© 2001 Macmillan Magazines Ltd

REVIEWS

Phosphorylation of cellular proteins is a versatile meansof rapidly and specifically modulating their activity,their interactions with other proteins or molecules, andtheir subcellular localization. Indeed, many proteinkinases — the enzymes that carry out the covalentmodification of proteins by transfer of phosphategroups from ATP to the substrate — initiate or propa-gate a range of cellular signals that are involved in manyfundamental physiological processes.

One vital process is the constant monitoring andmaintenance of chromosomal DNA quality — a prereq-uisite for the faithful duplication and segregation of thegenome from one generation to the next. This surveil-lance operates through a complex network of so-calledgenome-integrity or cell-cycle checkpoints1–3. These mol-ecular cascades detect and respond to incomplete DNAreplication or various forms of DNA damage that arecaused by genotoxic stress (FIG. 1). Given the enormoussize of the genome, the many errors that inevitably occurduring its replication, and the vast range of DNA-damag-ing insults that constantly attack our genes, well-func-tioning checkpoint mechanisms are essential for properdevelopment and survival. Such DNA-damaging insultsinclude environmental mutagens such as genotoxicchemicals, ultraviolet (UV) light or ionizing radiation,but also various endogenous reactive oxygen species,which arise during normal cellular metabolism4,5.

Perhaps the best way to appreciate the essential role ofthe checkpoint machinery is to consider the conse-quences of its failure, which results from naturally occur-

ring or experimentally induced mutations or EPIGENETIC

defects in crucial components of the checkpoint path-ways. Depending on the severity and timing, checkpointmalfunction can result in developmental malformations,embryonic lethality or the accumulation of mutationsthat could potentially lead to genetic disease, includingcancer6–9. The frequent occurrence of DNA lesionsrequires the rapid and efficient attention of DNA-repairmechanisms and temporary halting of cell-cycle progres-sion in proliferating cells, in order to provide time forrepair and prevent fixation and propagation of harmfulmutations. This is made possible by an efficient DNA-repair machinery as well as cell-cycle checkpoints.

Checkpoint mechanisms provide a rapid emergencyservice owing to the rapid amplification of signal trans-duction from DNA lesions down to checkpoint effec-tors, which delay cell-cycle progression and activateDNA repair. It is still not clear which molecules detectthe damaged DNA10–14, but the speed and breadth of sig-nal transduction is known to be accomplished throughchains of phosphorylation events which are carried outby protein kinases at two levels along the checkpoint cas-cades. The more upstream signal-transducing kinasesbelong to the phosphatidylinositol kinase (PIK)-relatedfamily and include the ‘ataxia-telangiectasia, mutated’(ATM) and ‘ataxia-telangiectasia and Rad3-related’(ATR) kinases in vertebrates, and their homologues inyeast15,16. At the next step, the DNA-damage signals aretransduced and amplified by two structurally unrelated,yet functionally complementary, serine/threonine kinases,

CHK2 KINASE — A BUSY MESSENGERJiri Bartek, Jacob Falck and Jiri Lukas

Checkpoint kinase 2 (Chk2) is emerging as a key mediator of diverse cellular responses togenotoxic stress, guarding the integrity of the genome throughout eukaryotic evolution.Recent studies show the fundamental role of Chk2 in the network of genome-surveillancepathways that coordinate cell-cycle progression with DNA repair and cell survival or death.Defects in Chk2 contribute to the development of both hereditary and sporadic humancancers, and earmark this kinase as a candidate tumour suppressor and an attractive targetfor drug discovery.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | DECEMBER 2001 | 877

Danish Cancer Society,Institute of Cancer Biology,Strandboulevarden 49, DK-2100 Copenhagen Ø,Denmark. Correspondenceto J.B. e-mail:bartek@biobase.dk

EPIGENETIC CHANGES

The alteration of geneexpression throughtranscriptional (due topromoter methylation) or post-transcriptional mechanisms,rather than ‘genetic’ alterationof sequences of bases ingenomic DNA.

M I L E S TO N E S

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cerevisiae, and they were defined as regulatory pathwaysthat can arrest cell-cycle progression in response toDNA damage to provide time for repair.

The founding member of the ‘Chk2 family’ of check-point kinases, called Rad53, was first identified in 1994as a kinase involved in many checkpoint responses inbudding yeast28. Homologues of Rad53 were subse-quently found in the fission yeast Schizosaccharomycespombe (named cds1)29 and in higher eukaryotes22–26,30–34

(FIG. 2). The identification of the human homologue wasreported by five laboratories in 1998/1999 (REFS 22–26),and the kinase was named Chk2 by the Elledge group,who first cloned the human gene22. As a result of inde-pendent cloning of these homologous kinases in differ-ent organisms, or in the same organism by different lab-oratories, the nomenclature is still confusing. Forsimplicity, and emphasis on recent findings about themammalian homologues, we refer to these kinasesgenerically as Chk2, and use the specific names onlywhen explaining specific features of the homologues inlower eukaryotes.

The overall structure of the Chk2 proteins is simi-lar in all eukaryotes (FIG. 2). The degree of overallhomology of Chk2 protein sequences across speciesroughly reflects the evolutionary distance among thedistinct organisms (FIG. 2). For example, human CHK2shows 83% and 82% amino-acid identity with the ratand mouse kinases, respectively, followed by 61%identity with the Xenopus laevis protein; 51% with thezebrafish; 34% with Drosophila melanogaster; 32%with both S. pombe and the nematode Caenorhabditiselegans; and 28% with S. cerevisiae Rad53.Interestingly, the budding yeast Rad53 kinase standsout as, so far, the only member of the Chk2 family tohave a large carboxy-terminal extension that alsoincludes a second forkhead-associated (FHA) domain(see overleaf).

Chk1 and Chk2, which target the downstream effectorsof the checkpoint pathways.

