Research Paper
1
A human homologue of the checkpoint kinase Cds1 directly
inhibits Cdc25 phosphatase
Alessandra Blasina*, Inez Van de Weyer†, Marc C. Laus†,
Walter H.M.L. Luyten†, Andrew E. Parker†‡ and Clare H. McGowan*
Background: In human cells, the mitosis-inducing kinase Cdc2 is inhibited by
phosphorylation on Thr14 and Tyr15. Disruption of these phosphorylation sites
abrogates checkpoint-mediated regulation of Cdc2 and renders cells highly
sensitive to agents that damage DNA. Phosphorylation of these sites is
controlled by the opposing activities of the Wee1/Myt1 kinases and the Cdc25
phosphatase. The regulation of these enzymes is therefore likely to be crucial
for the operation of the G2–M DNA-damage checkpoint.
Results: Here, we show that the activity of Cdc25 decreased following
exposure to ionizing radiation. The irradiation-induced decrease in Cdc25
activity was suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI)
3-kinases, and was dependent on the function of the gene that is mutated in
ataxia telangiectasia. We also identified two human kinases that phosphorylate
and inactivate Cdc25 in vitro. One is the previously characterized Chk1 kinase.
The second is novel and is homologous to the Cds1/Rad53 family of
checkpoint kinases in yeast. Human Cds1 was found to be activated in
response to DNA damage.
Addresses: *Department of Molecular Biology, The
Scripps Research Institute, La Jolla, California
92037, USA. †Department of Experimental
Molecular Biology, Janssen Research Foundation,
Turnhoutseweg 30, B-2340 Beerse, Belgium.
‡Present
address: Zeneca Pharmaceutical, Alderley
Edge, Cheshire, UK.
Correspondence: Clare H. McGowan
E-mail: chmcg@scripps.edu
Received: 11 September 1998
Revised: 27 November 1998
Accepted: 27 November 1998
Published: 17 December 1998
Current Biology 1999, 9:1–10
http://biomednet.com/elecref/0960982200900001
© Elsevier Science Ltd ISSN 0960-9822
Conclusions: These results suggest that, in human cells, the DNA-damage
checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1.
Background
In response to DNA damage, eukaryotic cells use a system
of checkpoint controls to delay cell-cycle progression.
Checkpoint delays provide time for repair of damaged
DNA prior to its replication in S phase and prior to segregation of chromatids in M phase [1]. In many cases, the
DNA-damage response pathways cause arrest by inhibiting the activity of the cyclin-dependent kinases that drive
cell-cycle progression [2]. A delay in G1 results from the
activation of p53 and consequent transcriptional induction
of the cyclin-dependent kinase inhibitor p21 [3–5]. In
human cells, the DNA-damage-induced G2 delay has
been shown to be largely dependent on inhibitory phosphorylation of Cdc2 [6–8] and is therefore likely to result
from a change in the activity of the opposing kinases and
phosphatases that act on Cdc2. Nevertheless, evidence
that the activity of these enzymes is substantially altered
in response to DNA damage is lacking [9].
In this paper, we report that the Cdc25-dependentdephosphorylation of the Cdc2–Cyclin B complex was significantly
reduced
following γ-irradiation.
This
irradiation-induced decrease in Cdc25 activity was suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinases, and was dependent on the function
of the gene that is mutated in ataxia telangiectasia (AT) —
the AT-mutated (ATM) gene. We also identified two
human kinases that phosphorylate and inactivate Cdc25 in
vitro. One is the previously characterized Chk1 kinase.
The second is a novel kinase with homology to the
Cds1/Rad53 family of checkpoint kinases in yeast. In contrast to the situation in fission yeast, we found that human
Cds1 is activated in response to DNA damage. Taken
together, these data support a model for the human DNAdamage checkpoint that involves the direct inactivation of
Cdc25, catalysed by Cds1 and/or Chk1.
Results and discussion
Dephosphorylation of Cdc2 is inhibited by DNA damage
The possibility that dephosphorylation of Cdc2 is downregulated in the presence of DNA damage was investigated. Three distinct Cdc25 proteins are expressed in
human cells [10–12]. Microinjection of specific antibodies
and the observation of distinct patterns of activation
suggest that Cdc25A has a role primarily in the G1–S transition [13,14], and that Cdc25B and Cdc25C have roles
primarily in the G2–M transition [15–17]. The exact contribution of Cdc25B and Cdc25C to M-phase progression
is not known. Therefore, an assay that allows direct analysis of the phosphatases acting on Cdc2–Cyclin B was used
to determine how Cdc2 is maintained in its phosphorylated state following irradiation. In the presence of EDTA,
the activity of Cdc2–Cyclin B from asynchronous HeLa
cell extracts was found to increase with time (Figure 1a).
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Current Biology, Vol 9 No 1
Figure 1
Kinase activity
(a) 100
Control
Irradiated
Anti-Cdc25 antibody
+ Vanadate
80
60
40
20
0
0
10
(b)
20
Time (min)
30
Control
+
Cdc25
Irradiated
p34 Cdc2
Time (min)
0
30
30
0
+
Cdc25
30
30
+
Cdc25
(c)
Cdc25C
1
2
CurrentBiology
Cdc25 is downregulated in response to DNA damage. (a) Cultures of
asynchronous HeLa cells were divided and either mock-treated
(control) or irradiated with 10 Gray, 1 h before harvesting. Cell lysates
were prepared and the dephosphorylation reaction was initiated by
addition of 10 mM EDTA. At the indicated times, Cdc2–Cyclin B was
immunoprecipitated and histone H1 kinase activity was assayed [6].
