1 Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA
2 Graduate Program in Cellular and Molecular Pharmacology, UMDNJ-Graduate School
of Biomedical Sciences and Rutgers, The State University of New Jersey,
Piscataway, NJ 08854, USA
3 Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
4 Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, St
Andrews Place, East Melbourne 3002, Australia
5 Department of Genetics, University of Melbourne, Parkville, Victoria 3010,
Australia
* Author for correspondence (e-mail: walworna{at}umdnj.edu)
Accepted 27 August 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Chk1, DNA damage, Checkpoint, Cell cycle
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell cycle progression in eukaryotes is mediated by the activation of a
highly conserved family of protein kinases, the cyclin-dependent kinases
(CDKs) (Pines, 1995). Much has
been learned in recent years regarding the interaction between Chk1 and the
core regulators of CDKs (O'Connell et al.,
2000
). Chk1 from fission yeast and Xenopus can
phosphorylate the proteins Wee1 and Cdc25 in vitro
(O'Connell et al., 1997
;
Peng et al., 1997
;
Sanchez et al., 1997
;
Zeng et al., 1998
;
Baber-Furnari et al., 2000
).
These proteins regulate phosphorylation of critical residues of Cdc2, the cdk
that controls entry into mitosis (MacNeill
and Nurse, 1997
). The Wee1 tyrosine kinases (Wee1 and Mik1 in
fission yeast, Wee1 in higher eukaryotes) phosphorylate Cdc2 at tyrosine 15
(Y15), and the Cdc25 phosphatases dephosphorylate this residue
(Tang et al., 1993
;
Lundgren et al., 1991
;
Millar et al., 1991
). In
higher eukaryotes, an additional kinase, Myt1, additionally phosphorylates
Cdc2 on threonine 14 (T14), though recent studies suggest that phosphorylation
of this residue is not involved in checkpoint control
(Fletcher et al., 2002
).
During G2, the phase of the cell cycle between S phase (DNA replication) and
mitosis, Cdc2 is regulated by cyclin B association and phosphorylation at the
negative regulatory sites, T14 and Y15 in vertebrates and Y15 alone in fission
yeast. Cdc2/cyclin B complexes become fully active when these residues are
dephosphorylated, allowing cells to progress into mitosis
(MacNeill and Nurse, 1997
). In
response to DNA damage, phosphorylation of Y15 of fission yeast Cdc2 is
maintained, probably through the activities of Wee1 and Mik1
(Kharbanda et al., 1994
;
O'Connell et al., 1997
;
Rhind et al., 1997
),
suggesting that regulators of this phosphorylation may be targets of the DNA
damage checkpoint pathway in fission yeast. One role of Chk1 may be to cause
cell cycle arrest in response to DNA damage by regulating the activity of Wee1
and Mik1 proteins via phosphorylation
(O'Connell et al., 1997
;
Baber-Furnari et al., 2000
).
Additionally, Chk1 may act by inhibiting the function of the Cdc25 phosphatase
(Peng et al., 1997
;
Sanchez et al., 1997
).
We have previously shown that a significant fraction of Chk1 is
phosphorylated following DNA damage induced by a variety of agents including
UV light, ionizing radiation (IR), reduced DNA ligase activity, and
camptothecin (CPT) (Wan et al.,
1999; Walworth and Bernards,
1996
). Phosphorylation results in a species of Chk1 with decreased
mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). This decrease in mobility is dependent on several components of
the DNA damage checkpoint pathway including Rad1, Rad3, Rad9, Rad17, Rad26,
Hus1 and Crb2 (Walworth and Bernards,
1996
; Saka et al.,
1997
). As a presumed kinase-inactive mutant of Chk1 was shown to
lack this mobility shift, we initially proposed that the shift might result
from autophosphorylation (Walworth and
Bernards, 1996
). However, a different kinase inactive mutant does
undergo the mobility shift suggesting that the phosphorylation event that
causes it is likely to be carried out by a distinct kinase
[(Wan and Walworth, 2001
) and
see below].
Vertebrate Chk1 is phosphorylated by ATR, the ATM- and Rad3-related protein
kinase (Zhao and Piwnica-Worms,
2001; Liu et al.,
2000
). ATM is the gene that is mutated in the human genetic
disorder Ataxia Telangiectasia (AT), which is characterized by
hypersensitivity to IR, neuronal degeneration, immune dysfunction, cancer
predisposition, and premature aging
(Savitsky et al., 1995
;
Lavin and Shiloh, 1997
). Cell
lines from AT patients have a defective checkpoint response to IR
(Kastan and Lim, 2000
). ATR is
an ATM-related protein that also has a role in the IR-induced DNA damage
checkpoint. Unlike ATM, however, ATR also has a role in the UV and hydroxyurea
(HU)-induced damage checkpoints (Cimprich
et al., 1996
). ATR, but not ATM, has been shown to be an essential
gene for mouse embryogenesis, as has Chk1
(Barlow et al., 1996
;
Xu and Baltimore, 1996
;
Brown and Baltimore, 2000
;
Liu et al., 2000
), though
expression of dominant negative mutants of ATR or Chk1 in cell lines
interferes with checkpoint responses, but not cellular viability
(Koniarias et al., 2001
).
However, a recent study suggests that conditional homozygous knockout of ATR
is not compatible with long-term somatic cell viability
(Cortez et al., 2001
).