Several recent reviews have highlighted the roles ofATM and ATR at the top of the checkpoint cas-cades10,17,18, and the functions of ATM in relation tocomplex symptoms of patients who suffer from ataxia-telangiectasia19,20 (a severe disorder caused by mutationof the ATM gene). By contrast, the Chk1 and Chk2kinases11,21 have received less attention, partly becausethey have only recently been identified in mammals22–26.Since then, however, an avalanche of exciting data hasdocumented the role of Chk2 as a crucial link betweenthe ATM/ATR kinases and the checkpoint effectors inthe cell-cycle and DNA-repair machinery. In addition,germline and somatic mutations of the CHK2 genehave been identified in human hereditary and sporadictumours, respectively.

So, Chk2 qualifies as a new tumour suppressor and apromising target for future cancer therapy strategies.These, and additional, functions of the Chk2 kinase andits homologues have been identified in other organisms— from yeast and worms, to flies, fish, amphibians andmammals. Here, we provide an overview of the evolu-tionary conservation, structure and activation of Chk2,as well as describing its role as a key messenger ingenome-integrity checkpoints, including its identifiedsubstrates and potential (patho)physiological signifi-cance, with particular reference to cancer.

Chk2 in evolutionAll organisms need to deal with problems that are asso-ciated with DNA damage or a replication block, so it isnot surprising to find that the main elements and overallstrategy of the cell-cycle checkpoints evolved early andremained highly conserved throughout eukaryotic evo-lution. The term ‘checkpoints’ was first coined27 basedon discoveries in budding yeast Saccharomyces

Figure 1 | Cell-cycle checkpoints. The cell-division cycle evolved to serve two main purposes — duplication of the genomeand segregation of the two identical sets of chromosomes. To ensure that these events are error free, cells have evolvedsurveillance mechanisms that are known as the cell-cycle checkpoints. These signal-transduction pathways ensure that eitherincompletely replicated (DNA-replication checkpoint) or damaged (DNA-damage checkpoint) chromosomal DNA is sensed, andthis signal is transmitted to effector molecules that delay cell-cycle progression and initiate repair. Checkpoint pathways alsocoordinate the resumption of cell-cycle progression, provided that the entire genome has been replicated and the DNA lesions(such as single- or double-stranded DNA breaks; SSBs and DSBs, respectively) are repaired. The DNA-damage response isunique among checkpoints in its ability to arrest/delay the cell cycle at several points, beginning from the G1/S transition andending in early prophase. As such, it allows cell-cycle progression to be modulated in cells that have passed the restriction point(R) and become committed to autonomous progression through the cell cycle, irrespective of extracellular signals.

Growth-factor- sensitive period

R

G1G1 S G2 Prophase Metaphase

DNA-replicationcheckpoint

Stalled replication forks

Cytokinesis

*

DNA lesions (SSB, DSB, base modifications)

Anaphase

DNA-damage checkpoint

© 2001 Macmillan Magazines LtdNATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | DECEMBER 2001 | 879

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ments, illustrated here using the human protein as anexample (FIG. 3b).

The SQ/TQ motif. The amino-terminal domain con-tains a series of seven serine or threonine residues fol-lowed by glutamine (SQ or TQ motifs). These areknown to be preferred sites for phosphorylation byATM/ATR kinases17. Indeed, three recent reports36–38

showed that, after DNA damage (which was caused byionizing radiation or UV light) or replication blockadeby hydroxyurea, Thr68 and other sites in this regionbecome phosphorylated by ATM/ATR, and this domainof Chk2 therefore seems to have a regulatory function.In fission yeast, phosphorylation of Thr11 in the TQmotif by Rad3 is required for activation of the cds1checkpoint kinase39.

The forkhead-associated domain. Another key domainof Chk2 is the FHA domain, first identified in fork-head transcription factors, and later shown to be pre-sent in a range of (mainly nuclear) proteins withdiverse functions40. Although the boundaries of theChk2 FHA domain (between amino-acid residues 115and 165) in FIG. 3b follow the original reports and defin-itions22,24, more recent studies indicate that functionalFHA domains can consist of up to 120–140 amino-acid residues41. FHA domains seem to bind phospho-threonine residues, and they are thought to beinvolved in protein–protein interactions42–45, which areprobably triggered by phosphorylation of proteinsthat are recognized by FHA-containing partners suchas Chk2. So, the FHA domain is a good candidate fordynamic interactions of Chk2 with its upstream regu-lators and/or downstream targets in cell-cycle-check-point signalling.

The kinase domain. The kinase domain occupiesalmost the entire carboxy-terminal half of Chk2, and its

Despite their overall structural homology andshared biological role as important transducers of cell-cycle checkpoint signals, there are functional differencesbetween the various Chk2 homologues. Whereas theyeast Rad53 and cds1 kinases are required for responsesto various forms of DNA damage as well as to replica-tion blocks15,21,28,29, the mammalian Chk2 seems torespond primarily to the most lethal type of DNA dam-age — double-stranded DNA breaks22–26, which arecaused by agents such as ionizing radiation andRADIOMIMETIC DRUGS14,18.

Another emerging difference is the role of some Chk2homologues in monitoring meiotic recombination ingerm cells. This function is particularly prominent for theChk2 homologue in C. elegans31–33, and is suggested bythe localization of Chk2 in germ cells in Drosophila34. Inbudding yeast, however, meiotic recombination is moni-tored by a more distantly related kinase called Mek1,rather than by Rad53 (REF. 35), and we do not know whichmammalian kinases are responsible for this function.

Delineation of the functional parallels among mem-bers of the Chk2 family in different organisms is furthercomplicated by the partial overlap, or differential usage,of the structurally distinct Chk1 kinase and its homo-logues. The functions of Chk1 kinases cooperate, overlapor alternate with those of the Chk2 kinases, dependingon the organism and the nature of the genotoxic insult.On the other hand, the human CHK2 gene complementsthe checkpoint defects in both budding and fission yeaststrains that are deficient in Rad53 (REF. 22) and cds1(REF. 26), respectively, which indicates the overall function-al conservation among the Chk2 family members.

