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The Chk2 protein kinase
DNA Repair 3 (2004) 1039–1047
Review
The Chk2 protein kinase Jinwoo Ahn1 , Marshall Urist1 , Carol Prives∗ Department of Biological Sciences, Columbia University, New York, NY 10027, USA Available online 22 April 2004
Abstract Checkpoint kinase 2 (Chk2) is a multifunctional enzyme whose functions are central to the induction of cell cycle arrest and apoptosis by DNA damage. Insight into Chk2 has derived from multiple approaches. Biochemical studies have addressed Chk2 structure, domain organization and regulation by phosphorylation. Extensive work has been done to identify factors that recognize and respond to DNA damage in order to activate Chk2. In turn a number of substrates and targets of Chk2 have been identified that play roles in the checkpoint response. The roles and regulation of Chk2 have been elucidated by studies in model genetic systems extending from worms and flies to mice and humans. The relationship of Chk2 to human cancer studies is developing rapidly with increasing evidence that Chk2 plays a role in tumor suppression. © 2004 Elsevier B.V. All rights reserved. Keywords: Checkpoint kinase 2; Apoptosis; Phosphorylation
1. Introduction
2. Domains of Chk2
Cells are exposed to constant endogenous and exogenous stresses that threaten DNA. To preserve the integrity of genetic information, an intricate network of proteins communicate with each other throughout the cell cycle to elicit appropriate cellular responses, such as DNA repair, cell cycle arrest or apoptosis in response to these insults (reviewed in [1,2]). The DNA damage checkpoint pathway has been of interest to the field of cancer biology, since checkpoint defects result in the accumulation of altered genetic information, a central feature of carcinogenesis (reviewed in [3,4]). The conservation of the pathway from yeast to human in eukaryotes has been beneficial to researchers in unraveling and identifying each component. Among the conserved DNA-damage activated kinases identified thus far, the Checkpoint kinase 2 (Chk2) plays a central role in implementing many aspects of the checkpoint response [5]. In this review, we summarize and discuss the results of biochemical, biophysical and biological studies of Chk2.
The Chk2 protein consists of three distinct functional domains (see the Small Molecular Architecture Research Tool database, http://smart.embl-heidelberg.de/); an SQ/TQ cluster domain (SCD, residues19–69), a forkhead associated domain (FHA, residues112–175), and a Ser/Thr kinase domain (residues 220–486) (Fig. 1A). The SCD contains five SQ and two TQ motifs, which satisfy the primary substrate motif for ataxia-telangectasia mutated (ATM) kinase [6]. Especially residues 67–70, STQE, are closely related to the refined substrate motif (LSQE) of ATM kinase, determined by oriented peptide library search [7], and T68 was identified to be a key phosphorylation site (see below). The FHA domain is an 80–100 amino acid phosphopeptide binding domain, conserved from yeast to human (reviewed in [8]). The domain consists of 11 beta sheets forming a structural core and connecting loops conferring diversity in phosphopeptide recognition as resolved by structural analyses of several FHA domains with and without phosphopeptides by X-ray crystallography [9–12] and nuclear magnetic resonance (NMR) spectroscopy [13–17]. FHA domains are found in proteins involved not only in DNA damage checkpoint pathways, but also kinesin family members, transcription factors, Ki-67, and nuclear inhibitors of protein phosphatase 1 (reviewed in [18]). The FHA domain functions in trans to modulate protein–protein interactions
∗ Corresponding author. Tel.: +1-212-854-2557; fax: +1-212-865-8246. E-mail address: [email protected] (C. Prives). 1 These authors contributed equally to this review.
1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.03.033
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Fig. 1. Functional Domain Architecture of Chk2 and a Model of Chk2 Activation. Landmarks of the Chk2 protein showing the SQ/TQ cluster domain (SCD) the forkhead associated domain (FHA) and the kinase domain. Shown below are previously identified phosphorylation sites phosphorylated by ATM or ATR kinases or resulting from dimerization induced autophosphorylation. A model for gamma irradiation induced formation of active dimers and monomers. After phosphorylation of inactive monomers at T68 by ATM, dimers form and undergo subsequent autophosphorylation and may also serve as substrates for other protein kinases. Phosphorylated dimers or monomers are active in phosphorylating substrates such as Cdc25C.
but also in cis to affect other functional domains within the protein itself. The kinase domain shares homology to other Ser/Thr or Tyr kinases with a Gly-rich region at the vicinity of Lys residues in the N-terminal part and Asp as a catalytic residue at the active site.
3. Chk2 and cancer Chk2 has established its position as a bona-fide tumor suppressor and the list of Chk2 mutations in cancer cells is continuously expanding (reviewed in [19]). The first indication of a tumor suppressor function for Chk2 came from the finding that a sub-population of families with the Li-Fraumeni syndrome (LFS), a familial cancer predisposition syndrome usually associated with germline TP53 mutation (reviewed in [20]), contain wild type p53 but mutant Chk2 suggesting that germline mutations in the CHEK2 gene phenocopy TP53 inactivation [21]. The CHEK2 allele containing the mutation 1100delC encodes a C-terminally truncated protein within the kinase domain and confers increased breast cancer risk independent of mutation in the breast cancer susceptibility associated (BRCA) loci [22–24]. Screening of cancer cell lines has revealed missense mutations in all three functional domains in a variety of tumor types [25–27]. The functional impact of CHEK2 mutation generally reflects its location. Thus, kinase domain mutants lead to faulty kinase activation after ionizing radiation [28], while FHA domain lesions may accelerate Chk2 degradation [29,30], or may induce structural changes conferring defects in protein–protein interaction [11,31,32]. It will be of interest to determine at what stage of tumor development CHEK2 mutations arise and the precise Chk2 functions that are selected against.
4. Regulation and activation of Chk2 Chk2 kinase activity is increased following DNA damage induced by ionizing radiation, chemotherapeutic agents, or other compounds that harm DNA directly or indirectly. Recently, senescence triggered by telomere erosion has been implicated as an activating signal for Chk2 [33,34]. Signals from damaged DNA are transmitted to the ATM kinase, which has a number of substrates involved in the checkpoint cascade [35–37]. Multiple lines of evidence establish a strong link between ATM and Chk2. First, Chk2 fails to be activated in A–T cells and restoration of a Chk2-dependent S-phase checkpoint was observed upon ectopic expression of ATM in A–T cells [5,38,39]. Second, ATM directly phosphorylates Chk2 at Thr68, a consensus site in the SCD domain in vitro [40,42] and alanine mutation of this residue prevents activation of Chk2 by gamma irradiation in vivo [40–42]. The Ataxia-Telangectasia-and-Rad3-related (ATR) kinase may similarly phosphorylate Chk2 after high levels of DNA damage or other stressors such as ultraviolet radiation (UV) or treatment with the ribonucleotide reductase inhibitor Hydroxyurea (HU) [41]. Chk2 exists as a monomer in unperturbed cells. Following DNA damage, Chk2 undergoes dimerization in which a region spanning phosphorylated Thr68 in one Chk2 molecule binds to the FHA domain of second molecule [43–45]. Dimerization is likely followed by multiple intermolecular phosphorylation events including Thr383 and Thr387 within the auto-inhibitory loop resulting in kinase activation (Fig. 1B) [46]. Autophosphorylation of Ser516 within the kinase domain was also recently identified, and is required for full kinase activation [47,48]. Furthermore, mutation of this residue prevents radiation-sensitization by
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Chk2, suggesting a functional role for autophosphorylation [48]. Phosphorylation at Thr68 is likely to be the initiating event for dimerization and activation since maintenance of Thr68 phosphorylation is not required for sustained kinase activity [45]. Phosphorylation of other sites are likely to mediate stress specific differences in the extent of kinase activation. Indeed, Chk2 purified after gamma radiation, UV, and HU treatment showed different kinase activities toward Cdc25C, one of its well validated substrates ([5] and Ahn and Prives, unpublished results). Moreover, the phospho-Thr68 to FHA domain intramolecular interaction mechanism may also function in stress induced association with other checkpoint proteins. For example, the MDC1 checkpoint protein FHA domain binds selectively to Chk2 phosphorylated at Thr68 [49]. The Polo Like Kinase 1 (PLK1), a centrosome-associated kinase implicated in mitotic progression, can interact with Chk2 and colocalize with Chk2 at centrosomes. Interestingly, PLK1 can phosphorylate residues in the N-terminus of Chk2 including Thr68 [50]. The functional import of this interaction for PLK1 or Chk2 function is not known. The related kinase PLK3 also interacts with Chk2 and phosphorylation of Chk2 and PLK3 by one another has been reported [51,52]. The subcelluar localization of Chk2 may be regulated by another recently described binding protein, karyopherin–alpha2 [53].
5. Substrates of Chk2 Upon activation Chk2 relays the checkpoint activation signal to a number of effectors, which mediate many of the phenotypic characteristics provoked by DNA damage including cell cycle arrest and apoptosis (Fig. 2). Use of oriented peptide libraries has shown Chk2 preferentially
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phosphorylates a consensus motif, L-X-R-X-X-S/T, [54,55]. However, these studies may only describe one aspect of Chk2 kinase specificity as they do not capture the in vivo complexity of kinase-substrate interaction. Nevertheless, many functionally important substrates bearing this motif have been identified. The first described and best validated of Chk2 substrates are two members of the cell division cycle 25 dual specificity phosphatase family, Cdc25A and Cdc25C. The Cdc25 family promotes cell cycle progression by activation of the cyclin-dependent kinases cdk2 and cdk1 through dephosphorylation of inhibitory phosphorylation at Thr14 and Tyr15 [5,32,38,56]. Cdc25A is destabilized by Chk2 phosphorylation at Ser123 [32,57] most likely by increasing susceptibility to the SCF-TCRP complex, which mediates Cdc25A turnover [58,59]. Chk2 phosphorylation of Ser216 in Cdc25C creates a binding site for 14-3-3 proteins [60] and this interaction is thought to result in persistent cytoplasmic Cdc25C localization thus preventing the G2/M transition as the cdk1/Cyclin B complex cannot be activated [61,62]. Taken together, phosphorylation of Cdc25A and Cdc25C by Chk2 in response to DNA damage explains in part how the checkpoint induces cell cycle arrest. Chk2 phosphorylates E2F-1 at Ser364 in response to the DNA-damaging agent etoposide, and the modification of that residue regulates both E2F-1 stabilization and transcriptional activity [63]. Given the observation that E2F-1 null thymocytes are resistant to etoposide-induced apoptosis [64] and that over-expression of E2F-1 can induce cell death [65], modulation of E2F-1 activity represents a potentially important pathway through which Chk2 may regulate DNA damage-induced apoptosis. Two checkpoint pathway scaffold proteins, BRCA1 and Promyelocytic Leukemia (PML), can interact with Chk2 and may be phosphorylated by Chk2 following DNA damage
Fig. 2. The Chk2 network. Chk2 integrates signals from upstream kinases and binding partners to phosphorylate key targets involved in cell cycle checkpoints. See text for details of Chk2 substrates and binding partners.
