Ataxia Telangiectasia Mutated (ATM) Kinase and ATM and Rad3
Related Kinase Mediate Phosphorylation of Brca1 at Distinct and
Overlapping Sites
IN VIVO ASSESSMENT USING PHOSPHO-SPECIFIC
ANTIBODIES*
Magtouf
Gatei
,
Bin-Bing
Zhou§,
Karen
Hobson
,
Shaun
Scott
,
David
Young
, and
Kum Kum
Khanna
¶
From the
Queensland Institute of Medical
Research, P.O. Royal Brisbane Hospital, Brisbane Qld 4029, Australia,
the § Department of Oncology Research, Glaxo SmithKline
Beecham, King of Prussia, Pennsylvania 19406, and the ¶ Department
of Pathology, University Of Queensland,
Brisbane Qld 4029, Queensland, Australia
Received for publication, December 26, 2000, and in revised form, February 12, 2001
 |
ABSTRACT |
Recent studies have provided
evidence that breast cancer susceptibility gene products (Brca1 and
Brca2) suppress cancer, at least in part, by participating in DNA
damage signaling and DNA repair. Brca1 is hyperphosphorylated in
response to DNA damage and co-localizes with Rad51, a protein involved
in homologous-recombination, and Nbs1·Mre11·Rad50, a complex
required for both homologous-recombination and nonhomologous end
joining repair of damaged DNA. Here, we report that there is a
qualitative difference in the phosphorylation states of Brca1 between
ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at
Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is
specifically phosphorylated after IR, and Ser-1457 is predominantly
phosphorylated after UV. These results suggest that different types of
DNA-damaging agents might signal to Brca1 in different ways. We also
provide evidence that the rapid phosphorylation of Brca1 at Ser-1423
and Ser-1524 after IR (but not after UV) is largely ataxia
telangiectasia mutated (ATM) kinase-dependent. The
overexpression of catalytically inactive ATM and Rad3 related (ATR)
kinase inhibited the UV-induced phosphorylation of Brca1 at these
sites, indicating that ATR controls Brca1 phosphorylation in
vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells
synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken
together, our results support a model in which ATM and ATR act in
parallel but somewhat overlapping pathways of DNA damage signaling but
respond primarily to different types of DNA lesion.
 |
INTRODUCTION |
Damage to DNA, which can occur from exogenous agents such as
IR,1 UV radiation, and
certain chemotherapeutic drugs or from endogenously generated reactive
oxygen species, poses a great threat to genomic stability. Cells
respond to DNA damage by activating a complex DNA damage-response
pathway that includes cell cycle arrest, transcriptional and
posttranscriptional regulation of genes associated with repair, and
under some circumstances, the triggering of programmed cell death.
Biochemically, the DNA damage-response pathway provides a mechanism for
transducing a signal from a sensor that recognizes the damage, through
a transduction cascade, to a series of downstream effector molecules,
which implement the appropriate response. At or close to the top of
this pathway in mammalian cells are the members of the phosphoinositide
kinase-related kinase (PIKK) family, which includes ATM (ataxia
telangiectasia mutated) and ATR (ATM and Rad3 related). These proteins
play important roles in signaling the presence of DNA damage,
activating cell cycle checkpoints, and repairing DNA (1). ATM is
homozygously mutated in the germline of patients with the
neurodegenerative and cancer predisposition syndrome, ataxia
telangiectasia (AT). Cells derived from AT patients are
hypersensitive to agents that cause double strand breaks in DNA, such
as IR, but retain normal resistance to UV and other damaging agents. AT
cells exhibit cell cycle defects at multiple checkpoints
(G1/S, intra S, and G2/M) in response to IR.
