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Molecular and Cellular Pathobiology
Autoregulatory Mechanisms of Phosphorylationof Checkpoint Kinase
1
Jingna Wang, Xiangzi Han, and Youwei Zhang
AbstractCheckpoint kinase 1 (Chk1), a serine/threonine protein
kinase, is centrally involved in cell-cycle checkpoints
and cellular response toDNAdamage. Phosphorylation of Chk1 at 2
Ser/Gln (SQ) sites, Ser-317 and Ser-345, by theupstream kinase ATR
is critical for checkpoint activation. However, the precise
molecular mechanismscontrolling Chk1 phosphorylation and subsequent
checkpoint activation are not well understood. Here, wereport
unique autoregulatory mechanisms that control protein
phosphorylation of human Chk1, as well ascheckpoint activation and
cell viability. Phosphorylation of Ser-317 is required, but not
sufficient, for maximalphosphorylation at Ser-345. The N-terminal
kinase domain of Chk1 prevents Chk1 phosphorylation at
theC-terminus by ATR in the absence of DNAdamage. Loss of the
inhibitory effect imposed by the N-terminus causesconstitutive
phosphorylation of Chk1 by ATR under normal growth conditions,
which in turn triggers artificialcheckpoints that suppress the
S-phase progression. Furthermore, two point mutations were
identified thatrendered Chk1 constitutively active, and expression
of the constitutively active mutant form of Chk1 inhibitedcancer
cell proliferation. Our findings therefore reveal unique regulatory
mechanisms of Chk1 phosphorylationand suggest that expression of
constitutively active Chk1 may represent a novel strategy to
suppress tumorgrowth. Cancer Res; 72(15); 3786–94. �2012 AACR.
IntroductionIn response to replication perturbation or DNA
damage,
cells activate elegant genome surveillance pathways,
calledcell-cycle checkpoints, to counter these assaults. Central
tothese surveillance pathways are 2 protein kinases, theupstream
kinase, ATR (ataxia telangiectasia mutated andRad3 related), and
its downstream target kinase, checkpointkinase 1 (Chk1). Complete
loss of CHK1 or ATR leads toembryonic lethality in mice (1–3). On
the other hand, partialloss of these 2 genes, for instance loss of
one copy of CHK1 ora hypomorphic mutation in ATR, increased genome
insta-bility and caused spontaneous cell death even in the
absenceof extrinsic stress (4–6). These findings suggest that these
2proteins play key roles in monitoring the DNA replicationand in
maintaining the genome integrity (7). Therefore,targeting ATR and
Chk1 has the potential to selectivelyenhance the antitumor effect
for tumors that undergoincreased replicative stress (8, 9).
Chk1 primarily responds to replication fork interference inthe S
phase and DNA damage at the G2 phase (10–12). Chk1 iscomposed of a
highly conserved kinase domain at the N-terminal half and a
regulatory region at the C-terminal half.The C-terminus contains a
Ser/Gln (SQ) motif and 2 highlyconserved motifs (CM1 and CM2, Fig.
1A). Recent studies fromthis laboratory and others showed a model
of Chk1 activationthat requires protein conformation change of
Chk1. Undernormal growth conditions, Chk1 seems to adopt a
"closed"conformation through an intramolecular interaction
betweenthe N-terminus and the C-terminus (13–15). This
closedconformation not only suppresses the kinase activity of
Chk1but also stabilizes the protein (14, 16). Upon DNA damage,Chk1
undergoes ATR-dependent phosphorylation on chroma-tin (17, 18).
This phosphorylation seems to disrupt the intra-molecular
interaction, leading to an "open" conformation ofChk1 followed by
checkpoint activation (16).
Phosphorylation of Chk1 at 2 conserved ATR sites, Ser-317and
Ser-345, has long been viewed as the gold standard for
theactivation of replication checkpoints. However, precise
molec-ular mechanisms controlling Chk1 phosphorylation are lesswell
understood. In this study, we uncover a number of novelmechanisms
buried within the Chk1 polypeptide that controlChk1 protein
phosphorylation, checkpoint activation, and themaintenance of cell
viability.
Materials and MethodsCell cultures, transfection, and cell
proliferation
HEK293T, HeLa, U2-OS, and A549 cells were cultured inDulbecco's
Modified Eagle's Medium with 10% FBS. Transfec-tion was carried out
with either calcium phosphate or
Authors' Affiliation: Department of Pharmacology, Case
ComprehensiveCancer Center, Case Western Reserve University,
Cleveland, Ohio
Note: Supplementary data for this article are available at
Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
J. Wang and X. Han contributed equally to this work.
Corresponding Author: Youwei Zhang, Department of
Pharmacology,Case Comprehensive Cancer Center, Case Western Reserve
University,2109 Adelbert Road,WoodBuildingW343A, Cleveland, OH
44106. Phone:216-368-7588; Fax: 216-368-1300; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-12-0523
�2012 American Association for Cancer Research.
CancerResearch
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Lipofectamine 2000 (Invitrogen). For the cell
proliferationassay, HEK293T cells were transfected with GFP,
GFP-Chk1wild type (WT), or the L449R mutant for 48 hours. The
cellswere reseeded at a density of 1 � 104 cells per well in
6-wellplates and cultured for 8 days. On each day, the number of
GFP-positive cells within a colony was counted under
fluorescencemicroscopy.
