From the
Division of Cancer Biology and Genetics, Queen's University Cancer
Research Institute, and the Departments of
Pathology,
¶Biochemistry, and
||Oncology, Queen's University, Kingston, Ontario
K7L 3N6, Canada
Received for publication, March 26, 2003
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ABSTRACT |
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INTRODUCTION |
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The hRad9 protein is the human homologue of Schizosaccharomyces
pombe Rad9, a member of the checkpoint Rad family of proteins. In fission
yeast, the checkpoint rad genes (rad1+,
rad3+, rad9+,
rad17+, rad26+, and
hus1+) are required for the S phase (DNA replication) and
G2 (DNA damage) checkpoints
(913).
Yeasts lacking these genes fail to inactivate Cdc2 and enter premature, lethal
mitosis when challenged with agents that inhibit DNA synthesis or damage DNA
(14,
15). Like its yeast
counterpart, hRad9 forms a ring-shaped, heterotrimeric complex with the hRad1
and hHus1 proteins
(1618).
Each member of the hRad9-hRad1-hHus1 complex (also known as the 9-1-1
complex), shares sequence homology with
PCNA,1 a homotrimer
that encircles DNA and tethers DNA polymerase during DNA synthesis
(19). PCNA is loaded onto DNA
by the pentameric protein complex replication factor C (RFC), which is
composed of one large subunit and four smaller subunits
(20). In a manner analogous to
PCNA and RFC, 9-1-1 is loaded onto DNA by a complex between hRad17 and the
four smallest subunits of RFC
(21). Since DNA damage induces
hRad17-dependent association of 9-1-1 with chromatin, the 9-1-1 complex is
believed to be involved in the direct recognition of DNA lesions during the
initial stages of the checkpoint response
(22). Also involved in this
recognition are two phosphatidylinositol 3-kinase-related kinases, ATM and
ATR, that regulate several cell cycle transitions and are central components
of the cell checkpoint machinery
(23). Even though these
kinases appear to respond to different types of DNA lesions, they share a long
list of common checkpoint substrates, including hRad17
(2426)
and hRad9 (27). In fission
yeast, Rad3 (which shares homology with both ATR and ATM) requires Rad9, Rad1,
Hus1, and Rad17 to phosphorylate certain substrates
(28). Similarly, in human
cells, phosphorylation of hRad17 by ATR requires hHus1
(22). These findings support a
model in which the 9-1-1 complex recruits substrates for ATM or ATR to sites
of DNA damage or stalled replication forks
(29).
In addition to interacting with hRad1 and hHus1, hRad9 also physically interacts with TopBP1, the human orthologue of Saccharomyces cerevisiae Dpb11, and Schizosaccharomyces pombe Cut5 (30). Each of these proteins contains multiple BRCA1 carboxyl-terminal (BRCT) domains, a putative protein-protein interaction motif common in cell cycle control and DNA repair. In addition to the checkpoint rad genes, cut5+ is also required for slowing S phase and delaying mitosis when DNA replication is challenged by DNA damage (13, 31). A similar requirement also exists for DPB11 in budding yeast (32), and a physical interaction between Dpb11 and Ddc1 (the budding yeast orthologue of hRad9) may play a role in this response (33).
hRad9 is a unique member of the 9-1-1 complex in that it contains a C-terminal region (of about 110 amino acids) that does not share homology with PCNA and is not believed to be directly involved in association with hRad1 or hHus1. This region of the protein is both constitutively and transiently phosphorylated at several amino acid residues (34), and hence represents a potential regulatory region for the effector functions of 9-1-1. We have characterized the extensive phosphorylation of hRad9 in this region and show that it is partially regulated by Cdc2. We also demonstrate that the C-terminal phosphorylation of hRad9 has roles in regulating both hRad9 interaction with TopBP1 and the cellular response to DNA damage in S phase.
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EXPERIMENTAL PROCEDURES |
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Drugs and IrradiationCells were irradiated using a
137Cs -irradiator at 0.78 grays/min. Thymidine (Sigma),
hydroxyurea (Sigma), and nocodazole (Sigma) were typically administered for 18
h at concentrations of 2 mM, 1 mM, and 0.1 µg/ml,
respectively.