ATM-deficient mice are viable but show growth retardation and a cancer
predisposition. In mammals, ATM is an upstream regulator of p53, c-Abl,
Chk2 (hCds1), Brca1, and nibrin in response to IR (2, 3). Less is known
about ATR because no ATR mutations have been detected in human
diseases. However, the overexpression of the dominant negative ATR
derivative in mammalian cells sensitizes them to a range of
DNA-damaging agents (4). The homozygous inactivation of ATR in mice
results in the early embryonic lethality associated with the loss of
cellular proliferative potential and chromosomal fragmentation leading
to mitotic catastrophe, a phenotype reminiscent of the phenotype of the
Brca1-null embryos (5, 6). Recent studies have provided evidence that
Brca1 suppresses cancer, at least in part, by participating in the
signaling of DNA damage and DNA repair (7, 8). Brca1 is
hyperphosphorylated after the exposure of cells to DNA-damaging agents
(9), indicating that it is a target of protein kinases activated by DNA
damage. We and others have recently provided evidence that ATM and
ATM-dependent kinase Chk2 mediate the phosphorylation of
Brca1 after IR (10-12). ATM is required only for the rapid and
immediate phosphorylation of Brca1 after IR because Brca1 is still
phosphorylated in ATM-null cells, albeit with delayed kinetics (11).
Furthermore, the phosphorylation of Brca1 after UV and
hydroxyurea is ATM-independent (13) and probably mediated by ATR
because ATM and ATR have been shown to have overlapping substrate
specificity in vitro (14). Despite the apparent overlap of
function between ATM and ATR, it has been clearly shown that the
immediate phosphorylation of p53 in response to IR is largely
ATM-dependent, whereas ATR mediates the UV-induced rapid
phosphorylation of p53. Recently, evidence has been presented that ATR
regulates the phosphorylation of human Chk1 in response to UV treatment
(15). Therefore, this study was designed to investigate the role of ATR
in UV-induced modifications of Brca1. During the preparation of this
manuscript, similar studies have suggested the role of ATR in Brca1
phosphorylation (13, 16). However, using phospho-specific antibodies,
our results provided further details of BRCA1 phosphorylations at
various sites by ATM and ATR. We found that ATM and ATR, in
addition to their shared phosphorylation sites, also phosphorylate
distinctively different sites in BRCA1 in vivo.
 |
EXPERIMENTAL PROCEDURES |
Constructs and Recombinant Proteins--
Epstein-Barr
virus-transformed lymphoblastoid cell lines generated from normal
controls and AT patients have been described previously (17). The
generation and characterization of GM847 cells that express wild-type
(wt) or kinase-dead (kd) ATR under the control of the bacterial
tet operator have been described previously (4). Lymphoblastoid
cell lines were cultured in RPMI 1640 medium supplemented with 10%
fetal calf serum at 37 °C in a humidified atmosphere of 5%
CO2. The SV40-transformed GM847 cells were grown in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
Following transfection, cells were grown in complete medium containing
G418 (Life Technologies, Inc.). The expression of wt and kd ATR was
induced by incubating the cells in culture medium containing 1 mM doxycycline (Sigma) for 48 h. Cells were grown to
80% confluence and then treated with either IR or UV light. The
indicated cells were irradiated (6 Gy) with a 137Cs
-ray
source or exposed to UV-C (80 J/m2).
Antisera and Plasmid Constructs--
Phospho-specific antibodies
against ATM phosphorylation sites in BRCA1 (anti-Ser-1423,
anti-Ser-1457, and anti-Ser-1387) have been described previously (11).
Anti-phospho-Ser-1524 rabbit antiserum was generated in this study by
immunizing with keyhole limpet hemocyanin-conjugated
phosphopeptide (QNRNYP-Phospho-Ser(P)-QEELIK). Bleeds were assayed by
enzyme-linked immunosorbent assay, and the serum was collected on days
corresponding to the peak antibody response and then affinity-purified
against the same peptide. The affinity-purified polyclonal antibodies
recognized the phosphorylated form of a Brca1 peptide but not the
unphosphorylated form employing an enzyme-linked immunosorbent assay.
Anti-BRCA1 monoclonal antibodies (Ab-1) and anti-ATR polyclonal
antibodies were from Oncogene Science, Inc. (Manhasset, NY). Anti-FLAG
was purchased from Sigma. A series of glutathione
S-transferase (GST)-BRCA1 fusion proteins was generated and
purified as described previously (11).
