From the Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut 06511
Received for publication, November 3, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Chk2 is a protein kinase intermediary in DNA
damage checkpoint pathways. DNA damage induces phosphorylation of Chk2
at multiple sites concomitant with activation. Chk2 phosphorylated at
Thr-68 is found in nuclear foci at sites of DNA damage (1).
We report here that Chk2 phosphorylated at Thr-68 and Thr-26 or
Ser-28 is localized to centrosomes and midbodies in the absence of DNA
damage. In a search for interactions between Chk2 and proteins with
similar subcellular localization patterns, we found that Chk2
coimmunoprecipitates with Polo-like kinase 1, a regulator of chromosome
segregation, mitotic entry, and mitotic exit. Plk1 overexpression
enhances phosphorylation of Chk2 at Thr-68. Plk1 phosphorylates
recombinant Chk2 in vitro. Indirect immunofluorescence (IF)
microscopy revealed the co-localization of Chk2 and Plk1 to centrosomes
in early mitosis and to the midbody in late mitosis. These
findings suggest lateral communication between the DNA damage and
mitotic checkpoints.
Successful completion of the cell division cycle requires that the
genome be duplicated accurately, and apportioned equally to daughter
cells. Defects in these processes cause genome instability and
predispose to cancer. DNA damage is induced by several mechanisms, including exposure to exogenous mutagens and endogenously produced reactive oxygen species. DNA checkpoints actuated by DNA damage or by
stalled replication delay cell cycle transitions, providing time for
DNA repair, and concomitantly promote DNA repair (2).
The protein kinase Chk2 is an important intermediary of vertebrate DNA
damage checkpoint signaling pathways. Chk2 is activated in response to
double-strand DNA breaks induced by ionizing radiation (IR)1 through a mechanism
primarily involving the phosphatidylinositol (3'-) kinase-like protein
kinase (PIKK) ATM (ataxia-telangiectasia-mutated) (3).
Chk2 is also activated in response to other DNA-damaging agents,
including ultraviolet (UV) light, and by interference with DNA
replication, for example by treatment with the ribonucleotide reductase
inhibitor hydroxyurea (HU) (3). Activation of Chk2 by these agents can
occur independent of ATM, probably through the related PIKK ATR (3-5).
The PIKK-dependent phosphorylation of Chk2 in response to
DNA damage is required for its activation. Chk2 transmits the
checkpoint signal to several downstream signaling pathways leading to
an arrest of the cell cycle in G1, S, or G2/M phases (reviewed in Ref. 6). Mouse Chk2 In addition to its importance in the regulation of tumor suppressor
genes TP53 and BRCA1, CHK2 is
apparently a tumor suppressor gene itself. CHK2 is defective
in a subset of families with variant Li-Fraumeni syndrome with
wild type TP53 (12). Males and females heterozygous for CHK2*1100delC have an elevated risk of
breast cancer (13). Reports of CHK2 mutations in sporadic
and familial human cancers are accumulating (14-20).
Chk2 plays a role in maintenance of G2 arrest after
exposure to IR, with Cdc25C being a likely effector in this pathway (3, 7). Since Cdc25C knockout mice have an intact G2 checkpoint arrest (21), it is possible that there are other Chk2 downstream effectors. Genetic studies suggest that the budding yeast Chk2 homolog
Rad53 prevents anaphase entry after checkpoint activation through
regulation of the Polo kinase Cdc5 (22). DNA damage induces
Rad53-dependent phosphorylation of Cdc5 (23). Rad53 and
Cdc5 regulate mitotic exit through independent modifications of Bfa1
(24).
There are three Cdc5-homologous Polo-like kinases (PLKs) in mammals:
Plk1, Snk, and Fnk/Prk/Plk3. Plk1 functions in mitosis and is most
likely to be the mammalian Cdc5 ortholog (reviewed in Ref. 25). Plk1
regulates many aspects of mitosis, including centrosome maturation and
orientation, chromosome adhesion, mitotic entry and exit (reviewed in
Ref. 26). Also, Plk1 is a target of the DNA damage checkpoint and is
inhibited in an ATM-dependent manner after DNA damage (27,
28).
We have examined the localization of phosphorylated and presumably
active forms of Chk2. The results suggest the existence of
unanticipated links between Chk2 and mitotic checkpoint regulation.
Cell Cultures, Transfections, and Treatments--
ATM-deficient
(GM5849C) human fibroblasts were obtained from Coriell Institute for
Medical Research, Camden, NJ. Other cell lines were obtained from
American Type Culture Collection. Cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum in
the presence of antibiotics in a humidified incubator at 37 °C.
Cells were treated with nocodazole (Sigma) (250 ng/ml) for 16 h
and adriamycin (Sigma) (0.5 µM) for 1 h. Cells were
irradiated in a Mark I 137Cs irradiator (Shepard). Cells were
transfected by calcium phosphate precipitation with 5 µg of plasmid
DNA and were analyzed after 40 h.
