ATP Activates Ataxia-Telangiectasia Mutated (ATM) in
Vitro
IMPORTANCE OF AUTOPHOSPHORYLATION*
Sergei
Kozlov
,
Nuri
Gueven
,
Katherine
Keating§,
Jonathan
Ramsay
¶, and
Martin F.
Lavin
**
From
The Queensland Institute of Medical Research, PO
Royal Brisbane Hospital, Herston Qld 4029, Australia,
§ EIRX Therapeutics Ltd., Cork, Airport Business Park, Cork,
Ireland, the ¶ Queensland Radium Institute, Mater Hospital,
Brisbane Qld 4101, Australia, and the
Department of Surgery,
University of Queensland, PO Royal Brisbane Hospital,
Herston QLD 4029, Australia
Received for publication, January 3, 2003
 |
ABSTRACT |
Ataxia-telangiectasia Mutated (ATM), mutated in
the human disorder ataxia-telangiectasia, is rapidly activated by DNA
double strand breaks. The mechanism of activation remains unresolved, and it is uncertain whether autophosphorylation contributes to activation. We describe an in vitro immunoprecipitation
system demonstrating activation of ATM kinase from unirradiated
extracts by preincubation with ATP. Activation is both time- and ATP
concentration-dependent, other nucleotides fail to activate
ATM, and DNA is not required. ATP activation is specific for ATM since
it is not observed with kinase-dead ATM, it requires Mn2+,
and it is inhibited by wortmannin. Exposure of activated ATM to
phosphatase abrogates activity, and repeat cycles of ATP and phosphatase treatment reveal a requirement for autophosphorylation in
the activation process. Phosphopeptide mapping revealed similarities between the patterns of autophosphorylation for irradiated and ATP-treated ATM. Caffeine inhibited ATM kinase activity for substrates but did not interfere with ATM autophosphorylation. ATP failed to activate either A-T and rad3-related protein (ATR) or
DNA-dependent protein kinase under these conditions,
supporting the specificity for ATM. These data demonstrate that ATP can
specifically induce activation of ATM by a mechanism involving
autophosphorylation. The relationship of this activation to DNA damage
activation remains unclear but represents a useful model for
understanding in vivo activation.
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INTRODUCTION |
Neurodegeneration, immunodeficiency, genome instability, and
cancer susceptibility represent the major hallmarks of the human genetic disorder ataxia-telangiectasia
(A-T)1 (1, 2). Extreme
sensitivity to radiation in patients undergoing radiotherapy for cancer
and in cells in culture are also characteristic of this disease (3-5).
The exact basis of the radiosensitivity in A-T cells remains unknown
but appears to be related to the failure to repair residual chromosomal
breaks (6, 7) or activation of the structural maintenance of
chromosomes (SMC1) protein in response to DNA damage, which is
ATM-dependent and implicated in modulating radiosensitivity
(8, 9). In A-T cells, faulty DNA damage recognition is also accompanied
by defective cell cycle checkpoint activation, which is likely to
contribute to the genetic instability and cancer susceptibility in this
syndrome (10, 11).
The gene defective in A-T, ATM (A-T mutated), encodes a
protein that is a member of the phosphatidylinositol 3-kinase
(PI3-kinase) family (12). This group includes the catalytic subunit of
DNA-dependent protein kinase (DNA-PKcs), A-T and
rad3-related protein (ATR), and proteins in different organisms
responsible for DNA damage recognition and cell cycle control (13).
Although there is some evidence for regulation of ATM at the
transcriptional and translational levels (14), it is primarily
activated as a pre-existing protein by ionizing radiation and other
agents that give rise to double strand breaks in DNA, suggesting that
post-translational modification may be important in its activation
(15-17). Changes in abundance of ATM protein in response to radiation
have not been observed (18). Once activated, ATM phosphorylates a host
of substrates, primarily involved in recognizing DNA damage or
signaling this damage to cell cycle checkpoints (19), but there is also
evidence for non-DNA damage activation (20). Among the ATM substrates involved in DNA damage recognition are Nbs1, defective in the Nijmegen
breakage syndrome (21-23), BRCA1 (24, 25), Rad51 (26), and BLM,
defective in Bloom's syndrome (27). ATM also plays a central role in
the activation of cell checkpoints in response to radiation damage (10,
11). It is of some interest that this regulation occurs at multiple
levels for a single checkpoint. In the case of the G1/S
checkpoint, activated ATM phosphorylates p53 on serine 15 (15-17), it
also phosphorylates and activates Chk2 to in turn phosphorylate p53 on
serine 20 (28), which may contribute to its stabilization at least in
some cell types, and by phosphorylating mdm2 on serine 395 (29), it
further ensures stabilization and activation of p53 to induce p21/WAF1
and the G1/S checkpoint (30, 31). ATM phosphorylates Chk2,
Nbs1, SMC1, and BRCA1 to achieve activation of the S phase checkpoint
in parallel pathways (8, 9, 32). Finally, Chk2 and BRCA1 are also substrates for ATM in the G2/M checkpoint (33).
Although multiple substrates and pathways controlled by ATM have been
identified, it remains unclear as to how double strand breaks in DNA
activate its kinase activity. In the case of the related
PI3-kinase, DNA-PKcs, it is recruited to DNA strand breaks and
activated by the Ku70/Ku80 heterodimer (34). Intriguingly, inositol
hexakisphosphate stimulates DNA-PK-dependent non-homologous DNA end joining in vitro, and this appears to be achieved by
specific interaction with Ku70/Ku80 (35). Another member of the
PI3-kinase family mTOR is altered in its activity by small molecules,
acting as a sensor of ATP concentration (36). Protein kinase activity can also be regulated by autophosphorylation, which has been observed with DNA damage signaling PI3-kinases (37). However, only DNA-PK autophosphorylation was demonstrated to have an effect on the functional activity of the enzyme in a DNA-dependent manner
(38). Autophosphorylation causes inactivation of DNA-PK kinase activity and disruption of the DNA-PKcs-Ku complex. Subsequently, it was shown
that phosphatase treatment activated DNA-PK (39). Autophosphorylation has also been observed with ATM immunoprecipitated from irradiated control cells and with ATM expressed in baculovirus (25, 40), and there
is evidence using 32P-labeling in vivo to
support this (23, 41), but its importance to ATM function has not been
addressed. The putative autophosphorylation sites on ATM were suggested
based on consensus motifs expected to be recognized by ATM in oriented
peptide libraries (42). These include Ser-2941, Ser-1635, and
Ser-440, but phosphorylation of these sites in vivo has not
been reported. To investigate the activation of ATM and the possible
involvement of autophosphorylation in this process, we have established
an in vitro system involving immunoprecipitation of ATM and
activation by ATP.