Immunoblotting, Co-immunoprecipitation, and Kinase
Assays--
Cellular extracts were prepared by resuspending the cells
in lysis buffer (50 mM Tris-HCL, pH 7.4, 150 mM
NaCl, 2 mM EGTA, 2 mM EDTA, 25 mM
NaF, 25 mM
-glycerophosphate, 0.1 mM sodium orthovandate, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/m1 leupeptin, 1 µg/ml aprotinin, 0.2% Triton X-100, and 0.3%
Nonidet P-40) and incubating on ice for 30 min. Supernatants were
collected following centrifugation at 14,000 × g for
15 min. 40 µg of protein/sample was then analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting with the
appropriate antibodies. For co-immunoprecipitations, protein samples
were precleared with protein A beads for 1 h at 4 C. The
supernatants were then incubated with the required antibody for 2 h. The immune complexes were collected with protein A- and G-Sepharose
beads. The complexes were washed twice with lysis buffer and then
fractionated by SDS-polyacrylamide gel electrophoresis for immunoblot
analysis. For kinase assays, cells were lysed in TGN buffer (50 mM Tris-HCl, pH 7.5, 50 mM
-glycerophosphate, 150 mM NaCl, 10% glycerol, 1% Tween
20, 1 mM NaF, 1 mM sodium orthovandate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 1 mM
dithiothreitol) as described previously (18). The immunoprecipitation
was carried out as described above using either anti-ATR or anti-FLAG
antibodies. The immunoprecipitates were washed twice with TGN buffer,
once with 100 mM Tris-HCl (pH 7.5) and 0.5 M
LiCl, and twice with kinase buffer (10 mM Hepes, pH 7.5, 50 mM
-glycerophosphate, 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2,
5 µM ATP, and 1 mM dithiothreitol). Kinase
reactions were prepared by resuspending washed beads in 30 µl of
kinase buffer containing 10 µCi of [
-32P]ATP and 1 µg of GST-BRCA1 fusion protein. Immune complex reactions were
incubated at 30 °C for 30 min and analyzed by SDS-polyacrylamide gel
electrophoresis followed by autoradiography.
Immunofluoresence Studies--
Cells were synchronized by a
double thymidine block at the G1/S transition and then
released into thymidine-free medium for a synchronous passage through S
phase. 2 h after release, cells were either left untreated or
irradiated at 10 Gy, and 4 h after exposure, cells were fixed in
methanol:acetone (1:1) at
20 °C, washed in phosphate-buffered
saline, and blocked with a blocking solution (3% bovine serum albumin,
0.1% Triton in phosphate-buffered saline). For double labeling with
rabbit (anti-ATR) and mouse (anti-Brca1) antibodies, cells were
incubated with a mixture of antibodies for 2 h at 4 °C. Cells
were then washed three times in phosphate-buffered saline and incubated
with a mixture of fluorescein-labeled anti-rabbit IgG (Zymed
Laboratories Inc.) and indocarbocyanine-labeled anti-mouse
IgG (Zymed Laboratories Inc.) for 30 min. After
extensive washing, samples were mounted onto glass slides with a drop
of anti-fade mounting reagent (Vectashield, Vector Laboratories). Samples were analyzed with a confocal laser scanning microscope (Bio-Rad). Images were digitally captured and overlaid using the RGB
function of Adobe Photoshop.
 |
RESULTS |
Ultraviolet or
Irradiation and Brca1
Phosphorylation--
We previously raised anti-phospho-specific
antibodies against three of the ATM phosphorylation sites in Brca1
(Ser-1387, Ser-1423, and Ser-1457). In this report, polyclonal
rabbit antibodies against an additional ATM phosphorylation site in
Brca1 (Ser-1524) were produced, affinity-purified, and termed
-1524.
In the present study, we monitored the phosphorylation of Brca1 in
intact cells by using the phospho-specific antibodies. Control and AT
cells were exposed to IR (6 Gy) or UV radiation (80 J/m2),
and the cells were harvested for Western blot analysis 30 min after
damage. The Brca1 protein level and phosphorylation state were then
analyzed by Western blotting by using anti-Brca1 (Ab-1, Oncogene
Science) or phospho-specific antibodies (
-1387,
-1423,
-1457,
and
-1524) against ATM phosphorylation sites in Brca1. In agreement
with our earlier study (11), Brca1 phosphorylation after IR but not
after UV was ATM-dependent (Fig.