Both ATM and ATR prefer to phosphorylate serine (S) or threonine (T)
residues that are followed by a glutamine (Q)
(Kim et al., 1999). The Chk1
amino acid sequence has several conserved SQ/TQ sites. In Xenopus
oocyte extracts, Chk1 phosphorylation in response to aphidicolin is dependent
on ATR. This phosphorylation is also dependent on the integrity of the
conserved SQ/TQ sites. A phosphoantibody was used to identify S344 as one of
the phosphorylated sites (Guo et al.,
2000
). Human Chk1 is phosphorylated by ATR at the analogous site,
S345, and another site, S317, in response to UV, IR, and HU
(Zhao and Piwnica-Worms,
2001
). In fission yeast, Rad3 and Chk1 can co-immunoprecipitate
following moderate overexpression, even in the absence of DNA damage,
suggesting that Rad3, like its human homolog ATR, may phosphorylate Chk1 in
response to DNA damage (Martinho et al.,
1998
). Rad3 can phosphorylate a fragment of fission yeast Chk1 in
vitro (Lopez-Girona et al.,
2001
).
The in vivo consequences of S317 or S345 phosphorylation are not yet clear.
Phosphorylated fission yeast Chk1 co-immunoprecipitates with 14-3-3 proteins,
small highly conserved proteins that may be involved in regulation of
intracellular localization and facilitation of protein-protein interactions
(Chen et al., 1999). Due to
conflicting results, it is unclear whether Chk1 phosphorylated in response to
DNA damage has increased activity. Two groups claim that human Chk1 kinase
activity is increased following treatment with UV, HU, or BNP 1350, a
topoisomerase I inhibitor (Zhao and
Piwnica-Worms, 2001
; Mailand
et al., 2000
); whilst one other group claims no change in activity
following genotoxic stress (Kaneko et al.,
1999
).
To determine which phosphorylation site(s) in fission yeast Chk1 is
important for the function of Chk1 in the DNA damage checkpoint pathway, we
constructed mutants in which serines or threonines within the SQ/TQ motifs of
S. pombe Chk1 were substituted with alanine or aspartic acid.
Consistent with the results of others
(Lopez-Girona et al., 2001),
we demonstrate that mutation of serine 345 to alanine abolishes the mobility
shift caused by exposure to DNA damaging agents. However, it is clear that
mutation of a variety of other residues in Chk1 also abolishes the mobility
shift [(Wan and Walworth,
2001
) and see below], and that this largely correlates with a
failure to localize to the nucleus. The serine 345 to alanine mutant, however,
is nuclear, eliminating mislocalization as a reason for the failure to shift.
More significantly, using antibody directed toward a peptide containing
phospho-serine 345, we report that serine 345 of fission yeast Chk1 is
phosphorylated in vivo in response to treatment with UV light or CPT, a
topoisomerase I poison. Furthermore, S345 phosphorylation is necessary for an
increase of basal Chk1 kinase activity in response to DNA damage that is
critical to the integrity of the DNA damage checkpoint pathway. DNA
damage-induced activation of Chk1 is impaired in a strain lacking Rad3
function. In sum, these data provide important in vivo evidence to support the
model of Chk1 activation by Rad3-mediated phosphorylation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment with camptothecin and UV light
Camptothecin lactone (CPT) was obtained from the Drug Synthesis and
Chemistry Branch, Developmental Therapeutics Program, Division of Cancer
Treatment, National Cancer Institute. Cells were treated with 40 µM CPT for
2 hours as described (Wan et al.,
1999). To determine the state of Chk1 phosphorylation after UV
treatment, cells were grown overnight to mid-log phase and
1x108 cells were spread on agar plates. Cells were exposed to
a dose of 100 J/m2 of UV light and harvested for lysate preparation
after 20 minutes. Survival following UV treatment was determined as described
(Walworth et al., 1993
) or by
spotting 5 µl 10-fold dilutions of a 1x107 cells/ml
culture on agar plates and exposing the cells to 100 J/m2 of UV
light. Plates were analyzed after 3 days. The ability of
chk1-:ep cells to arrest the cell cycle in response to UV
light treatment was determined by measuring the percentage of cells from a
synchronized population of cells exposed to UV light that have passed mitosis
at 20 minute intervals following treatment, as described
(Wan and Walworth, 2001
).
Lysate preparation, immunoprecipitation and immunoblotting
For lysate preparation, cells were harvested by centrifugation and lysed
using glass beads and a FastPrep (Bio 101) vortexing machine. Lysate was
centrifuged at 735 g for 1 minute and the supernatant was
collected for protein determination using the Bradford assay method (Bio-Rad).
For western blot analysis of total cell extracts, cells were lysed in
Phosphate Buffered Saline (PBS) + 1% Triton-X 100 + Complete Protease
Inhibitor (Roche Diagnostics), aliquots were run on 8% SDS-PAGE, transferred
to nitrocellulose (BA83, Schleicher and Schuell), and probed with 12CA5
antibody. Blocking of the membranes and all antibody incubations and washes
were done with 1% milk and 0.05% Tween-20 in PBS. 12CA5 antibody to the HA
epitope was used at a 1:500 dilution. A peroxidase-coupled secondary antibody
(Boehringer Mannheim) and the enhanced chemiluminescence detection system from
NEN-Dupont (Renaissance) were used to detect the immune complexes. Filters
were exposed to Kodak BioMax film for 2-60 minutes.
For Chk1 immunoprecipitation, cells were lysed in IP buffer (10 mM NaPO4 pH 7, 0.15 M NaCl, 1% NP-40, 10 mM EDTA, 50 mM NaF, 2 mM DTT, 50 mM PMSF, and Complete Protease Inhibitor (Roche Diagnostics) and 100 µl of F-7 anti-HA antibody (Santa Cruz) was added to 7-10 mg of protein. The IP volume was brought up to 500 µl using IP buffer and the IPs were incubated at 4°C on a rotator for 1-16 hours. 30 µl of recombinant protein A sepharose beads were added and the IPs were incubated for 1-2 hours. The IPs were then washed three times with IP buffer. 120 µl of 2x Laemmli sample buffer was added to the dry beads and boiled for 5 minutes. The IPs were split into 55 µl aliquots, run on 8% SDS-PAGE, transferred to nitrocellulose, and probed with SN252-1, antibody to S. pombe Chk1 phosphorylated on serine 345, at 1:4000 or Y-11 anti-HA antibody (Santa Cruz) at 1:1000. The rabbit polyclonal antibody, SN252-1, was raised against a phospho-serine 345 peptide, VEVYGALS(PO3) QPVQLNK by SynPep Corporation. Attempts to raise an antibody against a phospho-serine 367 peptide, DPSLS(PO3)QPVQLNK, that was useful for western blot analysis were unsuccessful. Blocking of the membranes and all antibody incubations and washes were done as described above for the 12CA5 antibody.