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EXPERIMENTAL PROCEDURES |
Materials and Reagents--
Cell culture medium RPMI 1640 was from Invitrogen. Fetal bovine serum was from JRH Biosciences.
Microcystin LR was from Biomol Research Laboaratories, Inc. (Sapphire
Bioscience).
-Protein phosphatase (
-PPase) was from New England
Biolabs. ATP, ADP, AMP, NADP, 5'-adenylylimidodiphosphate (AMP-PNP),
GTP, and okadaic acid were from Sigma. Wortmannin and caffeine were
from Fluka Chemie AG. Protein G-Sepharose 4 fast flow, protein
A-Sepharose CL-4B, and Redivue [
-32P]ATP (specific
activity ~3000 Ci/Mmol) were from Amersham Biosciences. Western
Lightning chemiluminescence reagent was from PerkinElmer Life Sciences.
Anti-mouse and anti-rabbit antibodies conjugated with peroxidase were
from Silenus/Chemicon Australia. For ATM Western blotting, ATM 2C1
monoclonal antibody (GeneTex) was used. For ATR Western blotting and
immunoprecipitations, ATR PA1-450 antibody (Affinity
Bioreagents) was employed. Western blotting and immunoprecipitations
for DNA-PK were performed with Ab-2 monoclonal antibody (Oncogene
Research Products). All other reagents were from standard suppliers or
as indicated in the text.
Cell Lines and Irradiation--
Lymphoblastoid cell lines
established from normal (C3ABR) and A-T patients (L3 and AT1ABR) were
used. We acknowledge Yosef Shiloh, Tel Aviv, Israel for the L3 cell
line that fails to produce ATM protein due to a homozygous truncation
mutation (C103T). AT1ABR is an A-T cell line homozygous for the
7636del9 mutation that produces near full-length ATM protein that is
kinase-dead (25). The Lymphoblastoid cell lines were cultured in RPMI
1640 medium with 10% fetal calf serum, 100 units/ml penicillin
(Invitrogen), and 100 units/ml streptomycin (Invitrogen). All
irradiations were performed at room temperature using an IBL437C
irradiator (2.4 Gy/min, Compagnie ORIS Industrie).
Antibody Production--
Construction of GST fusion proteins
containing fragments of ATM protein was described previously (17).
GST-ATM2 fusion protein (ATM amino acids 250-522) was expressed
according to standard protocol (43). Full-length GST-ATM2 was isolated
by preparative SDS-PAGE, eluted from gel slices, and used for sheep
immunization according to standard protocols. Antibodies were purified
from collected sheep sera by two consecutive affinity chromatography steps. GST-Affi-Gel 15 and GST-ATM2-Affi-Gel 15 affinity columns were
synthesized according to the manufacturer's instructions (Bio-Rad).
Firstly, serum diluted 1/10 in phosphate-buffered saline was applied to
a GST-Affi-Gel 15 column to deplete it from anti-GST antibodies.
Secondly, serum was passed through a GST-ATM2-Affi-Gel 15 column, and
ATM-specific antibodies were eluted, dialyzed against phosphate-buffered saline containing 0.05% sodium azide, and
concentrated by ultrafiltration on Ultrafree-4 BioMax 30K centrifugal
filter (Millipore). Affinity-purified antibodies were used in all
subsequent immunoprecipitations and kinase assays.
Immune Complex Protein Kinase Assays--
Cells were collected
by diluting with an equal volume of ice-cold phosphate-buffered saline
containing 1 mM Na3VO4 and 1 mM NaF followed by centrifugation. After a brief wash with
ice-cold phosphate-buffered saline, cells were lysed immediately for
use in immune complex kinase assays. ATM, ATR, and DNA-PK kinase assays were performed as described (16, 44, 45) with modifications. 0.2%
Tween 20 lysis buffer was modified by addition of 1 mM
Na3VO4, 1 mM NaF, 10 mM
Na2MoO4, 20 mM
-glycerophosphate, 5 µM microcystin-LR, 5 nM okadaic acid, 5 µg/ml each of aprotinin, leupeptin,
and pepstatin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. Cells were lysed by rotating for 45 min
at 4 °C followed by centrifugation at 13,000 × g
for 10 min. Cell lysates were precleared by constant mixing for 1 h with protein A/G-Sepharose. 1 mg of precleared lysate was used for
immunoprecipitation with ATM, ATR, or DNA-PK antibodies (see above)
overnight with constant mixing. Immune complexes were absorbed onto
protein G-Sepharose and washed. After two washes with ice-cold lysis
buffer, one wash with high salt buffer (lysis buffer with 0.5 M NaCl), and two washes with basic kinase buffer,
immunocomplexes were subjected to procedures outlined below or used
directly in kinase reactions. All washing buffers contained 1 mM Na3VaO4, 1 mM NaF,
and 1 mM phenylmethylsulfonyl fluoride. The
immunoprecipitates were resuspended in 30 µl of kinase buffer as
described (44), and kinase reaction was carried out for 30 min
at 30 °C either for autophosphorylation or for substrate
phosphorylation of 1 µg of GST-p53 1-44. Linear reaction
conditions were established for ATM kinase assays. The reaction was
stopped by addition of 5 µl of 6× SDS gel loading buffer. The
reaction products were separated by SDS-PAGE. A biphasic gel system
similar to one described by Ziv et al. (46) was used. The
top part of the separating gel (5%) was used for immunoblot analysis
of ATM, ATR, or DNA-PK to give an accurate estimation of the amount of
kinase present in the reaction. The bottom part of the gel (12%) was
stained with colloidal Coomassie G-250 to visualize the amount of p53
substrate present in the kinase reaction. Gels were dried onto a
Whatmann 3 MM filter paper and exposed to an x-ray film (Fuji Photo
Film Co., Ltd.) with an intensifying screen at
80 °C.
