From the Department of Molecular Medicine/Institute
of Biotechnology, The University of Texas Health Science Center, San
Antonio, Texas 78245-3207 and the § Center for Radiological
Research, Columbia University, New York, New York 10032
Received for publication, September 28, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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ATM (ataxia-telangiectasia-mutated) is a Ser/Thr
kinase involved in cell cycle checkpoints and DNA repair. Human Rad9
(hRad9) is the homologue of Schizosaccharomyces pombe Rad9
protein that plays a critical role in cell cycle checkpoint control. To
examine the potential signaling pathway linking ATM and hRad9, we
investigated the modification of hRad9 in response to DNA damage. Here
we show that hRad9 protein is constitutively phosphorylated in
undamaged cells and undergoes hyperphosphorylation upon treatment
with ionizing radiation (IR), ultraviolet light (UV), and
hydroxyurea (HU). Interestingly, hyperphosphorylation of hRad9
induced by IR is dependent on ATM. Ser272 of hRad9 is
phosphorylated directly by ATM in vitro. Furthermore, hRad9
is phosphorylated on Ser272 in response to IR in
vivo, and this modification is delayed in ATM-deficient cells.
Expression of hRad9 S272A mutant protein in human lung fibroblast VA13
cells disturbs IR-induced G1/S checkpoint activation and
increased cellular sensitivity to IR. Together, our results suggest
that the ATM-mediated phosphorylation of hRad9 is required for
IR-induced checkpoint activation.
In eukaryotic cells, DNA damage and stalled DNA replication forks
activate evolutionarily conserved checkpoint pathways, resulting in a
delay in cell cycle progression, initiation of DNA repair process, and
transcriptional regulation of specific genes (for reviews, see Refs.
1-4). These checkpoint controls prevent damaged DNA from being
replicated or distributed into daughter cells prior to the completion
of DNA repair, thus helping to maintain genomic integrity. Failure of
this cell cycle surveillance mechanism can cause genomic instability
that eventually leads to cancer formation in mammals (5, 6).
The DNA damage checkpoint pathways are conserved among
Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Drosophila melanogaster, Caenorhabditis elegans, and mammals
(7-9). In S. pombe, the protein products of the six
checkpoint rad genes (rad1+,
rad3+, rad9+,
rad17+, rad26+, and
hus1+) play crucial roles in sensing changes in DNA
structure. All of the six rad genes are required for
checkpoint activation (for reviews, see Refs. 10-12). Genetic studies
have placed the six checkpoint rad genes in the same
signaling pathways that monitor DNA damage and stalled replication
forks (13). Cells with mutations in any one of these genes are
hypersensitive to both replication blocks- and DNA damage-causing
agents in all cell cycle phases (14), suggesting that these genes are
involved in all cell cycle checkpoints. Rad1 is a putative exonuclease
(15) that forms a stable protein complex with Rad9 and Hus1 (16). Rad17
transiently interacts with the Rad1·Rad9·Hus1 complex (16,
17). There is a stable interaction between Rad3 and Rad26 (18). The
finding that Rad3, but not other checkpoint Rad proteins, is required for DNA damage-induced phosphorylation of Rad26 suggests that the
Rad3·Rad26 complex may be the "first sensor" of DNA damage (18).
Importantly, all six checkpoint Rad proteins are required for DNA
damage-induced phosphorylation of two downstream checkpoint protein
kinases, Cds1 and Chk1 (19, 20). Phosphorylation of Cds1 correlates
with its biological activity (19). These studies have provided a
framework for understanding the cell cycle checkpoint signal pathways.
Mammalian counterparts of all the checkpoint rad genes
except rad26+ have been identified (21-27). The
hRad9, hRad1, and hRad17 genes partially complement the DNA damage sensitivity of the counterpart yeast mutants (24, 27, 28), reinforcing the functional similarities between the human and yeast genes. Similar to their yeast homologues, hRad1, hRad9, and hHus1 form a stable complex (29-31). The exonuclease activity of both hRad9 and hRad1 has also been demonstrated (32, 33).