Complete dephosphorylation and activation was obtained by addition
of GST–Cdc25C purified from baculovirus-infected insect cells.
Histone H1 kinase activity was normalized to the plus GST–Cdc25
control (+ Cdc25). Results are the average of three data sets and are
representative of 10 separate experiments. (b) The phosphorylation
state of Cdc2 was confirmed by immunoblotting of Cyclin B
immunoprecipitates. (c) Cell lysates from asynchronous (lane 1) and
irradiated (lane 2) cells were examined for the presence of Cdc25C by
western analysis. No loss of Cdc25 protein from the soluble fraction
was seen following irradiation and no change in the electrophoretic
mobility (due to phosphorylation of Ser216) was detected.
Activation correlated with loss of the inhibited phosphorylated form of Cdc2, visualized as the slower migrating
species on SDS–PAGE (Figure 1b). There was no
increase in the levels of Cdc2 or Cyclin B protein, and
phosphorylation by Wee1 and Myt1 was blocked by the
presence of 10 mM EDTA (data not shown).
Activation was prevented by vanadate, an inhibitor of
Cdc25 and other tyrosine phosphatases. Furthermore,
immunodepletion with Cdc25C-specific antisera showed
that activation of Cdc2–Cyclin B was dependent on the
presence of Cdc25 (Figure 1a). Thus, activation of Cdc2 is
the result of its dephosphorylation and is a measure of total
Cdc25 activity in the extract. In lysates of asynchronous
HeLa cells, the endogenous Cdc25 phosphatase activity
was sufficient to dephosphorylate and activate more than
80% of the available Cdc2–Cyclin B within 30 minutes
(Figure 1a,b). Analysis of lysates of HeLa cells in which
the DNA had been damaged by exposure to 10 Gray γirradiation, 1 hour before harvesting, showed a significant
reduction in the rate of activation of Cdc2, such that less
than 25% of the available Cdc2–Cyclin B was activated
during the 30 minute incubation. The amount of
Cdc2–Cyclin B in complex was not significantly altered
and it was activated to the same extent as control
Cdc2–Cyclin B by addition of a glutathione-S-transferase
(GST) Cdc25C fusion protein (GST–Cdc25C; Figure 1a).
Irradiation with 10 Gray led to more than threefold reduction in the rate of Cdc2 dephosphorylation in the 10 time
courses examined. The possibility that loss of Cdc25 activity could be due to a reduction in the amount of Cdc25
protein present in the lysates was considered.
Western analysis of the supernatants used for the above
assays (that had been centrifuged at 14,000 × g) showed
that the extracts prepared from asynchronous and irradiated cells contained equal quantities of Cdc25 (Figure 1c).
Thus, the reduced rate of Cdc2 dephosphorylation following irradiation is not due to loss of Cdc25 protein from the
extracts. Cdc25 has previously been shown to be phosphorylated on Ser216 in asynchronous cultures [18] and phosphorylation of this site causes a decrease in electrophoretic
mobility of Cdc25 [19]. As shown in Figure 1c, Cdc25
from extracts of asynchronous cells runs as a closely
spaced doublet with the majority of the protein running as
the upper, Ser216-phosphorylated species [19]. The proportion of Cdc25 migrating at the lower unphosphorylated
position was not changed in response to irradiation, suggesting that the reduced rate of Cdc2 dephosphorylation
following irradiation does not depend on increased phosphorylation of Ser216.
Radiation-induced inactivation of Cdc25 requires the
activity of kinases related to PI 3-kinases
If the inactivation of Cdc25 measured above is part of the
DNA-damage checkpoint response in human cells, then
experimental conditions that override the DNA-damage
checkpoint might be expected to block radiation-induced
inhibition of Cdc25. Genetic data from a number of organisms has identified a family of related kinases that are
required for DNA-damage responses [20]. Structurally,
these enzymes are related to the PI 3-kinases, and two
members of the family — DNA–protein kinase and the
product of the ATM gene — have been shown to be sensitive to wortmannin in vitro [21,22]. The possibility that a
wortmannin-sensitive kinase is upstream of the radiationinduced delay in M-phase entry was therefore tested [23].
HeLa cells can be arrested in M phase by nocodazole,
and irradiation causes cells to delay in G2 prior to the
Research Paper Checkpoint kinases directly inactivate Cdc25 Blasina et al.
3
Figure 2
(b)
(a) 60
40
30
20
10
Control
Irradiated
80
60
40
20
0
C
R
0
C
R
Wortmannin
Control
(c)
0
γ-irradiated
15
Time (min)
30
+
Cdc25
UV-irradiated
100 ATM-minus
Kinase activity
100 ATM-plus
80
60
40
80
60
40
20
20
0
Wortmannin
100
Kinase activity
Mitotic index
50
kinase activity
Radiation-induced inactivation of Cdc25
requires the activity of kinases related to
PI 3-kinases. (a) Wortmannin overrides the
radiation-induced delay in entry to M phase.