1). The
-1524 and
-1423 antibodies
detected an increased level of phosphorylation of Brca1 after UV and
IR. The IR-induced phosphorylation was clearly ATM-dependent because ATM-deficient cells (L3)
failed to show increased phosphorylation at these sites; however,
UV-C-induced phosphorylation was ATM-independent. Immunoblotting with
-1457 also revealed dramatically increased levels of phosphorylated Brca1 in response to UV but not in response to IR (Fig. 1). Again, this
UV-C-induced increase was ATM-independent. However, previously (11) we have shown that
-1457 did detect phosphorylated Brca1 after
IR when Brca1 was immunoprecipitated from cell lysate (1 mg) with
anti-Brca1 antibody (Ab-1) followed by blotting with
-1457. These
results suggest that only a small fraction of Brca1 is phosphorylated
at this site after IR, which is below the threshold level of detection
after straight Western blot. However, a significant proportion of Brca1
is phosphorylated at Ser-1457 after UV. By contrast the
-1387
antibodies detected an increase in Brca1 phosphorylation after IR but
not after UV radiation.

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Fig. 1.
ATM is required for IR-induced
phosphorylation of Brca1 but is not required to phosphorylate Brca1
following UV treatment. Synchronously growing cultures from the
control lymphoblastoid cell line (C3ABR) and ATM-deficient
cell line (L3) were exposed to either IR (6 Gy) or UV-C (80 J/m2) and harvested at 30 min after treatment. The proteins
were analyzed by Western blotting (WB) with anti-BRCA1
(detects total pool of Brca1: phosphorylated and unphosphorylated) and
anti-phospho-specific Brca1 antibodies against ATM phosphorylation
sites in Brca1 ( -1524, -1457, -1423, and -1387).
|
|
Role of ATR in Damage-induced Phosphorylation of
Brca1--
Both ATM and ATR are members of the PIKK family and
contribute to the phosphorylation and accumulation of p53 after
cellular exposure to
and UV radiation, respectively (19, 20). No ATR mutations have so far been documented in human disease; therefore, human cell lines that carry mutant ATR are not available. Furthermore, a homozygous knockout of ATR in mice is embryonic lethal (5, 6).
Therefore, for this study we have used cells transfected with
FLAG-tagged kd ATR, which has previously been shown to exert a dominant
negative effect (4). Cells were induced with 1 mM doxycycline for 48 h to allow the expression of transfected
cDNA prior to harvesting. Western blotting of cell lysates using
anti-FLAG antibody revealed the presence of ectopic ATR protein induced with doxycycline (Fig. 2A).
This protein was not detected in untransfected GM847 cells.
Immunoblotting of cell lysates with the anti-ATR antibody, which
recognizes both endogenous and ectopic ATR, revealed an approximately
2-3-fold increase in ATR protein expression after the cells were
induced with doxycycline (Fig. 2A). The extracts from
uninduced and induced cells transfected with kd ATR, with and without
prior exposure to IR (6 Gy) and UV (80 J/m2), were analyzed
for damage-induced modifications of Brca1 by Western blotting with
-Brca1 (Ab-1) and various anti-phospho-specific antibodies
(
-1524,
-1457,
-1423, and
-1387) against Brca1. The
results, shown in Fig. 2B, clearly demonstrate that the
immediate UV-induced mobility shift of Brca1 is compromised in cells
overexpressing kd ATR protein. This is reflected as the markedly
reduced phosphorylation of Brca1 on serine residues 1524, 1423, and 1457 in these cells in response to UV (but not IR), suggesting
thereby that rapid UV-induced modifications of Brca1 are dependent upon
the presence of functional ATR kinase.

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Fig. 2.
Rapid UV-induced phosphorylation of Brca1 is
dependent upon the presence of functional ATR kinase.