The hRad9·hRad1·hHus1 complex may associate with chromatin upon DNA
damage (34). Murine cells lacking
Hus1 and nematode cells lacking MRT-2
(Rad1) have increased genomic instability and cellular
sensitivity to DNA damage (35-37). To date, two related mammalian
kinases, ATM1 (ataxia-telangiectasia mutated) and ATR
(ataxia-telangiectasia and Rad3-related) have been shown to share
structural and functional similarities with Rad3 in S. pombe
(21). ATM was identified as the gene mutated in the
ataxia-telangiectasia (A-T) patients (38). Cells established from A-T
patients are hypersensitive to IR but not UV or HU. They are defective
in multiple cell cycle checkpoints, including G1/S, S, and
G2/M. A-T cells exhibit radioresistant DNA
synthesis, likely equivalent to the DNA damage-induced S phase checkpoint defect (reviewed in Ref. 39). Similarly, ATR regulates G1/S, S, and G2/M checkpoint controls in
response to DNA damage and a block in DNA replication, thus
demonstrating functionally similarity to Rad3 in S. pombe
(40-42). ATM, ATR, and Rad3 are members of a Ser/Thr kinase family
that contains a serine/threonine kinase domain closely related to the
phosphatidylinositol 3-kinase at the carboxyl termini. ATM acts
upstream of Chk2 (43), the human homologue of Cds1 in S. pombe. ATR operates upstream of Chk1 in response to UV (44). In
mammalian cells, Chk1 and Chk2 phosphorylate Cdc25 phosphatase,
resulting in the nuclear export of Cdc25 and inhibition of Cdc2 kinase
activity (1, 45-47). Inactivation of Cdc2 eventually leads to cell
cycle arrest at the G2/M boundary (48). Taken together,
these studies highlight the evolutionary conservation of the checkpoint
signaling pathways.
In addition to the genes described, ATM-mediated signaling
pathways involve tumor suppressor genes such as p53,
BRCA1, and NBS1 (49-55). Molecular details of
how ATM regulates these tumor suppressors have been described, but
whether ATM regulates human checkpoint Rad proteins is unknown. Here,
we investigate the regulation of hRad9 by phosphorylation. We
demonstrate that ATM-mediated phosphorylation of hRad9 is critical for
ionizing radiation-induced checkpoint activation.
Antibodies--
Glutathione S-transferase (GST) and
full-length hRad9 or hRad9224-391 fusion proteins were
overexpressed in Escherichia coli and purified by affinity
chromatography following the manufacturer's instructions (Amersham
Pharmacia Biotech). BALB/c mice were immunized with GST-hRad9 or
GST-hRad9224-391 according to the standard procedure (56).
The rabbit Immunoblotting and Immunoprecipitation--
Cells were lysed in
ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40)
supplemented with protease inhibitors (10 µg
ml Phosphatase Treatment--
Immunoprecipitation was performed
using rabbit Mutagenesis and in Vitro Kinase Assay--
Substitution of
Ser272 of the hRad9 protein with Ala was generated using
the QuickChange site-directed mutagenesis kit according to the
manufacturer's instruction (Stratagene). A 120-base pair DNA fragment
containing Ser272 or Ala272 was generated by
polymerase chain reaction and fused in-frame with GST. Endogenous ATM
in HeLa cells mock-treated or IR-treated with 10-Gy radiation was
immunoprecipitated using Plasmid Construction--
pCMV2-FLAG-hRad9 was constructed by
subcloning the full-length hRad9 fragment generated by polymerase chain
reaction using pHRAD9-1 cDNA as template (24) into a
BamHI-XbaI-linearized pFLAG-CMV-2 (Kodak) vector.
The hRAD9 EcoRI-XhoI fragment from pGEX-5X-3-hRad9 was subcloned into pGEX-4T-1 to generate
pGEX-4T-1-hRad9224-391. Both pGEX-5X-3-hRad9 and
pGEX-4T-1-hRad9224-391 were used to produce GST fusion
proteins for immunization. pCMV-FLAG-His-ATMWT and
pCMV-FLAG-His-ATMD2870A expression vectors were as
described previously (55). pEGFP-N1 was purchased from
CLONTECH, Inc.
Cell Culture, Transfection, and Treatments for DNA Damage
Induction--
EBS (ATM-deficient) and YZ5 (ATM-complemented) cell
lines were kindly provided by Y. Shiloh. NBS1-LBI cells are SV40 T
antigen-immortalized fibroblasts established from NBS patients (59).
All other cell lines were from the American Type Culture Collection.
Cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 1%
penicillin-streptomycin (Life Technologies, Inc.). For cell cycle
studies, human bladder carcinoma T24 cells were synchronized by density
arrest. Extracts were prepared from cells at different cell cycle
stages as described previously (60). Transfections were
performed using LipofectAMINE (Life Technologies, Inc.) according to
the manufacturer's instructions. IR treatment was performed using a
137Cs DNA Damage-induced G1/S Checkpoint--
Cells
co-transfected with pEGFP and pCMV2-FLAG-hRad9 or
pCMV2-FLAG-hRad9S272A (1:10 ratio of EGFP to FLAG-hRad9)
were exposed to 20 Gy of Cellular Sensitivity to Ionizing Radiation--
Cells were
co-transfected with pEGFP and pCMV2-FLAG-hRad9 or
pCMV2-FLAG-hRad9S272A (as described above). Cells were
exposed to 5 Gy of Human Rad9 Is a Phosphoprotein--
In the Western blotting
analysis using mouse IR-induced Hyperphosphorylation of hRad9 Is Cell
Cycle-independent--
In S. pombe, the checkpoint
rad genes are required for the activation of all checkpoints
upon DNA damage. We examined whether IR-induced hyperphosphorylation of
hRad9 occurs in all cell cycle stages. Following synchronization of T24
cells by density arrest (60), extracts were prepared from cells in
G1, S, or G2/M, and the proteins were
fractionated by SDS-PAGE. The electrophoretic mobility of the hRad9
protein was constant throughout the cell cycle (Fig.