Cells were cultured in in the absence or
presence of 10 µM wortmannin for 1 h prior to
either mock treatment (C) or irradiation with
10 Gray (R); 100 ng/ml nocodazole was
added and, 14 h later, cells were fixed in 70%
ethanol, stained with Hoechst DNA stain and
mitotic index was scored. (b) Wortmannin
prevents the irradiation-induced
downregulation of Cdc25. Cells were cultured
in the presence of 10 µM wortmannin for 1 h
prior to either mock treatment (control) or
irradiation; extracts were prepared and
assayed as in Figure 1. (c) The ATM gene is
required for the downregulation of Cdc25
activity following γ-irradiation but not UV
irradiation. Kinase activity was assayed as
described for Figure 1a. The data shown are
the average of two data sets and are
representative of three independent
experiments.
0
15
Time (min)
30
+
Cdc25
0
0
15
Time (min)
30
+
Cdc25
Current Biology
nocodazole-sensitive M-phase block point. Thus, by
scoring the mitotic index of cells that are cultured in
nocodazole, it is possible to determine whether entry into
mitosis has been delayed. Control cells cultured in the
presence of nocodazole for 14 hours contained 60%
mitotic cells; the presence of wortmannin had little effect
on this number (Figure 2a). However, irradiation reduced
the number of cells that reached the nocodazole block
point to 10%. By contrast, irradiation in the presence of
wortmannin had only a modest effect on the number of
cells that reached the nocodazole block point (Figure 2a).
These results demonstrate that wortmannin overrides the
DNA-damage G2 checkpoint in HeLa cells. The effects
of wortmannin on the radiation-induced inactivation of
Cdc25 were therefore tested. Wortmannin had a minor
effect on the activation of Cdc2–Cyclin B in extracts prepared from non-irradiated cultures, although it greatly
diminished the irradiation-induced decrease in Cdc25
activity (Figure 2b).
A second experimental condition in which the radiationinduced G2 checkpoint is overridden is provided by cell
lines derived from patients with the genetic disorder AT.
ATM cells are defective in both the G1 and G2 checkpoints
following exposure to many, but not all, agents that damage
DNA [24–26]. The failure of ATM cells to delay in G1 correlates with a failure to upregulate the tumor suppressor
gene p53 [27] and with a failure to phosphorylate and activate the proto-oncoprotein c-Abl [28,29]. The molecular
basis of the failure to delay in G2 is not known. ATM cells
show greatly reduced responses to agents that generate
chromosomal breaks such as ionizing γ rays. Remarkably,
ATM cells have near-normal responses following the base
damage that is generated by irradiation with an ultraviolet
(UV) radiation source [26,30,31]. Therefore, the effects of
UV and γ-irradiation on the Cdc25 activity of normal
(ATM-plus) and ATM (ATM-minus) SV40-transformed
human fibroblast cell lines were investigated (Figure 2c).
ATM-minus cells respond to UV irradiation with a robust
reduction in the rate at which Cdc2 is dephosphorylated. In
contrast, γ-irradiation had only a modest effect on the rate
of dephosphorylation of Cdc2. In ATM-plus cells, the rate
of dephosphorylation of Cdc2 was significantly reduced following either ionizing radiation or UV radiation. Samples in
which Cdc2–Cyclin B was activated by addition of exogenous GST–Cdc25 showed that both cell types contained
similar quantities of Cdc2–Cyclin B following irradiation.
These data show that the ATM gene product is required for
the efficient inactivation of Cdc25 following γ-irradiation
and demonstrate a correlation between inactivation of
Cdc25 and delayed entry into M phase following DNA
damage [24–26].
Identification of a human homolog of fission yeast
checkpoint kinase Cds1
To identify potential mediators of the checkpoint-dependent inactivation of Cdc25, we searched for human
homologs of known yeast checkpoint genes. In view of the
4
Current Biology, Vol 9 No 1
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l
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s e
O tis
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Sm ry
C all i
ol nt
PB on est
in
L
e
(b)
2.2 kb–
Current Biology
similarity of mitotic control mechanisms in fission yeast
and mammals, our attention focused on two kinases, Chk1
and Cds1, that are required for the DNA-damage and
replication checkpoint in fission yeast [32,33]. An
expressed sequence tag (EST) with significant homology
to the cds1 gene of Schizosaccharomyces pombe was identified. The human cDNA predicts a translation product of
543 amino acids with a molecular weight of 61 kDa. The
Identification of a human homolog of Cds1. (a) A human cDNA
encoding an open reading frame of 534 amino acids with similarity to
checkpoint kinases from S. pombe and S. cerevisiae was identified.
Alignment of Cds1 homologs was generated using the CLUSTALW
program. Amino-acid identities are boxed, conservative changes are
shaded. Putative forkhead-associated domains are underlined.
HsCds1 is human Cds1; SpCds1 is a checkpoint kinase from the
fission yeast S. pombe; Rad53 and Dun1 are checkpoint kinases from
the budding yeast S. cerevisiae. (b) Northern analysis of HsCds1.
Blots containing 2 µg polyadenylated RNA from the indicated tissues
or cell lines were probed with an HsCds1 probe. PBL, peripheral
blood leukocytes.
predicted human Cds1 (HsCds1) protein is 28% identical
to the Cds1 protein of S. pombe, 28% identical to Rad53
and 27% identical to the Dun1 kinase of Saccharomyces
cerevisiae. Sequence alignment of these homologs
(Figure 3a) shows several regions of homology outside the
kinase domain, including conservation of the forkheadassociated domain [34]. The human protein shows the
same overall structure as Cds1 and Dun1 in that it lacks
the long carboxy-terminal extension found in Rad53.