A, inducible expression of ATR wt and kd in GM847
(GM) fibroblasts. Immunoblotting with anti-FLAG or anti-ATR
antibodies was carried out in the presence (+) or absence
( ) of 1 mM doxycycline
(dox) for 48 h prior to harvesting the cells.
WB, Western blot. B, ATR is required for the
UV-induced phosphorylation of Brca1. wt or kd ATR-transfected cells
were induced with 1 mM doxycycline (dox) for
48 h. After induction, the cells were treated with either IR (6 Gy) or UV-C (80 J/m2) and harvested 30 min
following damage. Proteins were fractionated by SDS-polyacrylamide gel
electrophoresis and immunoblotted with anti-Brca1 and
anti-phospho-specific Brca1 antibodies. GM, GM847.
WB, Western blot.
|
|
ATR Phosphorylates Brca1 Directly--
Recently it has been shown
that Brca1 exists as a part of a complex designated Brca1-associated
genome surveillance complex that includes putative DNA damage sensors
such as ATM (21). Therefore, we were interested in determining whether
ATR exists in complex with Brca1. To study this association, cell
lysates were co-immunoprecipitated with either anti-Brca1 or anti-ATR antibodies. The immunoblot analysis of the ATR immunoprecipitates with
anti-Brca1 antibody demonstrated an interaction between the two
proteins in control and AT cell lines, indicating that the interaction
between ATR and Brca1 is ATM-independent (Fig.
3A). Similar results were
obtained when Brca1 immunoprecipitates were blotted with the ATR
antibody. The specificity of this association was confirmed by
immunoprecipitation with nonspecific immunoglobulin, which failed to
show an ATR or Brca1 band after blotting with the respective antibodies
(results not shown). We next sought to examine whether ATR and Brca1
co-localize in cells with or without the prior treatment of cells with
DNA damage. Prior to DNA damage, the majority of cells stained with
anti-ATR antibodies indicated a nuclear diffuse pattern of staining.
However, about 15% of the cells exhibit a punctate pattern (foci) of
staining in the nucleus (data not shown). The synchronization of cells in S phase by a double thymidine block caused a dramatic increase in
the proportion of cells exhibiting ATR foci (Fig. 3B). In
synchronized cells, ATR foci overlapped Brca1 foci. The staining
pattern of ATR and Brca1 also overlapped extensively at 4 h after
the treatment of cells with IR and UV (Fig. 3B), indicating
that the damage-induced modification of Brca1 by ATR might contribute
to changes in Brca1 subnuclear localization after DNA damage. To
investigate this possibility, we sought to examine whether the
phosphorylated Brca1 is a part of the Brca1 foci induced after DNA
damage by using a phospho-specific antibody against Ser-1524. Most of
the undamaged cells (>85%) stained negative for the
-Ser-1524
antibody; however, by 4 h after the treatment of cells with IR or
UV, >80% of cells exhibited the presence of phosphorylated Brca1 as
nuclear foci (Fig. 3B). Most of the foci observed after
staining cells with the anti-Brca1 antibody (which detects both
phosphorylated and nonphosphorylated Brca1) co-localized with the foci
observed with the phospho-specific antibody, indicating that the
phosphorylation of Brca1 at Ser-1524 may be essential for regulating
the localization of Brca1.

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Fig. 3.
Direct interaction and
phosphorylation of BRCA1 by ATR. A, in vivo
association of ATR and Brca1. Total cell lysates were subjected to
immunoprecipitation (IP) with either anti-ATR or anti-Brca1
antibody, and the immunoprecipitated proteins were analyzed by
immunoblotting with anti-Brca1 and anti-ATR antibodies. WB,
Western blot. B, co-localization of ATR and Brca1 in cells
synchronized in S phase (top panel, middle
panel). Cells were synchronized by double thymidine block and then
released into the fresh medium and cultured for 2 h. The cells
were then either left untreated (top) or treated with either
IR (10 Gy) or UV-C (20 J/m2) (middle) in m2. 4 h after
treatment, cells were fixed, permeabilized, and stained with anti-ATR
polyclonal antibody (Ab-1, Oncogene Science) and anti-Brca1 monoclonal
antibody (Ab-1, Oncogene Science). Foci demonstrating coincident
labeling with both antibodies appear yellow in the merged
picture. Bottom panel, Brca1 phosphorylated on Ser-1524
localizes to nuclear foci. Normal human fibroblasts were fixed and
permeabilized at 4 h after exposure to 10 Gy. Cells were then
immunostained with a phospho-specific anti-Brca1 ( -1524) antibody
and anti-Brca1 monoclonal antibody (Ab-1) that detects both
unphosphorylated and phosphorylated Brca1. Foci that stain with both
antibodies appear yellow in the merged picture.