Plasmid construction and mutagenesisMyc- orGFP-tagged vectors
expressing Chk1WTormutants
were generated using standard PCRs. Point mutations werecarried
out using the Quick Change Mutagenesis Kit (Strata-gene). Primer
information will be provided upon request.
Cell-cycle analysis, immunoblotting, and antibodiesCell-cycle
analyses and immunoblotting were carried out as
previously described (17, 19). Anti-Chk1 (DCS-1310 and G4)and
anti-ATR (N-19) antibodies were from Santa Cruz.
Anti–phospho-S317-Chk1, anti–phospho-S345-Chk1,
anti–phospho-S1981-ATM, and anti–phospho-S216-Cdc25C were from
CellSignaling. Anti-MCM7 and anti–Cyclin B were from BD
Phar-mingen. Anti-Cdc25A was from NeoMarkers.
EdU stainingHeLa Tet/Off cells grown on glass cover slips were
trans-
fected with tetracycline-controlled GFP, GFP-Chk1 WT, or
theL449R mutant for 24 hours, synchronized at the G2–M phasewith
100 ng/mL nocodazole for 20 hours in the presence ofdoxcycline,
washed twice with 1 � PBS, and released intofresh medium without
doxcycline. After a 16-hour releasefrom nocodazole, cells were
pulse labeled with 10 mmol/L5-ethnyl-20-deoxyuridine (EdU), a
nucleotide analog, for20 minutes at 0-, 4-, 8-, and 10-hour time
period. Cells werethen washed with ice-cold PBS and fixed with 3.7%
formalde-hyde at room temperature for 10 minutes and followed
withthe Click-iT kit to measure EdU incorporation according tothe
manufacturer's instruction (Invitrogen).
ResultsThe kinase CM1 or CM2 domain is not essentialfor human
Chk1 phosphorylationThe kinase CM1 and CM2 domains of Chk1 (Fig.
1A) are
highly conserved among different species. However, whetherthese
domains are required for initiating Chk1 phosphoryla-tion at ATR
sites is unknown. To address this question, wegenerated Myc-tagged
mutants in which the kinase domain(theCmutant), theCM2domain (the
1-421mutant) or theCM1plus CM2 domain (the 1-368 mutant) of human
Chk1 weredeleted (Fig. 1A). All these mutants contain the 2 ATR
phos-phorylation sites, Ser-317 and Ser-345. HEK293T cells
expres-sing these mutants were treated with a DNA-damagingagent,
the topoisomerase 1 inhibitor, camptothecin (CPT), andimmunoblotted
with anti–phospho-Chk1 antibodies. Theresults showed that deletion
of the kinase CM2 or the CM1plus CM2 domain did not abolish Chk1
phosphorylation(Fig. 1B, lanes 10–12). These data suggested that
none of these3 conserved domains were essential for Chk1
phosphorylationat ATR sites.
Ser-317 is required for phosphorylation at Ser-345Previous
studies reported that mutating the Ser-317 to Ala
abolished phosphorylation at the Ser-345 site of Chk1 (15,
20).Here we asked whether the phospho-mimic S317E mutationwould
induce Chk1 phosphorylation at Ser-345. Consistentwith previous
publications, the S317Amutant failed to undergophosphorylation at
either the Ser-317 or the Ser-345 site (Fig.1B, lane 6). However,
the S317E mutant also failed to bephosphorylated at the Ser-345
site (Fig. 1B, lane 7), indicatingthat S317E is not a true
phosphomimicmutation or the Ser-317residue is critical for
phosphorylation at Ser-345. In contrast,mutating the Ser-345 site
to Ala or Glu only moderatelyreduced Chk1 phosphorylation at the
Ser-317 site comparedwith the Chk1 WT (Fig. 1B, lanes 2, 8–9).
To further test this idea, we examined protein phosphory-lation
of more refined mutations of the Chk1 C-terminusexpressing one or
both phosphorylation sites (C1 to C5in Fig. 1C) upon CPT treatment.
The small fragment C1, whichonly contains the Ser-317 site, was
phosphorylated at the Ser-317 site (Fig. 1D, lanes 3–4). However,
the C2, C4, or C5fragment, which only contains the Ser-345 site,
was not phos-phorylated at Ser-345 (Fig. 1D, lanes 5–6 and 9–12).
Only whenthe fragment contains both Ser-317 and Ser-345 sites
(i.e., theC3 fragment), can phosphorylation at Ser-345 be detected
(Fig.1D, lanes 7–8). These data suggested that either the
Ser-317residue or its phosphorylation is required for
phosphorylationat Ser-345, but not the other way around (15, 20,
21).
Ser-317 phosphorylation is not sufficient for
maximalphosphorylation at Ser-345
We recently reported that 3 highly conserved Arg
residues(R372/376/379, Supplementary Fig. S1B) in the CM1 region
ofChk1 play an important role in maintaining Chk1
proteinconformation (14). Thus, we asked whether mutating
theseresidues could affect Chk1 phosphorylation upon DNA dam-age.
