNA and Mg-ATP in
order to produce phosphorylated, inactive DNA-PK. Following this 10-min
incubation period, the purified catalytic subunit of either PP1 or PP2A
was added to the reactions, and DNA-PK protein kinase activity was
assayed. The addition of the catalytic subunit of either PP1 or PP2A
resulted in a complete (PP1) or partial (PP2A) restoration of DNA-PK
protein kinase activity (Fig. 1,
A and B, open symbols). In
contrast, when the catalytic subunit of PP1 or PP2A was preincubated
with microcystin, a potent inhibitor of both protein phosphatases, no
significant increase in DNA-PK protein kinase activity was observed
(Fig. 1, A and B, closed
symbols). Moreover, both PP1 and PP2A catalytic subunits were capable of removing phosphate from DNA-PKcs, Ku70, or Ku80 (Fig.
2, A and B). These
results strongly support our hypothesis that the loss of DNA-PK protein
kinase activity in vitro is due to reversible protein
phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was from PerkinElmer Life Sciences. Okadaic
acid (OA) and microcystin-LR were from Calbiochem. Dithiothreitol was
from BDH. Fostriecin was a kind gift from Dr. Michel Roberge
(University of British Columbia).
80 °C. Protein concentrations were
determined using a dye binding assay (Bio-Rad) with bovine serum
albumin as a standard.
1 cDNA was
polymerase chain reaction-amplified from a human (brain) cDNA
library (CLONTECH) incorporating an NdeI
site at the initiator codon (ATG) and a HindIII site
immediately following the stop codon. The polymerase chain reaction
product was cloned into pCRTOPO (Invitrogen), and the sequence was
verified by DNA sequencing. The modified cDNA was then cloned into
the NdeI/HindIII sites of the pCW vector (a kind
gift from Dr. F. W. Dalhqvist, University of Oregon) and introduced into Escherichia coli DH5
by standard techniques.
mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride and snap frozen in liquid
nitrogen and then stored at
80 °C. Frozen cells were thawed and
put through a French press cell (SIM Aminco) one time at 1100 p.s.i., and the extract was clarified by centrifugation at 35,000 × g for 30 min. The clarified extract was filtered through
a 0.45-µm filter and loaded onto a 20-ml column of SP-Sepharose
(Amersham Pharmacia Biotech) at 3 ml/min. The column was equilibrated
in buffer A (50 mM Tris-HCl, pH 7.5, 0.1 mM
EGTA, 5% glycerol, 0.1%
mercaptoethanol, and 1 mM
MnCl2). PP1
1 was eluted with a
gradient from 0-500 mM NaCl over 200 ml with 5-ml
fractions. PP1
1 was further purified on a
Mono-Q column equilibrated in buffer A. The column was developed at 1 ml/min from 0-500 mM NaCl over 40 ml, and 1-ml fractions
were collected. Peak fractions were dialyzed into buffer A containing
50% glycerol and 50 mM NaCl and stored at
20 °C. The
catalytic subunit of protein phosphatase 2A was purified from bovine
heart as described (21).
-mercaptoethanol, 1 mg/ml bovine
serum albumin) plus 10 µl of buffer C (50 mM Tris-HCl, pH
7.5 (20 °C), 0.1 mM EGTA, and 0.03% (v/v) Brij-35 plus
inhibitors/activators as required). The assays were initiated by the
addition of 10 µl of 30 µM 32P-labeled
glycogen phosphorylase a. One unit is defined as the amount
of enzyme that catalyzed the release of 1 µmol of phosphate in 1 min.
In all cases, dephosphorylation of substrates was kept less than 20%
to ensure the linearity of the assay. Rabbit skeletal muscle
inhibitor-2 was purified according to Cohen et al. (24) and
used at a final concentration of 200 nM in assays. All
assays that included protein phosphatase inhibitors were incubated at 30 °C for 15 min before initiating the assay with substrate.
Inhibitor-2 was diluted in assay buffer C immediately before use.
