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INTRODUCTION |
The maintenance of genomic integrity following DNA damage depends
on the coordination of DNA repair and the control of cell cycle
progression.
Chk21/hcds1, a
mammalian homolog of the Saccharomyces cerevisiae
rad53 and Schizosaccharomyces pombe
cds1 genes, plays a critical role in DNA damage
signaling pathways (1-5). Downstream of ATM in response to gamma
radiation (1, 4, 6), Chk2 directly phosphorylates and regulates the
functions of p53 and BRCA1 (7-9, 11). Moreover, heterozygous germline
mutations in Chk2 have been identified in a subset of
patients with Li-Fraumeni syndrome, a highly penetrant familial cancer
phenotype (12). These studies strongly suggest that Chk2 is
a tumor suppressor gene similar to p53.
Several mutations of Chk2 were identified in patients with Li-Fraumeni
syndrome and in sporadic colon cancer. Although it has been speculated
that these Chk2 mutants are defective in their tumor suppressor
functions (12), this possibility has not been addressed directly. Here,
we report the biochemical characterization of the four reported Chk2 mutations.
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MATERIALS AND METHODS |
Constructs--
Dr. Jann Sarkaria kindly provided plasmid for
the expression of HA-tagged Chk2 in mammalian cells (13). Site-directed
mutagenesis (Promega) was performed to introduce mutations into the
Chk2 coding sequence. For expressing wild-type or mutant
Chk2 as glutathione S-transferase (GST) fusion proteins in
insect cells, wild-type or mutant Chk2 coding sequences were
cloned into the pDONR201 vector (Life Technologies, Inc.). Gateway
cloning technology (Life Technologies, Inc.) was used to subclone these
coding sequences into pDEST20 vector, a vector for baculovirus
expression of GST fusion proteins. Recombinant baculoviruses encoding
GST-fused wild-type and mutants of Chk2 were generated using
Bac-to-Bac baculovirus system (Life Technologies, Inc.).
Cell Lines and Culture Conditions--
All cell lines were
obtained from American Tissue Culture Collection and cultivated in RPMI
1640 (Biofluids) supplemented with 10% fetal bovine serum. To
establish cell lines stably expressing HA-tagged wild-type or mutant
Chk2, HCT116 cells were transfected with plasmids encoding the
indicated HA-tagged sequences. G418 resistant clones were isolated and
analyzed by Western blotting using either anti-HA antibody (Babco) or
anti-Chk2 antibody. Clones that express HA-tagged Chk2 at levels
similar to that of endogenous Chk2 were used in this study. Where
indicated, cells were exposed to gamma radiation from a
137Cs source at a dose of 6.4 gray/min. Following
irradiation, cells were returned to the incubator and harvested 1 h later.
Sf9 insect cells were cultivated in Grace's insect media
supplemented with 10% fetal bovine serum. For protein expression, Sf9 cells were infected with baculoviruses encoding GST-fused wild-type or mutant Chk2. Cells were collected and lysed 48 h after viral infection. Wild-type or mutant Chk2 was purified using glutathione affinity chromatography.
Immunoprecipitation, Immunoblotting, and Kinase
Assays--
Preparation of cell lysates, immunoprecipitation, and
immunoblotting were performed as described previously (14). Antibodies against Chk2 were raised against GST fusion proteins containing full-length Chk2 (mAB no.7) or the C terminus of Chk2 (residues 193-543, anti-Chk2B). Anti-Chk2 Thr-68 phosphospecific antibodies were
provided by Dr. Bin-Bing Zhou. Chk2 kinase assays were performed as
described previously (13).
Size Fractionation of Native Chk2 Complexes--
HCT116 and
derivative cells were harvested and lysed in NETN buffer (150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.05% Nonidet P-40). Whole cell extracts were
loaded onto a Superdex 200 HR 10/30 (Amersham Pharmacia Biotech) column
equilibrated with NETN and run in the same buffer with a flow rate of
0.5 ml/min. For each run, a sample of 500 µl was injected, and
500-µl fractions were collected. For column equilibration, low and
high molecular weight gel filtration calibration kits (Amersham
Pharmacia Biotech) were used, and the column was run under identical conditions.