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[66,67]. Interestingly, both substrates follow a similar pattern in which Chk2 interacts with PML and BRCA1 in the absence of DNA damage but following gamma irradiation BRCA is phosphorylated at Ser988 and PML is phosphorylated at Ser117 in a Chk2-dependent fashion, resulting in subsequent inhibition of binding with Chk2. Alanine mutation of either phosphorylation site prevents release of Chk2 from BRCA1 or PML after DNA damage suggesting a relationship between activation of phosphorylation and inhibition of interaction. Functionally, BRCA1Ser988A fails to increase survival of BRCA1-null HCC1937 cells following IR and BRCA1Ser988A stably expressed in HCC1937 cannot mediate double strand break repair [68]. In contrast to wild-type PML, the mutant PMLSer117A does not sensitize U937 leukaemia cells to gamma irradiation- induced apoptosis suggesting functional importance for phosphorylation of Ser117 by Chk2. For both proteins, a mechanistic understanding of how modification impacts function is lacking. For example, it will be of interest to determine if the alanine mutants of either protein are able to inhibit downstream functions of Chk2. In an elegant recent paper, Lukas and colleagues demonstrated using both fluoresence-loss-in-photobleaching (FLIP) and fluoresence-return-after-photobleaching (FRAP) that unlike other another checkpoint protein Nbs1, Chk2 does not become immobilized at sites of damaged DNA but remains soluble after x-irradiation. Further, immobilization of Chk2 by fusion to Histone2B inhibits Chk2 function leading to the view that Chk2 acts as a “signal spreader” dispersing a checkpoint “on” signal to its various substrates implementing cell cycle arrest and apoptosis [69]. Given that both PML and BRCA1 form foci which collect many checkpoint proteins including Chk2’s principle activator ATM [70,71], perhaps PML and BRCA1 function in part to provide sites of Chk2 activation by ATM. Moreover, Thr68-Chk2 colocalizes with 53BP1, H2AX, and NBS1 in nuclear foci [72,73]. Once activated therein, Chk2 disengages from the complex by phosphorylation of PML, BRCA1, or other factors to then find and activate Chk2’s various substrates. Consistent with this model, BRCA1 is required for Thr68 phosphorylation by ATM [74]. A study, which found that closely associated mismatch repair factors (MMR) MSH2 and MLH1, members of the BASC or BRCA1-associated genome surveillance complex [71], bind Chk2 and ATM respectively and play an important role in Chk2 activation generally supports this hypothesis. Restoration of either MSH2 or MLH1 in colon tumor cell lines bearing mutations in these factors enhances Thr68 phosphorylation after DNA damage, restores Cdc25A destabilization by gamma irradiation, and complements the RDS phenotype, all hallmarks of increased Chk2 signaling [75]. Also, Chk2 from MSH−/− cells does not undergo Thr68 phosphorylation after gamma irradiation [76]. Moreover, Chk2 activation is defective in cells derived from Nijmegen Breakage Syndrome patients who carry inactivating mutations in Nbs1 a component of the Mre11–Rad50–Nbs1
complex which plays central role in DNA repair and is required for ATM activation after DNA damage [77,78–82] 53BP1 another checkpoint factor important in forming DNA damage signaling complexes with ATM and BRCA1 may also play a role in Chk2 activation. 53BP1 null MEFs show little if any defect in Chk2 phosphorylation as determined by mobility shift in polyacrylamide gels after IR [83,84] but focus formation of activated Chk2 as detected by an anti-phosphoT68 Chk2 antibody is compromised by 53BP1 knockdown [72]. Interestingly, 53BP1 is involved in the phosphorylation of other ATM substrates including Smc1 [85]. Also, Chk2 activation is independent of H2AX which is phosphorylated at sites of DNA damage [84]. A more recently discovered checkpoint factor also present in damage foci, NFBD1/MDC1 binds to phospho-Chk2 but its requirement for Chk2 Thr68 phosphorylation is controversial [49,86–89]. Detailed understanding of how the various signaling interactions required for Chk2 activation are integrated will be of great interest.
6. Chk2 knockout mice and the p53 connection Given the central role of both p53 and Chk2 in cell cycle arrest and apoptosis as well as their roles in tumor suppression, many reports have described connections between Chk2 and p53. Conclusions from several studies, however, have led to the realization that different approaches can provide widely varying results. Several groups placed Chk2 immediately upstream of p53 through studies involving Chk2 knockout mice as well as in vivo and in vitro experiments. Phosphorylation of p53 at Ser20 had been suggested to be a critical event for stabilizing p53 [90,91]. The search for a kinase capable of Ser20 phosphorylation seemed to end when it was reported that Chk2 could phosphorylate p53 at Ser20 in vitro and this event was sufficient to disrupt pre-complexed p53 and Mdm2 [92,93]. Introduction of a dominant negative Chk2 led to failure of U2OS cells to stabilize or phosphorylate endogenous p53 at Ser20 and Chk2 enhanced a p53 dependent G1 checkpoint [93]. In another study, it was reported that immunoprecipitated Chk2 is activated by IR to phosphorylate p53 and overexpression of Chk2 enhances the ability of p53 to transactivate a p53 responsive reporter plasmid [31]. Interestingly, Chk2 downregulation by antisense in 293 cells increases sensitivity to DNA double strand breaks [94]. Silencing of Chk2 by novel engineered zinc finger transcription factors decreases p53 transcriptional activation of the MDM2 gene and reduces p53 Ser20 phosphorylation [95]. However, other work has weakened the view that Chk2’s regulation of p53 is generally applicable. First, the region on p53 spanning Ser20 does not share homology with the consensus motif, L-X-R-X-X-S/T present in other known substrates of Chk2 and a peptide covering the p53 Ser20 region is a poor Chk2 substrate [54,55]. Interestingly, two regions of p53, aa 117–132 and aa 272–288, are proposed
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to provide Chk2 docking sites to allosterically regulate the kinase activity toward p53 and purified Chk2 supplemented with these docking-site motifs phosphorylated a Ser20 peptide by kinase assay [96]. A second group has identified another Chk2 binding domain in p53 centering on Thr329 [97]. In contrast to this work, Chk2 purified from tumor cell lines before and after DNA damage could not phosphorylate different forms of truncated or full-length p53 even though it was markedly activated to phosphorylate the domain of Cdc25C containing Ser216 [98]. Experiments in vivo similarly challenge the Chk2–p53 relationship. Introduction of Chk1 and Chk2 siRNAs into several human tumor cell lines failed to affect p53 stabilization, Ser20 phosphorylation or transcriptional activity despite preventing Ser216 phosphorylation of Cdc25C [98]. Even more, homozygous deletion of Chk2 in HCT-116 colon carcinoma cells yielded no phenotype with respect to p53, G1 or G2 cell cycle arrest, and apoptosis [99]. Two independently generated Chk2 knockout mice have supported a role for Chk2 in the regulation of p53 but diverge in their findings and proposed mechanisms. The first reported Chk2 null mouse from Hirao and colleagues [100] strikingly showed that thymocytes, splenic T cells, and MEFs lacking Chk2 are unable to stabilize p53 following gamma irradiation and this defect is corrected by reintroduction of Chk2. These cells are highly resistant to apoptosis consistent with a defect in p53 signaling [100]. A follow up study showed that Chk2 mice are highly resistant to apoptosis in multiple tissue compartments by whole body irradiation experiments and develop chemically-induced skin tumors more rapidly [101]. The G1/S checkpoint of these mice is compromised at low doses of gamma irradiation but cells arrest normally at doses above 5Gy. Interestingly, however, p53 null mice do not show any dose dependency in the same assay. In contrast to the original report a second study from this group showed that gamma irradiated Chk2−/− thymoctyes show a defect in transcriptional activation of Bax but only a slight depression in p21 upregulation [101]. A different group using MEFs from these same mice, however, found no defect in G1/S arrest or p21 mRNA induction at even low X-ray doses [102]. More recently a second Chk2 knockout mouse generated by another group was reported with somewhat different results [103]. Consistent with the first Chk2 null mouse, the second was resistant to apoptosis induced by gamma irradiation in many tissues strongly implicating Chk2 in DNA damage induced apoptosis [103]. No G2 or S-phase checkpoint defect was detected but these authors did observe a deficient G1/S arrest consistent with impaired p53 signaling. In contrast to Hirao et al, they reported only a 30–50% reduction in p53 stabilization and human p53 introduced into Chk2−/− MEFs was phosphorylated normally on Ser20 after IR, indicating that in murine tissue Chk2 makes a partial contribution to p53 stabilization but other factors clearly contribute. Perhaps the most striking result with respect to p53 was that, despite only a partial p53 stabilization defect, upon analysis of five p53 targets
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genes by quantitative RT–PCR in thymocytes and MEFs, the authors saw no mRNA upregulation in the Chk2−/− cells after IR providing a possible mechanistic explanation for the G1/S arrest and apoptotic defects in these cells [103]. However, the relationship between Chk2 and p53 is likely to be stimuli–as well as cell type-specific as cell death in primary neuronal cultures induced by etoposide or camptothecin was shown to be ATM-dependent but Chk2 independent [104]. To summarize the mouse studies to date, it is clear that Chk2 plays a central role in gamma irradiation induced apoptosis however the mechanism is unclear. Chk2 likely regulates p53 transcriptional activity independently of Ser20 phosphorylation or even stabilization in murine tissues after IR, but in other contexts yet to be described signaling pathways play a central role. It remains to be determined when Chk2 regulates p53, in what context, and what factors mitigate or compensate for Chk2 in cases where it is not required.
7. Chk2 in lower organisms As mentioned above, the Chk2 kinase is conserved throughout phylogeny including Caenorhabditis elegans and Drosophila melanogaster. Study of the Chk2 gene in these organisms has revealed novel functions not seen in human or mouse cells. The fly homolog of Chk2, Mnk or dmChk2, is highly expressed in ovaries during embryogenesis [105] and embryos lacking Chk2 are highly sensitive to gamma irradiation suggesting a checkpoint function [106]. Loss of Chk2 in fly embryos also leads to genomic instability, a phenotype enhanced by treatment with DNA damaging agents, that has been linked to abnormal centrosome function so that lethally damaged cells cannot be efficiently eliminated from the embryo in the absence of Chk2 [107,108]. Transgenic embryos expressing dominant-negative (DN) dmChk2 were resistant to radiation-induced apoptosis and prevented cell death caused by over-expressed dmp53 [109]. Also, dmChk2 regulates p53 phosphorylation status and transcriptional activity following gamma-irradiation [110]. RNA interference studies of the C. Elegans Chk2 homolog, Cds, revealed that this kinase is essential for meiotic recombination, a function not seen in other organisms [111–113]. Interestingly, a role for dmChk2 in a meiotic checkpoint has also been demonstrated suggesting that this function of Chk2 may be conserved [114].
8. Conclusions and future directions Future studies of Chk2 will certainly identify novel substrates, interacting partners, and functionally important phosphorylation sites in Chk2. As a picture of Chk2 function becomes clearer, it will be of particular interest to determine how Chk2 integrates its various functions to precisely determine the outcome of checkpoint activation. It will also be important to resolve differences in findings
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relating Chk2 and p53. Chk2’s relationship to modules in the checkpoint pathway controlling DNA repair, cell cycle arrest, and apoptosis places it at the nexus of the DNA damage response. Therefore, modulation of its activity might offer some future benefit to the development of cancer therapeutics.
Acknowledgements The work is supported by NIH grant CA87497.
References [1] B.B. Zhou, S.J. Elledge, The DNA damage response: putting checkpoints in perspective, Nature 408 (2000) 433–439. [2] J.M. Bradbury, S.P. Jackson, The complex matter of DNA double-strand break detection, Biochem Soc Trans 31 (2003) 40–44. [3] L.H. Thompson, D. Schild, Recombinational DNA repair and human disease, Mutat Res 509 (2002) 49–78. [4] B.B. Zhou, H.J. Anderson, M. Roberge, Targeting DNA checkpoint kinases in cancer therapy, Cancer Biol Ther 2 (2003) S16–S22. [5] S. Matsuoka, M. Huang, S.J. Elledge, Linkage of ATM to cell cycle regulation by the Chk2 protein kinase, Science 282 (1998) 1893–1897. [6] S.T. Kim, D.S. Lim, C.E. Canman, M.B. Kastan, Substrate specificities and identification of putative substrates of ATM kinase family members, J Biol Chem 274 (1999) 37538–37543. [7] T. O’Neill, A.J. Dwyer, Y. Ziv, D.W. Chan, S.P. Lees-Miller, R.H. Abraham, J.H. Lai, D. Hill, Y. Shiloh, L.C. Cantley, G.A. Rathbun, Utilization of oriented peptide libraries to identify substrate motifs selected by ATM, J Biol Chem 275 (2000) 22719–22727. [8] J. Li, G.I. Lee, S.R. Van Doren, J.C. Walker, The FHA domain mediates phosphoprotein interactions, J Cell Sci 113 (Pt 23) (2000) 4143–4149. [9] D. Durocher, J. Henckel, A.R. Fersht, S.P. Jackson, The FHA domain is a modular phosphopeptide recognition motif, Mol Cell 4 (1999) 387–394. [10] D. Durocher, I.A. Taylor, D. Sarbassova, L.F. Haire, S.L. Westcott, S.P. Jackson, S.J. Smerdon, M.B. Yaffe, The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms, Mol Cell 6 (2000) 1169–1182. [11] J. Li, B.L. Williams, L.F. Haire, M. Goldberg, E. Wilker, D. Durocher, M.B. Yaffe, S.P. Jackson, S.J. Smerdon, Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2, Mol Cell 9 (2002) 1045– 1054. [12] E.S. Stavridi, Y. Huyen, I.R. Loreto, D.M. Scolnick, T.D. Halazonetis, N.P. Pavletich, P.D. Jeffrey, Crystal structure of the FHA domain of the Chfr mitotic checkpoint protein and its complex with tungstate, Structure (Camb) 10 (2002) 891–899. [13] H. Liao, I.J. Byeon, M.D. Tsai, Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53, J Mol Biol 294 (1999) 1041–1049. [14] H. Liao, C. Yuan, M.I. Su, S. Yongkiettrakul, D. Qin, H. Li, I.J. Byeon, D. Pei, M.D. Tsai, Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9, J Mol Biol 304 (2000) 941–951. [15] P. Wang, I.J. Byeon, H. Liao, K.D. Beebe, S. Yongkiettrakul, D. Pei, M.D. Tsai II, Structure and specificity of the interaction between the FHA2 domain of Rad53 and phosphotyrosyl peptides, J Mol Biol 302 (2000) 927–940.