-32P]ATP (specific activity 500-1000 dpm/pmol) and
were started by the addition of purified DNA-PK proteins (concentration
as indicated). Reactions were at 30 °C for 5 min, and DNA-PK protein
kinase activity was calculated as nmol of phosphate incorporated into
the peptide substrate/min/mg of protein. Synthetic peptides were
synthesized and purified by high pressure liquid chromatography by the
Alberta Peptide Institute (Edmonton, Alberta, Canada).
80 °C and then probed with an
antibody to either Ku70 or DNA-PKcs as described in Fig. 6.
Approximately 2 × 107 cpm of lysate was used for each
immunoprecipitation reaction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
[in a new window]
Fig. 1.
Incubation of protein phosphatase 1 and 2A
catalytic subunits with phosphorylated inactivated DNA-PK complex
restores DNA-PK protein kinase activity. A, DNA-PK
complex consisting of DNA-PKcs (0.013 µg/µl) and Ku (0.004 µg/µl) (molar ratio 1:1) was incubated in a final volume of 33 µl, under standard assay conditions as described previously (15). At
0, 2, 5, and 10 min, 2-µl aliquots (equivalent to 0.1 µg of total
protein) were removed and assayed under standard assay conditions as
described previously (15). At 10 min, indicated by the
arrow, either recombinant PP1 (final concentration of 50 milliunits/ml (open squares)) or recombinant PP1
(final concentration of 50 milliunits/ml) that was preincubated with
microcystin-LR (1 µM final concentration)
(closed squares) was added to the incubation
mixture. After these additions, at timed intervals 2-µl aliquots were
removed and assayed under standard DNA-PK assay conditions as described
previously (15). B, DNA-PK was incubated exactly as
described for A; however, the catalytic subunit of PP2A
(purified from bovine heart) (50 milliunits/ml) was added in place of
recombinant PP1.
View larger version (35K):
[in a new window]
Fig. 2.
The catalytic subunits of PP1 and PP2A are
capable of dephosphorylating in vitro
autophosphorylated DNA-PK. DNA-PKcs (0.04 µg/µl) and Ku
(0.013 µg/µl) (1 µg of DNA-PK total protein) were preincubated in
50 mM Hepes-NaOH, pH 7.5, 50 mM KCl, 10 mM magnesium chloride, 1 mM dithiothreitol, 0.2 mM EDTA, 10 µg/ml calf thymus DNA, and 0.25 mM [ -32P]ATP in a total volume of 20 µl
for 10 min. Following the first incubation period, various
concentrations of recombinant PP1 catalytic subunit (A) or
PP2A catalytic subunit (purified from bovine heart) (B) were
added to the reaction at 15-s intervals (as shown), and incubations
were continued for a further 10 min. Following the second incubation
period, microcystin-LR (1 µM final) was added at 15-s
intervals to stop the dephosphorylation reactions, and samples were
boiled in SDS sample buffer. Samples were then analyzed by SDS-PAGE on
10% acrylamide gels followed by autoradiography. Shown are separate
exposures for DNA-PKcs (5 h at
80 °C with intensifying screens)
and Ku (overnight at
80 °C with intensifying screens).
The activity of most known protein phosphatases is regulated by
association with regulatory subunits that can target the protein phosphatase to certain subcellular locations or directly affect its
activity by allosteric or other effects. It was therefore important to
determine if the protein kinase activity of DNA-PK was affected by
phosphorylation in vivo. Specific protein phosphatases can
be inhibited by treatment of cells with particular cell-permeable protein phosphatase inhibitors. For example, treatment of human cells
with okadaic acid at 1 µM results in loss of PP2A
activity but not PP1 activity (27). Human lymphoblastoid (BT) cells
were incubated with either Me2SO (control), okadaic acid,
or fostriecin at PP2A-selective concentrations and were assayed for
protein phosphatase activity using radiolabeled phosphorylase
a as a substrate. Extracts were assayed either with no
additions (Fig. 3A,
solid bars), with the addition of the PP1
inhibitor, inhibitor-2, at 200 nM final (Fig.