Chk2 structure and activationThe human gene that encodes CHK2 spans 50 kilo-bases of genomic DNA and contains 14 exons (FIG. 3a).At the protein level, the structure of the Chk2 kinasesis dominated by several evolutionarily conserved ele-

Figure 2 | Chk2 in evolution. On the left is a phylogenetic tree showing the degree of amino-acid sequence similarity betweenthe Chk2 homologues in different organisms. (The alternative names of Chk2 in some species are indicated in parentheses.) Onthe right is an alignment of conserved domains in the Chk2 homologues: the SQ/TQ-rich domain (maroon); the forkhead-associated domain (FHA; blue); and the kinase domain (brown). The highly conserved activation loop is marked by an asterisk.Amino-acid (aa) sequence accession numbers are: human, AAC83693; mouse, AAC83694; rat, AAD55890; Xenopus laevis,AAF75829; zebrafish, AAK52419; Schizosaccharomyces pombe, Q09170; Saccharomyces cerevisiae, A39616; Caenorhabditiselegans, BAB15803; Drosophila melanogaster, BAA28755.

Human 543 aa

C. elegans 450 aa

S. pombe (cds1) 460 aa

S. cerevisiae (Rad53) 821 aa

D. melanogaster (Dmnk) 476 aa

Mouse 546 aa

Rat

X. laevis (Xcds1) 517 aa

Zebrafish 503 aa

545 aa

*

*

*

*

*

*

*

*

*

RADIOMIMETIC DRUGS

Drugs that have cellular effectsthat are similar to those ofionizing radiation, such as theinduction of double-strandedDNA breaks.

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R E V I E W S

regulator that is complexed with ATM and/or CHK2,such as BRCA1 or 53BP1 (REF. 48). The initial phosphoryla-tion in the SQ/TQ-rich domain might evoke a conforma-tional change in CHK2, which allows better access of thekinase domain to its own activation loop and therebyfacilitates the full activation of CHK2 (FIG. 4a).

In the model of budding yeast Rad53 activation47,DNA damage or replication block leads to rapid bind-ing of Rad53 with a dimer of Rad9, an importantcheckpoint regulator, which contains the characteristicBRCT HOMOLOGY DOMAIN that is found in checkpoint pro-teins in all eukaryotes49. The interaction of Rad9 withRad53 depends on pre-phosphorylation of Rad9 byMec1 (REF. 42), the budding yeast homologue of themammalian ATM kinase (FIG. 4b). These events bringtwo Rad53 molecules into close proximity, allowing intrans autophosphorylation of Rad53, the activated formof which is then released from its complex with Rad9.Interestingly, an adaptor protein, termed claspin, alsoseems to be required for the activation of Chk1 in aXenopus DNA-replication checkpoint50, and a claspin-likeprotein, Mrc1, has been proposed to serve as a replicativecounterpart of Rad9/crb2 in the activation of Rad53 andcds1 in budding and fission yeast, respectively51,52.

Given that there is no clear homologue of Rad9 inhigher eukaryotes, these two models of Chk2/Rad53activation seem distinct. However, in both cases,ATM/Mec1 are involved in the first activating phospho-rylation steps; the BRCT-containing (BRCA1, 53BP1) orclaspin-like adaptor protein might also contribute toactivation of mammalian Chk2; and both modelsinvolve autophosphorylation of Chk2 as the final acti-vating step.

Chk2 targets and functionsIn response to genotoxic insults, Chk2 is activated andpropagates the checkpoint signal along several pathways,which eventually causes cell-cycle arrest in the G1, S andG2/M phases; activation of DNA repair; and, in somecases, apoptotic cell death. So what are the pathways,and the crucial substrates of Chk2, that execute thesedownstream checkpoint effects?

The mammalian checkpoint effectors that are estab-lished as substrates of Chk2 in vivo include p53, BRCA1and two of the three members of the Cdc25 family ofphosphatases — Cdc25A and Cdc25C (FIG. 5). TheMdm2 protein — which promotes the turnover of p53and inhibits its activity as a transcription factor —seems to be phosphorylated by Chk1 (REF. 53) and is acandidate substrate for Chk2. Although this list willprobably grow, the functions of the known targets ofChk2 already give us an idea of how Chk2 affects theabove-mentioned diverse cellular processes (FIG. 5).

Phosphorylation of p53 on Ser20 by Chk2 and/orChk1 (probably together with phosphorylation ofMdm2) stabilizes the p53 protein53–55, which enhancesits potential to positively regulate the expression offactors that are involved in DNA repair, cell death andcell-cycle control56,57. Mouse Chk2-deficient cells that areexposed to ionizing radiation fail to stabilize and acti-vate p53 (REF. 58), as do cells that express kinase-defective

key functional elements — including the ACTIVATION LOOP

— have been identified based on their homology withother serine/threonine kinases (FIG. 3b). Mutating one ofthe key residues of this domain, Asp347, to alanine,results in a kinase-defective mutant22 that is used as aresearch tool.

Finally, an apparently unique feature of mam-malian Chk2 that is not conserved in lower eukaryotesis a c-Abl SRC HOMOLOGY-3 (SH3) DOMAIN-consensus bindingsequence, which is upstream of the FHA domain24. Thefunctional significance of this motif is not known; how-ever, both Chk2 and c-Abl are phosphorylated and acti-vated by ATM in response to DNA damage. Moreover,ATM and activated Chk2 often jointly target down-stream checkpoint effectors17, so Chk2 might also inter-act with c-Abl, either physically or functionally.

Models of Chk2 activation. Two plausible models ofChk2 activation in response to DNA damage havebeen proposed, which are based on experimentswith human CHK2 (REF. 46) and budding yeast Rad53(REF. 47), respectively.