[16] I.J. Byeon, S. Yongkiettrakul, M.D. Tsai, Solution structure of the yeast Rad53 FHA2 complexed with a phosphothreonine peptide pTXXL: comparison with the structures of FHA2-pYXL and FHA1-pTXXD complexes, J Mol Biol 314 (2001) 577–588. [17] C. Yuan, S. Yongkiettrakul, I.J. Byeon, S. Zhou, M.D. Tsai, Solution structures of two FHA1-phosphothreonine peptide complexes provide insight into the structural basis of the ligand specificity of FHA1 from yeast Rad53, J Mol Biol 314 (2001) 563–575. [18] D. Durocher, S.P. Jackson, The FHA domain, FEBS Lett 513 (2002) 58–66. [19] J. Bartek, J. Lukas, Chk1 and Chk2 kinases in checkpoint control and cancer, Cancer Cell 3 (2003) 421–429. [20] S.C. Evans, G. Lozano, The Li-Fraumeni syndrome: an inherited susceptibility to cancer, Mol Med Today 3 (1997) 390–395. [21] D.W. Bell, J.M. Varley, T.E. Szydlo, D.H. Kang, D.C. Wahrer, K.E. Shannon, M. Lubratovich, S.J. Verselis, K.J. Isselbacher, J.F. Fraumeni, J.M. Birch, F.P. Li, J.E. Garber, D.A. Haber, Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome, Science 286 (1999) 2528–2531. [22] P. Vahteristo, J. Bartkova, H. Eerola, K. Syrjakoski, S. Ojala, O. Kilpivaara, A. Tamminen, J. Kononen, K. Aittomaki, P. Heikkila, K. Holli, C. Blomqvist, J. Bartek, O.P. Kallioniemi, H. Nevanlinna, A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer, Am J Hum Genet 71 (2002) 432–438. [23] H. Meijers-Heijboer, J. Wijnen, H. Vasen, M. Wasielewski, A. Wagner, A. Hollestelle, F. Elstrodt, R. van den Bos, A. de Snoo, G.T. Fat, C. Brekelmans, S. Jagmohan, P. Franken, P. Verkuijlen, A. van den Ouweland, P. Chapman, C. Tops, G. Moslein, J. Burn, H. Lynch, J. Klijn, R. Fodde, M. Schutte, The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype, Am J Hum Genet 72 (2003) 1308–1314. [24] H. Meijers-Heijboer, A. van den Ouweland, J. Klijn, M. Wasielewski, A. de Snoo, R. Oldenburg, A. Hollestelle, M. Houben, E. Crepin, M. van Veghel-Plandsoen, F. Elstrodt, C. van Duijn, C. Bartels, C. Meijers, M. Schutte, L. McGuffog, D. Thompson, D. Easton, N. Sodha, S. Seal, R. Barfoot, J. Mangion, J. Chang-Claude, D. Eccles, R. Eeles, D.G. Evans, R. Houlston, V. Murday, S. Narod, T. Peretz, J. Peto, C. Phelan, H.X. Zhang, C. Szabo, P. Devilee, D. Goldgar, P.A. Futreal, K.L. Nathanson, B. Weber, N. Rahman, M.R. Stratton, Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations, Nat Genet 31 (2002) 55–59. [25] N. Sodha, S. Bullock, R. Taylor, G. Mitchell, B. Guertl-Lackner, R.D. Williams, S. Bevan, K. Bishop, S. McGuire, R.S. Houlston, R.A. Eeles, CHEK2 variants in susceptibility to breast cancer and evidence of retention of the wild type allele in tumours, Br J Cancer 87 (2002) 1445–1448. [26] M. Schutte, S. Seal, R. Barfoot, H. Meijers-Heijboer, M. Wasielewski, D.G. Evans, D. Eccles, C. Meijers, F. Lohman, J. Klijn, A. van den Ouweland, P.A. Futreal, K.L. Nathanson, B.L. Weber, D.F. Easton, M.R. Stratton, N. Rahman, Variants in CHEK2 other than 1100delC do not make a major contribution to breast cancer susceptibility, Am J Hum Genet 72 (2003) 1023–1028. [27] X. Dong, L. Wang, K. Taniguchi, X. Wang, J.M. Cunningham, S.K. McDonnell, C. Qian, A.F. Marks, S.L. Slager, B.J. Peterson, D.I. Smith, J.C. Cheville, M.L. Blute, S.J. Jacobsen, D.J. Schaid, D.J. Tindall, S.N. Thibodeau, W. Liu, Mutations in CHEK2 associated with prostate cancer risk, Am J Hum Genet 72 (2003) 270–280. [28] X. Wu, S.R. Webster, J. Chen Characterization of tumor-associated Chk2 mutations, J Biol Chem 276 (2001) 2971–2974. [29] S.B. Lee, S.H. Kim, D.W. Bell, D.C. Wahrer, T.A. Schiripo, M.M. Jorczak, D.C. Sgroi, J.E. Garber, F.P. Li, K.E. Nichols, J.M. Varley, A.K. Godwin, K.M. Shannon, E. Harlow, D.A. Haber, Destabilization of CHK2 by a missense mutation associated with Li-Fraumeni Syndrome, Cancer Res 61 (2001) 8062–8067. [30] S. Matsuoka, T. Nakagawa, A. Masuda, N. Haruki, S.J. Elledge, T. Takahashi, Reduced expression and impaired kinase activity of
J. Ahn et al. / DNA Repair 3 (2004) 1039–1047
[31]
[32]
[33]
[34]
[35]
[36]
[37] [38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
a Chk2 mutant identified in human lung cancer, Cancer Res 61 (2001) 5362–5365. J. Falck, C. Lukas, M. Protopopova, J. Lukas, G. Selivanova, J. Bartek, Functional impact of concomitant versus alternative defects in the Chk2- p53 tumour suppressor pathway, Oncogene 20 (2001) 5503–5510. J. Falck, N. Mailand, R.G. Syljuasen, J. Bartek, J. Lukas, The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis, Nature 410 (2001) 842–847. F. d’Adda di Fagagna, P.M. Reaper, L. Clay-Farrace, H. Fiegler, P. Carr, T. Von Zglinicki, G. Saretzki, N.P. Carter, S.P. Jackson, A DNA damage checkpoint response in telomere-initiated senescence, Nature 426 (2003) 194–198. H. Oh, S.C. Wang, A. Prahash, M. Sano, C.S. Moravec, G.E. Taffet, L.H. Michael, K.A. Youker, M.L. Entman, M.D. Schneider, Telomere attrition and Chk2 activation in human heart failure, Proc Natl Acad Sci USA 100 (2003) 5378–5383. S. Banin, L. Moyal, S. Shieh, Y. Taya, C.W. Anderson, L. Chessa, N.I. Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, Y. Ziv, Enhanced phosphorylation of p53 by ATM in response to DNA damage, Science 281 (1998) 1674–1677. C.E. Canman, D.S. Lim, K.A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E. Appella, M.B. Kastan, J.D. Siliciano, Activation of the ATM kinase by ionizing radiation and phosphorylation of p53, Science 281 (1998) 1677–1679. Y. Shiloh, ATM and related protein kinases: safeguarding genome integrity, Nat Rev Cancer 3 (2003) 155–168. A.L. Brown, C.H. Lee, J.K. Schwarz, N. Mitiku, H. Piwnica-Worms, J.H. Chung, A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage, Proc Natl Acad Sci USA 96 (1999) 3745–3750. P. Chaturvedi, W.K. Eng, Y. Zhu, M.R. Mattern, R. Mishra, M.R. Hurle, X. Zhang, R.S. Annan, Q. Lu, L.F. Faucette, G.F. Scott, X. Li, S.A. Carr, R.K. Johnson, J.D. Winkler, B.B. Zhou, Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway, Oncogene 18 (1999) 4047–4054. J.Y. Ahn, J.K. Schwarz, H. Piwnica-Worms, C.E. Canman, Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation, Cancer Res 60 (2000) 5934–5936. S. Matsuoka, G. Rotman, A. Ogawa, Y. Shiloh, K. Tamai, S.J. Elledge, Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro, Proc Natl Acad Sci USA 97 (2000) 10389– 10394. R. Melchionna, X.B. Chen, A. Blasina, C.H. McGowan, Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1, Nat Cell Biol 2 (2000) 762–765. J.Y. Ahn, X. Li, H.L. Davis, C.E. Canman, Phosphorylation of threonine 68 promotes oligomerization and autophosphorylation of the Chk2 protein kinase via the forkhead- associated domain, J Biol Chem 277 (2002) 19389–19395. X. Xu, L.M. Tsvetkov, D.F. Stern, Chk2 activation and phosphorylation-dependent oligomerization, Mol Cell Biol 22 (2002) 4419–4432. J. Ahn, C. Prives, Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation, J Biol Chem 277 (2002) 48418– 48426. C.H. Lee, J.H. Chung, The hCds1 (Chk2)-FHA domain is essential for a chain of phosphorylation events on hCds1 that is induced by ionizing radiation, J Biol Chem 276 (2001) 30537–30541. J.K. Schwarz, C.M. Lovly, H. Piwnica-Worms, Regulation of the Chk2 Protein Kinase by Oligomerization-Mediated cis- and trans-Phosphorylation, Mol Cancer Res 1 (2003) 598–609. X. Wu, J. Chen, Autophosphorylation of checkpoint kinase 2 at serine 516 is required for radiation-induced apoptosis, J Biol Chem 278 (2003) 36163–36168.
1045
[49] Z. Lou, K. Minter-Dykhouse, X. Wu, J. Chen, MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways, Nature 421 (2003) 957–961. [50] L. Tsvetkov, X. Xu, J. Li, D.F. Stern, Polo-like kinase 1 and Chk2 interact and co-localize to centrosomes and the midbody, J Biol Chem 278 (2003) 8468–8475. [51] S. Xie, H. Wu, Q. Wang, J. Kunicki, R.O. Thomas, R.E. Hollingsworth, J. Cogswell, W. Dai, Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2, Cell Cycle 1 (2002) 424–429. [52] M. Bahassi el, C.W. Conn, D.L. Myer, R.F. Hennigan, C.H. McGowan, Y. Sanchez, P.J. Stambrook, Mammalian Polo-like kinase 3 (Plk3) is a multifunctional protein involved in stress response pathways, Oncogene 21 (2002) 6633–6640. [53] L. Zannini, D. Lecis, S. Lisanti, R. Benetti, G. Buscemi, C. Schneider, D. Delia, Karyopherin–alpha2 protein interacts with Chk2 and contributes to its nuclear import, J Biol Chem 278 (2003) 42346– 42351. [54] T. O’Neill, L. Giarratani, P. Chen, L. Iyer, C.H. Lee, M. Bobiak, F. Kanai, B.B. Zhou, J.H. Chung, G.A. Rathbun, Determination of Substrate Motifs for Human Chk1 and hCds1/Chk2 by the Oriented Peptide Library Approach, J Biol Chem 277 (2002) 16102–16115. [55] G.J. Seo, S.E. Kim, Y.M. Lee, J.W. Lee, J.R. Lee, M.J. Hahn, S.T. Kim, Determination of substrate specificity and putative substrates of Chk2 kinase, Biochem Biophys Res Commun 304 (2003) 339– 343. [56] A. Blasina, I.V. de Weyer, M.C. Laus, W.H. Luyten, A.E. Parker, C.H. McGowan, A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase, Curr Biol 9 (1999) 1–10. [57] C.S. Sorensen, R.G. Syljuasen, J. Falck, T. Schroeder, L. Ronnstrand, K.K. Khanna, B.B. Zhou, J. Bartek, J. Lukas, Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A, Cancer Cell 3 (2003) 247–258. [58] L. Busino, M. Donzelli, M. Chiesa, D. Guardavaccaro, D. Ganoth, N.V. Dorrello, A. Hershko, M. Pagano, G.F. Draetta, Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage, Nature 426 (2003) 87–91. [59] J. Jin, T. Shirogane, L. Xu, G. Nalepa, J. Qin, S.J. Elledge, J.W. Harper, SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase, Genes Dev 17 (2003) 3062–3074. [60] B.B. Zhou, P. Chaturvedi, K. Spring, S.P. Scott, R.A. Johanson, R. Mishra, M.R. Mattern, J.D. Winkler, K.K. Khanna, Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity, J Biol Chem 275 (2000) 10342–10348. [61] C.Y. Peng, P.R. Graves, R.S. Thoma, Z. Wu, A.S. Shaw, H. Piwnica-Worms, Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216, Science 277 (1997) 1501–1505. [62] S.N. Dalal, C.M. Schweitzer, J. Gan, J.A. DeCaprio, Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site, Mol Cell Biol 19 (1999) 4465–4479. [63] C. Stevens, L. Smith, N.B. La Thangue, Chk2 activates E2F-1 in response to DNA damage, Nat Cell Biol 5 (2003) 401–409. [64] W.C. Lin, F.T. Lin, J.R. Nevins, Selective induction of E2F-1 in response to DNA damage, mediated by ATM-dependent phosphorylation, Genes Dev 15 (2001) 1833–1844. [65] J. DeGregori, G. Leone, A. Miron, L. Jakoi, J.R. Nevins, Distinct roles for E2F proteins in cell growth control and apoptosis, Proc Natl Acad Sci USA 94 (1997) 7245–7250. [66] J.S. Lee, K.M. Collins, A.L. Brown, C.H. Lee, J.H. Chung, hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response, Nature 404 (2000) 201–204. [67] S. Yang, C. Kuo, J.E. Bisi, M.K. Kim, PML-dependent apoptosis after DNA damage is regulated by the checkpoint kinase hCds1/Chk2, Nat Cell Biol 4 (2002) 865–870.