For co-immunoprecipitation of Chk1 with Rad24, 500 ml of mid-log phase
cells were or were not treated with 40 µM CPT for two hours, harvested, and
lysates were prepared by grinding in liquid nitrogen based on a method
described by Ansari et al. (Ansari et al.,
1999), as described (Chen et
al., 1999
). Immunoprecipitations were performed with anti-Rad24
antibody (UMDNJ 55) as described (Chen et
al., 1999
). The IPs were washed 3 times using lysing buffer and 60
µl 2x Laemmli sample buffer was added to the dry beads. 55 µl of
each IP was run on 8% SDS-PAGE, transferred to nitrocellulose, and probed with
12CA5 antibody as described above.
Localization of SQ/TQ mutants in S. pombe
To determine the localization of Chk1, Chk1D155A, Chk1S345A, Chk1S345D,
Chk1S367A and Chk1S367D, chk1::ura4 cells were transformed with pRep1
harboring chk1+ or chk1- cDNA fused to
the gene encoding GFP (Tatebe et al.,
2001). Cells were grown to mid-log phase in PMA containing 20
µM thiamine and analyzed using a wildtype GFP filter in a Zeiss microscope
equipped with a Zeiss Axiocam and OpenLab software. The localization of
Chk1K38A, Chk1G108S, Chk1N142D, Chk1L144P and Chk1D155G were determined by
visualizing those proteins fused in genomic constructs to GFP and expressed
from the Chk1 promoter in the plasmid pSP1
(Cottarel et al., 1993
) in a
chk1::ura4 background. Cells were visualized with an Olympus IX70
microscope and images captured with a MicroMax CCD camera from Princeton
Instruments using IP Lab Software.
Synthetic lethality of chk1 SQ/TQ mutants with
cdc17-K42
chk1- cdc17-k42 double mutants were obtained using
random spore analysis. Cells were grown to mid-log phase at 25°C and
resuspended in YEA to 1x107 cells/ml. 5 µl of 10-fold
dilutions were spotted onto YEA agar plates and incubated at 25°C,
32°C, or 36°C for 3 days. Pictures were analyzed to determine if
colonies were formed by the double mutant strains.
Kinase assay
Optimal assay conditions for Chk1 were determined by kinetic analysis of
recombinant protein, and defined conditions that were linear over time (U.J.
and M.O., data not shown). Chk1+:ep or
chk1-:ep cells were grown to mid-log phase, were or were
not treated with CPT, and harvested. Cells were lysed in IP buffer as
described above. Chk1 was immunoprecipitated from 2-3 mg of protein, in
triplicate, using F-7 anti-HA antibody and 20 µl of recombinant protein A
sepharose beads as described above. The IPs were washed 3 times with IP buffer
and 3 times with 2x kinase buffer (50 mM Tris pH 7, 1 mM DTT, 5 mM
MgCl2, 0.4 mM MnCl2, 25% SIGMA glycerol, 0.1% Triton
X-100, 100 µM ATP). 24 µl of kinase reaction (kinase buffer, 8-12 µg
peptide (RIARAASMAAALARK), and [-32P]ATP (3000 Ci/mmole))
was then added to each IP, incubated at 30°C for 10 minutes, and
immediately placed on ice. 20 µl of kinase reaction was spotted onto P81
phosphocellulose paper (Whatman), washed three times in 5% phosphoric acid,
rinsed with ethanol, dried, placed in a scintillation vial with scintillation
fluid, and counted for 30 seconds in a liquid scintillation counter.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Cells lacking chk1 have increased sensitivity to UV light as
compared to wildtype cells due to an inability to prevent mitotic entry prior
to completion of DNA repair (Walworth et
al., 1993). To determine if the SQ/TQ motifs are required for
cells to survive exposure to UV light, we inoculated decreasing concentrations
of wildtype, chk1::ura4, or chk1- cells onto agar
plates and exposed them to UV light (see Materials and Methods). Chk1S345A
cells exhibited an increased sensitivity to UV light, similar to that of a
chk1 deletion strain; however, cells harboring the chk1T323A
or chk1S367A mutant alleles remained wildtype for their UV response
(Fig. 1D). Taken together,
these results are consistent with the possibility that S345, but not T323 or
S367, is a phosphorylation site critical for the mobility shift of Chk1 in
response to DNA damage and for the function of Chk1 in the DNA damage
checkpoint pathway.
Mutation of non-phosphorylatable residues abolishes mobility shift of
Chk1
In a previous study, we isolated a number of DNA damage-sensitive alleles
of Chk1 with mutations in the catalytic domain that conferred checkpoint
sensitivity (Wan and Walworth,
2001). Several of these mutants did not undergo the mobility shift
normally observed for the wild-type protein, yet none of the mutations were
found to be in serine, threonine or tyrosine residues
(Wan and Walworth, 2001
).
Thus, the failure of the S345 mutant to undergo the mobility shift is not
necessarily attributable to its being a site of phosphorylation. Analysis of
the localization of the mutants identified in the aforementioned study
indicates that failure to undergo the mobility shift correlates with a failure
to localize efficiently to the nucleus
(Fig. 2A,B and data not shown).