Plasmids and Site-directed MutagenesisAll hRad9 point mutants were generated using the Transformer site-directed mutagenesis kit (BD Biosciences) according to the manufacturer's instructions. GST expression plasmids were generated by PCR subcloning segments of the hRad9 cDNA (wild-type and point mutants) into the BamHI and EcoRI restriction sites of the pGEX-2T vector.
AntibodiesRabbit polyclonal
-phospho-Ser272,
-phospho-Ser387, and
-phospho-Thr292 antibodies were raised against
phospho-Ser272 (SDTDSHpSQDLGS; where pS represents phosphoserine),
phospho-Ser387 (PVLAEDpSEGEG), and phospho-Thr292
(QLQAHSpTPHPDD; where pT represents phosphothreonine) hRad9 peptides,
respectively (Bethyl Laboratories, Montgomery, TX). Antisera were cleared of
nonspecific binding activity by passage over immobilized, nonphosphorylated
peptides and then affinity-purified with immobilized phosphorylated peptides.
-hRad9 polyclonal chicken antibodies have been described previously
(17).
-hHus1 and
-hRad1 polyclonal chicken antibodies were generated against bacterially
expressed and purified His-hHus1 and GST-hRad1, respectively (RCH Antibodies,
Kingston, Canada). Antibodies were cleared of GST reactivity and then
affinity-purified, as described previously
(17). Other antibodies used in
this study were mouse monoclonal antibodies directed against TopBP1 (BD
Biosciences), Myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), PCNA
(PC10; Santa Cruz Biotechnology), and Cdc2
(17) (Santa Cruz
Biotechnology).
Flow CytometryHeLa cells were fixed in 50% ethanol in phosphate-buffered saline for at least 30 min on ice. Cells were then collected by centrifugation, resuspended in phosphate-buffered saline with 50 µg/ml PI and 0.1 mg/ml RNase A, and analyzed using a flow cytometer (Beckman/Coulter EPICS ALTRA, Mississauga, Canada).
Immunoprecipitations and ImmunoblottingFor
immunoprecipitation reactions, cells were lysed in 250 mM NaCl, 1
mM EDTA, 20 mM Tris (pH 8.0), 0.5% Nonidet P-40, and 10%
glycerol, supplemented with 20 µg/ml aprotinin, 4 µg/ml leupeptin, 2
mM sodium orthovanadate, 20 mM -glycerophosphate,
and 0.2 mM sodium fluoride. Lysates were incubated on ice for 30
min and centrifuged at 13,000 x g. Supernatants were precleared
with
-chicken IgY-agarose (Promega, Madison WI) or with protein G
Sepharose (Amersham Biosciences) for 30 min at 4 °C prior to
immunoprecipitation. The immunoprecipitation was performed with 1 µg of
antibodies directed against the Myc epitope and 20 µl of protein
G-Sepharose or antibodies directed against hRad9 and
-chicken
IgY-agarose for 2 h at 4 °C. Immune complexes were washed four times with
1 ml of lysis buffer and then resuspended in SDS-PAGE loading buffer.
Immunoblotting was performed essentially as previously described
(17).
Protein PurificationGST and His-tagged protein expression
was induced in logarithmically growing BL21 bacteria, with 0.1 mM
isopropyl-1-thio--D-galactopyranoside for 3 h at 37 °C.
GST fusion proteins were batch-purified from bacterial lysates with
glutathione-Sepharose (Promega) using standard techniques. Bound proteins were
eluted with 20 mM reduced glutathione in 50 mM HEPES, 10
mM MgCl2, and 1 mM dithiothreitol (final pH
of 7.4). His-hHus1 was purified under denaturing conditions (6 M
guanidine HCl) on a nickel column.