Antibodies--
Rabbit polyclonal anti-phospho-Thr-68-Chk2
previously described (29) was affinity-purified by phosphopeptide
affinity chromatography. Rabbit polyclonal anti-Chk2 antibody was
produced by immunization with recombinant GST-Chk2 produced in
Escherichia coli. Mouse anti-Plk1 mAb mixture was obtained
from Zymed Laboratories; mouse anti-Chk2 mAb (clone 2CHK01) from
Neomarkers; and affinity-purified rabbit polyclonal
anti-phospho-Thr-26/PSer-28 antibody was a generous gift of Yi Tan
(Cell Signaling Technology). Mouse anti- Plasmids and Recombinant Proteins--
pcDNA-HAChk2 and
pcDNA-HAChk2T68A were previously described (29). Plk1 coding
sequences from an expressed sequence tag (EST) clone
(GenBankTM accession no. BE 900300, obtained from Research
Genetics) were amplified by PCR and cloned into pcDNA.3-3xFLAG
vector, resulting in pcDNA.3-FLAGPlk1. Plk1 deletion mutants were
generated by cloning of PCR-amplified DNA fragments into the
pcDNA.3-3xFLAG vector. Plk1T82A was derived from
pcDNA.3-FLAGPlk1 by PCR-based site-directed mutagenesis.
pGEX2TK-GSTChk2, pGEX2TK-GSTChk2 (D368A), pGEX2TK-Chk2-(1-221), and
His-FLAG-Chk2(D347A) previously described (29) were used for protein
expression in E. coli.
Immunoprecipitation and Immunoblotting--
HEK 293-T cells were
washed in PBS and solubilized in lysis buffer (50 mM
Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 120 mM NaCl) containing a protease inhibitor mixture (Roche Molecular Biochemicals). Cells were collected and centrifuged at 13 000 × g for
10 min at 4 °C. Immunoprecipitation was carried out by incubating
500 µg of protein lysate with 5 µg of anti-Chk2, 1.5 µg of
anti-Plk1, 3 µg of anti-HA, and 10 µl of anti-FLAG affinity agarose
gel (Sigma) at 4 °C overnight. For immunoblot (IB) analysis,
nitrocellulose membranes were blocked in 3% nonfat dry milk in TBST
(0.5% Tween-20, 120 mM NaCl, 50 mM Tris-HCl,
pH 7.5) for 1 h at room temperature, and incubated with either
anti-phospho-Thr-68-Chk2 (1:4000), anti-phospho-Thr-26/Ser-28-Chk2 (1:2000), anti-Plk1 antibody (1:500), mouse anti-Chk2 antibody (1:200),
anti-HA-HRP antibody (1:1000), or anti-FLAG-HRP antibody (1: 2000)
overnight at 4 °C. HRP-conjugated rabbit anti-mouse and goat
anti-rabbit IgG (Pierce)(1:10,000) were used as secondary antibodies.
Immunoblotted proteins were detected by ECLTM
chemiluminescence reagents (Amersham Biosciences).
Immunofluorescence Microscopy--
Cells grown on
poly-D-lysine coated culture slides (BD PharMingen) were
washed in PBS, fixed for 15 min in PBS containing 4 or 0.5%
paraformalaldehyde (PFA), and permeabilized in Triton buffer (0.5%
Triton X-100 in PBS). Fixed cells were blocked in blocking solution
(2% bovine serum albumin, 0.1% Tween, PBS) for 30 min at 37 °C in
a humidified chamber. Immunostaining was performed using
anti-PThr-68-Chk2 antibody (0.5 µg/ml), anti-Plk1 antibody (1: 100),
anti-
For phosphatase treatment, cells were permeabilized with 0.1% Triton
X-100 in PBS for 30 s at room temperature, followed by three
washes with PBS, and one with phosphatase buffer, and then incubated
with 4 units/µl In Vitro Kinase Assays--
Anti-Plk1 antibody (1:500) was
incubated at 4 °C overnight with 500 µg of protein lysate. Plk1
immunoprecipitates were incubated with 10 µg of
For detection of the kinase activity of exogenous FLAG-Plk1 protein,
with phosphospecific antibodies, 300 µg of protein lysate was
incubated with 10 µl of anti-FLAG affinity agarose gel. Anti-FLAG immunoprecipitates were incubated with 2 µg of recombinant
GST-Chk2-(1-221) or His-FLAG-Chk2D347A at 30 °C for 10 min in 20 µl of kinase buffer with 25 µM ATP. The samples were
analyzed by IB with anti-phospho-Thr-68-Chk2 antibody (1:4000).
Characterization of PThr-68-Chk2 Antibody--
Although Chk2
is activated by DNA damage, little is known about regulation of Chk2
during the normal cell cycle. The yeast homolog of Chk2, Rad53, is
required for normal delay of late-firing replication origins, and
timely induction of ribonucleotide reductase for the synthesis of
nucleotide precursors (30, 31). This suggests that mammalian
Chk2 may have important functions in normal cell cycle progression.
Damage-dependent activation of Chk2 is accompanied by the
phosphorylation of a cluster of SQ/TQ sites near the N terminus of Chk2
termed the SCD (32-34). These sites are consensus targets for
phosphorylation by PIKKs, including the Chk2 regulators Atm and Atr.
Phosphorylation of Chk2 at one or more of the SCD sites is required for
Chk2 activation after DNA damage (32-35).