3A, open bars) or inhibitor-2 (200 nM final concentration) plus 5 nM okadaic acid
(Fig. 3A, hatched bars). In this
experiment, the activity of both PP1 and PP2A is shown in assays with
no additions (solid bars), whereas in assays that contained inhibitor-2, only PP2A-like protein phosphatases would be
active (open bars), and in assays that contained
okadaic acid (at a concentration of 5 nM final) and
inhibitor-2 (hatched bars), neither of the
protein phosphatases would be active. These data show that of the total
phosphorylase phosphatase activity in BT cells, ~40% is PP1
protein phosphatase activity and 60% is PP2A-like protein phosphatase
activity (Fig. 3A, DMSO, open
bars compared with closed bars).
Treatment of cells with either okadaic acid or fostriecin, which, at
the specified concentrations used, is predicted to inhibit only
PP2A-like protein phosphatase activity, resulted in an ~50% loss of
phosphorylase phosphatase activity (Fig. 3A, OA or
fostriecin, solid bar). The further addition of inhibitor-2 (200 nM) to these extracts abolished the
majority of the remaining protein phosphatase activity, and this is
unaltered by the further addition of 5 nM OA to these
extracts. This indicates that treatment of cells with either OA or
fostriecin abolishes PP2A-like activity, leaving PP1 unaffected. Having
established this, the same extracts were used to assay for DNA-PK
protein kinase activity, and the DNA-PK protein kinase activity was
reduced by ~50% in the extracts from cells that had been treated
with either okadaic acid or fostriecin (Fig. 3B). No change
in the amount of DNA-PKcs protein was detected after treatment of cells with either okadaic acid or fostriecin (Fig. 3C). These data
support a model in which DNA-PK protein kinase activity is regulated by reversible protein phosphorylation in vivo and indicate
that, in vivo, protein phosphatases are required for
maintaining DNA-PK in a highly active state. Consequently, this model
would predict that DNA-PK is phosphorylated in vivo, and as
the phosphorylation state of DNA-PK increases, its activity
decreases.
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Our data therefore suggest that a protein phosphatase activity acts on
DNA-PK in vivo. It was therefore important to show that in
cell extracts there exists a protein phosphatase activity capable of
removing phosphate from DNA-PK. Purified DNA-PK was phosphorylated and
inactivated by incubation with DNA and radioactively labeled Mg-ATP.
The phosphorylation reactions were stopped by the addition of
wortmannin to 10 µM, and in vitro
phosphorylated DNA-PK protein was incubated with extracts from cells
that were either untreated, treated with okadaic acid, or treated with
fostriecin. The results show that extracts from untreated cells
contained an activity capable of removing phosphate from in
vitro phosphorylated DNA-PKcs and that the presence of inhibitor-2
(a PP1 inhibitor) had no inhibitory effect on the dephosphorylation
reaction (Fig. 4, compare lane
1 with lanes 2 and 3).
Significantly, when cells were treated with okadaic acid or fostriecin,
at PP2A-selective concentrations, no significant loss of DNA-PK
phosphorylation was observed (Fig. 4, lanes
4-7). Again, treatment of extracts with the PP1 inhibitor,
inhibitor-2, had no effect on dephosphorylation of DNA-PKcs, strongly
suggesting that the phosphatase in these extracts responsible for
dephosphorylating in vitro autophosphorylated DNA-PKcs is a
PP2A-like enzyme. Identical results were observed for Ku70 and Ku80
(data not shown). Although the data shown in Fig. 1 suggests that PP1
is more efficient than PP2A at dephosphorylating and activating DNA-PK
in vitro, it is important to note that these experiments
were carried out using the free catalytic subunits of either PP1 or
PP2A. These enzymes therefore lack their regulatory subunit(s) and
consequently would be expected to have different properties from the
naturally occurring enzymes.
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In order to determine whether cell extracts that had been treated with
okadaic acid or fostriecin were incapable of reactivating phosphorylated DNA-PK, purified DNA-PK was phosphorylated and inactivated in vitro and then incubated with extracts from
cells that were either untreated or treated with PP2A-selective
concentrations of okadaic acid or fostriecin. Since BT cells contain
abundant DNA-PK protein kinase activity, which would complicate
interpretation of results, a human cell line that lacks DNA-PKcs
protein (MO59J) was used for these experiments (28). Protein extracts
from untreated MO59J cells contained an activity that was capable of
reactivating DNA-PK, whereas treatment of MO59J cells with either
okadaic acid or fostriecin significantly decreased the ability of these
extracts to reactivate DNA-PK (Fig. 5).