Radioactivity was quantitated by excising ATM and p53 bands from the
gel and counting the incorporated 32P by liquid
scintillation counting.
Activation and Inhibition of Kinase Activities--
To
prephosphorylate ATM prior to kinase reaction, washed protein G-bound
ATM immunocomplexes were incubated with 1 mM ATP (or
concentrations as indicated in the figure legends) in the following
phosphorylation buffer: 20 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 10 mM MnCl2,
20 mM
-glycerophosphate, 1 mM
Na3VO4, 20 µM microcystin.
Reactions were incubated for 30 min at 30 °C, washed three times in
ice cold basic kinase buffer to remove the ATP, and then directly used
in kinase assays as described above. Treatment of ATM immunocomplexes
with ADP, AMP, NADP, AMP-PNP, GTP, dNTPs, and polyADP ribose was
performed as outlined for ATP. Wortmannin was dissolved in
Me2SO to make a 20 mM solution, which was
stored in aliquots at
80 °C. Wortmannin was used at 1 µM concentration in the reaction conditions described
above. Caffeine was prepared fresh as a 100 mM solution in
water. ATM immunocomplexes were incubated on ice with a range of
concentrations of caffeine as indicated in phosphorylation buffer for
15 min and then subjected to the reaction conditions described above.
Phosphatase Treatment of Immune Complexes--
Protein G-bound
ATM immunocomplexes were additionally washed twice with basic
phosphatase buffer (50 mM Tris-HCl, pH 7.5, 5 mM dithiothreitol, 2 mM MnCl2) and
then incubated with 100 units of
-protein phosphatase (New England
Biolabs) for 30 min at 30 °C. The reaction was stopped by addition
of ice-cold basic kinase buffer containing 10 mM
Na3VoO4 and 10 mM NaF.
Phosphatase-treated immune complexes were washed twice with basic
kinase buffer and used in kinase assays as described above.
Tryptic Phosphopeptide Mapping--
Protein G-bound
ATM-immunocomplexes from unirradiated, irradiated, and ATP-treated
samples were subjected to the autophosphorylation reaction conditions
described above. 32P-labeled ATM was separated by 5%
SDS-PAGE and visualized by autoradiography. The ATM band was excised
and subjected to in-gel digestion using trypsin (sequencing grade,
Promega). Peptides were extracted, and two-dimensional phosphopeptide
mapping on thin-layer cellulose plates was performed as described (47)
using electrophoresis at pH 1.9 in the first dimension followed by
chromatography in buffer containing
n-butanol/pyridine/acetic acid/water (15:10:3:12). Plates
were exposed to x-ray film with an intensifying screen at
70 °C to
detect phosphopeptides.
 |
RESULTS |
ATM Undergoes Autophosphorylation Post-irradiation--
We
initially determined whether ATM undergoes autophosphorylation in
response to ionizing radiation using immunoprecipitation with
anti-ATM antibody. Autophosphorylation of ATM was observed by 5 min after irradiation, and this did not appear to increase further up
to 15 min after irradiation (Fig.
1A). Indeed, no increase in
autophosphorylation was observed up to 1 h after irradiation (results not shown). On average, the extent of this increase was 2-fold
as determined by scintillation counting of the ATM bands. As expected,
ATM kinase activity toward p531-44 substrate increased in
parallel, with time (Fig. 1B). No change in the amount of
ATM protein occurred under these conditions. These data, together with
previous results showing that ATM is a phosphoprotein (41), would
predict that phosphatase treatment would abolish ATM kinase activity.
The results in Fig. 1C demonstrate that when ATM is immunoprecipitated from irradiated cell extracts and subsequently treated with phosphatase, most of its kinase activity is lost (lanes 3 and 4). This is not an artifact due to
residual phosphatase since addition of 32P-labeled p53 to
this incubation did not result in any release of free 32P
(results not shown). We also observed a decrease in the capacity of ATM
to autophosphorylate after pretreatment with phosphatase (Fig.
1D). Thus it appears that phosphorylation status of ATM is
critical to its kinase activity.

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Fig. 1.
Radiation-induced
activation of ATM. A, radiation-induced increase in ATM
autophosphorylation in vitro. ATM was immunoprecipitated
from unirradiated and irradiated (5- and 15-min incubation) control
C3ABR cells, and autophosphorylation was determined by incubation with
10 µCi of [ -32P]ATP, separation on 5% SDS-PAGE
followed by transfer onto a nitrocellulose membrane, and
autoradiography. The same membrane was immunoblotted with an anti-ATM
antibody to determine ATM loading. IR, 10 Gy of
ionizing radiation. IP, immunoprecipitate.
ATM-AutoP, autoradiogram of radioactive phosphate
incorporated into ATM. ATM, Western blot of the amount of
ATM protein in the autophosphorylation reaction.
32P-p531-44-GST, autoradiogram
of radioactive phosphate incorporated into the substrate peptide.
p531-44-GST, Coomassie-stained gel
indicating the amount of substrate in the reaction. B,
radiation-induced increase in ATM kinase activity in vitro.