Atm and Atr have different preferences for phosphorylation within the
SCD in vitro (34). The predominant site phosphorylated in
response to double-stranded breaks is Thr-68, which is required for
intact responses to DNA damage (33). ATM is the major Chk2 regulator in
this response, and preferentially phosphorylates Thr-68 in
vitro. In response to replication block or UV exposure, this site
is phosphorylated independently of ATM, probably by ATR (34).
In order to localize forms of Chk2 phosphorylated at this site, we
produced and affinity-purified a phosphospecific antibody,
DNA damage induces phosphorylation of Chk2 at Thr-68 in nuclear foci at
the sites of DNA damage (1). To determine if Phosphorylated Chk2 Is Localized to Centrosomes--
IF of
untreated 293 cells with
Additional controls were performed to verify specificity of the
antibody. IF background was low with control nonspecific IgG as primary
antibody (data not shown). Competition with the oligophosphopeptide used as antigen for production of
Two additional Chk2-reactive antibodies,
In addition to centrosome-associated staining, we detected
phosphorylated Chk2 with HA-Chk2 Localizes to Centrosomes--
Under basal conditions, the
bulk of Chk2 is not phosphorylated, and Chk2 is characterized by a
diffuse nuclear IF pattern. We hypothesized that a minor subset of Chk2
is phosphorylated without induced DNA damage, and it is this subset
that is associated with discrete basal localization. Fixation at
reduced PFA concentrations (1 and 0.5%) and pre-extraction of cells
with 0.1% Triton X-100 permitted visualization of a subpopulation of
overexpressed HA-Chk2 with centrosomal staining pattern and which
colocalizes with Chk2 Interacts with Plk1--
On the basis of localization of Chk2
and Plk1 and suggested communication between their signaling pathways
(22, 24, 37, 38), we determined whether Chk2 and Plk1 physically
interact. HA-tagged Chk2 and FLAG-tagged Plk1 coimmunoprecipitated when transiently expressed in 293T cells (Fig.
4A, lanes 3 and
6), and endogenous Chk2 and Plk1 co-immunoprecipitated from
293T cells (Fig. 4B, lanes 3 and 4). There was
more Plk1 protein in immunoprecipitates from nocodazole-treated cells,
but this may simply reflect the higher expression of Plk1 in mitotic
cells (Fig. 4B, lanes 9 and 10) (reviewed in Ref.
25). The Chk2/Plk1 interaction was not affected by Adriamycin
(doxorubicin)-induced DNA damage (Fig. 4B, lane 4). However,
Adriamycin induced the Chk2 mobility shift associated with
phosphorylation (Fig. 4B, lanes 2 and 4), and inhibited in vitro Plk1 kinase activity toward Plk1 Overexpression Induces Chk2 Phosphorylation--
To
understand further the functional interactions between Chk2 and Plk1,
we co-expressed HA-Chk2 and FLAG-Plk1 in 293T cells and analyzed the
cell lysates by IB with Plk1 Phosphorylates Chk2 in Vitro--
Immune complexes containing
FLAG-Plk1 transiently expressed in 293T cells phosphorylated
recombinant His-FLAG-Chk2-KD and GST-Chk2-(1-221) in vitro
(Fig. 6). This was due to Plk1 activity in the immune complexes, since FLAG-Plk1-KD was not associated with
kinase activity toward recombinant Chk2 (Fig. 6).
Chk2 and Plk1 Co-localize to Centrosomes and the Midbody--
Plk1
is associated with the kinetochores of condensed chromosomes, the
centrosome in prophase and metaphase, and the midbody, later in mitosis
(43, 44). Early in mitosis (prophase and metaphase) Plk1 and
PThr-68-Chk2 were apparently associated with the centrosomes (Fig.
7, A and B). Later
in mitosis, PThr-68-Chk2 was detected at the centrosomes and the
midbody, whereas the Plk1 signal was localized only to the midbody
(Fig. 7C). This suggests that Plk1 is not required to
maintain centrosomal localization of Chk2.
We found hat subpopulations of Chk2, phosphorylated at sites
required for DNA damage responses, co-localize with centrosomes and
midbodies. The results are surprising, because numerous studies have
implicated Chk2 and its yeast homologs in DNA damage responses and DNA
replication checkpoints, but Chk2 has not been directly linked to
activity of checkpoints that detect assembly of the mitotic spindle.
Potential activities of Chk2 at these sites include normal or
checkpoint regulation of the centrosome cycle and/or cytokinesis.
Alternatively, phospho-Chk2 could provide a lateral connection between
these processes and DNA damage and replication inhibition responses.
In the absence of DNA damage, most Chk2 molecules are not
phosphorylated. Activation of Chk2 by DNA damage or inhibition of replication coincides with Chk2 phosphorylation and requires Chk2 kinases Atm or Atr. Mutation of the phosphorylation site at Thr-68 impairs the regulation of Chk2 by DNA damage. The correlation of Chk2
phosphorylation at sites within its SCD and its activation suggests
that the Chk2 phosphospecific antibodies used here mark functionally
active forms of Chk2.