Northern blot analysis with HsCds1 identified a single
transcript of ~2.2 kb expressed in testis and in eight
human cancer samples examined (Figure 3b).
Cds1 and Chk1 fusion proteins phosphorylate and inhibit
Cdc25 in vitro
To facilitate biochemical analysis, amino-terminal histidine6 (His6)-tagged Cds1 and Chk1 fusion proteins were
expressed in insect cells, affinity purified and incubated in
extracts of HeLa cells in the presence of an ATP-regenerating system. After 30 minutes at 30°C, EDTA was added
to inhibit kinases in the extract, and the rate of dephosphorylation and activation of Cdc2–Cyclin B was monitored as
in Figure 1. Both His6–Cds1 and His6–Chk1 were found to
significantly reduce the activation of Cdc2–Cyclin B in
these assays (Figure 4a). The reduced activation of Cdc2
was dose dependent and required ATP (data not shown).
Confirmation that Cdc2 was not irreversibly inhibited by
His6–Chk1 or His6–Cds1 was shown by the activation that
resulted when excess GST–Cdc25C was added after
kinase treatment. Thus, both His6–Cds1 and His6–Chk1
can mimic the radiation-induced downregulation of Cdc25
seen in extracts. These experiments used HeLa cell
lysates that had been clarified by centrifugation; therefore
it seemed unlikely that changes in subcellular localization
could account for the inactivation of Cdc25. Indirect mechanisms of inhibition could, however, not be excluded by
this assay. We therefore used affinity-purified reagents
(Figure 4c) to determine whether His6–Cds1 or His6–Chk1
can directly phosphorylate and inhibit GST–Cdc25 activity. GST–Cdc25 was incubated with either His6–Cds1,
mock beads or His6–Chk1 in the presence of [γ-32P]ATP
for 15 minutes at 30°C. Proteins were resolved by
SDS–PAGE and visualized by autoradiography
Research Paper Checkpoint kinases directly inactivate Cdc25 Blasina et al.
5
Figure 4
(a)
No ATP
+ ATP
His6–Chk1 His6–Cds1
(d)
100
kDa
197–
97–
Untreated
20
43–
0
0 30 30 0 30 30 0 30 30 0 30 30 Time (min)
– – + – –
+ GST–Cdc25
+– –
+– –
20
0
68–
40
40
28–
GST
(b)
(e)
GST–Cdc25
+ His6–Chk1+PP2A
60
60
GST–Cdc25
+ His6–Cds1
GST–Cdc25
+ His6–Cds1+PP2A
GST–Cdc25
+ His6–Chk1
80
80
GST–Cdc25
100
Kinase activity
Kinase activity
G
120
ST
–
H Cdc
is
25
6–
H Ch
is
k1
6–
C
ds
1
(c)
His6–Chk1
Cdc2
GST–Cdc25
0 5 10 10 10 10 10
His6–Cds1
His6–Chk1
10 GST–Cdc25
His6–Cds1
Cdc2
+
–
+
+
–
–
+
+
–
–
–
+
– GST–Cdc25
+ His6–Cds1
– His –Chk1
6
5
10 10
10
10 10 10 10 GST–Cdc25
Current Biology
His6–Cds1 and His6–Chk1 phosphorylate and inhibit Cdc25 in vitro.
(a) His6–Cds1 and His6–Chk1 downregulate the dephosphorylation of
Cdc2 in HeLa cell extracts. Recombinant His6–Cds1 or His6–Chk1
purified from insect cells was incubated with HeLa cell extracts in the
presence of an ATP-regenerating system for 30 min at 30°C [57].
Control extracts incubated at 30°C in the presence of ATP (+ ATP) or
maintained on ice for 30 min (no ATP) show similar kinetics of
activation. Dephosphorylation and activation of Cdc2–Cyclin B was
initiated by addition of excess EDTA at the zero time point. Data are
the average of three experiments. (b) GST–Cdc25 purified from insect
cells was incubated with either His6–Cds1, mock beads or His6–Chk1
in the presence of [γ-32P]ATP for 15 min at 30°C. GST–Cdc25 bound
to GSH–sepharose was washed three times prior to addition of SDS.
Proteins were resolved by SDS–PAGE and visualized by
autoradiography. The authenticity of each band was confirmed by reprecipitation of the labeled bands with the relevant sera. (c) Affinitypurified GST–Cdc25, His6–Chk1 and His6–Cds1 were resolved by
SDS–PAGE and visualized by Coomassie blue staining.
(d) Both His6–Cds1 and His6–Chk1 inhibit purified GST–Cdc25.
GST–Cdc25c from insect cells was incubated with His6–Cds1,
His6–Chk1 or buffer in the presence of 1 mM ATP for 30 min at 30°C.
The sample was split and either incubated alone or with PP2A for
30 min at 30°C. GST–Cdc25 was washed three times with
phosphatase buffer containing 0.4 µM microcystin prior to incubation
with Cdc2–Cyclin B immunoprecipitates from asynchronous HeLa
cells. The histone H1 kinase activity of Cdc2–Cyclin B was
determined. (e) Reduced GST–Cdc25 activity after phosphorylation
by His6–Chk1 or by His6–Cds1. GST–Cdc25 was assayed by its
ability to convert Cdc2 to the faster-migrating dephosphorylated form.