3A, lanes 1,
3, and 5 and data not shown). The IR-induced mobility shift of hRad9 was observed at all cell cycle phases (Fig.
3A, lanes 2, 4, and 6).
Unlike its counterpart DDC1 in S. cerevisiae (61), no
mobility change of hRad9 was observed in the S phase of untreated
cells. Since not all phosphorylation events lead to protein mobility
shifts, it remains to be studied whether there are cell cycle-specific
modifications of hRad9 in mammalian cells. In S. pombe, the
checkpoint Rad proteins are required for IR-, UV-, as well as
replication block-induced checkpoint activation. We tested whether
hyperphosphorylation of hRad9 occurs upon different treatments. Similar
to the IR-induced response, UV and HU also caused hyperphosphorylation
of hRad9 (Fig. 3B). The IR-, UV-, and HU-induced
hyperphosphorylation of hRad9 was also detected in several other human
cell lines (data not shown). Collectively, these results demonstrate
that hRad9 hyperphosphorylation occurs in response both to different
types of DNA damage and to blockage of DNA replication, supporting the
notion that there may be several upstream pathways that trigger
hyperphosphorylation of hRad9.
IR-induced Hyperphosphorylation of hRad9 Is Dependent on
ATM--
Since the ATM kinase is activated, and hRad9 is
hyperphosphorylated upon IR, we tested whether the two proteins act in
the same signaling pathways. Treatment of A-T cells with 10-Gy
irradiation did not induce hyperphosphorylation of hRad9. Conversely,
hRad9 was hyperphosphorylated, in a dosage-dependent manner, in
A-T cells complemented with wild-type ATM cDNA (YZ5 cells, Fig.
4A). In contrast to the
IR-induced response, treatment with HU resulted in the
hyperphosphorylation of hRad9 in A-T cells (Fig. 4B),
indicating IR-induced, but not HU-induced, hyperphosphorylation of
hRad9 is ATM-dependent. Since the NBS shares similar
clinical and cellular phenotypes with the A-T disorder, we examined
whether hyperphosphorylation of hRad9 is defective in NBS1 cells. IR-,
UV-, and HU-induced hyperphosphorylations were normal in NBS cells
(Fig. 4C), demonstrating that NBS1 is not required for the
hyperphosphorylation of hRad9.
ATM Phosphorylates Ser272 of hRad9 in Vitro--
To
extend the above findings, we explored the possibility that ATM
phosphorylates hRad9 in vitro. Human Rad9 protein contains one SQ site, the preferred target site for the ATM kinase (62). GST
fused to hRad9 residues 255-295 (GST-hRad9255-295)
encompassing the Ser272 site was readily phosphorylated by
ATM immunoprecipitated from T24 cells (Fig.
5B, lane 2).
Interestingly, ATM immunoprecipitated from irradiated T24 cells
exhibited enhanced phosphorylation of GST-hRad9255-295
(Fig. 5B, lane 3). Substitution of
Ser272 to alanine abolished phosphorylation (Fig.
5B, lanes 5 and 6). In contrast,
GST-hRad9255-295 was not phosphorylated by ATR under
similar conditions (Fig. 5C). To further confirm the
kinase-substrate relationship between ATM and
GST-hRad9255-295, recombinant FLAG-tagged wild-type ATM
(pCMV-Flag-ATMWT) and kinase-inactive ATM
(pCMV-FLAG-ATMD2870A) were overexpressed in 293 cells. The
immunoprecipitated recombinant ATM was used as the kinase source for
the in vitro kinase assays. Wild-type recombinant ATM
phosphorylated the Ser272 site of hRad9 (Fig.
5D, lane 1), whereas the kinase-inactive form of
ATM did not (Fig. 5D, lane 2), providing further
evidence that Ser272 of hRad9 is phosphorylated by ATM but
not by a contaminating kinase. Examination of other hRad9 fragments
revealed that full-length (Fig. 5E),
NH2-terminal (1-223aa), and COOH-terminal (224-391aa) fragments of hRad9 (data not shown) could all be phosphorylated by both
ATM and ATR in vitro, indicating that there are likely to be
additional phosphorylation sites for these two kinases. Taken together,
these results demonstrate that Ser272 of hRad9 is a
substrate site specifically for ATM but not for ATR in
vitro.