ATM and ATR phosphorylate different sites in Chk2 SCD with overlapping
but distinct substrate specificity in vitro (34). The
presence of phosphorylated Chk2 at the mitotic structures that we
observed in AT cells indicates that there might be other kinases
involved, presumably including ATR. Centrosomes and the midbody are
sites of localization of non-PIKK protein kinases that might
phosphorylate Chk2. Although the Chk2 SQ/TQ sites monitored in these
experiments are sites for PIKK-dependent phosphorylation in
the DNA damage response, these same sites may also be targets for
PIKK-independent phosphorylation. Chk2 expressed in E. coli can transphosphorylate kinase-defective Chk2 at Thr-68 (29) and Thr-26
or Ser-28 (data not shown). The finding that at least one conventional
(non-PIKK) Ser/Thr kinase, Chk2, can phosphorylate this site suggests
that, in centrosomes and other loci with phospho-Chk2, other protein
kinases may be responsible for Chk2 phosphorylation. Recently it was
reported that another non-PIKK, Plk3 can phosphorylate the SCD of Chk2
in vitro and can contribute to its full activation (45). In
yeast and mammals, high local concentrations of Chk2 may enable
Chk2-Chk2 transphosphorylation, which may, in turn, be sufficient to
activate Chk2. In the DNA damage response, localization of Chk2 to
sites of DNA damage may be mediated by the FHA domain which itself
recognizes phosphopeptides. Recruitment of Chk2 to centrosomes could be
mediated through binding of the FHA domain of Chk2 to phosphoproteins
located there. This may deliver Chk2 in close proximity to activating
kinases at these sites. Non-PIKK Ser/Thr kinases localized to
centrosomes include Aurora family kinases (reviewed in Ref. 46), and at
some phases of the cell cycle, spindle checkpoint kinases Mps1, Bub1,
and BubR1 (reviewed in Ref. 47). Cell cycle-dependent
localization of PLKs to the centrosomes, kinetochores, and midbody has
been reported (43, 44).
Proper regulation of centrosome duplication and maturation is vital for
maintenance of a bipolar mitotic spindle. Aberrant centrosome and
spindle numbers are common hallmarks of aggressive cancers. Mutations
in genes associated with DNA damage responses, including p53, have been
implicated in aberrant partitioning of centrosomes after cell division,
and genomic instability. Cells lacking p53 or with mutated p53 have
abnormal numbers of centrosomes (48). Centrosome amplification in tumor
and cell lines is linked to mutations in p53 (48), abnormal expression
of MDM2 (49), and p21CIP1 (50). The mechanisms for these changes are
not completely understood, and may reflect direct regulation of
centrosome maturation and stability through these pathways, or indirect
effects on survival of changes resulting in multinucleation and
polyploidy (51).
A second Chk2 substrate, Brca1, partly co-localizes with Chk2 in
untreated cells (11), and displays centrosomal localization (52). Brca1
associates with the centrosome in mitotic cells and interacts with
The localization of Chk2 to components of the mitotic apparatus and its
physical association with Plk1 suggest that Chk2 functions in the
regulation of mitosis. In support of this, we observed the
Plk1-dependent phosphorylation of several sites within the SCD of Chk2. Plk1 is expressed in S, G2, and M phases of
cell cycle and undergoes phosphorylation-dependent
activation in G2 and M, with a peak in its activity in
mitosis (39). We speculate that Chk2 phosphorylation occurs locally at
the sites of the interaction between Plk1 and Chk2. These sites,
according to our results from colocalization experiments, could be the
centrosomes and midbody, although the possibility of Chk2-Plk1
interaction elsewhere cannot be ruled out. Another Polo-like kinase
Plk3, phosphorylates Chk2 after IR (45). The effect of IR on Plk1 and
Plk3 activities is diametrically opposite; Plk1 is inhibited while Plk3
is activated. Chk2 regulation by Plk1 is probably a part of a different
mechanism because we observed that Plk1 phosphorylated Chk2 without
exposure of the cells to DNA damaging agents. Also, the interaction
between Plk1 and Chk2 is not regulated by exposure of the cells to
Adriamycin, a DNA damaging agent. Overexpression of wild type Plk1,
kinase-defective Plk1, and an N-terminal-deleted Plk1-(330-CT), caused
the accumulation of transfected cells in G2/M and later a
mitotic catastrophe and apoptosis, which could activate DNA damage
checkpoint as a secondary event (36, 38, 39). This is unlikely to be
related to Chk2 regulation since we found that Chk2 phosphorylation
correlates with the overexpression of Plk1 mutant protein with normal
or elevated kinase activity but not with the overexpression of Plk1 proteins causing G2/M arrest. Definition of the
circumstances in which Plk1 phosphorylates Chk2 may reveal new Chk2
functions in the normal cell cycle, or in cross-talk between mitotic
regulation and DNA damage checkpoints.
Vertebrate and budding yeast Chk2 are important in the DNA
damage-dependent arrest of mitotic entry and exit. However,
Chk2 and Rad53 are not, apparently, regulated through or required for the mitotic spindle checkpoint (redundant functions have not been entirely ruled out). Moreover, unlike Plk1, it is not clear that Chk2
functions are required in a normal cell cycle, rather than being called
into play only after genotoxic damage. In human testes, Chk2 protein is
more abundant in spermatogonia (mitotic division), than in
spermatocytes (meiotic division), indicating a possible mitotic role
for Chk2 (55).