In the human model, the first activating phosphory-lation step of CHK2 depends on the integrity of its FHAdomain (REF. 46 and J.B., J.F. and J.L., unpublished obser-vations) and is carried out by the ATM kinase inresponse to double-stranded DNA breaks. This initialwave of phosphorylation targets Thr68 in particular,and also additional serines or threonines in the regula-tory SQ/TQ-rich domain of human CHK2 (REFS 36–38).Phosphorylation at Thr68 is a prerequisite for the sub-sequent activation step, which is attributable toautophosphorylation of CHK2 on residues Thr383 andThr387 in the activation loop of the kinase domain46.

How does the FHA domain contribute to Thr68phosphorylation by ATM, and how does this facilitate theautophosphorylation of CHK2? This is not known, butwe speculate that the FHA domain might mediate thedocking of CHK2 onto ATM itself or another checkpoint

Figure 3 | CHK2 genomic and protein structure. a | Structure of the human CHK2 gene.The CHK2 gene localizes to chromosome 22q12.1, which spans approximately 50 kilobases(kb), and consists of 14 exons (black boxes). Numbers below the bars indicate the base-pair(bp) range of each exon. Highly homologous fragments of the gene that include exons 11 to 14are found on chromosomes 2, 7, 10, 13, 15, 16, X and Y. b | Structure of the human CHK2protein, with the SQ/TQ-rich, forkhead-associated (FHA) and kinase domains shown inmaroon, blue and brown, respectively. Pinheads indicate putative ATM/ATR phosphorylationsites in the SQ/TQ-rich region. In vivo phosphorylations of CHK2 on threonine 68 (by ATM/ATR)and threonines 383 and 387 in the activation loop (autocatalytic) are indicated.

b

a

1 20 75 115 165 225 490 543Activation loop

T68

Kinase domainFHASQ/TQ

~50 kb

987654321 10 11 12 13 14

T383T387

1–31

9

320–

444

445–

592

593–

683

684–

793

794–

846

847–

908

909–

1008

1009

–109

5

1096

–125

9

1260

–137

5

1376

–146

1

1462

–154

2

1543

–163

2

ACTIVATION LOOP

A conserved structural motif inkinase domains, which needs tobe phosphorylated for fullactivation of the kinase.

SRC HOMOLOGY-3 (SH3)

DOMAIN

A non-catalytic homologyregion which mediatesprotein–protein interactionsand was first identified in Src-related protein kinases. SH3domains bind to proline (Pro)-rich peptides that contain theminimal consensusPro–X–X–Pro (in which X isany amino acid).

BRCA1

A checkpoint regulator andtumour suppressor, which ismutated with high incidence inhuman breast and ovariancancers.

BRCT HOMOLOGY DOMAIN

An evolutionarily conservedprotein–protein interactiondomain, first described in thecarboxy-terminal part of theBRCA1 tumour suppressor(BRCT; BRCA1 carboxyterminal), and subsequentlyidentified in other checkpointproteins.

© 2001 Macmillan Magazines Ltd

HOMOLOGOUS

RECOMBINATIONAL REPAIR

A mechanism for the repair ofdouble-stranded DNA breaks,which relies on the presence ofthe homologous, intact DNApartner as a template.

TRANSCRIPTION-COUPLED

REPAIR

Preferential removal of lesionsfrom the DNA strands in genesthat are actively transcribed byRNA polymerase II.

CHROMATIN REMODELLING

Dynamic changes of chromatinorganization, which arerequired for optimal executionof processes such as DNAreplication, gene transcription,DNA repair or chromosomesegregation.

14-3-3 PROTEINS

An evolutionarily conservedgroup of regulatory proteinsthat bind to discretephosphoserine-containingmotifs. 14-3-3 proteins seem tosequester their binding partnersand, in some cases, activelyexport them to the cytoplasm.

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The most recent addition to the list of Chk2 sub-strates is Cdc25A. This phosphatase activates the cyclinE/Cdk2 and cyclin A/Cdk2 kinases, which are requiredfor entry into, and progression through, S phase inmammalian cells. In response to ionizing radiation,Chk2 phosphorylates Ser123 of Cdc25A, and this mod-ification targets Cdc25A for rapid ubiquitin-dependent,proteasome-mediated degradation67. Cdc25A alsobecomes degraded in response to UV light68 and stalledDNA replication69, although the involvement of Chk2in these events is unclear. In every case, however, suchsilencing of the Cdc25A phosphatase results in persis-tent tyrosine phosphorylation of Cdk2, and thereforean inability to fire ORIGINS OF DNA REPLICATION. This, in turn,leads to delays in both late G1 and S phase67,68.

The recent description of the functional linkbetween Chk2 and Cdc25A — and, indeed, the identifi-cation of the ATM–Chk2–Cdc25A–Cdk2 axis, which isactivated in response to ionizing radiation — shedsmore light on a clinically important phenomenonknown as radioresistant DNA synthesis (RDS; BOX 1).Genetic manipulations that disrupt any component ofthe ATM–Chk2–Cdc25A–Cdk2 pathway give rise toRDS67, which highlights the significance of this check-point mechanism for maintaining genomic integrity.These results also raise the issue of potential functionallinks between the Chk2–Cdc25A interplay and theMre11–Nbs1–Rad50 checkpoint protein complex (BOX

1), defects in which also lead to RDS70,71.A common theme in checkpoint signalling has been

that ATM and ATR, which are preferentially activated inresponse to ionizing radiation and UV light or replica-tion defects, respectively10,11, often phosphorylate severalproteins in the same complex. Examples of such multi-ple phosphorylations include the p53–Mdm2 com-plex72–74, or BRCA1 and its inhibitory interactor CtIP(BRCA1 carboxy-terminal interacting protein)75–77. Thistrend for cooperative phosphorylations is furtherapparent from the fact that Chk2 often targets proteinsthat are also substrates of ATM, such as p53, BRCA1,and possibly also Mdm2 (REFS 24,53,55,77). It remains tobe seen whether this is also true for Cdc25A andCdc25C. One possibility is that the strategy of multiplephosphorylations applies mainly to targets withpleiotropic cellular effects (including cell death), such asp53–Mdm2 and BRCA1, rather than to the more ‘com-mitted’ cell-cycle effectors such as the Cdc25 phos-phatases (FIG. 5). Perhaps the former set of nodal effec-tors should be recruited and activated only when thecheckpoint responses are fully activated, to avoid anyspurious firing of their potentially harmful functions.