IR-induced Phosphorylation of Ser272 of hRad9 in Vivo
and Delayed Phosphorylation of Ser272 in A-T Cells--
To
further examine phosphorylation of hRad9 on Ser272 in
vivo, we used affinity-purified anti-phospho-Ser272
antibodies for immunoprecipitation. These antibodies immunoprecipitate hRad9 protein only after IR treatment in YZ5 and T24 cells (Fig. 6A and data not shown). The
hRad9 with p-Ser272 was detected 1 h post-IR, and the
amount of p-Ser272 was increased at 4 h post-IR. hRad9
levels remained constant following IR (Fig. 6A, middle
panel). In contrast, IR-induced phosphorylation of
Ser272 was delayed in ATM-deficient cells. The level of
p-Ser272 at 8 h post-IR was similar to that of YZ5
cells 1 h post-IR (Fig. 6B). This result suggests that
ATM phosphorylates Ser272 of hRad9 in response to IR
in vivo. In the absence of ATM, another kinase may
phosphorylate Ser272 in response to IR, but in a delayed
manner.
Expression of the hRad9S272A Mutant Protein Disturbs
G1/S Checkpoint Activation and Sensitizes Cells to
IR--
We next examined the effect of phosphorylation of the hRad9
Ser272 residue on checkpoint activation. Similar levels of
recombinant hRad9 were detected in cells transfected with either
pCMV2-FLAG-hRad9WT or pCMV2-FLAG-hRad9S272A
(Fig. 7A). The effects of
phosphorylation of Ser272 of hRad9 on G1/S
checkpoint activation was determined by co-transfecting human lung
fibroblast VA13 cells with EGFP, and hRad9WT or
hRad9S272A. In comparison with wild-type
pCMV2-FLAG-hRad9WT, expression of
pCMV2-FLAG-hRad9S272A resulted in defective checkpoint
activation (Fig. 7B). Since uptake of BrdUrd was scored in
these assays, the experiment mainly measured G1/S
checkpoint and may not reflect intra-S-delay. The effects of
hRad9S272A expression on cellular sensitivity to IR were
examined. VA13 cells co-transfected with EGFP and wild-type hRad9 or
mutant hRad9 (hRadS272A) were exposed to 5 Gy of Here we demonstrated that the anomalous molecular mass of
endogenous hRad9 is due to basal phosphorylation. This basal
phosphorylation is likely to occur at multiple sites, since the 60-kDa
form of hRad9 could be converted to 45 or 55 kDa molecular mass,
depending on the amounts of phosphatase used in CIAP treatment. While
the functional significance of the extensive phosphorylation of hRad9 is unknown, it is plausible that the basal phosphorylation of hRad9 is
either required for its interaction with hHus1 and/or hRad1 to form a
stable complex. Alternatively, phosphorylation may be required for the
stabilization of hRad9 within the cell (31).
Several observations described here lead to our conclusion that ATM
phosphorylates Ser272 of hRad9 in response to IR. First,
ATM specifically phosphorylated Ser272 in vitro,
but not other serines around it. Second, IR-induced hyperphosphorylation of hRad9 is diminished in A-T cells, and hyperphosphorylation of hRad9 is restored by the re-introduction of ATM cDNA into A-T cells. Third, IR-induced phosphorylation of
hRad9 on Ser272 was detected in vivo, and
ATM-deficient cells had delayed phosphorylation of Ser272.
Fourth, transient expression of hRad9S272A resulted in the
defective G1/S checkpoint activation upon DNA damage and
increased cellular sensitivity to IR, suggesting that the
hRad9S272A mutant may act in a dominant-negative manner.
The finding that hyperphosphorylation of hRad9 in response to IR is
ATM-dependent is consistent with the observation in yeast
species that DNA damage-induced phosphorylation of DDC1 or Rad9 is
dependent on MEC1 or Rad3, respectively (8, 61). While we demonstrated
that phosphorylation of Ser272 of hRad9 is required for
activation of the DNA damage-induced G1/S phase checkpoint,
the mechanism by which hyperphosphorylation of hRad9 activates
downstream effectors remains to be investigated.
Hyperphosphorylation of hRad9 was also observed upon UV and HU
treatment. It is likely that ATR mediates the UV- and HU-induced hyperphosphorylation of hRad9. In this regard, we found that ATR did
phosphorylate full-length hRad9 and NH2-terminal (amino
acids 1-223) and COOH-terminal (amino acids 224-391) hRad9 in
vitro (Fig. 5E and data not shown). Interestingly,
Ser272 was not a favorable substrate site for ATR in
vitro, suggesting that there may be unique phosphorylation sites
for ATM and ATR in the hRad9 protein. Studies using oriented peptide
libraries in the kinase assay have shown that some sequences are
preferentially phosphorylated by one kinase but not by the other (63).