Plk-1 is a multifunctional mitotic protein kinase involved in many
mitotic events, including centrosome maturation and formation of the
mitotic spindle (56). Plk1 regulates mitotic entry by promoting
translocation of cyclin B1 to the nucleus (57) and by inhibiting export
of Cdc25C from the nucleus (58). Plx may also directly enhance Cdc25
catalytic activity (59, 60). In contrast to Plk1, Chk2 is a negative
regulator of Cdc25C. DNA damage-activated Chk2 inhibits Cdc25C activity
through phosphorylation at a 14-3-3 binding site (3) that leads to
preferential accumulation of Cdc25C in the cytoplasm (61-63). Close
proximity of Chk2 and Plk1 suggests a possibility for mutual regulation
and regulation of other Cdc2 regulators, such as Myt1, Mik1, and Wee1.
At anaphase, phosphorylation of yeast Cdc5 and other PLKs are involved
in regulation of chromosome cohesion, and both entry and exit from
mitosis. In Xenopus, Plx1 promotes anaphase progression
through phosphorylation of APC/C and cohesin (64, 65).
CDC5 inhibits Rad53 in the process of adaptation to
unrepairable DNA damage (37, 38), and was implicated as a possible Rad53 effector (22). Recent work suggests that Cdc5 and Rad53 antagonistically regulate mitotic exit pathways through the common regulatory target Bfa1 (24). It is noteworthy that Bfa1 is located at
spindle pole bodies, where it regulates activity of TEM1, the limiting
regulator of CDK inactivation required for mitotic egress. Perhaps
localization of Chk2 to centrosomes and kinetochores (data not shown) reflects interaction with a similar mitotic exit complex.
Chk2 is an intermediary in pathways regulating DNA damage responses and
checkpoint arrest. The unexpected localization of phosphorylated forms
of Chk2 to centrosomes and midbodies; physical interaction and
colocalization with Plk1; and phosphorylation by Plk1 suggest hitherto
unexplored connections of these DNA damage checkpoint pathways with
chromosome mechanics. Since centrosome and spindle aberrations are
common in tumor cells with dysfunctional checkpoint pathways, these
findings shed new light on the basis of genomic instability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
ES cells are defective in
maintenance of G2 arrest and stabilization of p53 for
initiation of G1 arrest (7). In response to IR, Chk2
phosphorylates p53 at Ser-20 and interferes with p53 binding to MDM2,
thereby contributing to p53 stabilization and G1 arrest (8,
9). If DNA damage occurs in S phase, Chk2 phosphorylates Cdc25A and
targets it for degradation (10). Chk2 and the protein kinase Chk1
regulate the G2/M transition through phosphorylation of
their common target Cdc25C at Ser-216 (3). Checkpoint-induced
phosphorylation of Brca1 at Ser-988 by Chk2 also plays a role in cell
survival after DNA damage (11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin mAb (clone GTU-88)
and mouse anti-FLAG affinity agarose gel were obtained from
Sigma; mouse anti-hemagglutinin (HA) mAb (16B12) from BabCo/Covance;
horseradish peroxidase (HRP)-conjugated rat anti-HA mAb (3F10) and rat
anti-HA mAb (3F10) from Roche Molecular Biochemicals; and
HRP-conjugated secondary antibodies were obtained from Pierce.
-tubulin antibody (1:1000), anti-PThr-26/PSer-28-Chk2 antibody
(1:300), mouse anti-Chk2 antibody (1:50), mouse anti-HA mAb (16B12)
(1:300), or rat anti-HA mAb (3F10) (1:300) for 30 min at 37 °C in a
humidified chamber, followed by three washes in blocking buffer. Cells
were incubated with anti-mouse Rhodamine (Rd) (1:1000), anti-mouse
fluorescein isothiocyanate (FITC) (1:100), anti-rabbit-FITC (1:100)
secondary antibodies. DNA was stained with 6'-diamidino-2-phenylindole
(DAPI) in mounting solution (Vector Laboratories). IF microscopy was
performed using a Nikon Microphot-FX microscope using ×40 and ×60
Plan Apo objectives. Images were captured with a Spot digital camera
(Diagnostic Instruments Inc.) and processed using Adobe Photoshop.
-phosphatase (New England Biolabs) for 20 min at
30 °C. After treatment, cells were washed in PBS and fixed as in the
basic protocol.
-casein at
30 °C for 5 min in 20 µl of kinase buffer (20 mM HEPES
pH 7.4, 50 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, and 1 µM ATP) supplemented
with 5 µCi of [
-32P]ATP (>5000 Ci/mmol, AA0018,
Amersham Biosciences). The reactions were stopped with the addition of
20 µl of 2× Laemmli gel electrophoresis sample buffer. The samples
were separated by SDS-PAGE, and the gel was stained with Coomassie
Brilliant Blue, dried, and visualized by autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-PThr-68-Chk2, raised against a phosphopeptide containing the region
surrounding phospho-Thr-68. In lysates from irradiated 293T cells, but
not-mock-irradiated cells,
-PThr-68-Chk2 recognized endogenous Chk2
(lower band, Fig.
