Autoregulatory Mechanisms of Phosphorylation of …...Chk1undergoes ATR-dependent phosphorylation on...

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

www.aacrjournals.org Cancer Res; 72(15) August 1, 2012 3787



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,

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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

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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.





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

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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.

Wang et al.




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.

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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

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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.

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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.




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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