1046
J. Ahn et al. / DNA Repair 3 (2004) 1039–1047
[68] J. Zhang, H. Willers, Z. Feng, J.C. Ghosh, S. Kim, D.T. Weaver, J.H. Chung, S.N. Powell, F. Xia, Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair, Mol Cell Biol 24 (2004) 708–718. [69] C. Lukas, J. Falck, J. Bartkova, J. Bartek, J. Lukas, Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage, Nat Cell Biol 5 (2003) 255–260. [70] Z.X. Xu, A. Timanova-Atanasova, R.X. Zhao, K.S. Chang, PML colocalizes with and stabilizes the DNA damage response protein TopBP1, Mol Cell Biol 23 (2003) 4247–4256. [71] Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev 14 (2000) 927–939. [72] B. Wang, S. Matsuoka, P.B. Carpenter, S.J. Elledge, 53BP1, a mediator of the DNA damage checkpoint, Science 298 (2002) 1435– 1438. [73] I.M. Ward, X. Wu, J. Chen, Threonine 68 of Chk2 is phosphorylated at sites of DNA strand breaks, J Biol Chem 276 (2001) 47755– 47758. [74] N. Foray, D. Marot, A. Gabriel, V. Randrianarison, A.M. Carr, M. Perricaudet, A. Ashworth, P. Jeggo, A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein, Embo J 22 (2003) 2860–2871. [75] K.D. Brown, A. Rathi, R. Kamath, D.I. Beardsley, Q. Zhan, J.L. Mannino, R. Baskaran, The mismatch repair system is required for S-phase checkpoint activation, Nat Genet 33 (2003) 80–84. [76] A. Franchitto, P. Pichierri, R. Piergentili, M. Crescenzi, M. Bignami, F. Palitti, The mammalian mismatch repair protein MSH2 is required for correct MRE11 and RAD51 relocalization and for efficient cell cycle arrest induced by ionizing radiation in G2 phase, Oncogene 22 (2003) 2110–2120. [77] G. Buscemi, C. Savio, L. Zannini, F. Micciche, D. Masnada, M. Nakanishi, H. Tauchi, K. Komatsu, S. Mizutani, K. Khanna, P. Chen, P. Concannon, L. Chessa, D. Delia, Chk2 activation dependence on Nbs1 after DNA damage, Mol Cell Biol 21 (2001) 5214–5222. [78] T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex, Mol Cell 7 (2001) 1255–1266. [79] T. Uziel, Y. Lerenthal, L. Moyal, Y. Andegeko, L. Mittelman, Y. Shiloh, Requirement of the MRN complex for ATM activation by DNA damage, Embo J 22 (2003) 5612–5621. [80] C.T. Carson, R.A. Schwartz, T.H. Stracker, C.E. Lilley, D.V. Lee, M.D. Weitzman, The Mre11 complex is required for ATM activation and the G(2)/M checkpoint, Embo J 22 (2003) 6610–6620. [81] J.H. Lee, B. Xu, C.H. Lee, J.Y. Ahn, M.S. Song, H. Lee, C.E. Canman, J.S. Lee, M.B. Kastan, D.S. Lim, Distinct functions of Nijmegen breakage syndrome in ataxia telangiectasia mutated-dependent responses to DNA damage, Mol Cancer Res 1 (2003) 674–681. [82] T.A. Mochan, M. Venere, R.A. DiTullio Jr., T.D. Halazonetis, 53BP1 and NFBD1/MDC1-Nbs1 function in parallel interacting pathways activating ataxia-telangiectasia mutated (ATM) in response to DNA damage, Cancer Res 63 (2003) 8586–8591. [83] I.M. Ward, K. Minn, J. van Deursen, J. Chen, p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice, Mol Cell Biol 23 (2003) 2556–2563. [84] O. Fernandez-Capetillo, H.T. Chen, A. Celeste, I. Ward, P.J. Romanienko, J.C. Morales, K. Naka, Z. Xia, R.D. Camerini-Otero, N. Motoyama, P.B. Carpenter, W.M. Bonner, J. Chen, A. Nussenzweig, DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1, Nat Cell Biol 4 (2002) 993–997. [85] R.A. DiTullio Jr., T.A. Mochan, M. Venere, J. Bartkova, M. Sehested, J. Bartek, T.D. Halazonetis, 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer, Nat Cell Biol 4 (2002) 998–1002.
[86] G.S. Stewart, B. Wang, C.R. Bignell, A.M. Taylor, S.J. Elledge, MDC1 is a mediator of the mammalian DNA damage checkpoint, Nature 421 (2003) 961–966. [87] M. Goldberg, M. Stucki, J. Falck, D. D’Amours, D. Rahman, D. Pappin, J. Bartek, S.P. Jackson, MDC1 is required for the intra-S-phase DNA damage checkpoint, Nature 421 (2003) 952– 956. [88] A. Peng, P.L. Chen, NFBD1, like 53BP1, is an early and redundant transducer mediating Chk2 phosphorylation in response to DNA damage, J Biol Chem 278 (2003) 8873–8876. [89] Y.L. Shang, A.J. Bodero, P.L. Chen, NFBD1, a novel nuclear protein with signature motifs of FHA and BRCT, and an internal 41-amino acid repeat sequence, is an early participant in DNA damage response, J Biol Chem 278 (2003) 6323–6329. [90] S.Y. Shieh, Y. Taya, C. Prives, DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization, Embo J 18 (1999) 1815–1823. [91] T. Unger, T. Juven-Gershon, E. Moallem, M. Berger, R. Vogt Sionov, G. Lozano, M. Oren, Y. Haupt, Critical role for Ser20 of human p53 in the negative regulation of p53 by Mdm2, Embo J 18 (1999) 1805–1814. [92] S.Y. Shieh, J. Ahn, K. Tamai, Y. Taya, C. Prives, The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites, Genes Dev 14 (2000) 289–300. [93] N.H. Chehab, A. Malikzay, M. Appel, T.D. Halazonetis, Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53, Genes Dev 14 (2000) 278–288. [94] Q. Yu, J.H. Rose, H. Zhang, Y. Pommier, Antisense inhibition of Chk2/hCds1 expression attenuates DNA damage-induced S and G2 checkpoints and enhances apoptotic activity in HEK-293 cells, FEBS Lett 505 (2001) 7–12. [95] S. Tan, D. Guschin, A. Davalos, Y.L. Lee, A.W. Snowden, Y. Jouvenot, H.S. Zhang, K. Howes, A.R. McNamara, A. Lai, C. Ullman, L. Reynolds, M. Moore, M. Isalan, L.P. Berg, B. Campos, H. Qi, S.K. Spratt, C.C. Case, C.O. Pabo, J. Campisi, P.D. Gregory, Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity, Proc Natl Acad Sci USA 100 (2003) 11997– 12002. [96] A. Craig, M. Scott, L. Burch, G. Smith, K. Ball, T. Hupp, Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain, EMBO Rep 4 (2003) 787–792. [97] D. Qin, H. Lee, C. Yuan, Y. Ju, M.D. Tsai, Identification of potential binding sites for the FHA domain of human Chk2 by in vitro binding studies, Biochem Biophys Res Commun 311 (2003) 803–808. [98] J. Ahn, M. Urist, C. Prives, Questioning the role of Chk2 in the p53 DNA Damage Response, J Biol Chem 278 (2003) 20480–20489. [99] P.V. Jallepalli, C. Lengauer, B. Vogelstein, F. Bunz, The Chk2 tumor suppressor is not required for p53 responses in human cancer cells, J Biol Chem 278 (2003) 20475–20479. [100] A. Hirao, Y.Y. Kong, S. Matsuoka, A. Wakeham, J. Ruland, H. Yoshida, D. Liu, S.J. Elledge, T.W. Mak, DNA damage-induced activation of p53 by the checkpoint kinase Chk2, Science 287 (2000) 1824–1827. [101] A. Hirao, A. Cheung, G. Duncan, P.M. Girard, A.J. Elia, A. Wakeham, H. Okada, T. Sarkissian, J.A. Wong, T. Sakai, E. De Stanchina, R.G. Bristow, T. Suda, S.W. Lowe, P.A. Jeggo, S.J. Elledge, T.W. Mak, Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner, Mol Cell Biol 22 (2002) 6521–6532. [102] M.T. Jack, R.A. Woo, A. Hirao, A. Cheung, T.W. Mak, P.W. Lee, Chk2 is dispensable for p53-mediated G1 arrest but is required for a latent p53-mediated apoptotic response, Proc Natl Acad Sci USA 3 (2002) 3. [103] H. Takai, K. Naka, Y. Okada, M. Watanabe, N. Harada, S. Saito, C.W. Anderson, E. Appella, M. Nakanishi, H. Suzuki, K. Nagashima, H. Sawa, K. Ikeda, N. Motoyama, Chk2-deficient mice
J. Ahn et al. / DNA Repair 3 (2004) 1039–1047
[104]
[105]
[106]
[107]
[108]
[109]
exhibit radioresistance and defective p53-mediated transcription, Embo J 21 (2002) 5195–5205. E. Keramaris, A. Hirao, R.S. Slack, T.W. Mak, D.S. Park, Ataxia telangiectasia-mutated protein can regulate p53 and neuronal death independent of Chk2 in response to DNA damage, J Biol Chem 278 (2003) 37782–37789. I. Oishi, S. Sugiyama, H. Otani, H. Yamamura, Y. Nishida, Y. Minami, A novel Drosophila nuclear protein serine/threonine kinase expressed in the germline during its establishment, Mech Dev 71 (1998) 49–63. J. Xu, S. Xin, W. Du, Drosophila Chk2 is required for DNA damage-mediated cell cycle arrest and apoptosis, FEBS Lett 508 (2001) 394–398. J. Xu, W. Du, Drosophila chk2 plays an important role in a mitotic checkpoint in syncytial embryos, FEBS Lett 545 (2003) 209– 212. S. Takada, A. Kelkar, W.E. Theurkauf, Drosophila checkpoint kinase 2 couples centrosome function and spindle assembly to genomic integrity, Cell 113 (2003) 87–99. M. Peters, C. DeLuca, A. Hirao, V. Stambolic, J. Potter, L. Zhou, J. Liepa, B. Snow, S. Arya, J. Wong, D. Bouchard, R. Binari, A.S. Manoukian, T.W. Mak, Chk2 regulates irradiation-induced,
[110]
[111]
[112]
[113]
[114]
1047
p53-mediated apoptosis in Drosophila, Proc Natl Acad Sci USA 99 (2002) 11305–11310. M.H. Brodsky, B.T. Weinert, G. Tsang, Y.S. Rong, N.M. McGinnis, K.G. Golic, D.C. Rio, G.M. Rubin, Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage, Mol Cell Biol 24 (2004) 1219– 1231. I. Oishi, K. Iwai, Y. Kagohashi, H. Fujimoto, K. Kariya, T. Kataoka, H. Sawa, H. Okano, H. Otani, H. Yamamura, Y. Minami, Critical role of Caenorhabditis elegans homologs of Cds1 (Chk2)-related kinases in meiotic recombination, Mol Cell Biol 21 (2001) 1329– 1335. A. Higashitani, H. Aoki, A. Mori, Y. Sasagawa, T. Takanami, H. Takahashi, Caenorhabditis elegans Chk2-like gene is essential for meiosis but dispensable for DNA repair, FEBS Lett 485 (2000) 35–39. A.J. MacQueen, A.M. Villeneuve, Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2, Genes Dev 15 (2001) 1674–1687. U. Abdu, M. Brodsky, T. Schupbach, Activation of a meiotic checkpoint during Drosophila oogenesis regulates the translation of Gurken through Chk2/Mnk, Curr Biol 12 (2002) 1645–1651.