Chk2 and tissue biologyThe mission of genome-integrity checkpoints is to pre-serve the fidelity of genetic transmission during cell divi-sion. So the spectrum of checkpoint pathways that aredeployed, and the timing or extent of their response,might differ depending whether the cells proliferate at thetime of DNA damage.Another level of checkpoint adap-tation might reflect the cell- and tissue-type specialization.For example, lymphocytes need to deal with the enhanced

Chk2 mutants55. So, by modulating p53, Chk2 andChk1 help to enhance DNA repair, and cause sustainedG1 and G2/M cell-cycle blockade or apoptosis (FIG. 5).The downstream mediators of these p53-regulated cellu-lar effects include GADD45 and the p21CIP1/WAF1 inhibitorof cyclin-dependent kinases, apoptosis-promoting fac-tors such as Bax and Fas, and other targets whose roles inresponse to DNA damage are not yet known56,57.

BRCA1, another recently identified substrate ofChk2 (REF. 59), is one of the key checkpoint-controllingproteins, and a regulator of HOMOLOGOUS RECOMBINATIONAL

REPAIR and TRANSCRIPTION-COUPLED REPAIR, the cell cycle,CHROMATIN REMODELLING and, possibly, cell death60–62.Phosphorylation of Ser988 of human BRCA1 by CHK2(FIG. 5) is required for the release of BRCA1 from itscomplex with CHK2, and this event is important forcell survival after DNA damage59.

Apart from the involvement of Chk2/p53 in main-taining the G2 blockade, the G2/M cell-cycle arrestseems at least partly attributable to Chk2-mediatedphosphorylation of Ser216 on Cdc25C. This phospho-rylation both inhibits the phosphatase activity ofCdc25C and contributes to its cytoplasmic sequestra-tion by an interaction with 14-3-3 PROTEINS63–66. As removalof the inhibitory tyrosine phosphorylation from themitotic cyclin-dependent kinase 1 (Cdk1) by Cdc25C isa rate-limiting step in cell division, such prolongedphosphorylation of Ser216 of Cdc25C by Chk2 helpsprevent mitotic entry in cells with damaged DNA.

Figure 4 | Models of Chk2 activation. a | In mammalian Chk2, forkhead-associated (FHA)-domain-mediated phosphorylation by ATM/ATR on threonine 68 (T68) probably inducesconformational changes in the protein (dashed arrows). This makes threonines 383 and 387 inthe activation loop accessible for autophosphorylation — an event required for full activation ofChk2. This model accounts for the observed sequential order of phosphorylations in whichphosphorylation of T68 is required for subsequent phosphorylation of T383/387. b | Model ofRad53 activation in S. cerevisiae, in which Mec1-dependent, Rad9-mediated in transautophosphorylation of two Rad53 molecules leads to release of activated Rad53. Pinheadsindicate phosphorylations. A similar in trans autophosphorylation step in Chk2 activation inmammals cannot be excluded.

a b

Rad53

Rad53

Rad9

Rad9

Rad9

Rad9

Mec1

Rad53

ATM/ATR

T68

T383/T387

FHA

FHA

T68P

P P

T383/T387

Kinase domain

Kinase domain

Auto-phos

Rad53

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known examples of tissue-specific expression or subcel-lular localization of crucial checkpoint componentssuch as BRCA1 or ATM60,78.

Immunocytochemical analyses of Chk2 in culturedmammalian cells show that Chk2 is a predominantly —if not exclusively — nuclear protein25,48,59,79. This local-ization would be expected for a protein that is involvedin the regulation of the DNA-damage checkpoint (FIG. 6).Checkpoint proteins — including Chk2 — often local-ize to distinct subnuclear bodies or ‘foci’, and, indeed,ionizing radiation triggers the accumulation of Chk2 atsites of DNA strand breaks48. However, the identity,composition and dynamics of these structures inresponse to DNA damage is poorly defined48,59,62.

In contrast to other cell and tissue types, CHK2 hasa predominantly cytoplasmic localization in humanneuronal cells in situ79 (FIG. 6). This intriguing localiza-tion is reminiscent of that of the upstream regulator ofChk2, ATM78. This indicates that the ATM–Chk2 path-way might have a specialized cytoplasmic role, which ispossibly related to protection of sensitive neuronsagainst oxidative stress5,10. This possibility is consistentwith the fact that neurons that are exposed toenhanced reactive oxygen species undergo apoptoticdeath. In addition, patients with ataxia-telangiectasia,who lack functional ATM, suffer from severe neurode-generation, which probably reflects the elevated oxida-tive stress and progressive neuronal cell death5,10,19.

Early studies of mammalian Chk2 messenger RNAexpression were limited to a few tissues, and indicated a

frequency of double-stranded DNA breaks that are gener-ated during immunoglobulin or T-cell-receptor generearrangements,and germ cells must cope with the breaksthat are introduced in meiotic recombination.