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RESULTS |
Frameshift Mutations at the C Terminus of Chk2 Lead to the Loss of
Chk2 Kinase Activity--
Because Chk2 is a DNA damage-activated
protein kinase that participates in the phosphorylation of several
substrates including Cdc25C, p53, and BRCA1, we first examined the
kinase activity of Chk2 mutants. Using site-directed mutagenesis, four
Chk2 mutants were generated (Fig. 1) that
had been previously reported (12). Wild-type and mutant GST·Chk2
proteins were expressed in insect cells and purified using
glutathione-Sepharose beads, and kinase activities were assessed using
GST·Cdc25C (residues 200-256) as a substrate. As shown in Fig.
2, one FHA domain mutant (I157T) exhibited wild-type activity whereas the other FHA domain mutant (R145W) showed reduced catalytic activity, and the two frameshift mutants lacked kinase activity.

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Fig. 1.
Schematic diagram of Chk2 and its
mutants. S/TQ-rich, FHA, and
kinase domains are indicated, and corresponding Chk2
residues are labeled. Black boxes indicate unrelated protein
sequences caused by frameshift mutations.
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Fig. 2.
In vitro kinase activities of wild-type or
mutant Chk2. Wild-type or mutant GST·Chk2 proteins were
expressed and purified from insect cells. Upper, Coomassie
Blue-stained gel indicating amounts of GST·Chk2 proteins used.
Middle, autoradiograph showing the incorporation of
32P into the substrate GST·Cdc25C by input
kinases from the upper panel. Lower,
Coomassie blue-stained gel (the same gel as shown in the middle
panel) indicating equal levels of substrate in each kinase
reaction.
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The R145W Mutant of Chk2 Is Not Phosphorylated or Activated
Following Gamma Radiation--
Chk2 is activated following DNA damage
(1). Although Chk2 FHA domain mutants (R145W and I157T) retain some
kinase activity (Fig. 2), they may not be activated by DNA damage. To
explore this possibility, we have established HCT116 derivative cell
lines that stably express comparable levels of HA epitope-tagged
wild-type or mutant Chk2 (Fig.
3a). The expression levels of
HA-tagged Chk2 in these cells are similar to that of endogenous Chk2
(Fig. 3a). Like wild-type Chk2, the I157T Chk2 mutant was
activated following gamma radiation, as demonstrated by its ability to
autophosphorylate and to phosphorylate Cdc25C (Fig. 3b).
However, the R145W Chk2 mutant was not activated following DNA damage
(Fig. 3b).

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Fig. 3.
R145W mutant of Chk2 is not activated
following gamma radiation. a, HCT116 derivative cell
lines that stably express HA-tagged wild-type or mutant Chk2. Whole
cell extracts were prepared from indicated cell lines, and immunoblots
were probed with anti-Chk2 mAb or with anti-HA mAb HA11. b,
R145W mutant is not activated following gamma radiation. Extracts were
prepared from indicated cell lines before and 1 h after gamma
radiation. HA-tagged wild-type or mutant Chk2 were immunoprecipitated
using anti-HA antibodies, and kinase reactions were performed using
GST·Cdc25C as substrate. c, Thr-68 is not phosphorylated
in R145W mutant following gamma radiation. Extracts were prepared as
described above. Immunoprecipitates with anti-Chk2 or anti-HA
antibodies were immunoblotted with anti-phospho-Thr-68 antibodies and
anti-Chk2 or anti-HA antibodies.
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The increase of Chk2 kinase activity is coincident with its
phosphorylation following gamma radiation (1). Both activation and
phosphorylation of Chk2 depend on intact ATM kinase, strongly suggesting that ATM may phosphorylate Chk2 and activate its kinase activity following DNA damage (1). In vivo, Thr-68 of Chk2 is phosphorylated in an ATM-dependent manner following
gamma radiation (6). Thus, we examined whether the Chk2 mutants were
phosphorylated at Thr-68 following gamma radiation. Cells expressing
either wild-type or mutant HA-tagged Chk2 were irradiated. Wild-type
and mutant Chk2 were immunoprecipitated using either anti-Chk2 or
anti-HA antibody. Phosphorylation of Chk2 at Thr-68 was detected by
Western blotting using anti-Thr-68 phosphospecific antibody. In
agreement with its activation following gamma radiation, ectopically
expressed HA·Chk2 was phosphorylated at Thr-68 (Fig. 3c).