Immunoprecipitated ATM was used in a standard kinase reaction in the
presence of 10 µCi of [ -32P]ATP using
GST-p531-44 as substrate. ATM, Western blot of
the amount of ATM protein in the kinase reaction. As shown in
C, phosphatase treatment abrogates ATM kinase activity
in vitro. Immunoprecipitated ATM was incubated in
phosphatase buffer or phosphatase buffer containing 100 units of
-phosphatase for 30 min at 30 °C. After subsequent removal and
inactivation of phosphatase by extensive washing with kinase buffer
containing phosphatase inhibitors, ATM was subjected to a standard
kinase reaction in the presence of 10 µCi of
[ -32P]ATP using GST-p531-44 as substrate.
As shown in D, phosphatase treatment affects ATM
autophosphorylation in vitro. Immunoprecipitated ATM was
in- cubated in phosphatase buffer or phosphatase buffer containing
100 units of -phosphatase for 30 min at 30 °C. After subsequent
removal and inactivation of phosphatase by extensive washing with
kinase buffer containing phosphatase inhibitors, ATM was subjected to
autophosphorylation reaction in the presence of 10 µCi of
[ -32P]ATP.
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ATP Activates ATM Kinase--
Previous data have
revealed that when inactive forms of various protein kinases are
incubated with unlabelled ATP, they became activated by mechanisms
involving autophosphorylation (48-50). Since we have shown in Fig. 1
that autophosphorylation appears to be important for ATM activation, we
rationalized that ATM protein, immunoprecipitated from unirradiated
cell extracts, had the potential to be activated by significantly
higher concentrations of ATP than those utilized to measure ATM kinase
under standard conditions. Immunoprecipitated ATM protein from
unirradiated extracts was incubated with unlabelled ATP (1 mM) for 30 min at 30 °C followed by washing out excess
ATP and carrying out ATM kinase activity by standard assay. Under these
conditions, we observed a dramatic increase in ATM kinase
activity after preincubation with unlabelled ATP (Fig.
2).

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Fig. 2.
Preincubation of ATM from C3ABR cells with
ATP causes a marked increase in ATM kinase activity in
vitro. Immunoprecipitated ATM was activated by incubation for 30 min at 30 °C in kinase buffer with and without 1 mM
unlabelled ATP. After removal of ATP by extensive washing with kinase
buffer, ATM was subjected to a standard kinase reaction using
GST-p531-44 as substrate. IP,
immunoprecipitate.
32P-p531-44-GST, autoradiogram
of radioactive labeled substrate. ATM, Western blot of the
amount of ATM protein in the kinase reaction.
p531-44-GST, Coomassie-stained substrate
in the reaction.
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The amount of ATM kinase activity exceeded that observed with ATM from
irradiated extracts, 10-fold as compared with ~2-fold (Fig. 2,
lanes 2 and 3). A further increase in kinase
activity was also observed where ATM from irradiated extracts was
preincubated with unlabelled ATP, and the overall extent of activation
was approximately the same in both cases (Fig. 2, lanes 2 and 4). These results suggest that after irradiation, only a
proportion of ATM protein is activated, but in the presence of excess
ATP in vitro, all of the molecules are activated.
Activation Is ATM-specific--
It is well established that
in vitro ATM kinase activity has a requirement for
Mn2+ (15, 16) and that low concentrations of the PI3-kinase
inhibitor wortmannin inhibit ATM kinase (45). The results in Fig.
3A show that preincubation
with ATP activates ATM kinase from unirradiated cell extracts in the
presence of MnCl2, but in its absence, no activation is
observed.

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Fig. 3.
Specific activation of ATM kinase by ATP from
C3ABR cells. As shown in A, ATM kinase activation by
ATP from unirradiated extracts is
Mn2+-dependent. Immunoprecipitated ATM was
activated by incubation for 30 min at 30 °C in kinase buffer
containing 1 mM unlabelled ATP in the presence or absence
of Mn2+. After removal of ATP by extensive washing with
kinase buffer, ATM was subjected to a standard kinase reaction using
GST-p531-44 as substrate. IP,
immunoprecipitate.
32P-p531-44-GST, autoradiogram
of radioactive labeled substrate.
p531-44-GST, Coomassie-stained substrate
in the reaction. As shown in B, ATM kinase activation by ATP
in irradiated cell extracts is Mn2+-dependent.
ATM immunoprecipitated from cell extracts prepared form irradiated
cells was activated by incubation for 30 min at 30 °C in kinase
buffer containing 1 mM unlabelled ATP in the presence or
absence of Mn2+. After removal of ATP by extensive washing
with kinase buffer, ATM was subjected to a standard kinase reaction
using GST-p531-44 as substrate. IR, 10 Gy of
ionizing radiation. ATM, Western blot of the amount of ATM
protein in the kinase reaction. As shown in C, ATM kinase
activation by ATP is inhibited by wortmannin. Immunoprecipitated ATM
was incubated for 30 min at 30 °C in kinase buffer or kinase buffer
containing 1 µM wortmannin and/or 1 mM ATP.
After washing with kinase buffer, ATM was subjected to a normal kinase reaction using GST-p531-44 as substrate. As
shown in D, ATM kinase activation by ATP is absent in A-T
cell lines. Cell extracts were prepared from control (C3ABR)
and two A-T cell lines, L3 not expressing ATM protein and AT1ABR
expressing near full-length mutated ATM. Immunoprecipitates were
activated by incubation for 30 min at 30 °C in kinase buffer
containing 1 mM unlabelled ATP. After removal of ATP by
extensive washing with kinase buffer, ATM was subjected to a standard
kinase reaction using GST-p531-44 as substrate.
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Additional activation of ATM from irradiated extracts was seen with
ATP, and again, this was Mn2+-dependent (Fig.
3B). Wortmannin, when included in the preincubation step
with ATP, completely abolished activity (Fig. 3C). No
activation was obtained when ATP was incubated with immunoprecipitates
from an A-T cell line expressing mutant protein (Fig. 3D,
lanes 3 and 4). This A-T cell line has an
in-frame 9-nucleotide deletion (7636del9) upstream from the kinase
domain of ATM and expresses a less stable mutant form of ATM
(middle panel). A second A-T cell line, L3, homozygous for a
truncating mutation at nucleotide 103 and not producing ATM protein,
also failed to show a response to ATP (Fig. 3D, lanes
7 and 8), supporting specificity for ATM in this
activation. To define in more detail the dependence for ATP in ATM
activation, we carried out ATP concentration and time course
experiments. The results in Fig.