Kinase AssaysHeLa cells, arrested in mitosis with
nocodazole or in S phase with thymidine, were lysed in 1 ml each of kinase
lysis buffer (50 mM Tris (pH 7.4), 1 mM EDTA (pH 8.0),
25 mM NaCl, and 0.1% Nonidet P-40). The Cdc2 kinase was
immunoprecipitated from these extracts using 4 µg of mouse monoclonal
antibody directed against Cdc2. Immune complexes were washed three times with
1 ml of phosphate-buffered saline and then two times with 1 ml of kinase
reaction buffer (50 mM HEPES (pH 7.4), 10 mM
MgCl2, 1 mM dithiothreitol, 50 µM ATP)
Reactions were carried out in 60 µl of kinase reaction buffer in the
presence of 4 µCi of [-32P]ATP, and 3 µg of GST fusion
substrate for 30 min at 30 °C. Reactions were stopped by the addition of
30 µl of 3x SDS-PAGE loading buffer. 20 µl from each reaction was
subjected to SDS-PAGE (12%), and proteins were stained with Coomassie
Brilliant Blue. Gels were then dried and exposed to x-ray film (Eastman Kodak
Co.) for 24 h.
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RESULTS |
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We had also previously reported that mutation of Ser277 (a
constitutively phosphorylated residue in hRad9) to an alanine reduced the
efficiency of hRad9 hyperphosphorylation in mitosis
(34). To determine whether
Thr292 phosphorylation required prior phosphorylation at
Ser277 or any of the other constitutively phosphorylated residues
above, the S277A, S328A, S336G, and T355A mutants were also immunoblotted with
the -p292 antibodies. We found that whereas Thr292
phosphorylation was readily detectable in S328A, S336G, and T355A mutants
derived from mitotic extracts, it was severely reduced in the S277A mutant
(Fig. 1A). Similar
results were obtained when hRad9 proteins harboring multiple mutations were
analyzed (Fig. 1B).
Whereas hRad9 protein containing the S328A, S336G, and T355A mutations was
still efficiently phosphorylated at Thr292 in mitosis, the addition
of one or both of the S277A and T292A mutations inhibited this
phosphorylation. Hence, the mitotic phosphorylation of hRad9 at
Thr292 requires prior phosphorylation at Ser277.
hRad9 Phosphorylation Is Partially Dependent on Cdc2To
further characterize the mitotic phosphorylation of hRad9, we examined the
mobility of endogenous hRad9 as cells entered and exited nocodazole-induced
mitotic arrest. Consistent with our previous observations
(34), the predominant phospho
form of hRad9 in asynchronous cultures (hRad9) migrated at an apparent
molecular mass of
60 kDa. The discrepancy between the apparent molecular
mass of hRad9 and its predicted molecular mass of 43 kDa is largely due to
phosphorylation at Ser277, Ser328, Ser336,
and Thr355. Several less abundant, faster migrating phosphorylation
intermediates (hRad9
forms) represent hRad9 protein that is only
partially phosphorylated at these sites. When cells were blocked in mitosis by
treatment with nocodazole, the abundance of both hRad9
and hRad9
forms was reduced, and the majority of hRad9 existed as an even slower
migrating species, hRad9µ (see Fig.
2A). When the mobility pattern of hRad9 was examined at
increasing time points following the removal of nocodazole from the culture
medium, the emergence of normal hRad9 mobility coincided with the exit of
cells from mitosis, as defined by flow cytometry
(Fig. 2A, 120 and 400
min).
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The observations made in Figs. 1 and 2A, are consistent with each of Ser277, Ser328, Ser336, Thr355, and Thr292 being quantitatively phosphorylated during mitosis, although the vast majority of hRad9 is already phosphorylated at the four former residues prior to mitosis. Each of these five amino acids share the common consensus sequence ((S/T)PX(R/P)) and are thus potential targets for cyclin-dependent kinases. For these reasons, we sought to determine whether Cdc2, the cyclin-dependent kinase that controls the G2/M transition, could phosphorylate Ser277, Thr292, Ser328, Ser336, and Thr355 in vitro. To this end, we purified a series of hRad9 C-terminal peptides as GST fusion proteins that encompassed the amino acids indicated above, either left intact or mutated to alanine or glycine residues. Peptides were used as substrates for Cdc2, which was purified by immunoprecipitation from HeLa cells arrested in mitosis or S phase.