To determine whether mutation of S345 affects nuclear localization, the
Chk1S345A protein was fused to GFP and examined by microscopy. As shown in
Fig. 2C, the Chk1S345A protein
localizes to the nucleus in a manner that is indistinguishable from wild-type
Chk1.
|
A serine 345 phosphoantibody recognizes only phosphorylated Chk1
Failure of Chk1 to undergo the mobility shift is clearly not a suitable
assay for concluding that a residue is a phosphorylation site; therefore, we
produced antibodies against a serine 345 phosphopeptide (see Materials and
Methods) to determine if this antibody would recognize Chk1 that is
phosphorylated in response to the DNA damage produced upon CPT treatment. To
this end, we immunoprecipitated Chk1 or Chk1S345A and performed immunoblot
analysis of SDS-PAGE with antibody to the phosphopeptide (see Materials and
Methods). The serine 345 phosphoantibody strongly recognized Chk1 only upon
treatment with CPT and did not recognize Chk1S345A with or without CPT
treatment (Fig. 3A). We
conclude that serine 345 in fission yeast Chk1 is a site that is
phosphorylated in response to DNA damage and is important for the function of
Chk1 in the DNA damage checkpoint pathway.
|
Chk1S345A and Chk1S345D retain a basal level of kinase activity, but
this activity is not activated by DNA damage
To determine the role of S345 phosphorylation in regulating the activity of
Chk1, we performed a kinase assay using protein immunoprecipitated from
CPT-treated or untreated cells and a peptide substrate (see Materials and
Methods). The kinase activity of Chk1 was increased approximately two-fold in
response to CPT treatment. Activity associated with a presumed catalytically
inactive allele of Chk1, Chk1D155A, was minimal. To determine what role the
phosphorylation state of serine 345 might play in regulating Chk1 activity, we
assayed the kinase activity of the Chk1S345A mutant. In addition, we
constructed an allele of Chk1 in which S345 was changed to aspartic acid in an
attempt to mimic the negatively charged, phosphorylated state
(Li et al., 2000;
Huang and Erikson, 1994
), and
assayed its kinase activity as well. As shown in
Fig. 3B, the kinase activity of
Chk1S345A and Chk1S345D from treated or untreated cells was equivalent to that
of Chk1 from untreated cells, suggesting that Chk1S345A and Chk1S345D possess
a basal level of kinase activity that cannot be activated in response to CPT
(Fig. 3B). Since the activity
of the Chk1S345D mutant protein resembles that of the Chk1S345A mutant, we
assume that aspartic acid is not a good substitute for phosphorylated serine
in the context of Chk1.
Overexpression of Chk1, Chk1S345A, and Chk1S345D, but not Chk1D155A, causes
cell elongation [(Rhind et al.,
1997; Ford et al.,
1994
) and data not shown], providing further evidence that the
serine 345 mutants retain a basal level of kinase activity that can generate a
cell cycle arrest when expressed at high levels. Chk1D155A possesses a
mutation at position 155 of Chk1, which is within subdomain VII of the
catalytic domain. Structural data for several kinases suggest this conserved
residue helps to orient the
-phosphate of ATP by chelating the
activating Mg2+ ions that bridge the ß- and
-phosphates
(Hanks and Hunter, 1995
). This
mutation, therefore, is expected to compromise catalytic activity, an
expectation that is supported by its minimal level of kinase activity in this
assay (Fig. 3B). These results
suggest that Chk1 kinase activity is increased in response to CPT and that
this increase is dependent on the phosphorylation of serine 345.
Phosphorylation of Chk1 requires the kinase Rad3
(Walworth and Bernards, 1996).
To determine whether Rad3 function is required for activation of Chk1 kinase
activity in response to DNA damage, Chk1 was prepared from cells that have a
deletion of the rad3 gene. As shown in
Fig. 3C, full activation of
Chk1 is impaired in cells lacking Rad3 function. In this experiment Chk1
activity prepared from wild-type cells increased nearly two-fold in response
to CPT treatment, while an increase of
50% was seen in the rad3
deletion strain. The residual increase might simply reflect noise in the assay
resulting from co-precipitation of contaminating kinases. Alternatively, the
residual increase could reflect a Rad3-independent, but
phosphorylation-dependent activation of Chk1.
Checkpoint function and the ability to overcome DNA damage is
compromised in cells expressing Chk1S345A and Chk1S345D
To determine if the DNA damage checkpoint was intact in cells expressing
Chk1S345A and Chk1S345D, we analyzed mitotic entry of a synchronous population
of cells following exposure to UV light. The chk1S345A strain behaved
in a manner similar to a chk1::ura4 strain in that it entered mitosis
without a UV-induced arrest, indicating that the checkpoint arrest is
abolished in these cells. The chk1S345D strain delayed entry into
mitosis 40 to 60 minutes longer than a chk1::ura4 or
chk1S345A strain, but entered mitosis 100 to 120 minutes earlier than
a wildtype strain (Fig. 4A). We
conclude that the mutation of serine 345 to alanine or aspartic acid results
in the loss of the ability of cells to delay entry into mitosis for the length
of time needed for cells to cope with DNA damaged by UV light.
|
Activation of the DNA damage checkpoint and the transient block to mitotic
entry correlates with improved survival following DNA damage
(al-Khodairy and Carr, 1992).
Cells lacking chk1 fail to arrest the cell cycle in response to DNA
damage, enter mitosis with damaged DNA, and die
(Walworth et al., 1993
;
al-Khodairy et al., 1994
). To
characterize the UV sensitivity of cells expressing Chk1S345A and Chk1S345D,
we performed quantitative UV survival assays by plating 500 cells onto agar
plates and exposing them to UV light (see Materials and Methods). As shown in
Fig. 4B, cells expressing
Chk1S345A and Chk1S345D have increased UV sensitivity equivalent to that of a
chk1 deletion strain.