Other differences might relate to the longevity of dif-ferent cell types in higher eukaryotes, such as theextreme differences between the largely non-proliferat-ing neuronal cells, compared with continuously renew-ing cell populations, such as the epidermis or gastro-intestinal mucosal epithelium. Understanding thisadditional level of complexity is difficult given the needfor more demanding model systems, such as geneticallymodified animals or studies of tissues in situ. However,this issue is important for biomedicine, and there are

Figure 5 | Chk2 downstream effectors. Activated Chk2 induces rapid G1/S cell cycle arrest and/or S-phase delay byphosphorylating Cdc25A, an event that triggers ubiquitylation (Ub) and proteasome-dependent degradation of Cdc25A. Inaddition, activated Chk2 participates in maintaining the G2/M block by phosphorylating the mitosis-promoting Cdc25Cphosphatase. This phosphorylation generates a landing pad for 14-3-3 proteins, which are believed to sequester Cdc25C fromits substrates. Chk2 also phosphorylates the p53 tumour suppressor, which results in stabilization of p53 and transactivation ofp53’s target genes. The p53-specific ubiquitin ligase Mdm2 might also be a substrate for the Chk2 kinase. Concomitantphosphorylation of p53 and Mdm2 by ataxia-telangiectasia, mutated (ATM) kinase contributes to the stabilization of p53 and anincrease in its specific DNA-binding capacity. Hence, Chk2 and ATM cooperate to achieve maximal p53-dependent expressionof genes that are involved in sustained cell-cycle arrest (as long as there is a single unrepaired double-stranded DNA break;DSB); some forms of DNA repair; and promotion of apoptosis if the damage cannot be repaired. Finally, Chk2 and ATM jointlyphosphorylate the BRCA1 tumour suppressor. Chk2-dependent phosphorylation leads to dissociation of Chk2 from BRCA1, anevent that is required for efficient repair of DSBs and survival of cells that are exposed to ionizing radiation.

Chk2

Chk2

PUb S123Cdc25A

P S216 S15 S20Cdc25C

PCdc25C

p53

p53

PP

PP

P

? S395 S988P P PPPP

BRCA1

P PPPP

BRCA1

Mdm2

Chk2

S68

ATM

Rapid G1/S blockS-phase delay

G2/M block SustainedG1 and G2 arrest

Apoptosis DNA repair

S1387S1423S1457S1524

14-3-3

Box 1 | Radioresistant DNA synthesis (RDS)

This is the inability of cells to reduce the rate of DNA replication after exposure toionizing radiation. RDS was originally discovered in cells that were derived frompatients with ataxia-telangiectasia. It was later found that RDS is also associated withother genetically transmitted and cancer-prone diseases, such as Nijmegen breakagesyndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD). Cells from patientswith NBS and ATLD are deficient in the Mre11–Nbs1–Rad50 protein complex, whichis normally required for various aspects of the cellular response to DNA damage,including recombinational DNA repair. Interestingly, the Nbs1–Mre11–Rad50complex is itself targeted by the ATM kinase in response to ionizing radiation. RDSalso occurs in cancer cells with deficient BRCA1 or CHK2 tumour suppressors.Experimentally, RDS could be evoked by preventing radiation-induced degradationof the Cdc25A phosphatase.

ORIGINS OF DNA REPLICATION

Sites on chromosomal DNAwhere replicative DNAsynthesis is initiated. In someorganisms, such as yeast, theorigins of replication aredefined by a specific DNAsequence.

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chromosomes in mouse spermatocytes81, meioticrecombination might be monitored by Chk1 in mam-mals, possibly in concert with Chk2, which is alsoexpressed in the male germ cells (albeit at lower lev-els)80.

The survey of the abundance and localization ofCHK2 in human tissues and differentiating cellularmodels in vitro79 highlighted further differences betweenChk1 and Chk2 (BOX 2). With a few exceptions, Chk2 isexpressed in cells of proliferating, as well as terminallydifferentiated, non-proliferating tissue compartments79.As Chk1 is restricted to the S and G2 phases of proliferat-ing cells79,82, the propagation of DNA-damage signals inmost of the G1 phase, and in quiescent cells, might relyselectively on Chk2.

CHK2 as a tumour suppressorThe idea that genomic instability fuels the developmentof cancer is well established, and much research hasfocused on understanding the checkpoint defects thatcontribute to oncogenesis4,6–8,56,60,83. The identification ofthe human CHK2 gene22–26 allowed the search for itspotential tumour-associated aberrations. Indeed, thefirst CHK2 mutations were reported as early as in 1999,in both sporadic and hereditary human cancers84. Thesefindings indicated that CHK2 defects might explain thetumour-prone phenotype of at least a subset of familieswith LI–FRAUMENI SYNDROME, who do not have mutations inthe p53 tumour suppressor.

However, subsequent studies showed unexpected pit-falls in searching for CHK2 mutations at the genomicDNA level , owing to closely related, non-transcribedsequences, which are present in the human genome85.This has delayed the analysis of the status of CHK2 inhuman malignancies. However, recent studies86–88 haveconfirmed that CHK2 is mutated in a subset of familialbreast cancers, and identified rare defects of CHK2 insporadic lung and lymphoid tumours. The positionsand types of the published CHK2 mutations, relative toCHK2 protein structure, are shown in FIG. 7a.

Both the normal function of CHK2 in DNA-damagecheckpoints, and the fact that some of the mutations

broad but variable Chk2 distribution, with the highestlevels in the testis22,24. Given the predominant role ofChk2 in meiotic regulation in both C. elegans andDrosophila31–34, it is surprising that, in human testes,the CHK2 protein is more abundant in the mitoticallydividing SPERMATOGONIA rather than the SPERMATOCYTES,which undergo the meiotic cell cycles80. As the func-tionally analogous Chk1 kinase localizes to meiotic

Figure 6 | Subcellular localization and tissue biology ofhuman CHK2. a | Human CHK2 kinase is localized todistinct subnuclear foci (dashed line, nuclear boundary; scalebar, 10 µm). b | Human neurons are an exception as theabundant CHK2 protein is localized almost exclusively to thecytoplasm (indicated by green arrows; scale bar, 10 µm). c | In human colon mucosa, CHK2 is homogeneouslyexpressed (indicated by red arrows) in proliferativecompartments (crypt) as well as in the quiescentdifferentiated epithelium (lumen). d | Abundant accumulationof CHK2 in cell nuclei (red arrows) within the gastric mucosa.(Figure courtesy of Claudia Lukas and Jirina Bartkova.)