C, ATR phosphorylates Brca1 directly. Asynchronously growing
cells were either untreated or treated with UV-C (80 J/m2)
and then harvested at 30 min. ATR was immunoprecipitated using anti-ATR
antibodies and assayed with Brca1 fragments (B1F1-B1F7) as substrates.
D, wt but not kd ATR phosphorylates a fragment of Brca1.
Whole cell lysates from uninduced, induced (dox) plus
IR-treated, or induced plus UV-treated cells were immunoprecipitated
with anti-FLAG antibodies to precipitate ectopic ATR and assayed for
the phosphorylation of Brca1 (B1F6). GM, GM847.
WB, Western blot.
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|
To test whether Brca1 is a direct target for the protein kinase
activity of ATR, we used a series of recombinant GST-Brca1 fragments
(B1F1-B1F7) covering the entire open reading frame (11). Kinase
reactions were performed with purified immunoprecipitated ATR with or
without prior exposure to UV. Immunoprecipitated ATR from control
lymphoblastoid cell lines phosphorylated the region in Brca1, B1F6
(amino acids 1241-1530 (Fig. 3C)); the same region was
previously shown to be phosphorylated by ATM (11). This activity was
also observed against an additional Brca1 fusion protein not
phosphorylated by ATM, B1F3 (amino acids 521-757 (Fig. 3C)). None of the other Brca1-GST fusion proteins were
phosphorylated by ATR. Immunoprecipitated ATR from ATM-deficient cells
showed a similar phosphorylation of fragments 3 and 6 (data not shown), indicating that the activation of ATR is independent of ATM expression in cells. Although ATR is activated in vivo after the UV-C
treatment of cells (demonstrated in Fig. 2B), we were
experimentally unable to show the increased activation of ATR after
UV-induced DNA damage in vitro. Ectopically expressed
FLAG-tagged wt ATR revealed a similar phosphorylation of Brca1 at these
sites (B1F6 is shown in Fig. 3D), whereas kd ATR failed to
show any activity against Brca1, thus providing a specific control for
ATR kinase activity (Fig. 3D).
 |
DISCUSSION |
The present results demonstrate that there is a qualitative
difference in Brca1 phosphorylation between
radiation and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after both
IR and UV. However, Ser-1387 is specifically phosphorylated after IR,
and Ser-1457 is predominantly phosphorylated after UV. These different
responses in the phosphorylation of Brca1 after UV and IR exposure
correspond to the fact that the nature of DNA damage caused by
these two treatments is distinct; IR produces single or double strand
breaks followed by ligation or recombination repair, whereas UV
produces cyclobutane dimers followed by excision repair. The cellular
molecules that detect and repair these distinct DNA lesions are
different, which could well explain why the Brca1 response is also
different. For example, ATM-deficient cells are defective in the
cellular responses to double strand break-inducing agents (such
as IR and other radiomimetic agents) but have a normal response to UV
(22). However, there is not an absolute requirement for ATM in the
cellular response to double strand breaks because the stabilization and
phosphorylation of p53 (19, 22) is delayed but not abrogated in
ATM-deficient cells in response to IR. Recently, the ATM-related kinase
ATR was shown to mediate this late phosphorylation of p53 on Ser-15 in
response to IR and the early phosphorylation of p53 in response to UV
(20). These distinctions in kinetics may be a consequence of dynamic
changes in initiating damage during the cellular exposures to IR and
may dictate the need for particular components of the damage response
pathway at particular times. These experiments reveal that the primary
genetic damages induced by IR may initially activate ATM and,
subsequently, that ATR is required to recognize the late DNA strand
breakage. Moreover, these experiments also suggest that UV radiation
may up-regulate the catalytic activity of ATR efficiently rather
than regulating ATM activity. Although the mechanism by which ATM and
ATR kinases are selectively regulated by distinct DNA damage signals
remains unclear at present, it seems reasonable to propose that
distinct DNA damage detectors might act to identify different types of DNA damage and that these distinct detectors might signal to Brca1 in a
different way. ATR may be used immediately after UV damage, and ATM may
be used after IR to detect damage in the DNA and then add phosphate
groups to Brca1.