These analyses revealed that Cdc2 could phosphorylate each of the five
(S/T)PX(R/P) sites in vitro, with the exception of
Thr292 (Fig.
2B). Phosphorylation of hRad9 and the histone-positive
control was enhanced if Cdc2 was derived from mitotic rather than S phase
extracts, illustrating the specificity of the -Cdc2 antibody.
Phosphorylation of Ser277 was demonstrated by introduction of the
S277A mutation in GST-hRad9266301, which abolished
phosphorylation of this protein by Cdc2. Neither the S277A mutant nor the
S277A/T292A double mutant were phosphorylated at levels significantly above
background, indicating that Thr292 was not phosphorylated by Cdc2
in this assay. Phosphorylation of Ser328 and Ser336 was
shown using GST-hRad9314344. Whereas the S328A and S336G
single mutants of GST-hRad9314344 were still modestly
phosphorylated, the S328A/S336G double mutant showed only background levels of
32P incorporation. Thr355 was more efficiently
phosphorylated than any of the other hRad9 sites (see
GST-hRad9348391). There was no appreciable difference in
phosphorylation of T355A and P3A (T355A/S375A/S380G), indicating that
Ser375 and Ser380 are not phosphorylated by Cdc2 in
vitro. This is consistent with our previous work in which we found no
evidence for phosphorylation of these residues in vivo
(34). Furthermore, there did
not appear to be any significant phosphorylation of non-Cdk consensus sites in
hRad9, thus underscoring the specificity of Cdc2 toward Ser277,
Ser328, Ser336, and Thr355 of hRad9 in this
assay.
To determine whether Cdc2 phosphorylates hRad9 in vivo, we employed the Cdk inhibitor roscovitine. Roscovitine is a selective inhibitor of both Cdc2 and Cdk2 (35). When HeLa cells were arrested in mitosis with nocodazole and then treated with roscovitine (Ros) for 2 h, a drastic reduction in hRad9 phosphorylation was observed (Fig. 2C). Roscovitine had no effect however, on hRad9 phosphorylation in asynchronous cultures (Fig. 2C).
hRad9 Is Constitutively Phosphorylated at
Ser387Site-directed mutagenesis of potentially
phosphorylated residues in the C terminus of hRad9 identified a mutant, S387A,
which was not normally hyperphosphorylated in response to the DNA synthesis
inhibitor hydroxyurea (HU). To characterize this residue, phosphospecific
antibodies were raised against a phospho-Ser387 hRad9 peptide. The
quality of these antibodies was determined by testing their reactivity toward
phosphorylated and dephosphorylated wild-type and S387A hRad9. Although
transiently expressed wild-type and S387A hRad9 were indistinguishable in
terms of their SDS-PAGE mobility, they were clearly distinct with regard to
their detection when immunoblotted with -phospho-Ser387
(
-p387) antibodies (Fig.
3A). Whereas the
-p387 antibodies recognized all
differentially migrating forms of wild-type hRad9, they were completely
nonreactive toward the S387A mutant (Fig.
3A, right blot). In addition, the
-p387
antibodies did not detect wild type hRad9 that had been dephosphorylated with
calf intestinal phosphatase. Thus, hRad9 is phosphorylated at
Ser387, and the
-p387 antibodies are effective in
specifically recognizing Ser387-phosphorylated hRad9.
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To determine whether phosphorylation at Ser387 of hRad9 was
regulated in a cell cycle- or DNA damage-dependent manner, we collected HeLa
cells that were in G1, S, G2, and M phases of the cell
cycle, as we have previously described
(34). HeLa cells were
synchronized with a double thymidine block and then released and collected 2 h
(S), 7 h (G2), and 11 h (G1) later. One hour prior to
harvest, these cells were treated with 20 Gy of ionizing radiation (IR), as
indicated. Cells were arrested in mitosis (M) by treating asynchronous cells
with nocodazole. We also collected HeLa cells that were grown in the presence
of HU for 18 h or were harvested 18 h after a 20-Gy dose of IR. At the time of
harvest, these cells were arrested in early S phase and G2,
respectively. In all cases, cell cycle position was confirmed using flow
cytometry of propidium iodide-stained nuclei. Soluble cell lysates from these
cells were subjected to immunoblotting with -hRad9,
-p387, and
phosphospecific antibodies directed against phosphorylated Ser272
of hRad9 (
-p272), a site of DNA damage-dependent phosphorylation
(27).