Our results showed that Chk1 phosphorylation at both Ser-317 and
Ser-345 in the 3RE mutant was significantly reducedcompared with
the Chk1 WT (Fig. 1B, lanes 2 and 4). Thisseemed to be because of
the significantly increased cyto-plasmic localization of this 3RE
mutant (14). On the otherhand, the 3RA mutant is located mainly in
the nucleus like theWT (data not shown). Interestingly, although
the 3RA mutantwas highly phosphorylated at the Ser-317 site,
phosphorylationat the Ser-345 site was significantly reduced
comparedwith theChk1WT (Fig. 1B, lanes 2–3). We also noticed that
the Chk1 (1-421) mutant exhibited more profound reduction in
phosphor-ylation at the Ser-345 site than the Ser-317 site compared
withthe Chk1 WT (Fig. 1B, lanes 2 and 10). The Chk1 kinase
dead(D148A) mutant exhibited reduced phosphorylation at bothsites
(Fig. 1B, lane 5), probably because this mutant is lessstable than
the Chk1 WT (Supplementary Fig. S1A). Theseresults showed that high
level phosphorylation at Ser-317 doesnot necessarily correlate with
high-level phosphorylation atSer-345. Thus, even though the CM1 and
CM2 domains are notessential for initiating Chk1 phosphorylation,
they seem tocontribute to maximal phosphorylation at the Ser-345
site byDNAdamage. These data are consistent with yeast
Chk1whoseC-terminus contributed to the full activation of Chk1
(22).
Autoregulation of Chk1 Phosphorylation
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Together, these results indicated that phosphorylation atSer-317
is necessary, but not sufficient, for high level of
Chk1phosphorylation at the Ser-345 site. This is in line with the
ideathat phosphorylation at the Ser-345 site is the final
determi-nant of full activation of Chk1 (21). Therefore, we
focusedmainly on Chk1 phosphorylation at the Ser-345 site for
thefollowing studies.
ATR-dependent constitutive phosphorylationof the Chk1
C-terminus
During our analysis, we unexpectedly discovered that theChk1
C-terminus devoid of the kinase domain was constitu-tively
phosphorylated at both Ser-317 and Ser-345 sites (Fig. 1Dand 2A,
lane 1). CPT treatment did not further increase
proteinphosphorylation compared with the basal state (Fig. 1Dand
2A, lanes 1–2). We also noticed a weak constitutivephosphorylation
of the C3 fragment in the absence of DNAdamage (Fig. 3A lane 5 and
Fig. 3B lane 6), similar to the entireChk1 C-terminus (Fig. 3B,
lane 3). These results suggested thatthe N-terminal kinase domain
suppresses phosphorylation ofresidues located at the C-terminus of
Chk1 in the absence ofDNA damage. To understand the biologic
implications of this
constitutive phosphorylation of theChk1C-terminus, we
askedwhether this Chk1 C-terminus displayed properties similar
tothose that govern phosphorylation regulation as the
full-length(FL) Chk1.
First, we observed that the constitutive phosphorylation ofthis
fragment was completely abolished when Ser-317 wasmutated to Ala or
Glu (Fig. 2A, lanes 3–4). Mutating the Ser-345to Ala or Glu
significantly reduced the constitutive phosphor-ylation of the Chk1
C-terminus (Fig. 2A, lanes 5–6); however,treating these cells with
CPT increased Ser-317 phosphoryla-tion (data not shown). These
results suggested that the Chk1C-terminus undergoes phosphorylation
in a way similar to theChk1 FL protein (Fig. 1A). Second, we asked
whether ATR isalso responsible for this constitutive
phosphorylation of theChk1 C-terminus. Our results showed that
inhibiting ATR, andto a lesser extent, ATM, but not DNA-PK, reduced
the levels ofphosphorylated proteins of this Chk1 C-terminal
fragment(Fig. 2B). In parallel, similar effects were observed on
phos-phorylation of the Chk1 FL induced by CPT (Fig. 2C).
Further-more, we showed that depletion of ATR, but not ATM,
clearlyreduced the level of constitutive phosphorylation of the
Chk1C-terminal fragment under normal growth condition (Fig. 2D,
Kinase SQ CM1 CM2FL (1-476)
1-4211-368
C (265-476)
α-Myc
CPT+-WT(FL) S3
45E
S345
A
3RA
A
B
α-pS317
α-pS345
S317
E
S317
A
D148
A
3RE C
1-36
8
1-42
1
+ + + + + + + + + +
3R
1 2 3 4 5 6 7 8 9 10 11 12
S317 S345
SQ CM1 CM2
C: 265-476
Myc-Chk1
C
D
CPT
α-Myc
α-pS317
α-pS345
C1: 265-331C2: 331-368
C3: 265-368C4: 331-421
C5: 331-476
+-C
+-C1
+-C2
+-C3
+-C4
+-C5
S317 S345
1 2 3 4 5 6 7 8 9 10 11 12
N (1-264)
C6: 421-47675
50
3575
50
3575
50
35
35
20
35
20
35
20
(Myc-Chk1)
(Myc-Chk1)
(Myc-Chk1)
(Myc-Chk1)
Figure 1. Chk1 phosphorylation at ATR sites. A, schematic
diagram of human Chk1 and generation of deletion mutations. SQ,
Ser/Gln; CM, conservedmotif; 3R, Arg 372/376/379; FL, full-length
(1–476); N, N-terminus; C, C-terminus. B, HEK293T cells were
transfected with Myc-tagged Chk1 WT ormutants for 48 hours, treated
or not with 500 nmol/L CPT for 2 hours and immunoblotted with
indicated antibodies. 3RA and 3RE are Arg to Ala andGlu mutations,
respectively. Endogenous Chk1 proteins from lanes 2 to 12, which
were all CPT treated, attracted the phospho-antibodies to the
areaaround 50 kDa during immunoblotting, which caused an inability
to detect phosphorylation of overexpressed Chk1 proteins. Thus, the
areacorresponding to endogenous Chk1 was cut off from the membrane
as indicated by the dashed line. Nevertheless, this does not alter
our conclusionsfor phosphorylation of overexpressed Chk1 proteins.