We have shown here that ATM is involved in the immediate
phosphorylation of Brca1 at multiple sites (Ser-1387, Ser-1423, and Ser-1457) after IR but not after UV, which is consistent with accumulating data that ATM is primarily involved in the signaling of
double strand breaks in DNA. The finding that the overexpression of catalytically inactive ATR inhibits the UV-induced phosphorylation of Brca1 at Ser-1423, Ser-1457, and Ser-1524 strongly suggests that the
phosphorylation of BRCA1 in response to UV requires the presence of
functional ATR. The precise mechanism by which the ATM- and
ATR-dependent phosphorylation of Brca1 regulate Brca1 function is unclear at present. It is possible that phosphorylation might lead to a change in Brca1 subnuclear localization, which changes
in response to DNA damage. In this regard, we observed that the Brca1
pool in cells is phosphorylated on Ser-1524 in response to IR and UV,
and this phosphorylated form localizes to nuclear foci within 2 h
after DNA damage, suggesting that phosphorylation might regulate the
localization of Brca1. Interestingly, ATR also forms nuclear foci in
cells synchronized in S phase, and these foci co-localize with Brca1.
This observation is consistent with the essential roles of ATR and
BRCA1 during the normal cell cycle, possibly facilitating DNA
replication and preventing mitotic entry during S phase. A majority of
cells in asynchronous culture show ATR-containing nuclear foci when
exposed to DNA damage (IR and UV). Because an increase in ATR kinase
activity has not been observed after treatment with various
DNA-damaging agents, it is possible that ATR is regulated by its
location to its substrate, such as BRCA1, after DNA damage.
These results are also consistent with an earlier report that
demonstrated the co-localization of Brca1 and ATR onto asynapsed
regions of meiosis I chromosomes (23) and could represent the primary
response to early lesions, which might initiate meiotic recombination
and synapsis. It is quite conceivable that the ATR·Brca1 complex
might control homologous recombination and homology-directed repair in
somatic cells. The fact that ATR and BRCA1 mutant mice share similar
phenotypes (5) and that ATR controls BRCA1 phosphorylation suggests
that ATR is a key regulator of BRCA1 function. Uncovering the
biochemical details of signaling to Brca1 will be an important step
toward understanding why the defects in this gene product predispose people to breast cancer.
 |
ACKNOWLEDGEMENTS |
Special thanks are extended to Dr. S. Handeli
for providing cells expressing wild-type and dominant negative ATR.
 |
FOOTNOTES |
*
This work was supported by the National Health and
Medical Research Council (Australia), the Queensland Cancer Fund
(Australia), and the Susan G. Komen Breast Cancer Foundation.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.
Supported by a Senior Research Fellowship from the
Sylvia and Charles Viertel Foundation. To whom correspondence should be addressed. Tel.: 61-7-33620338; Fax: 61-7-33620106; E-mail:
kumkumK@qimr.edu.au.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011681200
 |
ABBREVIATIONS |
The abbreviations used are:
IR, ionizing
radiation;
ATM, ataxia telangiectasia mutated;
ATR, ATM and Rad3
related;
AT, ataxia telangiectasia;
wt, wild type;
kd, kinase dead;
Gy, gray;
UV-C, ultraviolet-C;
GST, glutathione
S-transferase;
PIKK, phosphoinositide kinase-related
kinase.
 |
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