These data again support our hypothesis that human cells contain a
PP2A-like enzyme that can both remove phosphate groups from in
vitro phosphorylated DNA-PK and reverse phosphorylation-induced
loss of protein kinase activity.
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Although our data show that a PP2A-like enzyme can dephosphorylate
DNA-PK that has been phosphorylated in vitro, it was
important to determine whether DNA-PK is phosphorylated in
vivo. Ku70 and Ku80 have previously been shown to be
phosphorylated on serine in vivo (18); however, to date,
in vivo phosphorylation of DNA-PKcs has not been reported.
Human lymphoblastoid (BT) cells were therefore incubated with
32P-labeled inorganic phosphate in phosphate-free RPMI in
the absence or presence of okadaic acid (1 µM final).
DNA-PKcs and Ku70/Ku80 subunits were immunoprecipitated with a
monoclonal antibody (42-27) and a polyclonal antibody to recombinant
Ku70 (Ab68) respectively, transferred to nitrocellulose membrane, and
exposed to x-ray film. Treatment with okadaic acid was found to cause a
dramatic increase in the in vivo phosphorylation of DNA-PKcs
(Fig. 6A), indicating that
DNA-PK undergoes reversible phosphorylation in vivo. In
addition, treatment of cells with okadaic acid increased the endogenous levels of phosphorylation of both Ku70 and Ku80 (Fig. 6C),
suggesting that both DNA-PKcs and Ku are modified by reversible
phosphorylation in vivo. Western blotting of the membrane
with antibodies to either DNA-PKcs (Figs. 6B) or Ku70 (Fig.
6D) shows that equal amounts of protein were
immunoprecipitated.
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DISCUSSION |
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Purified DNA-PKcs, Ku70, and Ku80 undergo DNA-dependent phosphorylation in vitro, and that phosphorylation is associated with loss of DNA-PK protein kinase activity (15). We have previously mapped the major in vitro Ku phosphorylation sites, and identification of the in vitro phosphorylation sites in DNA-PKcs is in progress.2 In this study, we show that the catalytic subunit of either PP1 or PP2A is capable of dephosphorylating DNA-PKcs, Ku70, and Ku80 in vitro and that this results in restoration of DNA-PK protein kinase activity. These data therefore strongly suggest that phosphorylation of one or more of the DNA-PK subunits is directly responsible for the loss of protein kinase activity. We estimate that in vitro, DNA-PKcs was phosphorylated on at least seven sites after 1 h of incubation under the conditions used in these experiments. Phosphorylation occurs predominantly on serine residues (15). Studies with synthetic peptides indicate that the protein phosphorylation consensus for DNA-PKcs is serine or threonine followed by glutamine (19). However, DNA-PK can also phosphorylate serines that are followed by tyrosine (16, 26), or valine (29), suggesting that the phosphorylation recognition motif may be more complex than originally proposed. The cDNA sequence of DNA-PKcs predicts 29 serine or threonine residues that are in an SQ or TQ motif, any of which could be a potential autophosphorylation site. The Ku heterodimer is also phosphorylated at several sites in vitro, one major site on Ku70 and at least three on Ku80 (16). At this time, it is not known if one single phosphorylation event is sufficient to inactivate DNA-PK or whether several phosphorylation events contribute to loss of protein kinase activity. We previously showed that DNA-PK-phosphorylated Ku70 is able to bind DNA and that phosphorylated DNA-PKcs dissociates from DNA-bound Ku (15), suggesting that the loss of kinase activity is due to inactivation of DNA-PKcs and not Ku. However, at this time, we cannot exclude the possibility that phosphorylation of Ku also plays a role in regulating the activity of DNA-PK.