C, generation of small fragments of the Chk1 C-terminus. D, HEK293T
cells were transfectedwith Myc-tagged Chk1 C-terminal fragments for
48 hours, treated or not with 500 nmol/L CPT for 2 hours, and
immunoblotted with indicated antibodies.Phosphorylated Chk1 small
fragments are illustrated in the oval.
Wang et al.
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lane 2). Owing to the fact that ATM-dependent activationof ATR
only occurs in the presence of DNA damage (7),these results
suggested that ATR is the predominant kinasethat causes the
constitutive phosphorylation of the Chk1C-terminal fragment in
nontreated cells.
Mechanisms suppressing Chk1 phosphorylation undernormal growth
conditionsPreviously, the C-terminus of Chk1 was proposed to
form
an intramolecular interaction with the N-terminal kinasedomain
(13–15), so that the catalytic activity of the open kinasedomain of
Chk1 is suppressed under normal conditions (ref. 16;see model in
Supplementary Fig. S5). Here we showed thatdeletion of the
N-terminal kinase domain led to constitutivephosphorylation of the
C-terminus of Chk1, suggesting thatanother purpose of this
intramolecular interaction might beto suppress Chk1 phosphorylation
in the absence of DNAdamage.If this hypothesis is correct, then we
would expect that
interrupting the interaction between the N-terminal kinasedomain
and the C-terminus of Chk1 should lead to
constitutivephosphorylation of endogenous Chk1 under normal
condi-tions. To address this issue, we overexpressed those
smallfragments of Chk1 (Fig. 1C) into HEK293T cells and
examinedphosphorylation of endogenous Chk1. This experimentaldesign
was based on the assumption that one or more than
one of those exogenous small Chk1 fragments will competewith the
C-terminus of endogenous Chk1 for the interactionwith the
N-terminal kinase domain of the same Chk1 moleculeand disrupt the
closed conformation of Chk1. As a result, theC-terminus of
endogenous Chk1 is now exposed to undergoconstitutive
phosphorylation at ATR sites (see model in Sup-plementary Fig. S5).
Our results showed that expression of theC5 fragment, and to a
lesser extent, the C6 fragment, but notother small fragments,
induced phosphorylation of endoge-nous Chk1 under normal growth
conditions (Fig. 3A, lanes 9and 11). Treatment with a replicative
stress, hydroxyurea, onlymoderately further increased the
phosphorylation signal ofendogenous Chk1 proteins in the presence
of the Chk1 C5fragment (Fig. 3A, lanes 9–10).
We further showed that overexpression of the C5 frag-ment, and
to a lesser extent, the entire C-terminus, inducedstrong
phosphorylation of endogenous Chk1 proteins inanother cell line,
HeLa (Fig. 3B, lanes 3 and 8), indicatingthat this is not a cell
line–specific effect. On the other hand,the Chk1 FL, the N-terminal
kinase domain, or other frag-ments failed to do so (Supplementary
Fig. S2A). The reasonwhy the C5 fragment, which contains the CM1
and CM2domains, caused the strongest phosphorylation of endoge-nous
Chk1 is probably because this fragment interacts moststrongly with
the N-terminal kinase domain (SupplementaryFig. S2C), thereby
providing the maximal interference of the
Myc-Chk1 C
Myc-Chk1 FL
A B
C
+ + + + +
S345
ES3
45A
S317
ES3
17A
++ + ++
++
CPT
α-Myc
α-pS317
α-pS345
α-ATR
α-ATM
ATM
ATR
Luc
Myc-Chk1 C
siRNA
α-Myc
α-pS317
α-pS345
D
α-Myc
α-pS317
α-pS345
DNA-
PKi
ATM
i
Caffe
ine
Cont
rol
Myc-Chk1 C
1 2 3 4 5 6
CPTCaffeineATMiDNA-PKi
α-Myc
α-pS317
α-pS345
1 2 3 4
1 2 3 4 5 1 2 3
Myc
-Chk
1 C
Myc
-Chk
1 C
Myc
-Chk
1 C
Myc
-Chk
1 F
L
Figure 2. Constitutive phosphorylation of the Chk1C-terminus at
ATR sites. A, HEK293T cells were transfectedwith vectors
expressingMyc-Chk1C-terminuswith(lanes 3–6) or without (lanes 1–2)
mutating Ser-317 or Ser-345 for 48 hours, treated or not with 500
nmol/L CPT for 2 hours and immunoblotted with indicatedantibodies.