Checkpoint function is also required for S. pombe cells to survive
a reduction of DNA ligase activity, which among its essential functions is
necessity to join Okazaki fragments following replication
(Walworth et al., 1993;
al-Khodairy and Carr, 1992
).
The cdc17-K42 allele of the gene that encodes DNA ligase is
completely inactive at its restrictive temperature of 36°C, but retains
some activity at lower temperatures
(Nasmyth, 1977
). A
chk1::ura4 cdc17-K42 strain cannot survive at the semi-permissive
temperature of 32°C, a temperature at which a chk1+
cdc17-K42 strain survives; however, it can survive at the permissive
temperature of 25°C. cdc17-K42 double mutant strains with both
S345 chk1- alleles survived at 25°C, but were unable
to survive at the semi-permissive temperature of 32°C
(Fig. 4C), indicating that both
S345 chk1- alleles abolish checkpoint function. Large
colonies appeared on patches derived from both strains indicating the
selection of suppressing mutations that rescue the viability of these strains.
The identity of these suppressors is currently under investigation.
Further evidence that phosphorylation of Chk1 is critical for Chk1 function
in vivo stems from an examination of the status of Chk1 in strain backgrounds
containing mutations in the Cdc2 tyrosine kinases Wee1 and Mik1. Cells with a
temperature sensitive allele of wee1, wee1-50, advance mitosis such
that they divide at a small cell size, but remain viable. However,
simultaneous inactivation of wee1 and a second tyrosine kinase in
fission yeast, mik1, results in rapid accumulation of cells with a
mitotic catastrophe phenotype (Lundgren et
al., 1991), while inactivation of wee1 in a checkpoint
mutant background results in a gradual accumulation of cells in mitotic
catastrophe (al-Khodairy and Carr,
1992
; Walworth et al.,
1993
). Since Chk1 is essential for viability in cells with
compromised Wee1 function, we examined the phosphorylation status of Chk1 in
such cells. As shown in Fig.
4D, Chk1 is constitutively phosphorylated in cells with mutations
in the Mik1 or Wee1 tyrosine kinases even at permissive temperature for the
wee1-50 mutant. Cells with mutations in both mik1 and
wee1 enter mitosis at a smaller size than cells with mutations in
either kinase alone and the fraction of phosphorylated Chk1 is highest in such
a strain (mik1::ura4 wee1-50). Indeed, a chk1::ura4 mik1::ura4
wee1-50 strain is barely viable even at 25°C (N.C.W., unpublished).
As is the case for DNA damage-induced phosphorylation of Chk1
(Walworth and Bernards, 1996
),
phosphorylation of Chk1 in the wee mutants is dependent on rad1.
Notably, a rad1-1 wee1-50 strain is inviable when wee1-50 is
inactivated at 36°C (al-Khodairy and
Carr, 1992
).
Chk1S345A and Chk1S345D do not co-immunoprecipitate with Rad24
Phosphorylated Chk1 co-immunoprecipitates with Rad24, a 14-3-3 protein
(Chen et al., 1999). This
interaction may be essential for checkpoint function
(Chen et al., 1999
). To
determine if Chk1S345D can constitutively co-immunoprecipitate with Rad24, we
immunoprecipitated Rad24, performed SDS-PAGE, and probed for HA-tagged Chk1
using an anti-HA antibody. Chk1S345D exhibits a mobility similar to that of
phosphorylated Chk1, but does not co-immunoprecipitate with Rad24
(Fig. 5). Chk1S345A also fails
to co-immunoprecipitate with Rad24 (Fig.
5). These results suggest that S345 phosphorylation is required
for Chk1 to associate with Rad24 and that aspartic acid at position 345 is not
sufficient to induce this association.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human Chk1 is phosphorylated in vivo at two SQ/TQ ATM or ATR consensus
phosphorylation sites, S317 and S345, in an ATR-dependent manner
(Zhao and Piwnica-Worms,
2001). Detection with a phosphospecific antibody indicates that
S345 of mouse Chk1 is phosphorylated in an ATR-dependent manner in vivo
(Liu et al., 2000
). Four SQ/TQ
consensus sites can be phosphorylated in vitro by Xenopus ATR and
one, S344, is phosphorylated in vivo (Guo
et al., 2000
). During the preparation of this manuscript and
consistent with our results, Lopez-Girona et al., reported that the S345A
mutation of fission yeast Chk1 abolishes the DNA damage-induced mobility shift
(Lopez-Girona et al., 2001
).
However, we have shown previously that mutation of amino acids that would
never be phosphorylated can also abolish the DNA damage-induced
phosphorylation of Chk1 (Wan and Walworth,
2001
), making it impossible to determine from mutagenesis data
alone that any particular amino acid is a site of phosphorylation. Therefore,
we used a S345 phosphospecific antibody to identify S345 as a site that is
phosphorylated in vivo in response to DNA damage
(Fig. 3). Phosphorylation at
this site is critical for checkpoint function and the association of Chk1 with
the 14-3-3 protein Rad24 (Figs
4,
5). Consistent with this,
Lopez-Girona et al., demonstrated that Rad3 can phosphorylate in vitro a 36
amino acid fragment of Chk1 encompassing this particular SQ site
(Lopez-Girona et al.,
2001
).
There has been controversy over the effect of phosphorylation on the kinase
activity of Chk1. It has recently been shown that human Chk1 kinase activity
is elevated in response to hydroxyurea and that this elevation requires
phosphorylation of serines 317 and 345
(Zhao and Piwnica-Worms,
2001). Mutation of either serine does not abolish a basal level of
Chk1 kinase activity (Zhao and
Piwnica-Worms, 2001
). Unphosphorylated fission yeast Chk1 also
maintains a basal level of kinase activity that does not appear to be
abrogated when serine 345 is mutated to alanine
(Fig. 3). Deletion of the
C-terminus of human and Xenopus Chk1 activates its intrinsic kinase
activity (Oe et al., 2001
;
Chen et al., 2000
).