In agreement with several previous reports
(16,
22,
27,
34,
36,
37), the constitutively
phosphorylated hRad9 protein underwent hyperphosphorylation under various
conditions (Fig. 3B,
top panel). Ionizing radiation induced rapid phosphorylation of hRad9
at Ser272 regardless of cell cycle position
(27). Whereas
Ser272 phosphorylation did produce a subtle mobility shift, it was
more easily visualized by immunoblotting with -p272 antibodies
(Fig. 3B, middle
panel). The
-p272 blot revealed that IR-induced phosphorylation at
Ser272 was not only rapid but also transient and dissipated by 18 h
following irradiation. The hRad9 protein from cells harvested 18 h after IR
exhibited a second IR-induced mobility shift. This IR-induced modification,
however, which persisted after phosphorylation at Ser272 had
dissipated, was less rapidly induced and was not readily detectable 1 h after
IR (Fig. 3B, top
panel). The hRad9 protein also underwent similar mobility shifts in
G2, in response to HU, and in mitosis.
In contrast to these dynamic phosphorylation changes, the extent of
Ser387 phosphorylation did not change significantly in response to
cell cycle perturbation or DNA damage. In fact, the reactivity of the
-p387 antibodies toward hRad9 seemed to mirror that of the
-hRad9 antibodies (Fig.
3B, bottom panel)in that they recognized all
differentially migrating forms of hRad9. We went on to show that
Ser387 of hRad9 is phosphorylated constitutively in normal
(noncancerous) cell lines and those with homozygous mutations in ATM
(Fig. 3C). Thus,
Ser387 represents a novel, ATM-independent, constitutive
phosphorylation site in hRad9. Ser387 is distinct from previously
identified constitutive sites in hRad9, however, in that it does not fit the
(S/T)PX(R/P) consensus sequence seen in these other sites
(Table I), and it is not
phosphorylated by Cdc2 in vitro
(Fig. 2B, peptide
348391).
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Ser387 Phosphorylation Regulates DNA Damage-dependent
Hyperphosphorylation of hRad9 We next examined the dependence of
DNA damage-induced hRad9 hyperphosphorylation events on hRad9 constitutive
phosphorylation. As described previously
(27) and as illustrated in
Fig. 3B, hRad9 becomes
hyperphosphorylated in a DNA damage-dependent manner at Ser272. We
had previously shown that this process occurs independently of constitutive
phosphorylation at (S/T)P sites
(34). Using -p272
antibodies, this observation was confirmed in
Fig. 4A, since a P4A
mutant (harboring the S277A, S328A, S336G, and T355A mutations) was still
inducibly phosphorylated at Ser272 1 h after a 20-Gy dose of IR.
Similarly, the S387A mutant was also inducibly phosphorylated at
Ser272 at levels comparable with that of wild-type hRad9
(Fig. 4A), indicating
that Ser387 phosphorylation is also not a prerequisite for DNA
damage-induced phosphorylation at Ser272.
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We had previously reported that hRad9 undergoes DNA damage-dependent
hyperphosphorylation that is distinct from phosphorylation at
Ser272 (34). This
second hyperphosphorylation event was illustrated in
Fig. 3B. When cells
were subjected to DNA damage in late S phase/G2, treated with
hydroxyurea, or harvested after extended periods of time following IR, hRad9
underwent a mobility shift that was similar to that observed in mitotic cells
(Fig. 3B). This
observation had initially led us to believe that this DNA damage-induced
phosphorylation of hRad9 was occurring at Thr292 (the site of
mitotic phosphorylation; Fig.
1). To address this directly, hRad9 protein was harvested from
cells grown under each of the above conditions and immunoblotted with
-p292 antibodies. As shown in Fig.
4B, only hRad9 derived from mitotic cells was
phosphorylated at Thr292. Thus, the location of
Ser272-independent, DNA damage-induced hRad9 phosphorylation
remains unclear.