We show, for the first time, that DNA-PKcs is phosphorylated in vivo. In preliminary experiments, low levels of phosphorylation of DNA-PKcs were observed in the absence of okadaic acid3; however, here we show that the addition of okadaic acid significantly increased the phosphorylation state of DNA-PKcs in vivo (Fig. 6A). These data suggest that DNA-PKcs is the target of protein phosphatases and protein kinases in vivo and that the activity of protein phosphatases is required to maintain DNA-PK in a highly active state. A prediction of this hypothesis is that DNA-PK would be expected to be largely dephosphorylated in its active form and highly phosphorylated in its inactive form. We have shown that, in vitro, autophosphorylation results in loss of DNA-PK protein kinase activity, again suggesting that DNA-PK is active in a dephosphorylated form and inactive in its phosphorylated form.
In vitro, co-immunoprecipitation experiments suggest that autophosphorylation of DNA-PKcs results in dissociation of phosphorylated DNA-PKcs from phosphorylated DNA-bound Ku (15). Again, this is consistent with phosphorylation resulting in an inactive form of DNA-PK. It remains to be seen whether DNA-PK is regulated by autophosphorylation or phosphorylation by other protein kinases in vivo. These observations also have implications for the repair of DNA double strand breaks by nonhomologous end joining in vivo. We speculate that Ku and DNA-PKcs assemble at the site of a DNA strand break in an ordered fashion, thus generating the active protein kinase. Subsequently, phosphorylation of DNA-PK, either by itself or by another protein kinase, may serve to inactivate DNA-PK and possibly remove DNA-PKcs from the site of the DNA lesion.
DNA-PKcs is a member of a growing family of
serine/threonine protein kinases that bears significant amino
acid similarity to the catalytic domain of phosphoinositide 3 kinases
(30-32). Interestingly, the p110 subunit of the class 1A
phosphoinositide-3 kinases has serine/threonine protein kinase
activity toward the Src homology 2 domain-containing targeting subunit,
p85, and increased incorporation of phosphate into p85 is associated
with a dramatic decrease in phosphoinositide 3-kinase activity (33).
Also, autophosphorylation of the p110 subunit of the class II
phosphoinositide 3 kinases down-regulates its lipid kinase activity
(34). Together, these data suggest that
phosphorylation-dependent inactivation may be a
characteristic of the phosphoinositide 3-kinase-like family of enzymes.
We have demonstrated the presence of a protein phosphatase activity in
crude extracts of human cells that is capable of dephosphorylating DNA-PKcs, Ku70, and Ku80 and restoring the kinase activity of phosphorylated inactivated DNA-PK. This activity has properties expected of a PP2A-like enzyme, since its activity is inhibited at
PP2A-selective concentrations of okadaic acid and fostriecin. Further
studies will be required in order to identify the protein phosphatase
responsible for dephosphorylating DNA-PK in vivo.
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ACKNOWLEDGEMENTS |
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We thank Seong-Cheol Son for supplying highly purified DNA-PKcs and Ku for these studies. Doug Chan is acknowledged for preliminary in vivo labeling experiments. We also thank Dr. Michel Roberge for providing a sample of fostriecin and Dr. Jim Lees-Miller for reading the manuscript.
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FOOTNOTES |
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* This work was supported by Canadian Institutes of Health Research Grant 13639 (to S. P. L. M.) and the Natural Sciences and Engineering Council of Canada (to G. M.).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.
Postdoctoral Fellow of the Alberta Heritage Foundation for Medical Research.
§ Senior Scholar of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed. Tel.: 403-220-7628; Fax: 403-289-9311; E-mail: leesmill@ucalgary.ca.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M011703200
2 Y. Yu, D. Chan and S. P. Lees-Miller, unpublished results.
3 D. Chan and S. P. Lees-Miller, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
PP1, PP2A, and
PP2B, protein phosphatase 1, 2A, and 2B, respectively;
DNA-PK, DNA-dependent protein kinase;
DNA-PKcs, DNA-PK catalytic
subunit;
OA, okadaic acid;
PAGE, polyacrylamide gel electrophoresis;
AMP-PNP, 5'-adenylyl-,
-imidodiphosphate.
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