Interestingly, delayed phosphorylation is also seen in p53 and NBS1 in
A-T cells (55, 65-67). Using Although hRad9 is a direct substrate of ATM in vitro and
in vivo, we were unable to show interaction between ATM and
hRad9 by co-immunoprecipitation. The absence of this evidence may
reflect a transient interaction or inefficient co-immunoprecipitaiton due to the presence of large protein complexes. The requirement of all
other checkpoint Rad proteins for DNA damage-induced phosphorylation of
DDC1/Rad9 (72)2 suggests that
the Rad protein complexes, not the DDC1/Rad9 itself, are likely to be
required for the interaction between MEC1/Rad3 and DDC1/Rad9 in yeast
species. Interestingly, in our 32P labeling experiment
(Fig. 2A) and GST-hRad9 pull-down assay (data not shown),
additional proteins were readily detectable, suggesting that hRad9 is
likely to be present in multiple protein complexes.
DNA damage-induced hyperphosphorylation of hRad9 appears to be normal
in NBS cells. There are two plausible explanations. Human Rad9 may
operate upstream of NBS1, alternatively, hRad9 may function separately
from NBS1 (Fig. 8). The checkpoint Rad proteins are hypothesized to play the roles of sensors in recognizing DNA structure changes (9). Other events, including DNA repair, transcription, and apoptosis, are likely downstream cellular
processes following the binding of these sensors to damaged DNA
(73-75). If this is true, it is most likely that hRad9 acts
upstream of NBS1. Further studies will be required to clarify the
relationship between Rad9 and NBS1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phospho-Ser272 antibodies were raised
against the keyhole limpet hemocyanin-conjugated hRad9 peptide,
CSDTDSHS(PO3)QDLG. The flow-through fractions of the antiserum in a
CSDTDSHSQDLG column were affinity-purified using the phosphopeptide
column to eliminate antibodies reacting with the nonphosphorylated
antigen peptide and nonspecific antigens. Antibodies against
-actin were from Sigma. Rabbit
-hRad9 polyclonal antibodies were purchased
from Santa Cruz Biotechnology.
-ATM monoclonal antibodies
were as described previously (57).
1 aprotinin, 50 µg
ml
1 leupeptin, 1 mM
phenylmethylsulfonyl fluoride) and phosphatase inhibitors (100 mM NaF and 1 mM
Na3VO4). Cell lysates containing 20-50 µg of
protein, as determined by Bradford assays, were subjected to SDS-7.5%
polyacrylamide gel electrophoresis followed by Western blotting. After
blocking with nonfat milk (5%) in TBST buffer (Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20), the membranes were incubated
with mouse
-hRad9 antibodies. The immune complexes in the
blot were detected by an enhanced chemiluminescence kit (Amersham
Pharmacia Biotech). For immunoprecipitation, 500-1,000 µg of
total protein (µg/µl) was incubated with 5 µl of mouse polyclonal antibodies or 5 µg of monoclonal antibody at 4 °C for 2 h,
followed by 1-h incubation with protein G-Sepharose. After five washes with ice-cold lysis buffer containing protease and phosphatase inhibitors, the immunoprecipitants were subjected to Western analysis as described above. For detection of p-Ser272 hRad9, cell
lysates containing 1 mg of protein were immunoprecipitated with 3 µg
of
-p-Ser272 antibodies followed by immunoblotting with
mouse
-hRad9 polyclonal antibodies.
-hRad9 polyclonal antibodies. The immunoprecipitants
were washed with ice-cold lysis buffer containing protease and
phosphatase inhibitors. A final wash was performed using phosphatase
buffer (Life Technologies, Inc.). The precipitates were equally
aliquoted into three tubes, each was subjected to mock, CIAP
(calf intestinal alkaline
phosphatase, from Life Technologies, Inc.) or CIAP plus
phosphatase inhibitor,
-glycerophosphate (Sigma) treatment,
respectively. The reaction was carried out at 30 °C for 30 min and
was stopped by addition of 4× SDS loading buffer. The resultant
products were analyzed by SDS-PAGE followed by immunoblotting with
mouse
-hRad9 polyclonal antibodies.
-ATM monoclonal antibody (3E8).
Recombinant FLAG-His-ATM was immunoprecipitated from transfected
293 cells using
-FLAG M2 mouse monoclonal antibody
(Eastman Kodak Co.). The kinase assays were conducted as described
previously (58). Briefly, the ATM protein in the protein
G-Sepharose beads was incubated with GST-hRad9 fusion protein (2-10
µg) in kinase buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM
dithiothreitol, 4 mM MnCl2, 6 mM MgCl2, 100 µM
NaVO4, 20 µM ATP, and 10 µCi of
[
-32P]ATP) at 30 °C for 30 min. The reaction was
stopped by addition of 4× SDS loading buffer. Proteins were separated
by SDS-PAGE and analyzed after Coomassie Blue staining and autoradiography.