Review
The Chk2 protein kinase Jinwoo Ahn1 , Marshall Urist1 , Carol Prives∗ Department of Biological Sciences, Columbia University, New York, NY 10027, USA Available online 22 April 2004
Abstract Checkpoint kinase 2 (Chk2) is a multifunctional enzyme whose functions are central to the induction of cell cycle arrest and apoptosis by DNA damage. Insight into Chk2 has derived from multiple approaches. Biochemical studies have addressed Chk2 structure, domain organization and regulation by phosphorylation. Extensive work has been done to identify factors that recognize and respond to DNA damage in order to activate Chk2. In turn a number of substrates and targets of Chk2 have been identified that play roles in the checkpoint response. The roles and regulation of Chk2 have been elucidated by studies in model genetic systems extending from worms and flies to mice and humans. The relationship of Chk2 to human cancer studies is developing rapidly with increasing evidence that Chk2 plays a role in tumor suppression. © 2004 Elsevier B.V. All rights reserved. Keywords: Checkpoint kinase 2; Apoptosis; Phosphorylation
1. Introduction
2. Domains of Chk2
Cells are exposed to constant endogenous and exogenous stresses that threaten DNA. To preserve the integrity of genetic information, an intricate network of proteins communicate with each other throughout the cell cycle to elicit appropriate cellular responses, such as DNA repair, cell cycle arrest or apoptosis in response to these insults (reviewed in [1,2]). The DNA damage checkpoint pathway has been of interest to the field of cancer biology, since checkpoint defects result in the accumulation of altered genetic information, a central feature of carcinogenesis (reviewed in [3,4]). The conservation of the pathway from yeast to human in eukaryotes has been beneficial to researchers in unraveling and identifying each component. Among the conserved DNA-damage activated kinases identified thus far, the Checkpoint kinase 2 (Chk2) plays a central role in implementing many aspects of the checkpoint response [5]. In this review, we summarize and discuss the results of biochemical, biophysical and biological studies of Chk2.
The Chk2 protein consists of three distinct functional domains (see the Small Molecular Architecture Research Tool database, http://smart.embl-heidelberg.de/); an SQ/TQ cluster domain (SCD, residues19–69), a forkhead associated domain (FHA, residues112–175), and a Ser/Thr kinase domain (residues 220–486) (Fig. 1A). The SCD contains five SQ and two TQ motifs, which satisfy the primary substrate motif for ataxia-telangectasia mutated (ATM) kinase [6]. Especially residues 67–70, STQE, are closely related to the refined substrate motif (LSQE) of ATM kinase, determined by oriented peptide library search [7], and T68 was identified to be a key phosphorylation site (see below). The FHA domain is an 80–100 amino acid phosphopeptide binding domain, conserved from yeast to human (reviewed in [8]). The domain consists of 11 beta sheets forming a structural core and connecting loops conferring diversity in phosphopeptide recognition as resolved by structural analyses of several FHA domains with and without phosphopeptides by X-ray crystallography [9–12] and nuclear magnetic resonance (NMR) spectroscopy [13–17]. FHA domains are found in proteins involved not only in DNA damage checkpoint pathways, but also kinesin family members, transcription factors, Ki-67, and nuclear inhibitors of protein phosphatase 1 (reviewed in [18]). The FHA domain functions in trans to modulate protein–protein interactions
∗ Corresponding author. Tel.: +1-212-854-2557; fax: +1-212-865-8246. E-mail address: [email protected] (C. Prives). 1 These authors contributed equally to this review.
1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.03.033
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Fig. 1. Functional Domain Architecture of Chk2 and a Model of Chk2 Activation. Landmarks of the Chk2 protein showing the SQ/TQ cluster domain (SCD) the forkhead associated domain (FHA) and the kinase domain. Shown below are previously identified phosphorylation sites phosphorylated by ATM or ATR kinases or resulting from dimerization induced autophosphorylation. A model for gamma irradiation induced formation of active dimers and monomers. After phosphorylation of inactive monomers at T68 by ATM, dimers form and undergo subsequent autophosphorylation and may also serve as substrates for other protein kinases. Phosphorylated dimers or monomers are active in phosphorylating substrates such as Cdc25C.
but also in cis to affect other functional domains within the protein itself. The kinase domain shares homology to other Ser/Thr or Tyr kinases with a Gly-rich region at the vicinity of Lys residues in the N-terminal part and Asp as a catalytic residue at the active site.
3. Chk2 and cancer Chk2 has established its position as a bona-fide tumor suppressor and the list of Chk2 mutations in cancer cells is continuously expanding (reviewed in [19]). The first indication of a tumor suppressor function for Chk2 came from the finding that a sub-population of families with the Li-Fraumeni syndrome (LFS), a familial cancer predisposition syndrome usually associated with germline TP53 mutation (reviewed in [20]), contain wild type p53 but mutant Chk2 suggesting that germline mutations in the CHEK2 gene phenocopy TP53 inactivation [21]. The CHEK2 allele containing the mutation 1100delC encodes a C-terminally truncated protein within the kinase domain and confers increased breast cancer risk independent of mutation in the breast cancer susceptibility associated (BRCA) loci [22–24]. Screening of cancer cell lines has revealed missense mutations in all three functional domains in a variety of tumor types [25–27]. The functional impact of CHEK2 mutation generally reflects its location. Thus, kinase domain mutants lead to faulty kinase activation after ionizing radiation [28], while FHA domain lesions may accelerate Chk2 degradation [29,30], or may induce structural changes conferring defects in protein–protein interaction [11,31,32]. It will be of interest to determine at what stage of tumor development CHEK2 mutations arise and the precise Chk2 functions that are selected against.
4. Regulation and activation of Chk2 Chk2 kinase activity is increased following DNA damage induced by ionizing radiation, chemotherapeutic agents, or other compounds that harm DNA directly or indirectly. Recently, senescence triggered by telomere erosion has been implicated as an activating signal for Chk2 [33,34]. Signals from damaged DNA are transmitted to the ATM kinase, which has a number of substrates involved in the checkpoint cascade [35–37]. Multiple lines of evidence establish a strong link between ATM and Chk2. First, Chk2 fails to be activated in A–T cells and restoration of a Chk2-dependent S-phase checkpoint was observed upon ectopic expression of ATM in A–T cells [5,38,39]. Second, ATM directly phosphorylates Chk2 at Thr68, a consensus site in the SCD domain in vitro [40,42] and alanine mutation of this residue prevents activation of Chk2 by gamma irradiation in vivo [40–42]. The Ataxia-Telangectasia-and-Rad3-related (ATR) kinase may similarly phosphorylate Chk2 after high levels of DNA damage or other stressors such as ultraviolet radiation (UV) or treatment with the ribonucleotide reductase inhibitor Hydroxyurea (HU) [41]. Chk2 exists as a monomer in unperturbed cells. Following DNA damage, Chk2 undergoes dimerization in which a region spanning phosphorylated Thr68 in one Chk2 molecule binds to the FHA domain of second molecule [43–45]. Dimerization is likely followed by multiple intermolecular phosphorylation events including Thr383 and Thr387 within the auto-inhibitory loop resulting in kinase activation (Fig. 1B) [46]. Autophosphorylation of Ser516 within the kinase domain was also recently identified, and is required for full kinase activation [47,48]. Furthermore, mutation of this residue prevents radiation-sensitization by
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Chk2, suggesting a functional role for autophosphorylation [48]. Phosphorylation at Thr68 is likely to be the initiating event for dimerization and activation since maintenance of Thr68 phosphorylation is not required for sustained kinase activity [45]. Phosphorylation of other sites are likely to mediate stress specific differences in the extent of kinase activation. Indeed, Chk2 purified after gamma radiation, UV, and HU treatment showed different kinase activities toward Cdc25C, one of its well validated substrates ([5] and Ahn and Prives, unpublished results). Moreover, the phospho-Thr68 to FHA domain intramolecular interaction mechanism may also function in stress induced association with other checkpoint proteins. For example, the MDC1 checkpoint protein FHA domain binds selectively to Chk2 phosphorylated at Thr68 [49]. The Polo Like Kinase 1 (PLK1), a centrosome-associated kinase implicated in mitotic progression, can interact with Chk2 and colocalize with Chk2 at centrosomes. Interestingly, PLK1 can phosphorylate residues in the N-terminus of Chk2 including Thr68 [50]. The functional import of this interaction for PLK1 or Chk2 function is not known. The related kinase PLK3 also interacts with Chk2 and phosphorylation of Chk2 and PLK3 by one another has been reported [51,52]. The subcelluar localization of Chk2 may be regulated by another recently described binding protein, karyopherin–alpha2 [53].
5. Substrates of Chk2 Upon activation Chk2 relays the checkpoint activation signal to a number of effectors, which mediate many of the phenotypic characteristics provoked by DNA damage including cell cycle arrest and apoptosis (Fig. 2). Use of oriented peptide libraries has shown Chk2 preferentially
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phosphorylates a consensus motif, L-X-R-X-X-S/T, [54,55]. However, these studies may only describe one aspect of Chk2 kinase specificity as they do not capture the in vivo complexity of kinase-substrate interaction. Nevertheless, many functionally important substrates bearing this motif have been identified. The first described and best validated of Chk2 substrates are two members of the cell division cycle 25 dual specificity phosphatase family, Cdc25A and Cdc25C. The Cdc25 family promotes cell cycle progression by activation of the cyclin-dependent kinases cdk2 and cdk1 through dephosphorylation of inhibitory phosphorylation at Thr14 and Tyr15 [5,32,38,56]. Cdc25A is destabilized by Chk2 phosphorylation at Ser123 [32,57] most likely by increasing susceptibility to the SCF-TCRP complex, which mediates Cdc25A turnover [58,59]. Chk2 phosphorylation of Ser216 in Cdc25C creates a binding site for 14-3-3 proteins [60] and this interaction is thought to result in persistent cytoplasmic Cdc25C localization thus preventing the G2/M transition as the cdk1/Cyclin B complex cannot be activated [61,62]. Taken together, phosphorylation of Cdc25A and Cdc25C by Chk2 in response to DNA damage explains in part how the checkpoint induces cell cycle arrest. Chk2 phosphorylates E2F-1 at Ser364 in response to the DNA-damaging agent etoposide, and the modification of that residue regulates both E2F-1 stabilization and transcriptional activity [63]. Given the observation that E2F-1 null thymocytes are resistant to etoposide-induced apoptosis [64] and that over-expression of E2F-1 can induce cell death [65], modulation of E2F-1 activity represents a potentially important pathway through which Chk2 may regulate DNA damage-induced apoptosis. Two checkpoint pathway scaffold proteins, BRCA1 and Promyelocytic Leukemia (PML), can interact with Chk2 and may be phosphorylated by Chk2 following DNA damage
Fig. 2. The Chk2 network. Chk2 integrates signals from upstream kinases and binding partners to phosphorylate key targets involved in cell cycle checkpoints. See text for details of Chk2 substrates and binding partners.