4A demonstrate that ATP
concentrations of 50 µM and above activate ATM kinase
after a 30-min preincubation. Activation was evident after a 5-min
preincubation with 1 mM ATP, reaching a maximum by
30 min (Fig. 4B). Comparison is made with the effect
of radiation (lanes 1 and 2)

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Fig. 4.
Concentration and
time-dependent ATM activation by ATP. A,
effect of ATP concentration on activation. Immunoprecipitated ATM was
incubated for 30 min at 30 °C in kinase buffer containing different
concentrations of unlabelled ATP as indicated. After removal of ATP by
extensive washing with kinase buffer, ATM was subjected to a standard
kinase reaction using GST-p531-44 as substrate.
IP, immunoprecipitate. ATM, Western blot of the
amount of ATM protein in the autophosphorylation reaction.
32P-p531-44-GST, autoradiogram
of radioactive phosphate incorporated into the substrate peptide.
p531-44-GST, Coomassie-stained gel
indicating the amount of substrate in the reaction. B, time
course of ATP preincubation for activation. Immunoprecipitated ATM was
incubated for different time intervals as indicated at 30 °C in
kinase buffer containing 1 mM unlabelled ATP. After removal
of ATP by extensive washing with kinase buffer, ATM was subjected to a
standard kinase reaction using GST-p531-44 as substrate.
Lanes 1 and 2 depict ATM kinase activity in
immunoprecipitates from unirradiated or irradiated samples without ATP
preincubation conditions. IR, 10 Gy of ionizing radiation.
As shown in C, immunoprecipitated ATM was activated by
incubation for 30 min at 30 °C in kinase buffer containing 1 mM unlabelled ATP. After removal of ATP by extensive
washing with kinase buffer, ATM was subjected to a standard kinase
reaction for various time intervals as indicated using
GST-p531-44 as substrate.
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Preincubation with ATP for 30 min followed by an
increasing time of incubation in [
32P[ATP led to a
gradual increase in p53 substrate phosphorylation (Fig. 4C).
To determine whether ATM activation required ATP hydrolysis, immunoprecipitated ATM was preincubated with a non-hydrolyzable ATP
analogue, AMP-PNP. In contrast to preincubation with ATP, AMP-PNP
failed to activate ATM kinase toward p53 substrate (Fig. 5, lane 4). Substitution of
ATP with GTP in the preincubation reaction did not cause any
significant change in the level of ATM kinase activity (data not
shown). Only ADP, dATP, and TTP had any appreciable effect on activity,
and these were considerably less effective than ATP (Fig. 5). Addition
of sonicated DNA to the preincubation step or to the incubation mix did
not alter the kinase activity (results not shown). In addition, DNase
treatment of the ATM immunoprecipitates did not prevent ATP activation
of ATM (results not shown).

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Fig. 5.
Activation of ATM by ATP analogues and other
nucleotides. Immunoprecipitated ATM was incubated for 30 min at
30 °C in kinase buffer containing ATP or other nucleotides as
indicated. After removal of ATP by extensive washing with kinase
buffer, ATM was subjected to a standard kinase reaction using
GST-p531-44 as substrate.
32P-p531-44-GST, autoradiogram
of radioactive phosphate incorporated into the substrate peptide.
ATM, Western blot of the amount of ATM protein in the kinase
reaction. p531-44-GST, Coomassie stained
gel indicating the amount of substrate in the reaction.
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ATM Is Activated by Autophosphorylation in Vitro--
To further
confirm that autophosphorylation is an inherent part of ATM activation,
we carried out successive treatments with ATP and phosphatase with
immunoprecipitated ATM from irradiated and unirradiated extracts. As
observed above, ATP activated ATM kinase from unirradiated extracts,
and subsequent phosphatase treatment abolished this capacity for
activation (Fig. 6A,
lanes 3 and 4). However, it was possible to
restore activity after phosphatase treatment and washing away excess
phosphatase by incubating again with ATP prior to carrying out the
kinase assay (Fig. 6A, lane 5). This was also the
case for immunoprecipitates from irradiated extracts (Fig.
6A, right panel). Increasing ATP concentrations over the range 0-2 mM failed to show a change in ATM
autophosphorylation either for irradiated or for unirradiated
immunoprecipitates (Fig. 6B). This can be explained by
having different levels of activation of ATM at different ATP
concentrations. In the less activated case, at lower ATP preincubation
concentrations, more sites would be available for 32P
incorporation in the subsequent autophosphorylation, whereas at higher
ATP concentrations, fewer sites would be unphosphorylated, but the
enzyme would have more activity. The end result would represent little
change in incorporation at the different ATP concentrations. To
demonstrate that autophosphorylation was indeed part of the mechanism
of activation, we carried out tryptic phosphopeptide mapping. For ATM
from irradiated cells, at least seven phosphopeptides (circled) were evident after tryptic digestion, indicating
that there are multiple phosphorylation sites on ATM (Fig.
6C). It is clear that when ATM kinase is activated by ATP,
substantial overlap occurs between the two phosphorylation patterns
(Fig. 6C). Notably absent in the ATP activated sample are
phosphopeptides 3 and 7. It should be noted that ATM isolated from
unirradiated cells has low basal activity, and this is reflected in
appearance of some of these phosphopeptides. In essence, these data
reveal that autophosphorylation appears to be essential for ATM kinase activity at least in vitro.

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Fig. 6.
ATM is activated by autophosphorylation.