-irradiator (Shepherd) at 2.55 Gy/min. UV
irradiation was performed using UV Stratalinker 2400 (Stratagene).
Hydroxyurea was added to cell culture medium at a final concentration
of 1 or 3 mM for 24 h. Extracts were prepared from
mock- or radiation-treated cells 1 h post-treatment unless
otherwise indicated.
-rays. After IR treatment, cells were
immediately incubated in fresh culture medium containing 10 µM BrdUrd for 2 h followed by immunostaining with
-BrdUrd monoclonal antibody (Becton-Dickinson). The percentage of EGFP/BrdUrd double-positive cells among the EGFP-positive population before and after IR was determined (55).
-irradiation 36 h post-transfection. The
viable EGFP-positive cells in a defined area were counted before and
72 h after IR treatment as described previously (55).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hRad9 antibodies, a single protein band with a
molecular mass of about 60 kDa was detected in extracts prepared from
human bladder carcinoma T24 cells (Fig.
1A). A protein band of similar
size was also detected after immunoprecipitation with rabbit
-hRad9 antibodies followed by Western blotting analysis
using mouse
-hRad9 antiserum (Fig. 1B). Upon
IR, this protein was detected as a slower migrating band. Together,
these results confirm that the 60-kDa protein is hRad9. Consistent with
a previous report, the molecular mass of the endogenous hRad9 protein
is much bigger than its predicted mass of 45 kDa, suggesting that hRad9
might undergo extensive post-translational modification (31). To
investigate the nature of modification of hRad9, extracts prepared from
T24 cells pulse-labeled with [32P]orthophosphoric acid
were subjected to immunoprecipitation using mouse
-hRad9
polyclonal antibodies. The detection of a labeled 60-kDa protein that
disappears following CIAP treatment indicates that hRad9 is a
phosphoprotein in undamaged cells (Fig. 2A, lanes 2 and
3). To further characterize the phosphorylation modification, hRad9 was immunoprecipitated from unlabeled cell extracts
and incubated with CIAP. Upon treatment with CIAP, 60-kDa hRad9 was
converted to faster migrating bands of either 45 or 55 kDa, depending
on the amount of CIAP used in the reaction (Fig. 2B,
lanes 2 and 4). In the presence of the CIAP
inhibitor
-glycerophosphate, the alternation in the hRad9
electrophoretic mobility was abolished (Fig. 2B, lane
5). These results demonstrate that the anomalous electrophoretic
mobility of endogenous hRad9 is due to phosphorylation. Similar to a
previous report (30), IR treatment resulted in the appearance of a slow
migrating form of hRad9 that could also be converted to a 45- or 55-kDa
band by CIAP treatment (Fig. 2B, lanes 6 and
7), indicating that IR resulted in hyperphosphorylation of
hRad9. Taken together, these results demonstrate that endogenous hRad9
is a heavily phosphporylated protein that undergoes
hyperphosphorylation after IR.
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Fig. 1.
Mouse -hRad9
antiserum recognizes a 60-kDa protein. A, detection of the hRad9
protein in cell extracts. Immunoblotting analysis of proteins in the
T24 cell extracts was performed using preimmune serum (lane
1) and mouse
-hRad9 antiserum generated against
GST-hRad9224-391 (lane 2). B,
specificity of mouse
-hRad9 polyclonal antibodies. Cell
extracts from mock (lane 2)- or IR (lane
3)-treated cells were immunoprecipitated (IP) using
rabbit
-hRad9 polyclonal antibodies. The IP products were subjected
to SDS-PAGE, followed by Western blotting analysis using mouse
-hRad9 antiserum. Human Rad9 in the total cell extracts is shown in
lane 1. hRad9pp indicates hyperphosphorylated
hRad9. R and M indicate rabbit and mouse
antibodies, respectively.
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Fig. 2.
IR-induced hyperphosphorylation of the
constitutively phosphorylated hRad9 protein. A, human
Rad9 is constitutively phosphorylated. T24 cells were labeled with
[32P]orthophosphoric acid, and cell lysates were
immunoprecipitated using preimmune serum (lane 1) and mouse
-hRad9 antiserum (lane 2). The
-hRad9
immunoprecipitants were incubated with CIAP (lane 3). The
arrows indicate unknown proteins in the immunoprecipitants.
B, reduction of the electrophoretic mobility of the hRad9
protein upon phosphatase treatment. Total cell extracts prepared from
mock- or IR-treated T24 cells were subjected to immunoprecipitation
using rabbit
-hRad9 antibodies. The
immunoprecipitants were incubated with phosphatase buffer only
(lanes 1, 3, and 6), CIAP (lanes
2, 4, and 7), or CIAP plus
-glycerophosphate (lanes 5 and 8). Resulting
proteins were analyzed by SDS-PAGE, followed by immunoblotting using
mouse
-hRad9 antibodies.