Furthermore, the crystal structure of the catalytic domain of human Chk1
revealed an open kinase conformation that enables kinase activity without
phosphorylation of the catalytic domain
(Chen et al., 2000
). This
evidence suggests that the C-terminus of Chk1 acts to regulate kinase activity
by suppressing activity when the kinase is in an unphosphorylated state. A
change in conformation upon phosphorylation of the C-terminus may cause a
derepression, or elevation, of kinase activity. In an attempt to construct a
mutant of Chk1 that mimics its phosphorylated state
(Huang and Erikson, 1994
;
Li et al., 2000
), we mutated
serine 345 to aspartic acid. Even though the mobility of this protein on
SDS-PAGE is similar to phosphorylated Chk1, it does not seem to possess the
properties of phosphorylated Chk1.
Fission yeast Chk1 kinase activity is elevated in response to DNA damage and this elevation requires serine 345 (Fig. 3). Mutating serine 345 to aspartic acid did not confer constitutively elevated Chk1 kinase activity as one might have expected. This mutant did, however, retain a basal level of kinase activity. A chk1S345D mutant behaved identically to chk1S345A except it seemed to initiate a checkpoint in response to UV light that could not be maintained (Fig. 4). Chk1S345D simply may not mimic phosphorylation of Chk1 at serine 345 or the cell may require both unphosphorylated and phosphorylated forms of Chk1 to maintain a checkpoint in response to DNA damage. However, expression of plasmid borne Chk1S345D in a chk1S345A background, or conversely, of Chk1S345A in a chk1S345D background, does not restore checkpoint function (H.C., H.R. and N.C.W., data not shown). It does not seem that the Chk1S345D mutation affects Chk1 function by causing a severe change in protein structure because the protein is stably expressed, localizes to the nucleus like wild-type Chk1 (Fig. 2) and is catalytically active (Fig. 3B).
We have shown previously that chk1 is essential for the viability
of wee mutants (Walworth et al.,
1993), which are characterized by having a shortened G2 period. We
show here that Chk1 is constitutively phosphorylated in these cells.
Presumably, the phosphorylated and, hence active, Chk1 elicits a delay in
these cells that is critical for preventing mitotic catastrophe when mitotic
entry is not under normal size control. As Wee1 may be an effector of
Chk1-mediated G2 arrest (O'Connell et al.,
1997
), the absence of functional Wee1 would explain why the
phosphorylated, active Chk1 is unable to cause permanent G2 arrest in these
cells. While it is not clear what signals Chk1 phosphorylation in cells with
the wee phenotype, one might assume that it is an unusual DNA damage-like
state or that it is the result of activation of Cdc2 in an inappropriate
context. Indeed, a recent study indicates that premature activation of the
Cdc2 homologue in S. cerevisiae, resulting from loss-of-function of a
Cdc2 inhibitor, Sic1, causes genomic instability due to alterations in S phase
progression (Lengronne and Schwob,
2002
). Similarly, activation of Cdc2 in S. pombe by
virtue of inactivation of the Wee1 or Mik1 kinases, may result in an
inappropriate combination of events that leads to a demand for checkpoint
function to ensure viability; hence, the phosphorylation of Chk1. The
essential nature of Chk1 in wee mutants may be related to the embryonic
lethality of chk1 mutations in mice and Drosophila, where
cells cycle rapidly and the major challenge is to coordinate S- and M-phases
(Fogarty et al., 1994
;
Fogarty et al., 1997
;
Takai et al., 2000
;
Liu et al., 2000
).
Here we have determined using a combination of mutational studies and the use of phosphospecific antibodies that fission yeast Chk1 is, importantly, phosphorylated in vivo on S345 in response to DNA damage and that this is essential for the function of Chk1 in the DNA damage checkpoint pathway. We have also shown that mutation of S345 abolishes a DNA damage-induced elevation in Chk1 protein kinase activity and the association of Chk1 with 14-3-3 protein. Although the mechanism by which S345 phosphorylation activates Chk1 is not known, genetic analyses of the mutants described here may help in deciphering this. These findings highlight the conserved nature of the checkpoint pathway in eukaryotic organisms and confirm that studies in fission yeast are relevant to higher eukaryotes.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
al-Khodairy, F. and Carr, A. M. (1992). DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J. 11,1343 -1350.[Abstract]
al-Khodairy, F., Fotou, E., Sheldrick, K. S., Griffiths, D. J. F., Lehmann, A. R. and Carr, A. M. (1994). Identification and characterization of new elements involved in checkpoints and feedback controls in fission yeast. Mol. Biol. Cell 5, 147-160.[Abstract]
Ansari, A., Cheng, T.-H. and Gartenberg, M. (1999). Isolation of selected chromatin fragments from yeast by site-specific recombination in vivo. Methods 17,104 -111.[CrossRef][Medline]
Baber-Furnari, B. A., Rhind, N., Boddy, M. N., Shanahan, P.,
Lopez-Girona, A. and Russell, P. (2000). Regulation of
mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint.
Mol. Biol. Cell 11,1
-11.
Barlow, C., Hirotsune, S., Paylor, R., Liyanage, M., Eckhaus, M., Collins, F., Shiloh, Y., Crawley, J. N., Ried, T., Tagle, D. et al. (1996). Atmdeficient mice: a paradigm of ataxia telangiectasia. Cell 86,159 -171.[Medline]
Basi, G., Schmid, E. and Maundrell, K. (1993). TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123,131 -136.[CrossRef][Medline]
Bentley, N. J., Holtzman, D. A., Flaggs, G., Keegan, K. S., DeMaggio, A., Ford, J. C., Hoekstra, M. and Carr, A. M. (1996). The S. pombe rad3 checkpoint gene. EMBO J. 15,6641 -6651.[Abstract]
Brown, E. J. and Baltimore, D. (2000). ATR
disruption leads to chromosomal fragmentation and early embryonic lethality.