In the presence of GST–Cdc25, the majority of Cdc2 is converted to
the lower band of the triplet. Prior phosphorylation of GST–Cdc25 by
His6–Chk1 or His6–Cds1 reduced the appearance of the
dephosphorylated form. GST–Cdc25 was phosphorylated with
increasing quantities of each kinase. The numbers refer to relative
quantities of proteins added and are arbitrary. The results are
representative of five independent experiments.
(Figure 4b). As previously shown [35], GST–Cdc25 was
phosphorylated by His6–Chk1. GST–Cdc25 was also phosphorylated by His6–Cds1 in vitro.
GST–Cdc25 by His6–Cds1 or by His6–Chk1 inhibited the
ability of GST–Cdc25 to activate Cdc2–Cyclin B
(Figure 4d). Inhibition was dependent on the presence of
ATP, was seen at a molar ratio of kinase to GST–Cdc25 of
1:100, and was reversed by treatment of GST–Cdc25 with
protein phosphatase 2A (PP2A; Figure 4d).
Experiments were performed to determine if Cdc25 phosphatase activity was effected by phosphorylation. In one
set of experiments, GST–Cdc25 was assayed by its ability
to activate the histone H1 kinase activity of
Cdc2–Cyclin B immunoprecipitates. Phosphorylation of
These results were somewhat surprising in the light of
previous reports that Chk1 does not inactivate Cdc25 [19]
6
Current Biology, Vol 9 No 1
dc
25
activity [37,38] and extend them by showing that the negative regulation involves inactivation of the phosphatase
activity and that a second checkpoint kinase also phosphorylates and inactivates Cdc25.
ST
–C
C
6–
Increased 14-3-3 binding is not required for inhibition of
Cdc25 in vitro
G
is
H
H
eL
a
ce
ll l
y
sa
te
(a)
hk
1
Figure 5
–14-3-3
–
–
(b)
–
+
–
–
+
+
+ His6–Chk1 (–ATP)
– HeLa (–ATP)
–GST–Cdc25
–14-3-3
–
– Cdc2
–
S
1
2
3
4
5
Current Biology
Inhibition of GST–Cdc25 correlates with Chk1 phosphorylation and
not with increased binding of 14-3-3 protein. (a) Purified GST–Cdc25
(~1 µg) and ~1 µg purified His6–Chk1 (see Figure 4) were probed
with a broad specificity anti-14-3-3 rabbit antiserum; 2 µg HeLa cell
lysate was run alongside for comparison. (b) GST–Cdc25C purified
from Sf9 cells was not treated (lane 1), mock phosphorylated (lanes
2,3), or phosphorylated with His6–Chk1 (lanes 4,5). Beads were
washed three times and either not treated (lane 1), or treated with
HeLa cell extracts in the presence of an ATP regenerating system for
30 min at 30°C (lanes 2,4), or in the presence of 10 mM EDTA (lanes
3,5). Samples were washed three times and divided into two parts for
the Cdc25 assay and for western analysis. The uppermost panel
shows that a constant amount of GST–Cdc25 was recovered in each
sample. The middle panel shows increased binding of 14-3-3 protein
in samples that were exposed to HeLa cell extracts. The left and right
hand sides of these panels have been cropped from a single gel to
reflect the same order as the lowermost panel, which shows that
dephosphorylation of Cdc2 is inhibited by Chk1 phosphorylation but
not by increased 14-3-3 binding. S corresponds to the starting
substrate. The data are representative of three separate experiments.
and that Cds1 phosphorylates Wee1 in fission yeast [36].
We therefore used a second assay system in which
dephosphorylation of Cdc2 was monitored by the disappearance of the slower migrating species of Cdc2 on gelmobility analysis (Figure 4e). In these assays, Cdc25
activity was measured in the presence of 10 mM EDTA
and in the absence of ATP, conditions that eliminate the
possibility of His6–Chk1 or His6–Cds1 phosphorylating
Cdc2 or Cyclin B. GST–Cdc25 catalysed a reduction in
the slower migrating phosphorylated forms of Cdc2. Prior
phosphorylation of GST–Cdc25 by His6–Chk1 or
His6–Cds1 led to a dose-dependent reduction in
GST–Cdc25 activity (Figure 4e). These data confirm
genetic predictions that Chk1 negatively regulates Cdc25
The 14-3-3 protein has been implicated in the regulation
of Cdc25 by Chk1 [19,35]. Therefore, the possibility that
14-3-3 binding is required for inactivation of Cdc25 was
tested. The GST–Cdc25 and the His6–Chk1 preparations
used in these experiments were examined for the presence of 14-3-3 proteins by western analysis. As shown in
Figure 5a, 14-3-3 protein was readily detectable in a
sample containing ~1 µg affinity-purified GST–Cdc25 but
not in the adjacent lane which contained ~1 µg
His6–Chk1; 2 µg whole HeLa cell lysate was run on the
same blot for comparison. The 14-3-3 proteins bind with
high affinity to specific phosphoserine-containing proteins
[39,40]; the absolute requirement for phosphorylation of a
serine residue within the 14-3-3-binding motif suggested
that GST–Cdc25 expressed in insect Sf9 cells is, at least
partially, phosphorylated at the 14-3-3-binding site. In
contrast, 14-3-3 proteins were not detected in the preparation of His6–Chk1. This finding suggested that inactivation of GST–Cdc25 occurs in the presence of a constant
amount of 14-3-3 protein. GST–Cdc25 was also purified
from bacteria free of 14-3-3 proteins; however, the very
low specific activity of this protein made activity assays
unreliable (data not shown).