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Fig. 3.
IR-induced hyperphosphorylation of hRad9
occurs in all cell cycle phases. A, Western blotting
analysis of hRad9 in synchronized cells. Density-arrested T24 cells
were released and collected at the indicated cell cycle phases. Cells
were harvested 1 h post 10-Gy irradiation. B, UV- and
HU-induced hyperphosphorylation of hRad9. T24 cells were treated with
IR (10 Gy), UV (50 µJ), or HU (0.03%). Analysis of the hRad9 protein
was as described in the legend to A. -Actin
serves as a protein loading control.
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Fig. 4.
IR-induced hyperphosphorylation of
hRad9 is ATM-dependent. A,
hyperphosphorylation of hRad9 in response to IR in cells with or
without ATM. AT22IJE-T/pEBS7 (A-T) cells and AT22IJE-T/pEBS7-YZ5 (A-T
cells complemented with ATM) were exposed to 0-, 10-, and 30-Gy
-rays, respectively. One hour post-treatment, cell lysates were
prepared and proteins analyzed by SDS-PAGE, followed by immunoblotting
using
-ATM,
-hRad9, and
-actin antibodies, respectively. B,
hyperphosphorylation of hRad9 in response to hydroxyurea in cells with
or without ATM. Cells were exposed to hydroxyurea (0, 1, or 3 mM) for 24 h. Analysis of hRad9 was as described in
the legend to A. C, hyperphosphorylation of hRad9
in NBS cells. Cell lysates were prepared and analyzed as described in
the legend to Fig. 3B.
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Fig. 5.
ATM phosphorylates Ser272 of the
hRad9 protein in vitro. A, schematic
representation of the GST-hRad9 fusion proteins. Asterisk
indicates Ser272, which is conserved between mouse (mRad9)
and human. Site-specific mutation from Ser272 to Ala was
confirmed by DNA sequencing. B, kinase assays using
immunoprecipitated (IP) ATM. GST-hRad9255-295
fusion proteins consisting of wild-type (WT) and mutant
(S272A) hRad9 sequences were incubated with ATM prepared from cells
mock- or IR-treated in the presence of [ -32P]ATP at
30 °C for 30 min. The kinase reaction products were separated by
SDS-PAGE and analyzed by Coomassie Blue staining and autoradiography.
C, kinase assays using immunoprecipitated (IP)
ATR. The substrates were as in B. The arrow
indicates unknown protein in the ATR immunoprecipitants. D,
kinase assays using the recombinant ATM protein. 293 cells transiently
transfected with FLAG-tagged wild-type (ATMWT) or
kinase-inactive mutant ATM (ATMD2870A) were exposed to
10-Gy
-rradiation or mock-treated. Recombinant FLAG-ATM was
immunoprecipitated 1 h post-treatment using
-FLAG
antibodies. IP-FLAG-ATMWT and IP-FLAG-ATMD2870A
were used as the kinase source, and the substrates were as in
B. E, IP-ATM and IP-ATR kinase assays using
GST-fused full-length hRad9 as substrate. Arrowheads
indicate IgG. p531-106 serves as a positive control.
View larger version (54K):
[in a new window]
Fig. 6.
ATM phosphorylates hRad9 on
Ser272 in vivo and delayed modification in
A-T cells. A, phosphorylation of Ser272 of
hRad9 upon IR. YZ5 cells (A-T cells complemented with ATM cDNA)
were untreated or treated with 30-Gy -irradiation. Cells were lysed
at different time points after IR as indicated. Upper panel,
whole cell lysates were immunoprecipitated with rabbit
-p-Ser272 antibodies followed by immunoblotting with
mouse
-hRad9 antibodies. Middle panel shows direct
Western blotting with
-hRad9 antibodies, and bottom panel
shows direct Western blotting with
-
-actin antibodies.
B, phosphorylation of Ser272 of hRad9 after IR
in A-T cells. Immunoprecipitation followed by Western blotting or
direct Western blotting of Rad9 as described in A.
-rays.
Transfection with hRadS272A significantly increased
cellular sensitivity to IR compared with wild-type controls when viable
EGFP-positive cells were scored 72 h after IR (Fig.
7B). These assays were performed using transient transfections, because prolonged overexpression of hRad9 induces apoptosis (64). As yet, it is unclear whether radiosensitivity of the
transfected cells is due to checkpoint or/and repair defect. Taken
together, these results suggest that phosphorylation of Ser272 of hRad9 is required for cell cycle checkpoint
activation in response to IR.
View larger version (34K):
[in a new window]
Fig. 7.
Effects of expression of
hRadS272A on G1/S checkpoint activation and
cellular sensitivity to IR. A, expression of the
recombinant hRad9 protein. VA13 cells were mock-transfected or
transfected with pCMV2-FLAG-hRad9WT or
pCMV2-FLAG-hRad9S272A. Proteins in the cell extracts were
separated by SDS-PAGE followed by immunoblotting (IB)
analysis using -FLAG monoclonal antibody (M2).