As mentioned earlier, our initial interest in Ser387 was sparked
from the observation that a S387A mutant was not efficiently
hyperphosphorylated in response to HU. Whereas this had initially led us to
believe that Ser387 phosphorylation was induced by HU, our analysis
with -p387 antibodies (Fig.
3) has indicated this to be incorrect. We therefore reasoned that
Ser387 phosphorylation was a prerequisite for HU-induced
hyperphosphorylation and may also be a prerequisite for hyperphosphorylation
of hRad9 under other conditions. To address this possibility, we examined the
response of wild-type, S272A, and S387A hRad9 protein to growth conditions
known to induce DNA damage-dependent/Ser272-independent
hyperphosphorylation. These proteins were transiently expressed at low levels
using the Tet-Off expression system and were Myc-tagged to distinguish them
from endogenous (wild-type) hRad9. In these experiments, transient expression
levels near that of endogenous hRad9 were necessary, since overexpressed hRad9
is not efficiently hyperphosphorylated in response to
HU.2 Unlike wild-type
and S272A hRad9, the S387A mutant was not efficiently hyperphosphorylated when
HeLa cells were synchronized in G2, harvested 18 h after 20 Gy of
IR, or treated with HU (Fig.
4C). We refer to this hyperphosphorylated form of hRad9
as hRad9
to distinguish it from mitotic hRad9 (hRad9µ), which is
readily observed in the S387A mutant. As shown in
Fig. 4D, neither the
S272A nor the S387A mutation prevented mitotic phosphorylation of hRad9 at
Thr292. A summary of the hRad9 phosphorylation sites described in
this paper is given in Table
I.
hRad9 Constitutive Phosphorylation Sites Regulate Association with
TopBP1hRad9 forms a ring-shaped, PCNA-like heterotrimeric complex
with hHus1 and hRad1
(1619).
The C terminus of hRad9, which does not exhibit sequence or structural
similarity to PCNA, has been shown to interact with the DNA damage-responsive
protein TopBP1 (30). Using
hRad9 C-terminal deletion mutants, we refined the region of hRad9 required for
the interaction with TopBP1 to 17 amino acids at the extreme C terminus of
hRad9.3 Because this
region encompasses Ser387, we hypothesized that phosphorylation at
Ser387 could be regulating the interaction between hRad9 and
TopBP1. To test this, HeLa cells were transiently transfected with plasmids
that overexpress wild-type, S272A, S387A, or P4A hRad9. hRad9-containing
complexes were immunoprecipitated from transfected cell lysates with
-hRad9 antibodies. Immunoprecipitated proteins were then
size-fractionated by SDS-PAGE and immunoblotted with
-TopBP1,
-hRad9,
-p387,
-hHus1, and
-hRad1 antibodies. Due
to high levels of transiently expressed hRad9 relative to endogenous hRad9,
the vast majority of the immunoprecipitated hRad9 protein was plasmid-derived.
Accordingly, the
-p387 immunoblot
(Fig. 5A, third
panel) does not exhibit any endogenous, Ser387-phosphorylated
hRad9. TopBP1 co-immunoprecipitated with wild-type, S272A, and to a lesser
extent P4A hRad9, but did not co-immunoprecipitate with the S387A mutant
(Fig. 5A, top
panel). This was confirmed in a similar experiment employing
epitope-tagged hRad9 proteins. Whereas TopBP1 co-immunoprecipitated with
Myc-tagged wild-type and S272A hRad9, it did not co-immunoprecipitate with
Myc-tagged S387A (Fig.
5B, top panel). Immunoblotting hRad9-containing
immune complexes with
-hRad1 and
-hHus1 antibodies revealed that
none of the mutations tested prevented hRad9 association with either hRad1 or
hHus1 (Fig. 5A,
bottom two panels).