Results for Myc-Chk1 C are shown. B, HEK293T cells were transfected
with Myc-Chk1 C for 48 hours, treated with 10 mmol/L caffeine or 1
mmol/Linhibitors against ATM or DNA-PK for 4 hours, and
immunoblotted the same as in A. Results of short exposure (� 1 sec)
for Myc-Chk1 C are shown. C,HEK293Tcells were
transfectedwithMyc-Chk1FL for 48 hours, pretreatedwith caffeineor
other inhibitors for 2 hours followedby500 nmol/LCPT for an
additional2 hours, and immunoblotted the same as inA. Results
forMyc-Chk1FL are shown. D, HEK293T cells were transfectedwith
siRNAs against luciferase control (Luc),ATR, or ATM for 24 hours,
retransfected with Myc-Chk1 C for an additional 48 hours, and
immunoblotted the same as in A. Results for Myc-Chk1 C are
shown.
Autoregulation of Chk1 Phosphorylation
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closed conformation of endogenous Chk1 (see the model
inSupplementary Fig. S5).
If the CM1 and CM2 domains were critical for
preventingphosphorylation of Chk1 under normal conditions, then
wewould expect to identify key residues within these domains,whose
mutation should disrupt the intramolecular interactionand lead to
constitutive phosphorylation of Chk1 in theabsence of DNA damage.
To address this issue, we generatedGFP-tagged FL Chk1 vectors, in
which essentially every residuewithin the CM1 and CM2 domains
wasmutated and examinedprotein phosphorylation with or without CPT
treatment. Themajority of these point mutations did not show
constitutivephosphorylation of Chk1 (data not shown). However,
mutatingone of 2 residues in the CM2 domain (G448 or L449) led
toconstitutive phosphorylation of GFP-Chk1 in the absence ofDNA
damage (Fig. 4, lanes 9 and 11). Phosphorylation ofendogenous
proteins, including Chk1 and ATM, was notobserved in cells
expressing these 2 mutants (Fig. 4, endoge-nous Chk1), indicating
the lack of a pan-cellular DNA damageresponse. CPT treatment
moderately increased the phosphor-ylation signal of these 2mutants
(Fig. 4, lanes 9–12). These datasuggested that these 2 residues
(G448 and L449) play importantroles in suppressing constitutive
phosphorylation of Chk1under normal conditions.
The L449R mutant undergoes the same regulationas endogenous
Chk1
To further understand the physiologic relevance of
theconstitutive phosphorylation of these Chk1 mutants, we first
asked whether it is a cell line–specific effect or not.
Weconsistently detected high levels of constitutive
phosphoryla-tion of Chk1 mutants in HeLa, U2-OS, A549, or HCT116
celllines (Fig. 5A in HeLa cells, lanes 2–3; data not shown).
Incontrast, phosphorylation of the Chk1 WT was only detectedby CPT
treatment (Fig. 5A, lanes 1 and 4). Thus,
constitutivephosphorylation of these 2 Chk1 mutants is not
restricted toone system or cell line.
Second, we asked whether this constitutive phosphorylationis
also ATR dependent. Inhibiting ATR, but not other kinases,reduced
the level of constitutive phosphorylation of the Chk1L449Rmutant
(Fig. 5B, lane 2). Considering that cells were onlytreatedwith
caffeine for hours although they had expressed theL449Rmutant for
days, the reduction in Chk1 phosphorylationis significant.
Depletion of ATR significantly reduced phos-phorylation of the Chk1
L449R mutant, in a way similar toendogenous Chk1 (Fig. 5C).
Together, these data stronglyindicated that constitutive
phosphorylation of the Chk1L449R mutant is ATR dependent.
Third, we asked whether the L449Rmutant follows a
similarcell-cycle–dependent expression pattern as endogenous
Chk1whose expression peaks in the S to G2 phase (23). The
resultsshowed that indeed the level of the GFP-Chk1 L449R mutantwas
the highest from S to G2 phase, similar to endogenousChk1 (Fig. 5D,
compare the anti-GFP and the anti-Chk1 blots).No phosphorylation
was detected for endogenous Chk1 inthe absence of DNA damage;
however, phosphorylation of theL449Rmutant was detected throughout
the cell cycle, with thehighest in the S phase (Fig. 5D, lane 3 in
the anti-pS345 blot).Together, these data suggested that the Chk1
L449R mutantundergoes the same regulation as the endogenous
Chk1protein.
Constitutive activation of Chk1 in the absence ofDNA damage
reduces cell viability
To understand the biologic significance of the
constitutivephosphorylation of Chk1, we first asked whether it
inducedan artificial S-phase checkpoint. To this end, we
transfectedHeLa Tet/Off cells with tetracycline-regulated
expression
A
B
Myc-Chk1FLControl C C1 C2 C3 C4 C5
Endo Chk1
Exo Chk1 C3
α-pS345
α-Chk1
α-Myc
1 2 3 4 5 6 7 8
Endo Chk1Exo Chk1 FL
Exo Chk1 FL
Exo Chk1 C
α-pS345Endo Chk1∗
α-Myc
1 2 3 4 5 6 7 8 9 10 11 12
Myc-Chk1HU+-
C1+-
C2+-
C3+-
C4+-
C5+-
C6
α-Chk1 (Endo)Exo Chk1 C3
Figure 3. Constitutive phosphorylation of endogenousChk1. A,
HEK293Tcells were transfected with Myc-tagged Chk1 C-terminal
fragments for48 hours, treated or not with 2 mmol/L hydroxyurea
(HU) for 1 hour,and immunoblotted with indicated antibodies. The
asterisk indicates anon-Chk1 protein cross-reacted with the
anti–p-Ser-345 antibodies afterhydroxyurea treatment. B, HeLa cells
were transfected with Myc-taggedChk1 FL or truncated mutants for 48
hours and immunoblotted withindicated antibodies. Overexpressed
(Exo) and endogenous (Endo) Chk1proteins were shown by arrows.