A, effect of successive phosphorylation and
dephosphorylation on ATM kinase activity. Immunoprecipitated ATM was
incubated for 30 min at 30 °C in kinase buffer or kinase buffer
containing 1 mM ATP. After removal of ATP by extensive
washing with kinase buffer, ATM was incubated in phosphatase buffer or
phosphatase buffer containing 100 units of -phosphatase for 30 min
at 30 °C. After subsequent removal of phosphatase by extensive
washing with kinase buffer, ATM was again incubated in kinase buffer or
kinase buffer containing 1 mM ATP for 30 min at 30 °C.
After additional washing with kinase buffer, ATM was subjected to a
normal kinase reaction using GST-p531-44 as substrate.
Left panel, immunoprecipitates (IP) from
unirradiated extracts (lanes 3-5). Right panel,
immunoprecipitates from irradiated extracts (lanes 8-10).
IR, 5 Gy of ionizing radiation. ATP
(1), first round of incubation with ATP. ATP
(2), second round of incubation with ATP.
32P-p531-44-GST, autoradiogram
of radioactive phosphate incorporated into the substrate peptide.
ATM, Western blot of the amount of ATM protein in the kinase
reaction. p531-44-GST, Coomassie-stained
gel indicating the amount of substrate in the reaction.
ATM-AutoP, autoradiogram of radioactive phosphate
incorporated into ATM. B, effect of pretreatment with ATP on
ATM autophosphorylation. Immunoprecipitated ATM was incubated for 30 min at 30 °C in kinase buffer or kinase buffer containing ATP. After
removal of ATP by extensive washing with kinase buffer, ATM was
subjected to autophosphorylation reaction in the presence of 10 µCi
of [ -32P]ATP. Lane 1 depicts ATM
autophosphorylation in immunoprecipitates from irradiated (two
upper lanes) or unirradiated (two lower lanes)
samples without ATP preincubation conditions. Ig ATM,
immunoprecipitated and stained ATM. C, tryptic
phosphopeptide mapping of ATM, autophosphorylated in vitro
from unirradiated (UNIRR), irradiated, and ATP-activated
ATM. Immunoprecipitates were subjected to ATM autophosphorylation
conditions as described under "Experimental Procedures" followed by
5% SDS-PAGE. The labeled ATM protein band was enzymatically digested
with trypsin. The resulting peptides were separated in the first and
second dimension by electrophoresis and chromatography, respectively.
The origins are marked by diamonds, and the positions of the
phosphopeptides are indicated by circles.
|
|
Previous results reveal that preincubation of cells with caffeine, a
compound that overrides cell cycle checkpoints and radiosensitizes cells by inhibiting both ATM and ATR, did not affect radiation-induced ATM kinase when subsequently assayed in vitro (44). These
data were interpreted to mean that the pathway leading to ATM
activation in irradiated cells is insensitive to caffeine and that this
activation does not involve autophosphorylation. These results appear
to be at odds with the ATP-induced autophosphorylation of ATM observed here. To address this, we preincubated ATM immunoprecipitates from
unirradiated cells with caffeine, prior to incubation with unlabelled
ATP and subsequent washing out before measuring kinase activity. When
caffeine (1 mM) was added with unlabelled ATP in the
preincubation step to immunoprecipitates, it failed to interfere with
activation of ATM (Fig. 7A). A
small stimulation of activity was observed with caffeine alone.
However, when caffeine was added to the kinase reaction with p53
substrate, as expected, it inhibited substrate phosphorylation by ATM
(Fig. 7B). Caffeine concentrations up to 4 mM
failed to interfere with in vitro activation (results not
shown). It is evident from the results in Fig. 7C that
preincubation of cells with caffeine did not interfere with the
activation of ATM kinase, as observed by Sarkaria et al.
(44). Again caffeine on its own had some stimulatory activity.
Incubation of ATM immunoprecipitates with 1 mM caffeine in
the kinase reaction had only a minimal effect on autophosphorylation
(results not shown). These data suggest that although caffeine inhibits
ATM kinase activity, it fails to interfere with autophosphorylation of
ATM, thus distinguishing between the two types of activity.

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|
Fig. 7.
Effect of caffeine pretreatment on ATM kinase
activity. As shown in A, immunoprecipitated ATM was
incubated for 30 min at 30 °C in kinase buffer or kinase buffer
containing 1 mM caffeine and/or 1 mM ATP. After
washing with kinase buffer, ATM was subjected to a standard kinase
reaction using GST-p531-44 as substrate. IP,
immunoprecipitate.
32P-p531-44-GST, autoradiogram
of radioactive labeled substrate. ATM, Western blot of the
amount of ATM protein the kinase reaction.
p531-44-GST, Coomassie-stained substrate
in the reaction. B, effect of caffeine in the kinase
reaction. Immunoprecipitated ATM was incubated for 30 min at 30 °C
in kinase buffer or kinase buffer containing 1 mM ATP.
After washing with kinase buffer to remove the ATP, ATM was subjected
to a kinase reaction in the presence of 1 mM caffeine, 10 µCi of [ -32P]ATP and GST-p531-44 as
substrate. C, ATM kinase activity after treatment of cells
with caffeine. Control (C3ABR) cells were treated with 3 mM caffeine for 1 h before irradiation. ATM was
subsequently immunoprecipitated and subjected to a standard kinase
assay using GST-p531-44 as substrate. IR, 5 Gy
of ionizing radiation.
|
|
Since small molecules have also been shown to alter the
autophosphorylation capacity and activity of other PI3-kinase family members (35, 36, 38, 39) we determined whether ATP activation might
extend to DNA-PK and ATR in vitro. Incubation of ATP with immunoprecipitates of DNA-PK and ATP failed to significantly activate these kinases (Fig. 8, A and
B), indicating that this was not some nonspecific effect
capable of stimulating other family members because they possessed a
protein kinase domain and an ATP-binding region.

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[in a new window]
|
Fig. 8.