B, G1/S checkpoint activation upon IR. The ratio
of BrdUrd- and EGFP double-positive cells to EGFP-positive cells was
determined in mock- and IR-treated cells. Bar represents the ratio of
the IR-treated cells divided by that in the mock-treated cells. At
least 1,000 cells were counted in each group. The mean and S.D. values
were calculated from three separate plates. C, cellular sensitivity to
IR. EGFP-positive cells were counted before and 72 h after 5-Gy
irradiation. Cellular sensitivity was represented by dividing the
number of cells recovered from mock-treated by the number of cells
recovered from the IR-treated populations. The cellular sensitivity of
mock-treated cells is defined as 1. Over 1,000 cells were counted in
each plate. The mean and S.D. values were derived from three separate
plates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphopeptide antibodies, we showed
that Ser272 of hRad9 was phosphorylated in vivo
upon DNA damage (Fig. 6). IR-induced phosphorylation of
Ser272 was delayed in ATM-deficient cells, suggesting that
ATR or another kinase may phosphorylate Ser272 of hRad9 in
the absence of ATM. Recent studies showed that Hus1-deficient mouse
cells are sensitive to UV but not IR, while C. elegans mrt2 mutants (S. pombe rad1+ homologue) are sensitive
to IR but less sensitive to UV (36, 37), suggesting that members of the
checkpoint Rad complex may be selectively activated by a specific type
of DNA damage. In addition, our results on hRad9, together with
other's results on p53, also indicate that different sites of the same
protein could be targeted by different upstream kinases in response to DNA damage or stalled DNA replication forks (68-71). Thus, multiple levels of regulation may provide fine tuning of cellular responses to
different DNA structure changes.
View larger version (15K):
[in a new window]
Fig. 8.
Model for ATM/hRad9 signaling pathways.
DNA double strand break induced by IR activates ATM, which
phosphorylates hRad9. Hyperphosphorylated hRad9 and its interacting
proteins hRad1 and hHus1, possibly together with the hRad17 complex,
bind to a DNA break site. Induction of hRad9 phosphorylation on
Ser272 upon treatment with UV and HU are likely to be
mediated by ATR. IR may also induce activation of ATR in a delayed
manner. The Nbs1·Mre11·Rad50 complex is activated by ATM and is
involved in the S phase checkpoint. The hRad9·hRad1·hHus1 and
Nbs1·Mre11·Rad50 complexes may mediate separate signaling cascades;
alternatively, the hRad1·hRad9·hHus1 complex may function upstream
of the Nbs1·Mre11·Rad50 complex.
Our studies provide the first demonstration that the mere presence of
the hRad9 protein may not be sufficient to activate checkpoint
controls. Instead, phosphorylation of Ser272 of hRad9
per se is required to activate the cellular response to IR.
Given that the main functions of the six checkpoint Rad proteins are
conserved among yeast and mammals, we anticipate that human checkpoint
Rad proteins, including ATM, hRad1, hRad9, hHus1, hRad17, and ATR, may
form a multiprotein sensor detecting and processing aberrant DNA
structures or stalled replication forks (Fig. 8). The integrity and the
tight coordination among these checkpoint Rad proteins underlie their
roles in maintaining genomic stability and in preventing cancer development.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Wen-Hwa Lee and Alan Tomkinson for critical reading of the manuscript and Dr. Tony Carr for sharing unpublished information. We are grateful to Dr. Gopal Dasika, Dr. Song Zhao, Sean Post, and K. Wyatt McMahon for their helpful advice and critical discussion. We also thank all the members in Dr. Eva Lee's laboratory for their help and encouragement.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA81020 and NS37381 (to E. L.) and GM52493 (to H. B. L.).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.
¶ Current address: Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Rd., Tarrytown, NY 10591-6707.
To whom correspondence should be addressed. Tel.:
210-567-7326; Fax: 210-567-7324; E-mail:
leee@uthscsa.edu.
Published, JBC Papers in Press, February 28, 2000, DOI 10.1074/jbc.M008871200
2 A. Carr, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ATM, ataxia-telangiectasia-mutated; ATM, ataxia-telangiectasia-mutated gene; A-T, ataxia-telangiectasia; ATR, ataxia-telangiectasia and Rad3-related; HU, hydroxyurea; IR, ionizing radiation; hRad, the human Rad proteins; GST, glutathione S-transferase; CIAP, calf intestinal alkaline phosphatase; PAGE, polyacrylamide gel electrophoresis; Gy, gray(s); NBS, Nijmegen breakage syndrome; BrdUrd, bromodeoxyuridine; EGFP, enhanced green fluorescent protein.
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