α-pS345
Endo Chk1
α-Chk1
1 2 3 4 5 6 7 8 9 10 11 12
GFP-Chk1CPT+-
WT+-
L354R+-
M377R+-
L443R+-
G448D+-
L449R
Exo Chk1
Endo Chk1
Exo Chk1
Figure 4. Roles of the CM2 domain in Chk1 phosphorylation.
HEK293Tcells were transfected with GFP-tagged full-length Chk1 WT
or pointmutants for 48 hours, treated or not with 500 nmol/L CPT
for 2 hours, andimmunoblotted with indicated antibodies.
Overexpressed (Exo) andendogenous (Endo) Chk1 proteins are shown by
arrows.
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vectors for GFP, GFP-Chk1 WT, or the L449R mutant andexamined
expression of Cdc25A. Cdc25A is a key Chk1 down-stream target that
plays a crucial role in regulating both theentry and the
progression of S phase (24). Activation of Chk1leads to the
proteasome-dependent degradation of Cdc25Afollowed by S-phase
progression inhibition (24). The resultsshowed that the Chk1 WT
only slightly reduced the level ofCdc25A compared with GFP alone
(Fig. 6A, lanes 3–4), inagreement with our previous report (14). In
contrast, expres-sion of the L449R mutant almost completely blocked
Cdc25Aexpression (Fig. 6A, lane 5), as did the DNA damage
agenthydroxyurea (Fig. 6A, lane 2). Consistent with the reduction
ofCdc25A, only hydroxyurea treatment and the L449R mutantshowed
Chk1 phosphorylation at Ser-345 (Fig. 6A, lanes 2 and5). In vitro
kinase assay showed that the L449R mutant exhib-ited much stronger
autophosphorylation than the Chk1 WT(Supplementary Fig. S2D). These
results showed that the L449Rmutant is functionally more active
than the Chk1 WT, both invitro and in vivo.Subsequently, we tested
the effect of the Chk1 L449Rmutant
on the S-phase progression. HeLa Tet/Off cells blocked at
theG2–M phase by nocodazole were released into the cell cyclewith
concomitant expression of GFP, GFP-Chk1 WT, or theL449R mutant. Our
preliminary data showed that a 16-hourrelease after nocodazole
treatment would allow normal HeLacells to start entering the S
phase (data not shown). Thus, wemonitored DNA synthesis over a
10-hour period beginning at16 hours of nocodazole release
bymeasuring the incorporationof EdU, a nucleotide analog (see
Supplementary Fig. S3A forexperimental design). Fluorescence
microscopy revealed thatthe GFP-Chk1 WT and the L449R mutant were
nearly equallyexpressed at the end of the 16 hours of release
(Supplementary
Fig. S3B). Importantly, we found that less cells expressing
theGFP-Chk1 L449R were incorporating EdU compared with theGFP-Chk1
WT or the GFP alone (Fig. 6B, 0 hour), indicating adelayed S-phase
entry. During the subsequent 10-hour chaseperiod, the number of
cells that incorporated EdU in the GFP-Chk1 WT or GFP control group
dropped much more signif-icantly than in the L449R group (Fig. 6B,
4–10 hours). Thisindicated that at a time point when control cells
are exiting Sphase, GFP-Chk1 L449R-expressing cells remain in the S
phase.We also noticed a slightly less reduction in EdU-positive
cells inthe GFP-Chk1 WT group than the GFP alone (Fig. 6B). This
isconsistent with the Cdc25A expression profile (Fig. 6A).
Thesedata indicated a delayed S-phase entry and prolonged
S-phaseprogression caused by the L449Rmutant, and to a much
lesserextent, the Chk1 WT, compared with the GFP control.
To confirm the prolonged S-phase progression, we analyzedthe
percentage of late S-phase cells during that 10-hour chaseperiod.
Whereas early S-phase cells had a pan-nuclear EdUstaining pattern,
late S-phase cells exhibited punctuate or morefocal EdU staining
pattern (Supplementary Fig. S3C). Theresults showed that GFP-Chk1
L449R-expressing cells had asignificantly lower percentage of late
S-phase cells than theGFP-Chk1WTor theGFP control, especially at
later time points(Fig. 6C). Again, the S-phase progression in the
GFP-Chk1 WTwas slower than the GFP control (Fig. 6C, more obvious
at0–4 hour). Cell-cycle analyses confirmed that the
L449Rmutant-expressing cells progressed through S phase muchmore
slowly than GFP control or the Chk1WT (SupplementaryFig. S4).
Together, these data showed that constitutive phos-phorylation of
Chk1 leads to prolonged S-phase progression.
The cell-cycle analyses showed significantly increased deadcell
population for cells expressing the Chk1 L449Rmutant (the
Figure 5. Constitutivephosphorylation of the Chk1 L449Rmutant.