a b

c

d

Lumen

Lumen

Crypt

Crypt

Box 2 | Chk2 versus Chk1

Chk2 is a relatively stable protein (with a half-life longer than six hours), which is expressed and can be activated in allphases of the cell cycle, including G0 (quiescence evoked either by depletion of growth factors or by contactinhibition). Moreover, Chk2 is present — and can be activated — in at least some differentiated cells and tissues. Bycontrast, Chk1 is an unstable protein (with a half-life of less than two hours), and its expression is restricted to the S and G2 phases of the cell cycle. Chk1 is also absent in differentiated cells. There is indirect evidence that Chk1expression (unlike that of Chk2) might be regulated by E2F TRANSCRIPTION FACTORS. The Chk1 and Chk2 proteins arestructurally distinct, and Chk1 lacks the FHA domain that is found in Chk2.

In terms of an immediate response (within two hours of DNA damage), the ATM–Chk2 pathway is specificallyactivated by the generation of DNA double-strand breaks that are induced by ionizing radiation or radiomimeticdrugs. The ATR–Chk1 pathway, by contrast, is activated in response to stalled DNA-replication forks that are inducedby either UV light (which stalls replication by generating nucleotide dimers) or drugs such as hydroxyurea (whichdepletes the cellular deoxyribonucleotide pool) and aphidicolin (which inhibits DNA polymerase α).

Homozygous disruption of either the ATR or Chk1 genes causes early embryonic lethality. Hence, both genes seemto be essential and cannot be replaced by redundant mechanisms. By contrast, ATM-deficient organisms (bothhumans and mice) do not show gross developmental defects, although deficiency of ATM predisposes to severalsymptoms, including cancer. The consequences of homozygous Chk2 disruption in mice are unknown.

SPERMATOGONIA AND

SPERMATOCYTES

Successive developmental stagesof male germ-cell maturation.Mitotically proliferatingspermatogonia mature intospermatocytes, which undergomeiotic divisions, followed byfunctional maturation intospermatids and spermatozoa.

LI–FRAUMENI SYNDROME

A highly cancer-prone familialdisorder (clinically defined byLi and Fraumeni in 1988), thatis caused by germline mutationsin TP53 or other tumour-suppressor genes, includingCHK2.

E2F TRANSCRIPTION FACTORS

A family of six proteins thatregulate expression of genesthat are required for DNAreplication.

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cycle checkpoint regulators that act in common path-ways with CHK2 (FIG. 7b). On the other hand, concomi-tant loss-of-function mutations in CHK2 and its sub-strate, the p53 tumour suppressor, were identified in asporadic colon carcinoma cell line84,89. This combinationof mutations in CHK2 and TP53 (the gene that encodesp53 in humans) might provide additional selectiveadvantage to tumour cells, compared with cells that har-bour defects in either CHK2 or p53 alone89.

Such a cooperative effect could be explained by thefact that, apart from their common function in the samecheckpoint pathway, CHK2 and p53 also have mutuallyindependent functions. Furthermore, these results indi-cate that there might be simultaneous defects of CHK2with p53 or other checkpoint regulators in humanmalignancies. Epigenetic mechanisms are also known toundermine the expression of some tumour suppressors,and it remains to be seen if silencing by promoter methy-lation, or defects in pathways that regulate transcriptionor turnover of CHK2, might be targeted in oncogenesis.Screening human tumour biopsies for reduction or lossof CHK2 protein seems feasible, as documented by therecent identification of subsets of human testiculartumours with reduced CHK2 (REF. 80), and the reducedprotein stability of some of the identified CHK2mutants80,90,91. Immunohistochemical screening could befacilitated by the often ubiquitous expression of CHK2 inmany normal tissues79, which makes it easier to findtumours with a significant loss of CHK2.

Finally, CHK2 might turn out to be an attractive tar-get for drug discovery, and several pharmaceutical com-panies are searching for small-molecule inhibitors of thiskinase. As cancer cells often lack one or more genome-integrity checkpoints, inhibition of the remaining check-point(s) could make tumour cells selectively more sensi-tive to anticancer therapies, such as γ-radiation orDNA-damaging drugs. Whereas normal cells wouldstill activate their other checkpoint(s) and recover fromthe temporary cell-cycle arrest that is evoked by thetreatment, cancer cells deprived of most or all check-points would be more likely to die. Encouraging exam-ples of such selective effects of checkpoint inhibi-tion92–94 raise the possibility of clinically applicablecancer treatments in the future. For example, blockingthe activity of ATM/ATR by caffeine92,94, inhibition ofCHK1 by UCN-01 (REFS 95,96), or competition for sub-strates of CHK1/CHK2 with CDC25C-derived pep-tides93, resulted in checkpoint abrogation and preferen-tial cancer-cell death after exposure to DNA-damagingtreatments, particularly in tumour cells that were alreadydeficient in their p53-dependent checkpoint pathway.

Conclusions and future challengesThe cloning of the human CHK2 gene22–26 marked therecent explosion of research on the regulation, functionand malfunction of this busy and versatile messenger,which is involved in several genome-integrity check-points. Despite the observed checkpoint failure inmouse embryonic stem cells that are genetically defi-cient for Chk2 (REF. 58), the phenotype of such animals isnot yet known. Such animal models should also help us

were expected to result in a truncated — and thereforeprobably defective — protein, were consistent with theidea that CHK2 might be a tumour suppressor. On theother hand, the originally published mutations were allheterozygous, calling for a biochemical assessment ofthe mutant CHK2 proteins. Such analyses confirmedthat both truncated and missense mutants of CHK2have lost their ability to interact with, and efficientlyphosphorylate, substrates such as CDC25A or p53 (REFS

67,89,90). Ectopic expression of the missense mutants dis-rupted the function of the wild-type CHK2 in humancells, which is consistent with a dominant-negativeeffect67. So, the identified heterozygous mutations ofCHK2 might undermine genomic stability by eitherpartial loss of function (haploinsufficiency), dominant-negative effects of some mutants, or a combination ofboth. Collectively, these results confirmed the loss-of-function nature of the tumour-associated CHK2 muta-tions, and strongly supported the candidacy of thischeckpoint kinase as a tumour suppressor.