1A, lanes 2 and
4) and transiently expressed HA-Chk2 (upper band,
Fig. 1A, lane 2). However, this antibody did not recognize
transiently expressed HA-Chk2T68A, in which the target phosphorylation
site has been replaced with A (Fig. 1A,
lane 4). In addition, the
-PThr-68-Chk2 antibody recognized
endogenous Chk2 as a single band that increased in intensity with
escalating radiation dose, while
-Chk2 antibody detected
approximately equal amounts of Chk2 protein in all samples (Fig.
1B). Recombinant Chk2 undergoes autophosphorylation at
multiple sites when expressed in bacteria (29).
-PThr-68-Chk2
recognized GST-Chk2-WT, but not GST-Chk2D368A, which has a mutation
that eliminates kinase catalytic activity (Fig. 1C).
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Fig. 1.
Characterization of -PThr-68-Chk2
antibody. A, extracts from HEK 293T cells transiently
expressing HA-Chk2-wild type (lanes 1 and 2) or
HA-Chk2T68A (lanes 3 and 4) were mock-irradiated
(lanes 1 and 3) or irradiated with 5 Gy
(lanes 2 and 4) and immunoblotted with
-PThr-68-Chk2,
Chk2, or
HA antibodies. B, IB of
extracts from HEK 293T cells exposed to indicated doses of IR analyzed
by immunoblotting with
-PThr-68-Chk2, or
-Chk2. C, IB
of purified recombinant GST-Chk2-WT and kinase-defective GST-Chk2 D368A
fusion proteins, produced in bacteria, and immunoblotted with
-PThr-68-Chk2 or
-Chk2. D, IF analysis of
non-irradiated (0 Gy) and irradiated (4 Gy) HEK-293, HT-1080, and
GM5849C cells with
-PThr-68-Chk2 antibody. Bar is 10 µm
long.
-PThr-68-Chk2 can
detect these foci, we immunostained irradiated 293T, HT-1080 and
GM5849C cells. Immunoreactive foci were formed following exposure to IR
in 293T and HT-1080 cells, but not in GM5849C cells, which lack
functional ATM (Fig. 1D).
-PThr-68-Chk2 antibody produced a staining
pattern characteristic of proteins associated with centrosomes (Fig.
2A). Signals generally
consisted of single or paired nuclear dots in interphase cells,
separated dots flanking condensed chromatin in metaphase, and single
dots adjacent to chromatin in telophase (Fig. 2A). Similar
staining patterns with
-PThr-68-Chk2 were observed with three
additional cell lines: WI38, HT-1080, U2OS, and GM5849C (AT cells)
(data not shown). The signal is ATM-independent since it was similar in
ATM-deficient GM5849C cells.
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[in a new window]
Fig. 2.
Centrosomal localization of phosphorylated
Chk2. A, IF analysis of HEK-293 cells with -PThr-68-Chk2
antibody detected with FITC. DAPI staining marks nuclei. B,
IF analysis of U2-OS cells with
-PThr-68-Chk2 (FITC) and
-tubulin (rhodamine). C, centrosome and midbody
localization of phosphorylated Chk2. IF of U2-OS cells with
-PThr-68-Chk2 (red) and
-PThr-26/PSer-28-Chk2
(green) antibodies, DAPI (blue). D,
phosphopeptide immunogen used to elicit
-PThr-68-Chk2 antibody was
used for competition with the antigen in IF analysis of U2-OS cells
with
-PThr-68-Chk2 and
-tubulin antibodies. E,
double IF of U2-OS cells with
-PThr-68-Chk2 and
-tubulin
antibodies after in situ permeabilization and treatment with
4 units/µl
-phosphatase or buffer for 30 min at 25 °C.
Bars are 10 µm long.
-PThr-68-Chk2 eliminated the IF
signal (Fig. 2D).
-tubulin nucleates microtubule assembly and is concentrated at centrosomes (36). To confirm the localization of
Chk2 to the centrosome, we performed double IF using
-PThr-68-Chk2 and
-tubulin antibodies in U2-OS cells (Fig. 2B). The
PThr-68-Chk2 and
-tubulin fluorescence signals overlapped at the
strong, centrosome-like foci seen with
-PThr-68-Chk2, providing
direct evidence for centrosomal localization of phosphorylated Chk2
(Fig. 2, B and D). However, in contrast to
PThr-68-Chk2 signals, the
-tubulin signal was not competed with the
PThr-68-containing phosphopeptide (Fig. 2D). In order to
verify that the
-PThr-68-Chk2 signal is a result of Chk2
phosphorylation, rather than high local concentration of Chk2 or other
artifact, we treated permeabilized U2-OS cells with
-phosphatase.
Phosphatase treatment eliminated the PThr-68-Chk2 signal, but the
-tubulin signal remained although slightly reduced (Fig.
2E). Incubation with buffer did not change either
-tubulin or PThr-68-Chk2 signals (data not shown).
Chk2, which should
recognize all forms of Chk2, and
-PThr-26/PSer-28-Chk2, were tested.
Anti-PThr-26/PSer-28-Chk2 should recognize additional Chk2 regulatory
sites in the SCD (34). Although S28 does not seem to be a good
substrate for either ATM or ATR, Thr-26 is evidently a good ATR
substrate in vitro (34). Treatment of 293 and U2OS cells
with IR, UV, or hydroxyurea (HU)-induced reactivity of exogenously expressed HA-Chk2 with
-PThr-26/PSer-28-Chk2 antibody analyzed by IB
(data not shown). The antibody detects endogenous Chk2 after exposure
to IR and doxorubicin, but not in untreated cells (data not shown).