Moreover, consistent with the above findings that HA·Chk2 containing
the I157T mutation but not the R145W mutation can be activated by DNA
damage (Fig. 3b), the I157T mutant but not the R145W mutant
was phosphorylated at Thr-68 (Fig. 3c). In addition,
expression of the R145W mutant did not affect the phosphorylation of
endogenous Chk2 in these cells (Fig. 3c), suggesting that
the mutant protein may not exhibit dominant-negative activity.
FHA Domain Mutants of Chk2 Localize Normally in Nuclei--
Chk2
normally localizes to the nuclei. We examined the subcellular
localization of wild-type and mutant HA-tagged Chk2 stably expressed in
HCT116 derivative cell lines. Immunostaining using anti-HA antibodies
revealed that wild-type and the R145W and I157T mutants of Chk2 all
localized normally to nuclei (Fig. 4).
Furthermore, the localization of wild-type or mutant Chk2 did not
change following gamma radiation (data not shown).

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Fig. 4.
Nuclear localization of exogenously expressed
wild-type or mutant Chk2. HCT116 and its derivative cells were
permeabilized, fixed, and stained with DAPI (left panels)
and anti-HA mAb HA11 (right panels) to reveal the
localization of HA·Chk2 proteins.
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Chk2 Exists As a Protein Complex of Apparent Mr
~200,000 in the Cell--
Because Chk2 R145W and I157T mutants are
missense mutations within the FHA domain (involved in protein-protein
interaction, Refs. 10 and 15), we speculated that these Chk2 mutations might affect the association of Chk2 with other proteins. To
investigate this possibility, we first used size-exclusion
chromatography to determine the native size of Chk2 in HeLa and HCT116
cells. As shown in Fig. 5, endogenous
Chk2 eluted from a Superdex 200 column mainly as a protein complex with
an apparent Mr ~200,000, although a
smaller portion of Chk2 eluted as a protein complex of
Mr ~600,000. Because only a very small amount
of Chk2 eluted where monomeric Chk2 is predicted to elute, we conclude
that the majority of Chk2 exists in complex(es) with other proteins.
Alternatively, Chk2 may exist as multimers.

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Fig. 5.
Native sizes of wild-type or mutant Chk2 in
the cell. Extracts prepared from HeLa, HCT116, and HCT116
derivative cells were analyzed by size-exclusion chromatography.
Immunoblots were probed with either anti-Chk2 or anti-HA
antibody.
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We then examined the elution profiles of the stably expressed
Chk2 mutants in these HCT116 derivative cell lines. As a control, HA-tagged wild-type Chk2 eluted with an apparent molecular weight identical to endogenous Chk2 (Fig. 5). The Chk2 I157T mutant eluted in
fractions similar to that of wild-type Chk2. In contrast, the Chk2
R145W mutant eluted as a much larger protein complex (Fig. 5),
suggesting that this mutant may affect the association of Chk2 with
other proteins.
Chk2 Expression Is Down-regulated in HCT15 Cells--
The R145W
mutation of Chk2 was identified in a colon cancer cell line HCT15 (12).
Based on sequence analysis, HCT15 carries one mutant allele (R145W) and
one wild-type allele of Chk2 (12). It is speculated that the Chk2
mutation in HCT15 may contribute to tumorigenesis either as a result of
reduced gene dosage or through a dominant-negative effect (12).