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[66,67]. Interestingly, both substrates follow a similar pattern in which Chk2 interacts with PML and BRCA1 in the absence of DNA damage but following gamma irradiation BRCA is phosphorylated at Ser988 and PML is phosphorylated at Ser117 in a Chk2-dependent fashion, resulting in subsequent inhibition of binding with Chk2. Alanine mutation of either phosphorylation site prevents release of Chk2 from BRCA1 or PML after DNA damage suggesting a relationship between activation of phosphorylation and inhibition of interaction. Functionally, BRCA1Ser988A fails to increase survival of BRCA1-null HCC1937 cells following IR and BRCA1Ser988A stably expressed in HCC1937 cannot mediate double strand break repair [68]. In contrast to wild-type PML, the mutant PMLSer117A does not sensitize U937 leukaemia cells to gamma irradiation- induced apoptosis suggesting functional importance for phosphorylation of Ser117 by Chk2. For both proteins, a mechanistic understanding of how modification impacts function is lacking. For example, it will be of interest to determine if the alanine mutants of either protein are able to inhibit downstream functions of Chk2. In an elegant recent paper, Lukas and colleagues demonstrated using both fluoresence-loss-in-photobleaching (FLIP) and fluoresence-return-after-photobleaching (FRAP) that unlike other another checkpoint protein Nbs1, Chk2 does not become immobilized at sites of damaged DNA but remains soluble after x-irradiation. Further, immobilization of Chk2 by fusion to Histone2B inhibits Chk2 function leading to the view that Chk2 acts as a “signal spreader” dispersing a checkpoint “on” signal to its various substrates implementing cell cycle arrest and apoptosis [69]. Given that both PML and BRCA1 form foci which collect many checkpoint proteins including Chk2’s principle activator ATM [70,71], perhaps PML and BRCA1 function in part to provide sites of Chk2 activation by ATM. Moreover, Thr68-Chk2 colocalizes with 53BP1, H2AX, and NBS1 in nuclear foci [72,73]. Once activated therein, Chk2 disengages from the complex by phosphorylation of PML, BRCA1, or other factors to then find and activate Chk2’s various substrates. Consistent with this model, BRCA1 is required for Thr68 phosphorylation by ATM [74]. A study, which found that closely associated mismatch repair factors (MMR) MSH2 and MLH1, members of the BASC or BRCA1-associated genome surveillance complex [71], bind Chk2 and ATM respectively and play an important role in Chk2 activation generally supports this hypothesis. Restoration of either MSH2 or MLH1 in colon tumor cell lines bearing mutations in these factors enhances Thr68 phosphorylation after DNA damage, restores Cdc25A destabilization by gamma irradiation, and complements the RDS phenotype, all hallmarks of increased Chk2 signaling [75]. Also, Chk2 from MSH−/− cells does not undergo Thr68 phosphorylation after gamma irradiation [76]. Moreover, Chk2 activation is defective in cells derived from Nijmegen Breakage Syndrome patients who carry inactivating mutations in Nbs1 a component of the Mre11–Rad50–Nbs1
complex which plays central role in DNA repair and is required for ATM activation after DNA damage [77,78–82] 53BP1 another checkpoint factor important in forming DNA damage signaling complexes with ATM and BRCA1 may also play a role in Chk2 activation. 53BP1 null MEFs show little if any defect in Chk2 phosphorylation as determined by mobility shift in polyacrylamide gels after IR [83,84] but focus formation of activated Chk2 as detected by an anti-phosphoT68 Chk2 antibody is compromised by 53BP1 knockdown [72]. Interestingly, 53BP1 is involved in the phosphorylation of other ATM substrates including Smc1 [85]. Also, Chk2 activation is independent of H2AX which is phosphorylated at sites of DNA damage [84]. A more recently discovered checkpoint factor also present in damage foci, NFBD1/MDC1 binds to phospho-Chk2 but its requirement for Chk2 Thr68 phosphorylation is controversial [49,86–89]. Detailed understanding of how the various signaling interactions required for Chk2 activation are integrated will be of great interest.
6. Chk2 knockout mice and the p53 connection Given the central role of both p53 and Chk2 in cell cycle arrest and apoptosis as well as their roles in tumor suppression, many reports have described connections between Chk2 and p53. Conclusions from several studies, however, have led to the realization that different approaches can provide widely varying results. Several groups placed Chk2 immediately upstream of p53 through studies involving Chk2 knockout mice as well as in vivo and in vitro experiments. Phosphorylation of p53 at Ser20 had been suggested to be a critical event for stabilizing p53 [90,91]. The search for a kinase capable of Ser20 phosphorylation seemed to end when it was reported that Chk2 could phosphorylate p53 at Ser20 in vitro and this event was sufficient to disrupt pre-complexed p53 and Mdm2 [92,93]. Introduction of a dominant negative Chk2 led to failure of U2OS cells to stabilize or phosphorylate endogenous p53 at Ser20 and Chk2 enhanced a p53 dependent G1 checkpoint [93]. In another study, it was reported that immunoprecipitated Chk2 is activated by IR to phosphorylate p53 and overexpression of Chk2 enhances the ability of p53 to transactivate a p53 responsive reporter plasmid [31]. Interestingly, Chk2 downregulation by antisense in 293 cells increases sensitivity to DNA double strand breaks [94]. Silencing of Chk2 by novel engineered zinc finger transcription factors decreases p53 transcriptional activation of the MDM2 gene and reduces p53 Ser20 phosphorylation [95]. However, other work has weakened the view that Chk2’s regulation of p53 is generally applicable. First, the region on p53 spanning Ser20 does not share homology with the consensus motif, L-X-R-X-X-S/T present in other known substrates of Chk2 and a peptide covering the p53 Ser20 region is a poor Chk2 substrate [54,55]. Interestingly, two regions of p53, aa 117–132 and aa 272–288, are proposed
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to provide Chk2 docking sites to allosterically regulate the kinase activity toward p53 and purified Chk2 supplemented with these docking-site motifs phosphorylated a Ser20 peptide by kinase assay [96]. A second group has identified another Chk2 binding domain in p53 centering on Thr329 [97]. In contrast to this work, Chk2 purified from tumor cell lines before and after DNA damage could not phosphorylate different forms of truncated or full-length p53 even though it was markedly activated to phosphorylate the domain of Cdc25C containing Ser216 [98]. Experiments in vivo similarly challenge the Chk2–p53 relationship. Introduction of Chk1 and Chk2 siRNAs into several human tumor cell lines failed to affect p53 stabilization, Ser20 phosphorylation or transcriptional activity despite preventing Ser216 phosphorylation of Cdc25C [98]. Even more, homozygous deletion of Chk2 in HCT-116 colon carcinoma cells yielded no phenotype with respect to p53, G1 or G2 cell cycle arrest, and apoptosis [99]. Two independently generated Chk2 knockout mice have supported a role for Chk2 in the regulation of p53 but diverge in their findings and proposed mechanisms. The first reported Chk2 null mouse from Hirao and colleagues [100] strikingly showed that thymocytes, splenic T cells, and MEFs lacking Chk2 are unable to stabilize p53 following gamma irradiation and this defect is corrected by reintroduction of Chk2. These cells are highly resistant to apoptosis consistent with a defect in p53 signaling [100]. A follow up study showed that Chk2 mice are highly resistant to apoptosis in multiple tissue compartments by whole body irradiation experiments and develop chemically-induced skin tumors more rapidly [101]. The G1/S checkpoint of these mice is compromised at low doses of gamma irradiation but cells arrest normally at doses above 5Gy. Interestingly, however, p53 null mice do not show any dose dependency in the same assay. In contrast to the original report a second study from this group showed that gamma irradiated Chk2−/− thymoctyes show a defect in transcriptional activation of Bax but only a slight depression in p21 upregulation [101]. A different group using MEFs from these same mice, however, found no defect in G1/S arrest or p21 mRNA induction at even low X-ray doses [102]. More recently a second Chk2 knockout mouse generated by another group was reported with somewhat different results [103]. Consistent with the first Chk2 null mouse, the second was resistant to apoptosis induced by gamma irradiation in many tissues strongly implicating Chk2 in DNA damage induced apoptosis [103]. No G2 or S-phase checkpoint defect was detected but these authors did observe a deficient G1/S arrest consistent with impaired p53 signaling. In contrast to Hirao et al, they reported only a 30–50% reduction in p53 stabilization and human p53 introduced into Chk2−/− MEFs was phosphorylated normally on Ser20 after IR, indicating that in murine tissue Chk2 makes a partial contribution to p53 stabilization but other factors clearly contribute. Perhaps the most striking result with respect to p53 was that, despite only a partial p53 stabilization defect, upon analysis of five p53 targets
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genes by quantitative RT–PCR in thymocytes and MEFs, the authors saw no mRNA upregulation in the Chk2−/− cells after IR providing a possible mechanistic explanation for the G1/S arrest and apoptotic defects in these cells [103]. However, the relationship between Chk2 and p53 is likely to be stimuli–as well as cell type-specific as cell death in primary neuronal cultures induced by etoposide or camptothecin was shown to be ATM-dependent but Chk2 independent [104]. To summarize the mouse studies to date, it is clear that Chk2 plays a central role in gamma irradiation induced apoptosis however the mechanism is unclear. Chk2 likely regulates p53 transcriptional activity independently of Ser20 phosphorylation or even stabilization in murine tissues after IR, but in other contexts yet to be described signaling pathways play a central role. It remains to be determined when Chk2 regulates p53, in what context, and what factors mitigate or compensate for Chk2 in cases where it is not required.
7. Chk2 in lower organisms As mentioned above, the Chk2 kinase is conserved throughout phylogeny including Caenorhabditis elegans and Drosophila melanogaster. Study of the Chk2 gene in these organisms has revealed novel functions not seen in human or mouse cells. The fly homolog of Chk2, Mnk or dmChk2, is highly expressed in ovaries during embryogenesis [105] and embryos lacking Chk2 are highly sensitive to gamma irradiation suggesting a checkpoint function [106]. Loss of Chk2 in fly embryos also leads to genomic instability, a phenotype enhanced by treatment with DNA damaging agents, that has been linked to abnormal centrosome function so that lethally damaged cells cannot be efficiently eliminated from the embryo in the absence of Chk2 [107,108]. Transgenic embryos expressing dominant-negative (DN) dmChk2 were resistant to radiation-induced apoptosis and prevented cell death caused by over-expressed dmp53 [109]. Also, dmChk2 regulates p53 phosphorylation status and transcriptional activity following gamma-irradiation [110]. RNA interference studies of the C. Elegans Chk2 homolog, Cds, revealed that this kinase is essential for meiotic recombination, a function not seen in other organisms [111–113]. Interestingly, a role for dmChk2 in a meiotic checkpoint has also been demonstrated suggesting that this function of Chk2 may be conserved [114].
8. Conclusions and future directions Future studies of Chk2 will certainly identify novel substrates, interacting partners, and functionally important phosphorylation sites in Chk2. As a picture of Chk2 function becomes clearer, it will be of particular interest to determine how Chk2 integrates its various functions to precisely determine the outcome of checkpoint activation. It will also be important to resolve differences in findings
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relating Chk2 and p53. Chk2’s relationship to modules in the checkpoint pathway controlling DNA repair, cell cycle arrest, and apoptosis places it at the nexus of the DNA damage response. Therefore, modulation of its activity might offer some future benefit to the development of cancer therapeutics.
Acknowledgements The work is supported by NIH grant CA87497.
References [1] B.B. Zhou, S.J. Elledge, The DNA damage response: putting checkpoints in perspective, Nature 408 (2000) 433–439. [2] J.M. Bradbury, S.P. Jackson, The complex matter of DNA double-strand break detection, Biochem Soc Trans 31 (2003) 40–44. [3] L.H. Thompson, D. Schild, Recombinational DNA repair and human disease, Mutat Res 509 (2002) 49–78. [4] B.B. Zhou, H.J. Anderson, M. Roberge, Targeting DNA checkpoint kinases in cancer therapy, Cancer Biol Ther 2 (2003) S16–S22. [5] S. Matsuoka, M. Huang, S.J. Elledge, Linkage of ATM to cell cycle regulation by the Chk2 protein kinase, Science 282 (1998) 1893–1897. [6] S.T. Kim, D.S. Lim, C.E. Canman, M.B. Kastan, Substrate specificities and identification of putative substrates of ATM kinase family members, J Biol Chem 274 (1999) 37538–37543. [7] T. O’Neill, A.J. Dwyer, Y. Ziv, D.W. Chan, S.P. Lees-Miller, R.H. Abraham, J.H. Lai, D. Hill, Y. Shiloh, L.C. Cantley, G.A. Rathbun, Utilization of oriented peptide libraries to identify substrate motifs selected by ATM, J Biol Chem 275 (2000) 22719–22727. [8] J. Li, G.I. Lee, S.R. Van Doren, J.C. Walker, The FHA domain mediates phosphoprotein interactions, J Cell Sci 113 (Pt 23) (2000) 4143–4149. [9] D. Durocher, J. Henckel, A.R. Fersht, S.P. Jackson, The FHA domain is a modular phosphopeptide recognition motif, Mol Cell 4 (1999) 387–394. [10] D. Durocher, I.A. Taylor, D. Sarbassova, L.F. Haire, S.L. Westcott, S.P. Jackson, S.J. Smerdon, M.B. Yaffe, The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms, Mol Cell 6 (2000) 1169–1182. [11] J. Li, B.L. Williams, L.F. Haire, M. Goldberg, E. Wilker, D. Durocher, M.B. Yaffe, S.P. Jackson, S.J. Smerdon, Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2, Mol Cell 9 (2002) 1045– 1054. [12] E.S. Stavridi, Y. Huyen, I.R. Loreto, D.M. Scolnick, T.D. Halazonetis, N.P. Pavletich, P.D. Jeffrey, Crystal structure of the FHA domain of the Chfr mitotic checkpoint protein and its complex with tungstate, Structure (Camb) 10 (2002) 891–899. [13] H. Liao, I.J. Byeon, M.D. Tsai, Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53, J Mol Biol 294 (1999) 1041–1049. [14] H. Liao, C. Yuan, M.I. Su, S. Yongkiettrakul, D. Qin, H. Li, I.J. Byeon, D. Pei, M.D. Tsai, Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9, J Mol Biol 304 (2000) 941–951. [15] P. Wang, I.J. Byeon, H. Liao, K.D. Beebe, S. Yongkiettrakul, D. Pei, M.D. Tsai II, Structure and specificity of the interaction between the FHA2 domain of Rad53 and phosphotyrosyl peptides, J Mol Biol 302 (2000) 927–940.