A, HeLa cells weretransfected with GFP-Chk1 WT,G448D, or L449R
mutant for48 hours, treated or not with500 nmol/L CPT for 2 hours,
andimmunoblotted with indicatedantibodies. Results for GFP-Chk1are
shown. B, HEK293T cells weretransfected with GFP-Chk1 L449Rmutant
for 48 hours, treated with10 mmol/L caffeine or 1 mmol/Lkinase
inhibitors for 4 hours, andimmunoblotted as in A. Results
forGFP-Chk1 are shown. C, HEK293Tcells were cotransfected with
GFP-Chk1 L449R and lentivirus vectorcontrol or shATR for 72
hours,treated or not with 500 nmol/L CPTfor 2 hours, and
immunoblotted as inA. D, HeLa cells were transfectedwith GFP-Chk1
L449R vector for 24hours, synchronized at G2–M phaseby 100 ng/mL
nocodazole treatmentfor 20 hours, released into differentcell-cycle
stages, andimmunoblotted with indicatedantibodies.
A
GF
P-C
hk1
α-GFP
α-pS345
WTL4
49R
G448
DWT L4
49R
G448
DCPT
GFP-Chk1
+ ++
B
α-pS345
α-GFP
Cont
rol
ATM
i
Caffe
ine
CDKi
Chk1
i
DNA-
PKi
GFP-Chk1 L449R
Asyn. G1 S MG2
Endo.
Exo.
GFP-Chk1 L449R
- + - + CPT
α-GFP
α-pS345
α-CyclinB
α-pS345
α-ATR
Control shATR
α-Chk1
C
α-MCM7
GFP-Chk1 L449R
D
1 2 3 4 5 6 1 2 3 4 5 6
1 2 3 41 2 3 4 5
Endo.
Exo.α-Chk1
GF
P-C
hk1
GF
P-C
hk1
Autoregulation of Chk1 Phosphorylation
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-
sub-G1 population in Supplementary Fig. S4), indicating
thatexpression of constitutively active Chk1 is counterproductiveto
cell viability. To further test this idea, we transfectedHEK293T
cells with vectors expressing the GFP control,GFP-Chk1 WT or
GFP-Chk1 L449R mutant, and countedGFP-positive cell numbers in each
clone over 8 days. Theresults showed that although cells expressing
GFP controlexpanded exponentially, cells expressing GFP-Chk1 WT
hada significant delay in expansion; however, no clone expansionwas
observed for cells expressing the GFP-Chk1 L449R mutant(Fig. 7).
Similar results were observed for HeLa, U2-OS, andHCT116 cell
lines. These data suggested that constitutiveactivation of Chk1
suppresses tumor cell growth.
DiscussionIn this study, we provide evidence that the Chk1
polypeptide
contains a number of critical regulatory mechanisms mediat-ing
its own phosphorylation, checkpoint activation, and cellviability.
Checkpoint activation requires phosphorylation ofChk1 at both
Ser-317 and Ser-345 residues. However, whereasSer-345 is essential
for cell viability, Ser-317 is not (21). This ledto the idea that
Ser-345 phosphorylation is the final determi-nant of checkpoint
activation. Our data support this idea byshowing that DNA
damage–induced Ser-345 phosphorylationof Chk1 is tightly regulated.
First, either the phosphate group or
the serine residue at the position of Ser-317 is critical
forinducing phosphorylation of Chk1 at Ser-345 (15, 20, 21).
Sec-ond, phosphorylation of Ser-317 is not sufficient for
maximalphosphorylation at Ser-345. It seems that the relief from
the
A B
C
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Late
/tota
l S c
ells
Edu
-pos
itive
cel
ls %
0 4 8 10 h
0 4 8 10 h
α-GFP
α-pS345
α-Chk1
α-ATM
α-Cdc25A
Endo.
Exo.
- + HUWT L4
49R
Non
Transf
ected
---
GFP-Chk1
1 2 3 4 5
WT
L449R
GFP
WT
L449R
GFP
GFP
Figure 6. Checkpoint activation by constitutive phosphorylation
of Chk1. A, HeLa Tet/Off cells were transfected or not with
tetracycline-inducible GFP, GFP-Chk1WT, or the L449Rmutant for 48
hours in the absence of doxycycline, treated or not with 2mmol/L
hydroxyurea (HU) for 1 hour, and immunoblotted
withindicatedantibodies. B,HeLaTet/Off cellswere transfectedwith
tetracycline-inducibleGFP,GFP-Chk1WT, or the L449Rmutant and
cultured in the presenceof 1 mg/mL doxcycline for 24 hours,
synchronized at the G2–M phase by 100 ng/mL nocodazole for 20
hours, released into doxcycline-free medium for16 hours, and then
chased for additional 0, 4, 8, or 10 hours. During the last 20
minutes of each chase period, cells were labeled with 10 mmol/L
EdU,fixed, and analyzed as described in Materials and Methods. Data
represent mean and SD of EdU-positive cells from 3 independent
experiments. C,EdU-positive cells from Bwere further divided into
late versus early S phase as shown in Supplementary Fig. S3C. The
ratio of late versus total S-phase cellswas measured. Data
represent mean and SD from 3 independent experiments.