To test further the requirement for 14-3-3 binding in the
inactivation of GST–Cdc25, we took advantage of the
observation that GST–Cdc25 becomes phosphorylated on
Ser216 and binds 14-3-3 protein on incubation in eukaryotic cell extracts ([18,41] and data not shown). As shown in
Figure 5b, approximately fivefold more 14-3-3 protein was
found in association with GST–Cdc25 following incubation in HeLa cell lysates in the presence of ATP, and no
decrease in Cdc25 activity was detected in these samples.
By contrast, GST–Cdc25 that was phosphorylated using
purified His6–Chk1 was inhibited whether or not
increased 14-3-3 protein was bound. Thus, inactivation of
Cdc25 correlates with Chk1 phosphorylation and not with
increased binding of 14-3-3 protein.
The observations presented here — that irradiation does
not lead to increased phosphorylation of Ser216 and that in
vitro inactivation of Cdc25 does not require increased 14-33 binding — suggest that inactivation of Cdc25 may result
from phosphorylation of sites other than Ser216. Previous
in vitro mapping studies using mammalian Chk1 and
Cdc25C showed that Ser216 is the major in vitro site of
phosphorylation of Cdc25C by Chk1 [35]. Mapping experiments in vitro, using Chk1 and Cdc25 from other species,
showed that phosphorylation of 14-3-3-binding sites by
Research Paper Checkpoint kinases directly inactivate Cdc25 Blasina et al.
7
Figure 6
(a)
His6–Cds1–
–p64Cds1
Cds1 is activated in response to DNA damage
Having determined that His6–Cds1 inactivates Cdc25 in
vitro and that Cdc25 is inactivated in vivo following DNA
damage, we were interested in determining whether DNA
damage might lead to modification or activation of human
Cds1. To facilitate analysis of endogenous human Cds1,
polyclonal antisera were raised against His6–Cds1. The
specificity of the antisera was tested by immune-precipitation kinase assay in the presence of [γ-32P]ATP. The
immunoprecipitates were examined by autoradiography
following SDS–PAGE. A signal corresponding to
His6–Cds1 was readily detected in immune-precipitation
kinase assays from extracts of Sf9 cells infected with baculovirus encoding His6–Cds1 (Figure 6a), but not in cells
infected with virus encoding His6–Chk1 nor in uninfected
cells. A very weak signal corresponding to HsCds1 was
detected in the immunoprecipitate from asynchronous
HeLa cells; increased phosphorylation of HsCds1 was
seen following irradiation. That this band represents Cds1
5
6
8
9
le
–
+
–
+
H
yd
ro
xy
az
o
id
in
e
Th
ym
As
yn
ch
ro
– +
7
ur
ea
4
oc
od
3
no
us
(b)
–
+ γ-irradiation
p64Cds1–
GST–Cdc25–
(c)
100
G2/M
S
G1
80
60
40
Hydroxyurea
Hydroxyurea
(irradiated)
Nocodazole
Nocodazole
(irradiated)
Thymidine
Thymidine
(irradiated)
Asynchronous
(irradiated)
Asynchronous
20
0
Chk1 is highly conserved [42,43]. Nevertheless, Cdc25 is
phosphorylated by Chk1 at a number of sites in Xenopus
and fission yeast [42,43]. Future experiments will be necessary to determine whether inactivation of human Cdc25
is the result of phosphorylation of novel sites that are not
readily detected by in vitro mapping experiments.
Although these data indicate that 14-3-3 binding is not
required for inactivation of Cdc25, an important role for 143-3 binding in determining the localization or sequestration
of Cdc25 [19,44] is not precluded by this in vitro analysis.
2
N
1
Percentage of cells
Activation of HsCds1 in response to DNA damage. (a) Anti-Cds1
antisera immunoprecipitates prepared from 100 µg (unless otherwise
indicated) Sf9 cell extract were incubated in the presence of 10 µCi
[γ-32P]ATP for 15 min at 30°C. Proteins were resolved by SDS–PAGE
and visualized by autoradiography. Lane 1: uninfected Sf9 cells; lane
2: cells infected with recombinant virus encoding His6–Chk1; lane 3:
cells infected with recombinant virus encoding His6–Cds1; lanes 4,5:
400 µg irradiated HeLa cell extract (lane 4) and asynchronous HeLa
cell extract (lane 5); lanes 6,7: 400 µg irradiated HeLa cell extract
boiled in 4% SDS, diluted 10-fold in phosphate-buffered saline
containing 2% Triton X-100 and reprecipitated with anti-Cds1
antiserum in the absence (lane 6) or the presence (lane 7) of ~2 µg
purified His6–Cds1; lanes 8,9: 5 and 10%, respectively, of the initial
immune-complex kinase assay. (b) Anti-Cds1 antibody
immunoprecipitates prepared from 400 µg HeLa cell extract were
incubated in the presence of 10 µCi [γ-32P]ATP for 15 min at 30°C.