Effect of ATP on activation of other
PI3-kinases. Effect of ATP pretreatment on ATR and DNA-PKcs kinase
activity. Immunoprecipitated ATR (A) and DNA-PKcs
(B) were subjected to ATP pretreatment (0-2 mM
as described above) before kinase activity was determined by standard
kinase reaction in the presence of 10 µCi of
[ -32P]ATP using GST-p531-44 as substrate.
The amount of kinase in the reaction was determined by Western blot
against ATR or DNA-PK, respectively. The specificity of the
immunoprecipitation was verified by using nonspecific antiserum
(lane ns). IP, immunoprecipitate.
32P-p531-44-GST, autoradiogram
of radioactive labeled substrate. DNA-PKcs, Western blot of
the amount of DNA-PKcs protein in the kinase reaction.
ATR, Western blot of the amount of ATR protein in the kinase
reaction. p531-44-GST, Coomassie-stained
substrate in the reaction.
|
|
 |
DISCUSSION |
The data described here provide good evidence that ATM can be
activated in vitro by an autophosphorylation mechanism to
phosphorylate downstream substrates such as p53. When
immunoprecipitated from unirradiated cell extracts, ATM has a low basal
level of kinase activity that is markedly enhanced by prior incubation
with unlabelled ATP. The extent of ATM activation exceeds that observed
in irradiated samples, suggesting that ATP is titrating out all the ATM
activity. Further enhancement of ATM activation by ATP in
immunoprecipitates from irradiated cells by ATP supports this. Previous
data reveal that ATM is only activated 2- to 3-fold above the basal
level by exposure to radiation doses as high as 10 Gy (15, 16, 21, 24,
25). These results suggest that not all of the ATM is activated
in vivo by radiation. Although it is not clear how ATM is
activated in irradiated cells, it appears to be due to recognition of
double strand breaks in DNA either by direct binding through a complex
with other proteins such as the Brca1-associated genome surveillance
complex (BASC) complex (51) or perhaps by responding to changes in the
superhelicity of chromatin remote from the actual site of the break.
The activation of only some of the ATM protein after irradiation is
consistent with recent data demonstrating heterogeneous distribution of
ATM within the nucleus (52). A subset of the ATM pool appears to
rapidly associate with chromatin and the nuclear matrix at sites of
double strand breaks (52). Most of the ATM is loosely tethered to the
nucleus; however, both fractions contain at least some active ATM kinase.
It is evident from our data that activation of ATM from unirradiated
cells is only observed at concentrations of ATP from 0.05 mM up to physiological concentrations. Under kinase
reaction conditions, the concentration of ATP is 5-10
µM, which clearly distinguishes between
immunoprecipitates from irradiated and unirradiated cells (15, 16). The
difference between irradiated and unirradiated could be due to greater
access of ATP to the kinase active site in the former case. Radiation
damage to DNA could alter the conformation of ATM or its association
with an interacting protein to provide unrestricted access to the
active site. This phenomenon has been observed with TAK1, a member of
the mitogen-activated kinase kinase kinase family, which is activated
in vivo by different cytokines (53). When
phosphatase-treated immunoprecipitates of TAK1 are subsequently
incubated with cold ATP (1 mM) and reassayed, kinase activity is greatly increased (54). How do we explain activation by
higher concentrations of ATP? Activation could simply be due to an
excess of a charged molecule, which caused a switch from basal to
active states. This seems unlikely since a variety of nucleotide
analogs failed to appreciably raise activity, and poly ADP-ribose also
failed to activate ATM (results not shown). More likely, the
Km for ATP binding to the unirradiated ATM is
considerably higher than that for irradiated, possibly as a consequence
of the ATP/substrate-binding site being occluded by an inhibitory
domain of ATM itself or due to interaction with another protein. This
may be related to the behavior of ATM during purification where it
binds tightly to
-phosphate-linked ATP-Sepharose, but it is readily
eluted from a metal-chelating Sepharose resin as a constitutively
active phosphoprotein (45).
We have demonstrated here that autophosphorylation is a key event in
the activation of ATM kinase in vitro and that the pattern of autophosphorylation induced by ATP bears considerable overlap with
that seen in radiation-activated ATM. It appears that multiple autophosphorylation sites exist on ATM after both treatments. Treatment
of ATP-activated ATM kinase with phosphatase abrogated activity, but
this was fully restored upon subsequent incubation with ATP. Evidence
for ATM autophosphorylation has been observed previously, but its
importance was not highlighted (23, 25). In one of these reports, a
radiation-induced increase in ATM phosphorylation in controls was not
observed in an A-T cell line (25). [32P]orthophosphate
labeling in vivo provides evidence for ATM
autophosphorylation in response to radiation damage supported by a
failure to observe this effect in cells expressing kinase-dead ATM
(23). The activation observed here was specific for ATM since it was
absent in immunoprecipitates from A-T patients expressing mutant
protein, was dependent on the presence of Mn2+, and was
inhibited by wortmannin. These data suggest that at least in
vitro, no other kinase is required for the activation of ATM, and
this may also be the case in irradiated cells. These observations
appear to be contradictory to those of Sarkaria et al. (44),
who reported that preincubation of cells with caffeine prior to
radiation exposure failed to prevent the radiation-induced activation
of ATM, as measured in immunoprecipitates, but prevented radiation-induced phosphorylation of p53 in the cell. They interpreted these results to mean that autophosphorylation was not part of the
mechanism for ATM activation. We made the same observations as Sarkaria
et al. (44) with pretreatment of cells with caffeine, but we
described a differential effect of caffeine on autophosphorylation and
its capacity to phosphorylate added substrate. Caffeine did not
interfere with ATP-dependent activation of ATM kinase
(i.e. autophosphorylation) at concentrations up to 4 mM, but it did prevent substrate phosphorylation when
included in the kinase reaction. Thus the data are not contradictory
but rather demonstrate that autophosphorylation of ATM, whether it be
induced in vivo by radiation or in vitro by ATP,
is not inhibited by caffeine, but the capacity of ATM to phosphorylate
substrates such as p53 in vivo or in vitro is
prevented by caffeine. Although caffeine has been shown to interfere
with cell cycle checkpoints and radiosensitize cells, the exact
mechanism of action remains unknown. This compound inhibits both ATM
and ATR kinase activities (44, 55), but it is not clear whether this
purine analog competes with ATP or inhibits the enzymes by some other
mechanism. The differential effect of caffeine on ATM
autophosphorylation and substrate phosphorylation suggests that it is
not simply competition with ATP for active site binding that causes
inhibition. In this context, it is of considerable interest that
several of the ATM substrates including p53, BRCA1, and BLM not only
bind to the kinase domain in vitro but also bind to a region
close to the N terminus of the protein (17, 25, 27). In addition, p53
and BRCA1 have been shown by co-immunoprecipitation to bind
constitutively to ATM (17, 56). Thus, although two sites may be
required for substrate phosphorylation, this would not be expected in
the case of autophosphorylation and could explain differential
inhibitory effects as observed here for caffeine. A greater
understanding of the mechanism of ATM autophosphorylation will allow
for the identification of more specific inhibitors than caffeine.