Expression of this R145W mutant in HCT116 cells did not affect the
phosphorylation (see Fig. 3c) or the activation (data not shown) of endogenous Chk2, arguing that this mutant may not behave as a
dominant-negative mutant. We also observed that, when the same amount
of DNA encoding either wild-type or R145W mutant of Chk2 were used in
transient transfection experiments, the expression level of R145W
mutant was only 10-20% that of wild-type Chk2 (data not shown). These
results suggest that the R145W mutation of Chk2 may affect the
stability of this mutant Chk2 protein. Thus, it is possible that this
mutant contributes to tumorigenesis because of haploid insufficiency.
To examine whether mutation in HCT15 cells results in reduced levels of
Chk2 protein, we compared Chk2 protein levels in HCT15 cells with that
in K562, HCT116, or HeLa cells. If the presumed wild-type allele of
Chk2 in HCT15 cells were expressed normally, we would expect to observe
at most a 2-fold reduction in Chk2 protein levels. However, as shown in
Fig. 6a, Chk2 protein was barely detectable in the extract of HCT15 cells, whereas Chk2 protein
was readily detected in extracts of K562, HeLa, and HCT116 cells. We
estimate that the steady-state level of Chk2 in HCT15 cells is only
5-10% of that in other cell lines. Additionally, Chk2 kinase activity
was undetectable in HCT15 cells (Fig. 6b and data not
shown). These data strongly suggest that the Chk2 expression from the
second Chk2 allele is greatly reduced, if not absent, in HCT15 cells.
The mechanism for the down-regulation of Chk2 expression is unknown.
Because HCT15 carries inactivating mutations in both hMSH6 alleles, it
is also possible that genomic instability in these cells may lead to
the mutation in the second Chk2 allele. Such mutation, either at the
promoter or in the coding sequence of Chk2, could result in reduced
levels of Chk2 protein.

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Fig. 6.
Down-regulation of Chk2 in HCT15 cells.
a, Chk2 expression is down-regulated in HCT15 cells.
Extracts prepared from HeLa, HCT15, HCT116, and K562 cells were
subjected to Western blotting using two independent anti-Chk2
antibodies (anti-Chk2 no. 7 and anti-Chk2B). b, HCT15 has no
detectable Chk2 kinase activity before or after gamma radiation.
Extracts were prepared from indicated cell lines before and 1 h
after gamma radiation. Kinase reactions were performed using anti-Chk2B
immunoprecipitates.
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DISCUSSION |
In this study, we have characterized the four reported Chk2
mutations. Two mutations identified in Li-Fraumeni patients that lead
to frameshifts at the C-terminal kinase domain result in loss of kinase
activity. In contrast, the R145W Chk2 mutant still retains some kinase
activity in vitro but is incapable of being activated
following gamma radiation in vivo, most likely because it is
not phosphorylated at Thr-68 by ATM kinase. This mutant also behaves
differently from wild-type Chk2 in size-fractionation experiments,
suggesting that this mutation may also affect associations of Chk2
with other cellular proteins.
The I157T Chk2 mutant behaves similar to wild-type Chk2 in all the
assays used in this study. The I157T mutation may be a rare
polymorphism that does not affect Chk2 functions. Alternatively, this
mutation may affect associations of Chk2 with certain cellular proteins
in a way that does not result in apparent changes in the sizes of
Chk2-containing protein complexes as revealed by size-exclusion
chromatography. Identification of Chk2-associated proteins will provide
us with some insights in this regard.
It is interesting that the two mutations with the Chk2 FHA domain
behave differently in our assays. The FHA domain of Chk2 may have
multiple functions. FHA domain is involved in protein-phosphoprotein interaction (10, 15). These interactions may be essential for
transmitting DNA damage signals to Chk2. Any alternation of association
of Chk2 with upstream signaling proteins could lead to the failure of
Chk2 activation following DNA damage, as in the case of R145W mutant.
In addition, the FHA domain may also mediate transmitting signals from
Chk2 to downstream effectors such as p53, BRCA1, and Cdc25C. It is
reasonable to speculate that the I157T mutant may be defective in this
aspect of Chk2 function. One could examine whether any
Chk2-dependent events, such as stabilization of p53
following gamma radiation (8), are defective in cells that carry only
the I157T mutant of Chk2. Such experiments will provide insights into
the mechanism of this Chk2 mutant.