The upper panel shows the phosphorylation of Cds1. The lower panel
shows phosphorylation of GST–Cdc25 by Cds1 immunoprecipitates.
GST–Cdc25 here corresponds to the first 145 amino acids of fission
yeast Cdc25 expressed as a GST fusion protein in bacteria. No
phosphorylation of GST–Cdc25 was detected when pre-immune
serum was used (data not shown). Cells were accumulated with
unreplicated DNA by addition of 2 mM thymidine for 17 h, or by
addition of 2 mM hydroxyurea for 17 h. Cells were accumulated in M
phase by growth in the presence of 100 ng/ml nocodazole for 18 h.
Where indicated, cultures were exposed to 10 Gray ionizing radiation,
1 h prior to harvesting. (c) Cell-cycle position was determined by flow
cytometric analysis of DNA content.
Current Biology
and not an associated protein was confirmed as we could
reprecipitate the protein following denaturation in 4%
SDS in the absence, but not in the presence, of excess
purified His6–Cds1 (Figure 6a).
The phosphorylation of Cds1 in vitro most likely represents autophosphorylation, in which case the increased
signal reflects an increase in activity following irradiation,
and the increased autophosphorylation should be paralleled by increased phosphorylation of an exogenous substrate. Cds1 was therefore immunoprecipitated and
assayed both for autophosphorylation activity and for the
ability to phosphorylate an exogenous substrate. For practical reasons, fission yeast Cdc25 fused to GST and
expressed in bacteria provided a convenient substrate (data
not shown). As shown in Figure 6b, Cds1 was activated by
irradiation of asynchronous cells. The increased phosphorylation of p64Cds1 suggests that, like Rad53 and Dun1
[45–47], HsCds1 is activated in response to DNA damage.
Fission yeast Cds1 has recently been shown to be activated
in response to incompletely replicated DNA [36,48]. We
therefore examined the consequences of arresting DNA
synthesis on the activity of HsCds1. No significant increase
in autophosphorylation or phosphorylation of GST–Cdc25
8
Current Biology, Vol 9 No 1
Figure 7
γ-irradiation
ATM
Cds1/Chk1
Cdc25
Cdc2–Cyclin B
Inactive
Cdc2–Cyclin B
Active
Current Biology
Model of the DNA-damage G2 checkpoint in human cells. The product
of the ATM gene is required for the efficient inactivation of Cdc25 in
response to γ-irradiation. By analogy with model systems, the ATM
protein is assumed to be upstream of Chk1 and Cds1 [2].
Phosphorylation of Cdc25 by Cds1 and/or Chk1 inhibits its activity and
thereby prevents dephosphorylation and activation of Cdc2–Cyclin B.
was seen in response to thymidine or hydroxyurea
(Figure 6b). Both agents blocked DNA replication and led
to the accumulation of cells with a 2N content of DNA
(Figure 6c). Increased phosphorylation of p64Cds1 and
GST–Cdc25 was detected following irradiation of thymidine-arrested or hydroxyurea-arrested cells.
Finally, the effect of damaging DNA in cells that have
been predominantly arrested outside S phase was tested.
Cells were cultured in the presence of nocodazole for
20 hours prior to irradiation. Again a weak, but
detectable, signal was seen in the non-irradiated sample,
whereas irradiation of nocodazole-arrested cells led to
increased activity (Figure 6b). In all circumstances
tested, the increased in vitro phosphorylation of HsCds1
was closely paralleled by increased phosphorylation of
the exogenous substrate. These data strongly suggest
that Cds1 is activated in response to DNA damage. The
possibility that the signal is due to an associated kinase,
which also phosphorylates GST–Cdc25, cannot be totally
excluded. Nevertheless, the observation that HsCds1 is
modified or activated in response to DNA damage, but
not in the presence of unreplicated DNA, contrast with
the situation in fission yeast, suggesting a role for human
Cds1 in the DNA-damage checkpoint rather than in the
replication checkpoint.
This study was based, in part, on the premise that checkpoint control proteins are conserved between organisms.
That assumption has facilitated the cloning of a number of
human checkpoint genes ([35] and this work). Despite the
structural homology, some these proteins may not be true
functional homologs; for example, in fission yeast, chk1
mutants are highly sensitive to DNA damage and are
resistant to agents that block DNA replication [49],
whereas mutants in the homologous Drosophila gene,
grapes, respond normally to DNA damage but are sensitive
to delayed DNA synthesis [50,51]. Likewise, for Cds1,
fission yeast mutants are sensitive to agents that block
replication but have normal responses to DNA damage
[32], whereas the budding yeast homologs, Rad53 and
Dun1, have functions in both the replication and DNAdamage responses [45,52]. In this study, we have defined
a mechanism by which both human Cds1 and Chk1 might
contribute to the G2–M checkpoint (Figure 7). The possibility that Cds1 carries out some of the other
checkpoint/repair functions undertaken by its structural
homologs remains to be explored.