Preincubation of cells with wortmannin, an inhibitor of ATM kinase,
suggested that autophosphorylation is not required for the nuclear
retention of ATM kinase (52). However, it should be pointed out that in
that report, the bulk of ATM kinase was not tightly bound to chromatin
but was nevertheless activated by radiation.
It is well established that serine/threonine protein kinases are
activated due to conformational change in the molecule as a consequence
of binding to a variety of ligands or by phosphorylation by upstream
kinases (57). This alters the conformation of the protein, allowing
increased access to ATP and substrate. In the present case, it is
conceivable that a substrate such as p53 (e.g. as a
tetramer), which is capable of binding to the two ends of the ATM
molecule simultaneously, would effectively close off the active site by
excluding access to ATP and maintaining the kinase in an inactive
state. In the case of immunoprecipitates, removal of ATM and its
associated proteins from their nuclear location may be sufficient to
alter the conformation to an extent at which access to ATP occurs at
higher concentrations. An alternative possibility for activation is
that ATP binds at another site on ATM or on an associated protein and
alters its conformation to allow more favorable access to the
ATP-binding site within the active site. Previous data have
demonstrated that ATP molecules bind co-operatively to the
Escherichia coli chaperonin GroEL and cause long range
conformational changes that determine the orientations of remote
substrate-binding sites (58). ATP has also been shown to induce an
increase in Ca 2+-dependent K+
channel (hIK1) activity, whereas several of its hydrolysable or
non-hydrolysable analogs failed to do so (59). Another member of the
P13-kinase family, mTOR, plays an important role in ribosome biogenesis
and cell growth (36). Recent data demonstrate that the intracellular
concentration of ATP alters the activity of mTOR independent of amino
acid-induced changes (36). As ATP increases up to 1 mM, the
activity of mTOR, detected as S6K1 activation, increased. These data
were interpreted to mean that mTOR is a direct sensor of ATP in the
cell. It is also of interest that mTOR bound to ATP agarose is
efficiently eluted off the matrix with excess cold ATP (60).
Phosphatidic acid also interacts with mTOR, which is positively
correlated with the ability of mTOR to activate downstream substrates
(61). Another small molecule inositol hexakisphosphate has been shown
to stimulate the kinase activity of DNA-PK by interacting specifically
with the Ku7u/80 heterodimer (35).
In summary, we have shown that ATP activates ATM kinase in
vitro by a mechanism involving autophosphorylation. The process of
autophosphorylation is resistant to caffeine inhibition, whereas substrate phosphorylation is sensitive to this agent. Activation is
specific for ATM and is not observed to a significant extent with
either ATR or DNA-PK. This is in agreement with previous results
employing purified DNA-PK in which preincubation with unlabelled ATP in
the absence of DNA failed to appreciably alter the activity (62). When
DNA was included in the preincubation, autophosphorylation of DNA-PKcs
reduced its capacity to phosphorylate exogenous substrates (38). It is
intriguing that autophosphorylation has the opposite effect on the
activities of these two P13-kinase family members that recognize double
strand breaks in DNA. It is proposed that a similar mechanism for
activation occurs in the cell, where DNA double strand breaks
either indirectly alter the conformation of ATM or indirectly alter its
association with an interacting protein, allowing unrestricted access
to ATP for autophosphorylation and substrate phosphorylation. Clearly,
it is important to determine how the observations made here impact upon
in vivo activation of ATM kinase. Identification of the
in vivo phosphorylation sites on ATM are critical to an
overall understanding of the mechanism of activation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Maria Malanga and Dr. Felix
Althus, Institute of Pharmacology and Toxicology, Zurich for Poly
ADP-ribose, Tracey Laing for typing the manuscript, and Aine Farrell
for assistance with cell culture.
 |
FOOTNOTES |
*
This work was supported by grants from the Australian
National Health and Medical Research Council, the Queensland Cancer Fund, and the A-T Children's Project, Florida.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: The Queensland Cancer
Fund Research Unit, The Queensland Institute of Medical Research,
P. O. Box Royal Brisbane Hospital, Herston, Brisbane 4029, Australia.
Tel.: 617-3362-0341; Fax: 617-3362-0106; E-mail: martinL@qimr.edu.au.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M300003200
 |
ABBREVIATIONS |
The abbreviations used are:
A-T, ataxia-telangiectasia;
ATM, ataxia-telangiectasia mutated;
ATR, ataxia-telangiectasia and Rad3-related;
DNA-PK, DNA-dependent protein kinase;
DNA-PKcs, DNA-PK catalytic
subunit;
BLM, defective in Bloom's Syndrome;
Nbs1, defective in
Nijmegen breakage syndrome;
GST , glutathione
S-transferase;
Gy, gray;
PI3-kinase, phosphatidylinositol
3-kinase;
mTOR, mammalian target of rapamycin;
AMP-PNP, 5'-adenylylimidodiphosphate.
 |
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