0
50
100
150
200
250
1 2 3 4 5 6 7 8
Cel
l nu
mb
er p
er c
olo
ny
Day
WT
L449R
GFP
Figure 7. Growth suppression by constitutively active Chk1.
HEK293Tcells plated in 12-well plates were transfected with vectors
expressingGFP, GFP-Chk1 WT, or GFP-Chk1 L449R mutant for 48 hours.
Thecells were reseeded into 6-well plates in triplicate at a
density of 10,000cells per well, cultured for 1 to 8 days. On each
day, the number ofGFP-positive cells within each colony was counted
under fluorescencemicroscope. Data represent mean and SD from 3
independentexperiments.
Wang et al.
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-
N-terminal kinase domain and the involvement of the CM1 andCM2
domains are also required for high-level phosphorylationof Chk1 at
Ser-345 (see the model in Supplementary Fig. S5).TheC-terminal
CM1andCM2motifs seem to have dual roles inregulating Chk1
phosphorylation. In the absence of DNAdamage, the CM1 and CM2
domains contribute to the inhib-itory effect on Chk1
phosphorylation by interacting with the N-terminal kinase domain.
On the other hand, the CM1 and CM2domain contributes to high-level
phosphorylation of Chk1 inresponse to DNA damage, which might be
through providing aproper conformation (22) for maximal Ser-345
phosphoryla-tion by ATR.Previously, Chk1 has been proposed to adopt
a closed
conformation through an intramolecular interaction betweenthe
N-terminal kinase domain and the C-terminal regulatorydomain
(13–15). This conformation not only suppresses thecatalytic
activity of Chk1, but also stabilizes the protein (14, 16).Data
presented in this study added further insights into thefold-back
structure of Chk1 and its roles in checkpoint regu-lation. These
new data indicated that under normal circum-stances, while the
C-terminus of Chk1 masks the kinasedomain, the N-terminal kinase
domain of Chk1 simultaneouslysuppresses Chk1 phosphorylation by
ATR. Thesemutual inhib-itory effects may provide much safer
mechanisms to ensurethat no accidental activation of checkpoints,
through either theexposure of the catalytic domain or the
phosphorylation of theSQ sites of Chk1, will be achieved under
normal growthconditions.It has long been known that inadequate Chk1
phosphory-
lation leads to checkpoint defects, and consequently loss of
cellviability (25). These new results indicate that cells may
haveevolved mechanisms to prevent accidental Chk1
phosphory-lationwhen not needed. In agreement with this idea,
activationof ATR by expressing the ATR-activating domain of TopBP1
ortethering TopBP1 or claspin to DNA led to artificial
checkpointactivation in the absence of DNA damage. As a result,
cellsundergo permanent cell-cycle arrest or senescence (26,
27).However, one potential caveat of these methods is that
theyactivated the entire ATR pathway. In this study, we used
theconstitutively active Chk1 mutant (L449R) as a model to showthat
activation of Chk1 only, but not the entire ATR pathway,
isdetrimental to cell viability without exogenous DNA damage.
The existence of constant S phase checkpoints posed by
thisconstitutively active Chk1 mutant is likely to eventually
stopcell division and growth. Similarly,mutating the
correspondingLeu residue in budding yeast Chk1 (L506) activated
check-points in the absence of DNA damage (28, 29), indicating
thatmechanisms preventing Chk1 from being accidentally
phos-phorylated might be highly conserved.
An interesting question is how mutation of G448 or L449leads to
constitutive Chk1 phosphorylation at ATR sites? Apossible
explanation is that these 2 residues sit at the keyinterface
between the N-terminal kinase domain and the C-terminal domain of
Chk1. Therefore, mutating one of themfully exposes the Ser-345 site
to ATR, leading to its phosphor-ylation in the absence of DNA
damage (Supplementary Fig. S5).Clearly, structural studies are
needed to provide answers tothis question and other questions that
are related to theconformational change of Chk1.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Authors' ContributionsConception and design: Y. ZhangDevelopment
of methodology: J. Wang, X. HanAcquisition of data (provided
animals, acquired and managed patients,provided facilities, etc.):
J. Wang, X. HanAnalysis and interpretation of data (e.g.,
statistical analysis, biostatistics,computational analysis): J.
Wang, Y. ZhangWriting, review, and/or revision of the manuscript:
Y. ZhangStudy supervision: Y. Zhang
AcknowledgmentsThe authors thank Tony Hunter for discussion and
suggestions, Zhenghe
Wang and Paul MacDonald for critical reading of the manuscript,
and alsothank James Jacobberger and the core facility at Case
Comprehensive CancerCenter for the help in cell-cycle analyses.
Grant SupportY. Zhang is funded by the NCI Howard Temin Career
Development Award
(R00CA126173), NCI R01CA163214, and a pilot grant from the
American CancerSociety (IRG-91-022-15).
The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby markedadvertisement in accordance with 18 U.S.C. Section
1734 solely to indicate thisfact.
Received February 10, 2012; revised April 24, 2012; accepted May
9, 2012;published August 1, 2012.
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2012;72:3786-3794. Cancer Res Jingna Wang, Xiangzi Han and
Youwei Zhang Kinase 1Autoregulatory Mechanisms of Phosphorylation
of Checkpoint
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