Materials and methods
Cell lines
Normal (GM637G) and AT-deficient (GM5849C) SV40-transformed
human fibroblasts obtained from Coriell Institute for Medical Research,
(Camden, NJ) were grown in DMEM supplemented with 15% fetal
bovine serum, 100 µg/ml penicillin and streptomycin. For γ-irradiation
experiments, cells were irradiated with a cesium137 source in a Gamma
Cell 1000 at a rate of 3.8 Gray/min. For UV irradiation experiments,
cells were washed once with PBS and irradiated at 254 nm in a UV
Stratalinker 1800.
Assays and western analysis
Cells were lysed in ice-cold lysis buffer (50 mM Tris pH 7.4 containing
2 mM MgCl2, 1 mM phenylmethylsulphonyl fluoride, and 5 µg/ml leupeptin, pepstatin and aprotinin). Lysates were cleared by centrifugation
at 14,000 × g for 10 min and the protein concentration of the supernatants determined using the Lowry assay. Dephosphorylation of Cdc2
was initiated by addition of 10 mM EDTA and incubation at 30°C. At the
indicated time, the activity of Cdc2–Cyclin B was assayed by measuring
the histone H1 kinase activity present in anti-Cyclin B immunoprecipitates [6]. For immunoblots, 400 µg cell lysate was immunoprecipitated
using anti-Cyclin B antibody and resolved on 11% acrylamide–SDS
gels. Monoclonal antibody against the PSTAIRE motif of Cdc2 was
used to detect the different phospho-forms of Cdc2 (gift of S. Reed,
TSRI). Polyclonal anti-14-3-3 serum (k-19) was from Santa Cruz
Biotechnology. For flow cytometric analysis, cells were fixed in 70%
ethanol prior to treatment with 100 µg/ml RNAse and 40 µg/ml propidium iodide as recommended by Becton Dickinson. Cells were analyzed
on a Becton Dickinson FACSort. The proportion of cells in G1, S or
G2/M was determined using CellQuest software.
Recombinant baculoviruses and protein production
Recombinant viruses encoding His6–Chk1, His6–Cds1, His6–Wee1,
His6–Myt1, His6–Cdc2 and GST–Cdc25C were generated using the
Bac-to-Bac expression system from Gibco/BRL. His6-fusion proteins
were purified following the procedure described in [53].
GSH–sepharose beads were incubated for 15 min in Sf9 extracts. The
beads were collected by centrifugation and washed three times with
lysis buffer (50 mM Tris pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.1%
NP40, 5% glycerol, 0.1% β-mercaptoethanol and protease inhibitors).
Beads were washed twice with kinase assay buffer (50 mM Tris pH 7.4
10 mM MgCl2) prior to phosphorylation reactions or twice with phosphatase assay buffer (50 mM imidazole pH 7.4, 5 mM EDTA and 0.1%
β-mercaptoethanol) prior to phosphatase assays. Phosphorylated
Cdc2 was purified from Sf9 cells that had been simultaneously
infected with recombinant baculoviruses encoding His6–Cdc2,
His6–Wee1, His6–Myt1 and GST–Cyclin B [54]. His6–Cdc2 complexed to Cyclin B was purified using GSH beads using the conditions
described for GST–Cdc25 except that 1 mM VO4 was included in the
lysis buffer. The catalytic subunit of PP2A was purified from rabbit
Research Paper Checkpoint kinases directly inactivate Cdc25 Blasina et al.
skeletal muscle [55]. Western analysis showed that quadruple infection
resulted in phosphorylation of the majority of Cdc2–GST–Cyclin B at
one or both inhibitory sites.
Cloning
A search for sequences similar to S. pombe cds1+ was carried out
using the TBLASTN program. A human EST (No. 864164) was identified in the proprietary LifeSeq® database (Incyte Pharmaceuticals Inc.,
Palo Alto, USA). Sequence analysis of the 1.3 kb insert revealed an
incomplete open reading frame which was highly similar to the S.
pombe cds1. Approximately 650 nucleotides of novel 5′ DNA
sequence were obtained by 5′ rapid amplification of cDNA ends
(RACE). Termination codons were present in all three reading frames in
the 120 nucleotides immediately 5′ to the putative HsCds1 initiation
codon, indicating that the complete coding region had been isolated.
The sequence shown here is identical to two partial sequences in the
National Centre for Biotechnology Information (NCBI) databases. The
EST (AA285249) and genomic sequence (H55451) most likely
encode the same protein product. The EMBL accession number for
HsCds1 is AJ131197.
A human EST encoding a protein with homology to S. pombe Chk1
[33] and to Drosophila Grapes [51] was identified by BLAST analysis
of the NCBI database. Clones were retrieved from ATCC (IMAGE
Clone ID 663485) and sequenced. The human Chk1 homolog used
here is identical to the one described by Sanchez et al. [35], with the
exception of three amino-acid substitutions (H/N at amino acid 64, R/H
at 141 and N/M at 410 using single-letter amino-acid code).
Antibodies
Antibodies to HsCds1 were generated by immunizing a rabbit with
His6–Cds1 purified from Sf9 cells according to [56]. The resulting sera
immunoprecipitates an active kinase of the expected molecular weight
from Sf9 cells infected with His6–Cds1 virus but not from uninfected
Sf9 cells or from cells infected with His6–Chk1 virus (data not shown).
Acknowledgements
We thank Beth Furnari and Paul Russell for providing GST–Cdc25. This
work was supported by DOD grant DAMD1794-J-4227 grant to C.H.McG.
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