Finally,
-PThr-26/PSer-28-Chk2 antibody recognizes wild-type HA-Chk2
but not HA-Chk2T26A/S28A with amino acid substitutions at the
immunogenic sites (data not shown). IF with
-PThr-26/PSer-28-Chk2 in
U2OS cells yielded a centrosome-like pattern (Fig. 2C). In contrast,
Chk2 detected a diffuse nuclear and cytoplasmic
localization that overlapped with focal
-PThr-68-Chk2 fluorescence
at centrosomes (Fig.
3A).
View larger version (38K):
[in a new window]
Fig. 3.
Localization of Chk2 to the centrosome.
A, dual IF of U2-OS cells with -PThr-68-Chk2 and
Chk2
antibodies. Arrowheads mark coincident staining between
-PThr-68-Chk2 and a subset the
Chk2 signal. B, dual IF
of 293T cells transiently expressing HA-Chk2 with
-tubulin
(red) and
HA (green) antibodies after mild
fixation (0.5% PFA) and Triton extraction. DAPI (blue)
staining marks nuclei. C, co-IF of 293T cells transiently
expressing HA-Chk2 with
-PThr-68-Chk2 (green) and
HA
(red) antibodies after mild fixation (0.5% PFA) and Triton
extraction. Bars are 10 µm long.
-PThr-68-Chk2 and
-PThr-26/PSer-28-Chk2 antibodies at the midbody, the central part of the cytokinetic bridge,
in telophase (Fig. 2C).
-tubulin (Fig. 3B). Under these
conditions,
HA and
-PThr-68-Chk2 signals overlapped, including at
the centrosomes (Fig. 3C). Staining for HA-Chk2 with two
different
HA antibodies yielded the same localization pattern
(data not shown).
-casein
(Fig. 4B, line 10).
View larger version (25K):
[in a new window]
Fig. 4.
Chk2 interacts with Plk1. A,
co-immunoprecipitation of HA-Chk2 and FLAG-Plk1. Whole cell extracts
from HEK 293T cells transiently expressing HA-Chk2 (lanes
1, 2, 3, 5, and 6)
and/or FLAG-Plk1 (lanes 2-6) were immunoprecipitated with
mouse IgG (lanes 2 and 5), FLAG antibody
(lanes 1 and 3), or
HA antibody (lanes
4 and 6). Western blot analysis of immunoprecipitates
was performed with
FLAG (lower panel) and
-HA
antibodies (upper panel). B, co-IP of endogenous
Chk2 and Plk1. HEK 293-T cells were treated with 0 (lanes
1, 2, 7, and 8) or 250 ng/ml
nocadazole (lanes 3, 4, 5,
6, 9, and 10) for 16 h and with 0 (lanes 1, 3, 5, 6,
7, and 9) or 0.5 µM (lanes
2, 4, 8, and 10) Adriamycin for
1 h. Extracts from the cells were IP with preimmune serum
(lane 5), mouse IgG (lane 6),
Chk2 antibody
(lanes 1-4), and
Plk1 antibody (lanes 7-10).
Western blot analyses were performed to detect Chk2 (lower left
panel) and Plk1 (upper left and upper right
panels) in the immunoprecipitates. Portions of Plk1
immunoprecipitates were used for in vitro kinase assays.
Kinase activity of Plk1 was detected by incorporation of
[
-32P]ATP into
-casein, used as a substrate
(middle right). Amounts of Plk1 used for in vitro
kinase assay were monitored by IB analysis of Plk1 immunoprecipitates
(upper right).
-Casein was detected by Coomassie
Brilliant Blue staining (lower right).
-PThr-68-Chk2 and
-PThr-26/PSer-28-Chk2 (Fig. 5B). Co-expression of
HA-Chk2 and FLAG-Plk1 promoted phosphorylation of least at 2 sites
(Thr-26 and/or Ser-28 and Thr-68) in the SCD of HA-Chk2 (Fig.
5B). We next expressed full-length and truncated forms of
FLAG-Plk1 in 293-T cells. FLAG-Plk1-(1-330), FLAG-Plk1-(1-408), and
FLAG-Plk1-(1-480) have deletions in the C terminus, that enhance their
kinase activity (39-42) (Fig. 5A, lanes 4-6). Two other
proteins, FLAG-Plk1(K82A), a kinase defective (KD) mutant and
FLAG-Plk1(330-CT), with a deleted kinase domain, have no apparent
kinase activity (Fig. 5A, lanes 3 and
7) (39-42). Fluorescence-activated sorter analysis of 293T
cells co-expressing green fluorescence protein (GFP) and FLAG-Plk1 wild
type, FLAG-Plk1-KD and Plk1-(330-CT), showed accumulation of 34, 37, and 56% of transfected cells in G2/M in comparison to 19%
of cells expressing GFP only (data not shown). However, expression of
FLAG-Plk1-(1-330), FLAG-Plk1-(1-408), and FLAG-Plk1-(1-480) had no
effect on cell cycle distribution of transfected cells, consistent with
previous reports (39-42). Expressed FLAG-Plk1 wild type,
FLAG-Plk1-(1-330), FLAG-Plk1-(1-408), and FLAG-Plk1-(1-480), which
have normal or enhanced kinase activity, increased phosphorylation of
endogenous Chk2 at Thr-68 (Fig. 5A, lines
2, 4, 5, and 6). In contrast,
phosphorylation of Chk2 Thr-68 was unchanged in the cells with
expressed FLAG-Plk1-KD or FLAG-Plk1-(330-CT), which have no associated
kinase activity (Fig. 5A, lanes 3 and 7). The correlation of Thr-68 phosphorylation with the
kinase activity of FLAG-Plk1 mutants, but not with G2/M
cell arrest, indicates that Thr-68 phosphorylation is dependent upon
FLAG-Plk1 kinase activity.