[16] I.J. Byeon, S. Yongkiettrakul, M.D. Tsai, Solution structure of the yeast Rad53 FHA2 complexed with a phosphothreonine peptide pTXXL: comparison with the structures of FHA2-pYXL and FHA1-pTXXD complexes, J Mol Biol 314 (2001) 577–588. [17] C. Yuan, S. Yongkiettrakul, I.J. Byeon, S. Zhou, M.D. Tsai, Solution structures of two FHA1-phosphothreonine peptide complexes provide insight into the structural basis of the ligand specificity of FHA1 from yeast Rad53, J Mol Biol 314 (2001) 563–575. [18] D. Durocher, S.P. Jackson, The FHA domain, FEBS Lett 513 (2002) 58–66. [19] J. Bartek, J. Lukas, Chk1 and Chk2 kinases in checkpoint control and cancer, Cancer Cell 3 (2003) 421–429. [20] S.C. Evans, G. Lozano, The Li-Fraumeni syndrome: an inherited susceptibility to cancer, Mol Med Today 3 (1997) 390–395. [21] D.W. Bell, J.M. Varley, T.E. Szydlo, D.H. Kang, D.C. Wahrer, K.E. Shannon, M. Lubratovich, S.J. Verselis, K.J. Isselbacher, J.F. Fraumeni, J.M. Birch, F.P. Li, J.E. Garber, D.A. Haber, Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome, Science 286 (1999) 2528–2531. [22] P. Vahteristo, J. Bartkova, H. Eerola, K. Syrjakoski, S. Ojala, O. Kilpivaara, A. Tamminen, J. Kononen, K. Aittomaki, P. Heikkila, K. Holli, C. Blomqvist, J. Bartek, O.P. Kallioniemi, H. Nevanlinna, A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer, Am J Hum Genet 71 (2002) 432–438. [23] H. Meijers-Heijboer, J. Wijnen, H. Vasen, M. Wasielewski, A. Wagner, A. Hollestelle, F. Elstrodt, R. van den Bos, A. de Snoo, G.T. Fat, C. Brekelmans, S. Jagmohan, P. Franken, P. Verkuijlen, A. van den Ouweland, P. Chapman, C. Tops, G. Moslein, J. Burn, H. Lynch, J. Klijn, R. Fodde, M. Schutte, The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype, Am J Hum Genet 72 (2003) 1308–1314. [24] H. Meijers-Heijboer, A. van den Ouweland, J. Klijn, M. Wasielewski, A. de Snoo, R. Oldenburg, A. Hollestelle, M. Houben, E. Crepin, M. van Veghel-Plandsoen, F. Elstrodt, C. van Duijn, C. Bartels, C. Meijers, M. Schutte, L. McGuffog, D. Thompson, D. Easton, N. Sodha, S. Seal, R. Barfoot, J. Mangion, J. Chang-Claude, D. Eccles, R. Eeles, D.G. Evans, R. Houlston, V. Murday, S. Narod, T. Peretz, J. Peto, C. Phelan, H.X. Zhang, C. Szabo, P. Devilee, D. Goldgar, P.A. Futreal, K.L. Nathanson, B. Weber, N. Rahman, M.R. Stratton, Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations, Nat Genet 31 (2002) 55–59. [25] N. Sodha, S. Bullock, R. Taylor, G. Mitchell, B. Guertl-Lackner, R.D. Williams, S. Bevan, K. Bishop, S. McGuire, R.S. Houlston, R.A. Eeles, CHEK2 variants in susceptibility to breast cancer and evidence of retention of the wild type allele in tumours, Br J Cancer 87 (2002) 1445–1448. [26] M. Schutte, S. Seal, R. Barfoot, H. Meijers-Heijboer, M. Wasielewski, D.G. Evans, D. Eccles, C. Meijers, F. Lohman, J. Klijn, A. van den Ouweland, P.A. Futreal, K.L. Nathanson, B.L. Weber, D.F. Easton, M.R. Stratton, N. Rahman, Variants in CHEK2 other than 1100delC do not make a major contribution to breast cancer susceptibility, Am J Hum Genet 72 (2003) 1023–1028. [27] X. Dong, L. Wang, K. Taniguchi, X. Wang, J.M. Cunningham, S.K. McDonnell, C. Qian, A.F. Marks, S.L. Slager, B.J. Peterson, D.I. Smith, J.C. Cheville, M.L. Blute, S.J. Jacobsen, D.J. Schaid, D.J. Tindall, S.N. Thibodeau, W. Liu, Mutations in CHEK2 associated with prostate cancer risk, Am J Hum Genet 72 (2003) 270–280. [28] X. Wu, S.R. Webster, J. Chen Characterization of tumor-associated Chk2 mutations, J Biol Chem 276 (2001) 2971–2974. [29] S.B. Lee, S.H. Kim, D.W. Bell, D.C. Wahrer, T.A. Schiripo, M.M. Jorczak, D.C. Sgroi, J.E. Garber, F.P. Li, K.E. Nichols, J.M. Varley, A.K. Godwin, K.M. Shannon, E. Harlow, D.A. Haber, Destabilization of CHK2 by a missense mutation associated with Li-Fraumeni Syndrome, Cancer Res 61 (2001) 8062–8067. [30] S. Matsuoka, T. Nakagawa, A. Masuda, N. Haruki, S.J. Elledge, T. Takahashi, Reduced expression and impaired kinase activity of
J. Ahn et al. / DNA Repair 3 (2004) 1039–1047
[31]
[32]
[33]
[34]
[35]
[36]
[37] [38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
a Chk2 mutant identified in human lung cancer, Cancer Res 61 (2001) 5362–5365. J. Falck, C. Lukas, M. Protopopova, J. Lukas, G. Selivanova, J. Bartek, Functional impact of concomitant versus alternative defects in the Chk2- p53 tumour suppressor pathway, Oncogene 20 (2001) 5503–5510. J. Falck, N. Mailand, R.G. Syljuasen, J. Bartek, J. Lukas, The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis, Nature 410 (2001) 842–847. F. d’Adda di Fagagna, P.M. Reaper, L. Clay-Farrace, H. Fiegler, P. Carr, T. Von Zglinicki, G. Saretzki, N.P. Carter, S.P. Jackson, A DNA damage checkpoint response in telomere-initiated senescence, Nature 426 (2003) 194–198. H. Oh, S.C. Wang, A. Prahash, M. Sano, C.S. Moravec, G.E. Taffet, L.H. Michael, K.A. Youker, M.L. Entman, M.D. Schneider, Telomere attrition and Chk2 activation in human heart failure, Proc Natl Acad Sci USA 100 (2003) 5378–5383. S. Banin, L. Moyal, S. Shieh, Y. Taya, C.W. Anderson, L. Chessa, N.I. Smorodinsky, C. Prives, Y. Reiss, Y. Shiloh, Y. Ziv, Enhanced phosphorylation of p53 by ATM in response to DNA damage, Science 281 (1998) 1674–1677. C.E. Canman, D.S. Lim, K.A. Cimprich, Y. Taya, K. Tamai, K. Sakaguchi, E. Appella, M.B. Kastan, J.D. Siliciano, Activation of the ATM kinase by ionizing radiation and phosphorylation of p53, Science 281 (1998) 1677–1679. Y. Shiloh, ATM and related protein kinases: safeguarding genome integrity, Nat Rev Cancer 3 (2003) 155–168. A.L. Brown, C.H. Lee, J.K. Schwarz, N. Mitiku, H. Piwnica-Worms, J.H. Chung, A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage, Proc Natl Acad Sci USA 96 (1999) 3745–3750. P. Chaturvedi, W.K. Eng, Y. Zhu, M.R. Mattern, R. Mishra, M.R. Hurle, X. Zhang, R.S. Annan, Q. Lu, L.F. Faucette, G.F. Scott, X. Li, S.A. Carr, R.K. Johnson, J.D. Winkler, B.B. Zhou, Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway, Oncogene 18 (1999) 4047–4054. J.Y. Ahn, J.K. Schwarz, H. Piwnica-Worms, C.E. Canman, Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation, Cancer Res 60 (2000) 5934–5936. S. Matsuoka, G. Rotman, A. Ogawa, Y. Shiloh, K. Tamai, S.J. Elledge, Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro, Proc Natl Acad Sci USA 97 (2000) 10389– 10394. R. Melchionna, X.B. Chen, A. Blasina, C.H. McGowan, Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1, Nat Cell Biol 2 (2000) 762–765. J.Y. Ahn, X. Li, H.L. Davis, C.E. Canman, Phosphorylation of threonine 68 promotes oligomerization and autophosphorylation of the Chk2 protein kinase via the forkhead- associated domain, J Biol Chem 277 (2002) 19389–19395. X. Xu, L.M. Tsvetkov, D.F. Stern, Chk2 activation and phosphorylation-dependent oligomerization, Mol Cell Biol 22 (2002) 4419–4432. J. Ahn, C. Prives, Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation, J Biol Chem 277 (2002) 48418– 48426. C.H. Lee, J.H. Chung, The hCds1 (Chk2)-FHA domain is essential for a chain of phosphorylation events on hCds1 that is induced by ionizing radiation, J Biol Chem 276 (2001) 30537–30541. J.K. Schwarz, C.M. Lovly, H. Piwnica-Worms, Regulation of the Chk2 Protein Kinase by Oligomerization-Mediated cis- and trans-Phosphorylation, Mol Cancer Res 1 (2003) 598–609. X. Wu, J. Chen, Autophosphorylation of checkpoint kinase 2 at serine 516 is required for radiation-induced apoptosis, J Biol Chem 278 (2003) 36163–36168.