Genes Dev. 14,397
-402.
Chen, L., Liu, T.-H. and Walworth, N. C.
(1999). Association of Chk1 with 14-3-3 proteins is stimulated by
DNA damage. Genes Dev.
13,675
-685.
Chen, P., Luo, C., Deng, Y., Ryan, K., Register, J., Margosiak, S., Tempczyk-Russell, A., Nguyen, B., Myers, P., Lundgren, K. et al. (2000). The 1.7 Å crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation. Cell 100,681 -692.[Medline]
Cimprich, K. A., Shin, T. B., Keith, C. T. and Schreiber, S.
L. (1996). cDNA cloning and gene mapping of a candidate human
cell cycle checkpoint protein. Proc. Natl. Acad. Sci.
USA 93,2850
-2855.
Cortez, D., Guntuku, S., Qin, J. and Elledge, S. J.
(2001). ATR and ATRIP: Partners in checkpoint signaling.
Science 294,1713
-1716.
Cottarel, G., Beach, D. and Deuschle, U. (1993). Two new multi-purpose multicopy Schizosaccharomyces pombe shuttle vectors, pSP1 and pSP2. Curr. Genet. 23,547 -548.[Medline]
Fletcher, L., Cheng, Y. and Muschel, R. J.
(2002). Abolishment of the Tyr-15 inhibitory phosphorylation site
on cdc2 reduces the radiation-induced G(2) delay, revealing a potential
checkpoint in early mitosis. Cancer Res.
62,241
-250.
Fogarty, P., Kalpin, R. F. and Sullivan, W.
(1994). The Drosophila maternal-effect mutation grapes causes a
metaphase arrest at nuclear cycle 13. Development
120,2131
-2142.
Fogarty, P., Campbell, S. D., Abu-Shumays, R., Phalle, B. S., Yu, K. R., Uy, G. L., Goldberg, M. L. and Sullivan, W. (1997). The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity. Curr. Biol. 7,418 -426.[Medline]
Ford, J. C., al-Khodairy, F., Fotou, E., Sheldrick, K. S., Griffiths, D. J. and Carr, A. M. (1994). 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast. Science 265,533 -535.[Medline]
Guo, Z., Kumagai, A., Wang, S. X. and Dunphy, W. G.
(2000). Requirement for Atr in phosphorylation of Chk1 and cell
cycle regulation in response to DNA replication blocks and UV-damaged DNA in
Xenopus egg extracts. Genes Dev.
14,2745
-2756.
Hanks, S. K. and Hunter, T. (1995). The
eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and
classification. FASEB J.
9, 576-596.
Hartwell, L. H. and Weinert, T. A. (1989). Checkpoints: controls that ensure the order of cell cycle events. Science 246,629 -634.[Medline]
Huang, W. and Erikson, R. L. (1994). Constitutive activation of Mek1 by mutation of serine phosphorylation sites. Proc. Natl. Acad. Sci. USA 91,8960 -8963.[Abstract]
Kaneko, Y. S., Watanabe, N., Morisaki, H., Akita, H., Fujimoto, A., Tominaga, K., Terasawa, M., Tachibana, A., Ikeda, K., Nakanishi, M. et al. (1999). Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase. Oncogene 18,3673 -3681.[CrossRef][Medline]
Kastan, M. B. and Lim, D. S. (2000). The many substrates and functions of ATM. Nat. Rev. Mol. Cell Biol. 1,179 -186.[CrossRef][Medline]
Keegan, K. S., Holtzmann, D. A., Plug, A. W., Christenson, E. R., Brainerd, E. E., Flaggs, G., Bentley, N. J., Taylor, E. M., Meyn, M. S., Moss, S. B. et al. (1996). The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes. Genes Dev. 10,2423 -2437.[Abstract]
Kharbanda, S., Saleem, A., Datta, R., Yuan, Z. M., Weichselbaum, R. and Kufe, D. (1994). Ionizing radiation induces rapid tyrosine phosphorylation of p34cdc2. Cancer Res. 54,1412 -1414.[Abstract]
Kim, S. T., Lim, D. S., Canman, C. E. and Kastan, M. B.
(1999). Substrate specificities and identification of putative
substrates of ATM kinase family members. J. Biol.
Chem. 274,37538
-37543.
Koniarias, K., Cuddihy, A. R., Christopolous, H., Hogg, A. and O'Connell, M. J. (2001). Inhibition of Chk1-dependent G2 DNA damage checkpoint radiosensitizes p53 mutant human cells. Oncogene 20,7453 -7463.[CrossRef][Medline]
Lavin, M. F. and Shiloh, Y. (1997). The genetic defect in Ataxia-Telangiectasia. Annu. Rev. Immunol. 15,177 -202.[CrossRef][Medline]
Lengronne, A. and Schwob, E. (2002). The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G1. Mol. Cell 9,1067 -1078.[Medline]
Li, M., Bermak, J. C., Wang, Z. W. and Zhou, Q. Y.
(2000). Modulation of dopamine D(2) receptor signaling by
actin-binding protein (ABP-280). Mol. Pharmacol.
57,446
-452.
Liu, Q., Guntuku, S., Cui, X.-S., Matsuoka, S., Cortez, D.,
Tamai, K., Luo, G., Carattini-Rivera, S., DeMayo, F., Bradley, A. et al.
(2000). Chk1 is an essential kinase that is regulated by Atr and
required for the G2/M DNA damage checkpoint. Genes
Dev. 14,1448
-1459.