View larger version (46K):
[in a new window]
Fig. 5.
Plk1 overexpression induces Chk2
phosphorylation. A, lysates from 293T vector-transfected
cells (lane 1) or cells transiently expressing wild type
FLAG-Plk1 (lane 2), FLAG-Plk1-KD (lane 3),
FLAG-Plk1-(1-330) (lane 4), FLAG-Plk1-(1-480) (lane
5), FLAG-Plk1-(1-408) (lane 6), and FLAG-Plk1 (330-CT)
were analyzed by IB with -PThr-68-Chk2,
Chk2, and
FLAG
antibodies. The kinase activity of the exogenous Plk1 was measured by
in vitro kinase assay with
FLAG immunoprecipitates from
the same lysates and 10 µg of
-casein substrate in presence of
[
-32P]ATP.
-Casein was detected by Coomassie
Brilliant Blue staining. B, lysates from 293T cells
transiently expressing HA-Chk2 only or HA-Chk2 and FLAG-Plk1 were
analyzed by IB with
-PThr-68-Chk2,
-PThr-26/PSer-28-Chk2,
HA,
and
FLAG antibodies.
View larger version (32K):
[in a new window]
Fig. 6.
Plk1 phosphorylates Chk2 in vitro.
A, FLAG immunoprecipitates from lysates from 293T
vector-transfected cells or cells transiently expressing FLAG-Plk1 or
FLAG-Plk1-KD 293T cells were incubated with 2 µg of recombinant
GST-Chk2-(1-221) (A) or His-FLAG-Chk2D347A (B)
under kinase assay conditions with ATP. Proteins from the reactions
were separated by SDS-PAGE, and immunoblotted with
-PThr-68-Chk2
antibody for detection of phosphorylation at Thr-68 Chk2,
FLAG
antibody for FLAG-Plk1 and His-FLAG-Chk2D347A, and
Chk2 antibody for
GST-Chk2-(1-221).
View larger version (48K):
[in a new window]
Fig. 7.
Co-localization of Chk2 and Plk1 to the
centrosome and the midbody. Co-immunostaining of U2-OS cells with
-PThr-68-Chk2 (green) and
Plk1 (red)
antibodies. Signals of
-PThr-68-Chk2 and
Plk1 antibodies overlap
at the centrosome in prophase (A) and metaphase
(B) cells and at the midbody later in mitosis
(C). DAPI staining marks the location of DNA.
Bars are 10 µm long.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin. It is interesting that the Brca1 fraction interacting with
-tubulin is hypophosphorylated (52). Brca1 disassociation from
centrosomes could be phosphorylation dependent and mediated by
Chk2 phosphorylation. Mutations in the DNA repair protein Brca2 (53) or
overexpression of the Chk2 kinase ATR (54) also affect centrosome number.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Marc Schwartz, Jonathan McMenamin-Balano, Soo-Jung Lee, and other members of the Stern laboratory for helpful comments. We are grateful to J. Rose for providing the IF microscope.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Army Medical Research and Materiel Command (USAMRMC) DAMD 17-98-1-8272, U. S. Public Health Service (USPHS) R01CA82257, Anna Fuller fellowship in molecular oncology (to L. T.), Susan G. Komen Breast Cancer Foundation Fellowship PDF2000 719 (to L. T.), and USAMRMC DAMD 17-01-1-0464 (to L. T.), USAMRMC DAMD 17-01-1-0465 (to X. X.).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 USAMRMC Predoctoral Training Program in Breast Cancer
Research DAMD 17-99-1-9461.
§ To whom correspondence should be addressed: Dept. of Pathology, School of Medicine, Yale University, 310 Cedar St., BML 342, New Haven, CT 06511. Tel.: 203-785-4832; Fax: 203-785-7467; E-mail: Df.stern@yale.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M211202200
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ABBREVIATIONS |
---|
The abbreviations used are: IR, ionizing radiation; IF, immunofluorescence; PIKK, phosphatidylinositol (3') kinase-like kinase; ATM, ataxia-telangiectasia mutated; HU, hydroxyurea; SCD, SQ/TQ cluster domain; PLKs, Polo-like kinases; GST, glutathione S-transferase; HA, hemagglutinin; IB, immunoblot; PFA, paraformaldehyde; FITC, fluorescein isothiocyanate; DAPI, 6'-diamidino-2-phenylindole; GFP, green fluorescent protein; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; PThr, phospho-Thr; PS, phospho-Ser; Gy, Gray; mAb, monoclonal antibody.
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