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Overexpression of hRad9 Constitutive Phosphorylation Mutants Results in Prolonged G2/M Arrest following IRWe used siRNA directed against the hRad9 transcript to determine whether reduction of hRad9 protein levels would impair the response of cells to DNA damage. Immunoblotting revealed that the hRad9 siRNA duplex was successful in reducing steady state hRad9 protein levels, relative to mock- and luciferase (Luc) siRNA-transfected controls (Fig. 6A, top). Sixteen hours after exposure of cells to 20 Gy of IR, the majority of mock-, hRad9 siRNA-, and Luc siRNA-transfected cells were arrested with a G2/M DNA content (Fig. 6A, bottom). When these cells were monitored for exit from G2/M arrest, hRad9-siRNA-transfected cells exhibited prolonged G2/M arrest in relation to the control cells (Fig. 6A). No measurable changes in cell cycle distribution were observed in unperturbed hRad9 siRNA transfected cells relative to control cells.2
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We next wanted to determine if cells overexpressing the hRad9 constitutive phosphorylation mutants exhibited the prolonged G2/M arrest defect observed in hRad9 hypomorphic cells. To this end, HeLa cells were transiently transfected with expression plasmids encoding wild-type, S272A, S387A, or P4A hRad9. In all cases, the transfection efficiency was about 50%, based on indirect immunofluorescence/flow cytometry. In this experiment, cells were arrested in early S phase with thymidine, released from arrest, and then irradiated 15 min later with 10 Gy of IR. Cells were then collected at the indicated time points, fixed, and stained with propidium iodide (Fig. 6B). By 9 h after irradiation, a time at which unirradiated cells had surpassed the G2/M transition (34), most irradiated cells were arrested with a G2/M DNA content. Even though the effect was not as pronounced as that of the hRad9-siRNA-transfected cells (Fig. 6A), cells overexpressing S387A and P4A exited G2/M at a reduced rate when compared with cells overexpressing wild-type or S272A hRad9 (Fig. 6B, 13-h time point). Thus, overexpression of hRad9 mutants that cannot be phosphorylated at Ser387 or constitutive (S/T)P sites results in prolonged G2/M arrest following IR, a defect that is also exhibited by cells with reduced hRad9 protein levels.
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DISCUSSION |
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ATM phosphorylates hRad9 at Ser272 in response to ionizing radiation (27). hRad9 also undergoes a second DNA damage-regulated hyperphosphorylation that occurs on an amino acid residue other than Ser272 (Fig. 4C). Whereas both of these events are dependent on DNA damage, they differ significantly in their regulation in a number of ways. Ser272 phosphorylation is response to IR occurs rapidly (27) (Fig. 3A), independently of cell cycle position (27) (Fig. 3A), independently of constitutive phosphorylation at Cdk sites and Ser387 (Fig. 4A), and is not limited when hRad9 is overexpressed (Fig. 4A). Ser272-independent IR-induced phosphorylation, on the other hand occurs less rapidly (Fig. 3B), requires prior phosphorylation at Ser387 (Fig. 4C), and does not occur efficiently when hRad9 is overexpressed.2 The fact that a similar Ser387-dependent hRad9 mobility shift is observed in cells that are exposed to hydroxyurea or synchronized in G2 (Fig. 4C) suggests that this event is also cell cycle-dependent and may be a response to DNA damage detected during S phase. Others have reported that hyperphosphorylation of hRad9 under similar conditions requires the activity of phosphatidylinositol 3-kinase-related kinases (37). Further experimentation will be required to determine whether this modification is the same as the one reported here.
Our results also demonstrate that the constitutive phosphorylation of hRad9 at (S/T)PX(R/P) sites and Ser387 is dispensable for interaction with hHus1 and hRad1 (Fig. 5A). These results are in agreement with a previous report, which demonstrated that constitutive phosphorylation of hRad9 does not influence the stability of the 9-1-1 complex (38). In contrast, a recent report has suggested that hRad9 phosphorylation is important for 9-1-1 stability (39). Analysis of a hRad9 mutant, in which phosphorylation is completely ablated, will be required to fully reconcile these seemingly conflicting results. We have shown, however, that hRad9 interaction with TopBP1 is partially compromised in the P4A mutant and undetectable in the S387A mutant. Since the majority of cellular hRad9 exists as part of the 9-1-1 complex (40), our data are consistent with hRad9 phosphorylation regulating protein interactions with the 9-1-1 complex rather than interactions between 9-1-1 members.