The relative paucity of cancer-associated mutationsof CHK2 identified so far can be explained by alterna-tive cancer-promoting defects, which target other cell-

Figure 7 | CHK2 as a tumour suppressor. a | Structure of human CHK2, which shows thepositions of the mutations that have been identified so far. Aberrations include missensemutations (R145W, I157T, D311V and A507G) and a single nucleotide deletion (del1100C),which leads to a frameshift in the CHK2 coding sequence. b | Interestingly, both ATM (requiredfor initiation of CHK2 activation) and most downstream targets of CHK2 are also deregulatedin cancer and could be classified as tumour suppressors (TP53, BRCA1) and proto-oncogenes (CDC25A, MDM2), respectively. A recent report81 showed concomitant mutation ofboth CHK2 and TP53 in one tumour cell line. This finding indicates that, even in the absence offunctional TP53, the cells can gain a proliferative advantage by disrupting the CHK2–CDC25Aaxis, which regulates the rapid cell-cycle arrest in response to DNA damage. FHA, forkhead-associated domain; ATM, ataxia-telangiectasia, mutated kinase; TP53, the gene that encodesp53 in humans.

b

del1100C

D311V

R145W

l157T A507G

ATM

CHK2

BRCA1

TP53

MDM2

CDC25A

Mutated (ataxia-telangiectasia)

Mutated (breast and ovarian cancer)

Mutated; functionally compromised (multiple tumour types)

Amplified; overexpressed (osteosarcomas)

Mutated (colorectal carcinomas, Li–Fraumeni syndrome)

Overexpressed (breast, lung,

head and neck carcinomas)

Tumour suppressors Proto-oncogenes

a

1 20 75 115 165 225 490 543Activation loop

Kinase domainFHASQ/TQ

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required for its full activation and efficient amplificationof the checkpoint-signalling cascade (FIG. 4).

The complexity of the involvement of Chk2 in cell-cycle arrest is also far from being fully appreciated.Given that Cdc25C knockout mice have no obviousdeficiency in their G2 checkpoint response97, the bio-chemical basis and involvement of Chk2 in the G2/Marrest following DNA damage has to be carefully re-evaluated. So, searching for additional physiologicalsubstrates that are targeted by Chk2 in response togenotoxic stress should be a rewarding line of research.

In terms of the role of Chk2 in molecular oncogenesis,the extent of its inactivating mutations in diverse formsof familial versus sporadic cancer should be clarified.Furthermore, studies in many types of human malig-nancy should find additional types of mutations inChk2. These will provide clues to the function of thetargeted motifs within the protein and allow assessmentof the potential effects of such mutations on the sensi-tivity of the Chk2-deficient tumours towards existingradiotherapy and chemotherapy protocols.

Structure–function analysis and the search for small-molecule inhibitors of CHK2 would greatly benefit fromknowledge of its crystal structure.Such detailed structuralinformation, along with potentially specific newinhibitors, should not only aid further analysis of the mol-ecular signalling pathways that are mediated by Chk2,butmight also offer a new generation of drugs for sensitizingtumours to DNA-damaging therapies in the clinic.

to work out how much redundancy there is betweenChk2 and Chk1, and the potential tissue-specific func-tions of Chk2. The roles of Chk2 in the nervous system— as characterized by the unorthodox cytoplasmiclocalization of the kinase — and in lymphocytic andgerm cells, both of which are physiologically exposed toa high frequency of double-stranded DNA breaks,would be of particular interest.

We should soon know more about the involvementof the Chk2 FHA domain in protein–protein interac-tions, along with the composition and dynamics of theChk2-containing nuclear foci. These issues are beingaddressed using fluorescence-based technologies tostudy protein–protein interactions and subcellulartransport directly in live cells during real time. It wouldalso be interesting to corroborate the structural/func-tional interplay between CHK2 and BRCA1. BRCA1 hasbeen proposed as a master molecule that assembles andintegrates the many components of the early checkpointresponse into a dynamic multicomponent complexcalled the BASC (BRCA1-associated genome surveil-lance complex)62. Whether CHK2 is a genuine compo-nent of the BASC remains to be established.Nevertheless, BRCA1 and CHK2 do physically interact59,and this might either bring CHK2 in close proximity tothe upstream ATM/ATR kinases that are required to ini-tiate CHK2 activation, or it might operate in a similarmanner to yeast Rad9 and assemble CHK2 molecules toundergo auto- and/or trans-phosphorylation, which is

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Online links

DATABASESThe following terms in this article are linked online to:Interpro: http://www.ebi.ac.uk/interpro/FHALocusLink: http://www.ncbi.nlm.nih.gov/LocusLinkATR | Bax | CtIP | cyclin E | GADD45 | p21CIP1/WAF1

OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMataxia-telangiectasia | ataxia-telangiectasia-like disorder |Nijmegen breakage syndromeSaccharomyces Genome Database: http://genome-www.stanford.edu/Saccharomyces/Mec1 | Mek1 | Mrc1 | Mre11 | Rad3 | Rad9 | Rad50 | Rad53 Swiss-Prot: http://genome-www.stanford.edu/Saccharomyces/53BP1 | ATM | BRCA1 | c-Abl | Cdc25A | Cdc25C | Chk1 | Chk2 |Mdm2 | p53 Access to this interactive links box is free online.