Lopez-Girona, A., Tanaka, K., Chen, X. B., Baber, B. A.,
McGowan, C. H. and Russell, P. (2001). Serine-345 is required
for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in
fission yeast. Proc. Natl. Acad. Sci. USA
98,11289
-11294.
Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M. and Beach, D. (1991). mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell 64,1111 -1122.[Medline]
MacNeill, S. A. and Nurse, P. (1997). Cell cycle control in fission yeast. In The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology, Vol. 3 (ed. J. R. Pringle, J. R. Broach and E. W. Jones), pp. 697-763. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker,
M., Bartek, J. and Lukas, J. (2000). Rapid destruction of
human Cdc25A in response to DNA damage. Science
288,1425
-1429.
Martinho, R. G., Lindsay, H. D., Flaggs, G., DeMaggio, A. J.,
Hoekstra, M. F., Carr, A. M. and Bentley, N. J. (1998).
Analysis of Rad3 and Chk1 protein kinases defines different checkpoint
responses. EMBO J. 17,7239
-7249.
Maundrell, K. (1990). nmt1 of fission yeast. A
highly transcribed gene completely repressed by thiamine. J. Biol.
Chem. 265,10857
-10864.
Maundrell, K. (1993). Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123,127 -130.[CrossRef][Medline]
Millar, J. B. A., McGowan, C. H., Lenaers, G., Jones, R. and Russell, P. (1991). p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast. EMBO J. 10,4301 -4309.[Abstract]
Moreno, S., Klar, A. and Nurse, P. (1991). Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.Methods Enzymol. 194,795 -823.[Medline]
Nasmyth, K. A. (1977). Temperature-sensitive lethal mutants in the structural gene for DNA ligase in the yeast Schizosaccharomyces pombe. Cell 12,1109 -1120.[Medline]
O'Connell, M. J., Raleigh, J. M., Verkade, H. M. and Nurse,
P. (1997). Chk1 is a wee1 kinase in the G2 DNA damage
checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO
J. 16,545
-554.
O'Connell, M. J., Walworth, N. C. and Carr, A. M. (2000). The G2-phase DNA-damage checkpoint. Trends Cell Biol. 10,296 -303.[CrossRef][Medline]
Oe, T., Nakajo, N., Katsuragi, Y., Okazaki, K. and Sagata, N. (2001). Cytoplasmic occurrence of the Chk1/Cdc25 pathway and regulation of Chk1 in Xenopus oocytes. Dev. Biol. 229,250 -261.[CrossRef][Medline]
Peng, C. Y., Graves, P. R., Thoma, R. S., Wu, A., Shaw, A. S.
and Piwnica-Worms, H. (1997). Mitotic and G2 checkpoint
control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on
serine-216. Science 277,1501
-1505.
Pines, J. (1995). Cyclins and cyclin-dependent kinases: a biochemical view. Biochem J. 308,697 -711.[Medline]
Rhind, N., Furnari, B. and Russell, P. (1997). Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast. Genes Dev. 11,504 -511.[Abstract]
Saka, Y., Esashi, F., Matsusaka, T., Mochida, S. and Yanagida,
M. (1997). Damage and replication checkpoint control in
fission yeast is ensured by interactions of Crb2, a protein with BRCT motif,
with Cut5 and Chk1. Genes Dev.
11,3387
-3400.
Sanchez, Y., Wong, C., Thoma, R. S., Richman, R., Wu, Z.,
Piwnica-Worms, H. and Elledge, S. J. (1997). Conservation of
the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk
regulation through Cdc25. Science
277,1497
-1501.
Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L., Tagle, D. A., Smith, S., Uziel, T., Sfez, S. et al. (1995). A single Ataxia Telangiectasia gene with a product similar to PI-3 kinase. Science 268,1749 -1753.[Medline]
Takai, H., Tominaga, K., Motoyama, N., Minamishima, Y. A.,
Nagahama, H., Tsukiyama, T., Ikeda, K., Nakayama, K. and Nakanishi, M.
(2000). Aberrant cell cycle checkpoint function and early
embryonic death in Chk1-/- mice. Genes
Dev. 14,1439
-1447.
Tang, Z., Coleman, T. R. and Dunphy, W. G. (1993). Two distinct mechanisms for negative regulation of the Wee1 protein kinase. EMBO J. 12,3427 -3436.[Abstract]
Tatebe, H., Goshima, G., Takeda, K., Nakagawa, T., Kinoshita, K. and Yanagida, M. (2001). Fission yeast living mitosis visualized by GFP-tagged gene products. Micron 32, 67-74.[CrossRef][Medline]
Walworth, N., Davey, S. and Beach, D. (1993). Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2. Nature 363,368 -371.[CrossRef][Medline]
Walworth, N. C. and Bernards, R. (1996). rad-Dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint. Science 271,353 -356.[Abstract]
Wan, S., Capasso, H. and Walworth, N. C. (1999). The topoisomerase I poison camptothecin generates a Chk1-dependent DNA damage checkpoint signal in fission yeast. Yeast 15,821 -828.[CrossRef][Medline]
Wan, S. and Walworth, N. C. (2001). A novel genetic screen identifies checkpoint-defective alleles of Schizosaccharomyces pombe chk1. Curr Genet. 38,299 -306.[CrossRef][Medline]
Xu, Y. and Baltimore, D. (1996). Dual roles of ATM in the cellular response to radation and in cell growth control. Genes Dev. 10,2401 -2410.[Abstract]
Zeng, Y., Forbes, K. C., Wu, Z., Moreno, S., Piwnica-Worms, H. and Enoch, T. (1998). Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1. Nature 395,507 -510.[CrossRef][Medline]
Zhao, H. and Piwnica-Worms, H. (2001).
ATR-mediated checkpoint pathways regulate phosphorylation and activation of
human Chk1. Mol. Cell. Biol.
21,4129
-4139.