Depletion of hRad9 protein using hRad9-directed siRNA results in a prolonged accumulation of cells in G2/M following irradiation (Fig. 6A). Furthermore, overexpressing hRad9 mutant proteins that cannot be phosphorylated at Ser387 or (S/T)PX(R/P) constitutive sites (P4A) produces a similar, albeit less pronounced, effect (Fig. 6B). Prolonged G2/M accumulation following irradiation is a common phenotype of S phase checkpoint-defective cells, such as those lacking ATM, BRCA1, Nbs1, and Smc1 (41, 42). It has been proposed to be a compensatory mechanism that provides cells that failed to appropriately arrest DNA synthesis extra time to deal with replicated damaged DNA (41). Therefore, S phase checkpoint dysfunction may be a plausible explanation for the cell cycle defects associated with hRad9 protein depletion and S387A or P4A mutant overexpression. The fact that these mutants are also defective in their ability to interact with TopBP1 further suggests that the hRad9/TopBP1 interaction plays a functionally significant role in the S phase checkpoint. This is supported by work in S. cerevisiae, which indicates an important role for the Dpb11/Ddc1 interaction in responding to genotoxins (33). The abrogation of DNA damage-dependent, Ser272-independent phosphorylation of hRad9 (Fig. 4C) may also be a contributing factor to the prolonged G2/M phenotype. Mapping the site(s) of this phosphorylation will be required to fully resolve this possibility.
Finally, the data reported here suggest that hRad9 is regulated in part by
Cdc2, a kinase generally thought to represent the ultimate target of
hRad9-associated signaling pathways. Whereas the maintenance of basal
phosphorylation of the hRad9 C terminus appears to be essential for
hRad9-mediated DNA damage response, the hyperphosphorylation of hRad9 at
Thr292 in mitosis is particularly intriguing and could potentially
represent a means of resetting hRad9 activity for future cell cycles. The fact
that this phosphorylation is exclusively a mitotic event is consistent with
Thr292 being an in vivo substrate of Cdc2. Whereas
Thr292 is the only amino acid in hRad9 with the
(S/T)PX(R/P) consensus sequence that was not phosphorylated by Cdc2
in vitro (Fig.
2B), this is probably due to the dependence of
Thr292 phosphorylation on prior phosphorylation at
Ser277 (Fig. 1, A and
B). The extent to which Ser277,
Ser328, Ser336, and Thr355 are phosphorylated
outside of mitosis suggests that these sites may be regulated by other kinases
in addition to Cdc2. In fact, we have observed that Cdk2 can also
phosphorylate each of these four residues in vitro.2 In
addition, protein kinase C has recently been shown to directly regulate
hRad9 phosphorylation, although the location of protein kinase
C
-mediated phosphorylation in hRad9 remains unclear
(39).
The non-PCNA-like, C-terminal region of hRad9 represents an ideal regulatory domain for the effector functions of the 9-1-1 complex. Its extensive phosphorylation may enable hRad9 and 9-1-1 to coordinate multiple functions during the DNA damage response. The complex nature of hRad9 phosphorylation, however, as illustrated by the large number of phosphorylation sites and the interdependencies between these sites, has made deciphering the precise role of individual phosphates a formidable task. The results presented here advance the current understanding of how hRad9 phosphorylation contributes to checkpoint signaling.
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FOOTNOTES |
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Recipient of a National Cancer Institute of Canada studentship.
** A Cancer Care Ontario Scientist. To whom correspondence should be addressed: Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-6923; Fax: 613-533-6830; E-mail: sd13{at}post.queensu.ca.
1 The abbreviations used are: PCNA, proliferating cell nuclear antigen;
siRNA, small interfering RNA; GST, glutathione S-transferase; HU, hydroxyurea;
IR, ionizing radiation.
2 R. P. St.Onge, B. D. A. Besley, J. L. Pelley, and S. Davey, unpublished
results.
3 Greer, D., Besley, B. D. A., Kennedy, K., and Davey, S. (2003) Cancer
Res., in press.
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ACKNOWLEDGMENTS |
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REFERENCES |
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