1045
[49] Z. Lou, K. Minter-Dykhouse, X. Wu, J. Chen, MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways, Nature 421 (2003) 957–961. [50] L. Tsvetkov, X. Xu, J. Li, D.F. Stern, Polo-like kinase 1 and Chk2 interact and co-localize to centrosomes and the midbody, J Biol Chem 278 (2003) 8468–8475. [51] S. Xie, H. Wu, Q. Wang, J. Kunicki, R.O. Thomas, R.E. Hollingsworth, J. Cogswell, W. Dai, Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2, Cell Cycle 1 (2002) 424–429. [52] M. Bahassi el, C.W. Conn, D.L. Myer, R.F. Hennigan, C.H. McGowan, Y. Sanchez, P.J. Stambrook, Mammalian Polo-like kinase 3 (Plk3) is a multifunctional protein involved in stress response pathways, Oncogene 21 (2002) 6633–6640. [53] L. Zannini, D. Lecis, S. Lisanti, R. Benetti, G. Buscemi, C. Schneider, D. Delia, Karyopherin–alpha2 protein interacts with Chk2 and contributes to its nuclear import, J Biol Chem 278 (2003) 42346– 42351. [54] T. O’Neill, L. Giarratani, P. Chen, L. Iyer, C.H. Lee, M. Bobiak, F. Kanai, B.B. Zhou, J.H. Chung, G.A. Rathbun, Determination of Substrate Motifs for Human Chk1 and hCds1/Chk2 by the Oriented Peptide Library Approach, J Biol Chem 277 (2002) 16102–16115. [55] G.J. Seo, S.E. Kim, Y.M. Lee, J.W. Lee, J.R. Lee, M.J. Hahn, S.T. Kim, Determination of substrate specificity and putative substrates of Chk2 kinase, Biochem Biophys Res Commun 304 (2003) 339– 343. [56] A. Blasina, I.V. de Weyer, M.C. Laus, W.H. Luyten, A.E. Parker, C.H. McGowan, A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase, Curr Biol 9 (1999) 1–10. [57] C.S. Sorensen, R.G. Syljuasen, J. Falck, T. Schroeder, L. Ronnstrand, K.K. Khanna, B.B. Zhou, J. Bartek, J. Lukas, Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A, Cancer Cell 3 (2003) 247–258. [58] L. Busino, M. Donzelli, M. Chiesa, D. Guardavaccaro, D. Ganoth, N.V. Dorrello, A. Hershko, M. Pagano, G.F. Draetta, Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage, Nature 426 (2003) 87–91. [59] J. Jin, T. Shirogane, L. Xu, G. Nalepa, J. Qin, S.J. Elledge, J.W. Harper, SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase, Genes Dev 17 (2003) 3062–3074. [60] B.B. Zhou, P. Chaturvedi, K. Spring, S.P. Scott, R.A. Johanson, R. Mishra, M.R. Mattern, J.D. Winkler, K.K. Khanna, Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity, J Biol Chem 275 (2000) 10342–10348. [61] C.Y. Peng, P.R. Graves, R.S. Thoma, Z. Wu, A.S. Shaw, H. Piwnica-Worms, Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216, Science 277 (1997) 1501–1505. [62] S.N. Dalal, C.M. Schweitzer, J. Gan, J.A. DeCaprio, Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site, Mol Cell Biol 19 (1999) 4465–4479. [63] C. Stevens, L. Smith, N.B. La Thangue, Chk2 activates E2F-1 in response to DNA damage, Nat Cell Biol 5 (2003) 401–409. [64] W.C. Lin, F.T. Lin, J.R. Nevins, Selective induction of E2F-1 in response to DNA damage, mediated by ATM-dependent phosphorylation, Genes Dev 15 (2001) 1833–1844. [65] J. DeGregori, G. Leone, A. Miron, L. Jakoi, J.R. Nevins, Distinct roles for E2F proteins in cell growth control and apoptosis, Proc Natl Acad Sci USA 94 (1997) 7245–7250. [66] J.S. Lee, K.M. Collins, A.L. Brown, C.H. Lee, J.H. Chung, hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response, Nature 404 (2000) 201–204. [67] S. Yang, C. Kuo, J.E. Bisi, M.K. Kim, PML-dependent apoptosis after DNA damage is regulated by the checkpoint kinase hCds1/Chk2, Nat Cell Biol 4 (2002) 865–870.
1046
J. Ahn et al. / DNA Repair 3 (2004) 1039–1047
[68] J. Zhang, H. Willers, Z. Feng, J.C. Ghosh, S. Kim, D.T. Weaver, J.H. Chung, S.N. Powell, F. Xia, Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair, Mol Cell Biol 24 (2004) 708–718. [69] C. Lukas, J. Falck, J. Bartkova, J. Bartek, J. Lukas, Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage, Nat Cell Biol 5 (2003) 255–260. [70] Z.X. Xu, A. Timanova-Atanasova, R.X. Zhao, K.S. Chang, PML colocalizes with and stabilizes the DNA damage response protein TopBP1, Mol Cell Biol 23 (2003) 4247–4256. [71] Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev 14 (2000) 927–939. [72] B. Wang, S. Matsuoka, P.B. Carpenter, S.J. Elledge, 53BP1, a mediator of the DNA damage checkpoint, Science 298 (2002) 1435– 1438. [73] I.M. Ward, X. Wu, J. Chen, Threonine 68 of Chk2 is phosphorylated at sites of DNA strand breaks, J Biol Chem 276 (2001) 47755– 47758. [74] N. Foray, D. Marot, A. Gabriel, V. Randrianarison, A.M. Carr, M. Perricaudet, A. Ashworth, P. Jeggo, A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein, Embo J 22 (2003) 2860–2871. [75] K.D. Brown, A. Rathi, R. Kamath, D.I. Beardsley, Q. Zhan, J.L. Mannino, R. Baskaran, The mismatch repair system is required for S-phase checkpoint activation, Nat Genet 33 (2003) 80–84. [76] A. Franchitto, P. Pichierri, R. Piergentili, M. Crescenzi, M. Bignami, F. Palitti, The mammalian mismatch repair protein MSH2 is required for correct MRE11 and RAD51 relocalization and for efficient cell cycle arrest induced by ionizing radiation in G2 phase, Oncogene 22 (2003) 2110–2120. [77] G. Buscemi, C. Savio, L. Zannini, F. Micciche, D. Masnada, M. Nakanishi, H. Tauchi, K. Komatsu, S. Mizutani, K. Khanna, P. Chen, P. Concannon, L. Chessa, D. Delia, Chk2 activation dependence on Nbs1 after DNA damage, Mol Cell Biol 21 (2001) 5214–5222. [78] T. Usui, H. Ogawa, J.H. Petrini, A DNA damage response pathway controlled by Tel1 and the Mre11 complex, Mol Cell 7 (2001) 1255–1266. [79] T. Uziel, Y. Lerenthal, L. Moyal, Y. Andegeko, L. Mittelman, Y. Shiloh, Requirement of the MRN complex for ATM activation by DNA damage, Embo J 22 (2003) 5612–5621. [80] C.T. Carson, R.A. Schwartz, T.H. Stracker, C.E. Lilley, D.V. Lee, M.D. Weitzman, The Mre11 complex is required for ATM activation and the G(2)/M checkpoint, Embo J 22 (2003) 6610–6620. [81] J.H. Lee, B. Xu, C.H. Lee, J.Y. Ahn, M.S. Song, H. Lee, C.E. Canman, J.S. Lee, M.B. Kastan, D.S. Lim, Distinct functions of Nijmegen breakage syndrome in ataxia telangiectasia mutated-dependent responses to DNA damage, Mol Cancer Res 1 (2003) 674–681. [82] T.A. Mochan, M. Venere, R.A. DiTullio Jr., T.D. Halazonetis, 53BP1 and NFBD1/MDC1-Nbs1 function in parallel interacting pathways activating ataxia-telangiectasia mutated (ATM) in response to DNA damage, Cancer Res 63 (2003) 8586–8591. [83] I.M. Ward, K. Minn, J. van Deursen, J. Chen, p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice, Mol Cell Biol 23 (2003) 2556–2563. [84] O. Fernandez-Capetillo, H.T. Chen, A. Celeste, I. Ward, P.J. Romanienko, J.C. Morales, K. Naka, Z. Xia, R.D. Camerini-Otero, N. Motoyama, P.B. Carpenter, W.M. Bonner, J. Chen, A. Nussenzweig, DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1, Nat Cell Biol 4 (2002) 993–997. [85] R.A. DiTullio Jr., T.A. Mochan, M. Venere, J. Bartkova, M. Sehested, J. Bartek, T.D. Halazonetis, 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer, Nat Cell Biol 4 (2002) 998–1002.
[86] G.S. Stewart, B. Wang, C.R. Bignell, A.M. Taylor, S.J. Elledge, MDC1 is a mediator of the mammalian DNA damage checkpoint, Nature 421 (2003) 961–966. [87] M. Goldberg, M. Stucki, J. Falck, D. D’Amours, D. Rahman, D. Pappin, J. Bartek, S.P. Jackson, MDC1 is required for the intra-S-phase DNA damage checkpoint, Nature 421 (2003) 952– 956. [88] A. Peng, P.L. Chen, NFBD1, like 53BP1, is an early and redundant transducer mediating Chk2 phosphorylation in response to DNA damage, J Biol Chem 278 (2003) 8873–8876. [89] Y.L. Shang, A.J. Bodero, P.L. Chen, NFBD1, a novel nuclear protein with signature motifs of FHA and BRCT, and an internal 41-amino acid repeat sequence, is an early participant in DNA damage response, J Biol Chem 278 (2003) 6323–6329. [90] S.Y. Shieh, Y. Taya, C. Prives, DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization, Embo J 18 (1999) 1815–1823. [91] T. Unger, T. Juven-Gershon, E. Moallem, M. Berger, R. Vogt Sionov, G. Lozano, M. Oren, Y. Haupt, Critical role for Ser20 of human p53 in the negative regulation of p53 by Mdm2, Embo J 18 (1999) 1805–1814. [92] S.Y. Shieh, J. Ahn, K. Tamai, Y. Taya, C. Prives, The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites, Genes Dev 14 (2000) 289–300. [93] N.H. Chehab, A. Malikzay, M. Appel, T.D. Halazonetis, Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53, Genes Dev 14 (2000) 278–288. [94] Q. Yu, J.H. Rose, H. Zhang, Y. Pommier, Antisense inhibition of Chk2/hCds1 expression attenuates DNA damage-induced S and G2 checkpoints and enhances apoptotic activity in HEK-293 cells, FEBS Lett 505 (2001) 7–12. [95] S. Tan, D. Guschin, A. Davalos, Y.L. Lee, A.W. Snowden, Y. Jouvenot, H.S. Zhang, K. Howes, A.R. McNamara, A. Lai, C. Ullman, L. Reynolds, M. Moore, M. Isalan, L.P. Berg, B. Campos, H. Qi, S.K. Spratt, C.C. Case, C.O. Pabo, J. Campisi, P.D. Gregory, Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity, Proc Natl Acad Sci USA 100 (2003) 11997– 12002. [96] A. Craig, M. Scott, L. Burch, G. Smith, K. Ball, T. Hupp, Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain, EMBO Rep 4 (2003) 787–792. [97] D. Qin, H. Lee, C. Yuan, Y. Ju, M.D. Tsai, Identification of potential binding sites for the FHA domain of human Chk2 by in vitro binding studies, Biochem Biophys Res Commun 311 (2003) 803–808. [98] J. Ahn, M. Urist, C. Prives, Questioning the role of Chk2 in the p53 DNA Damage Response, J Biol Chem 278 (2003) 20480–20489. [99] P.V. Jallepalli, C. Lengauer, B. Vogelstein, F. Bunz, The Chk2 tumor suppressor is not required for p53 responses in human cancer cells, J Biol Chem 278 (2003) 20475–20479. [100] A. Hirao, Y.Y. Kong, S. Matsuoka, A. Wakeham, J. Ruland, H. Yoshida, D. Liu, S.J. Elledge, T.W. Mak, DNA damage-induced activation of p53 by the checkpoint kinase Chk2, Science 287 (2000) 1824–1827. [101] A. Hirao, A. Cheung, G. Duncan, P.M. Girard, A.J. Elia, A. Wakeham, H. Okada, T. Sarkissian, J.A. Wong, T. Sakai, E. De Stanchina, R.G. Bristow, T. Suda, S.W. Lowe, P.A. Jeggo, S.J. Elledge, T.W. Mak, Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner, Mol Cell Biol 22 (2002) 6521–6532. [102] M.T. Jack, R.A. Woo, A. Hirao, A. Cheung, T.W. Mak, P.W. Lee, Chk2 is dispensable for p53-mediated G1 arrest but is required for a latent p53-mediated apoptotic response, Proc Natl Acad Sci USA 3 (2002) 3. [103] H. Takai, K. Naka, Y. Okada, M. Watanabe, N. Harada, S. Saito, C.W. Anderson, E. Appella, M. Nakanishi, H. Suzuki, K. Nagashima, H. Sawa, K. Ikeda, N. Motoyama, Chk2-deficient mice
J. Ahn et al. / DNA Repair 3 (2004) 1039–1047
[104]
[105]
[106]
[107]
[108]
[109]
exhibit radioresistance and defective p53-mediated transcription, Embo J 21 (2002) 5195–5205. E. Keramaris, A. Hirao, R.S. Slack, T.W. Mak, D.S. Park, Ataxia telangiectasia-mutated protein can regulate p53 and neuronal death independent of Chk2 in response to DNA damage, J Biol Chem 278 (2003) 37782–37789. I. Oishi, S. Sugiyama, H. Otani, H. Yamamura, Y. Nishida, Y. Minami, A novel Drosophila nuclear protein serine/threonine kinase expressed in the germline during its establishment, Mech Dev 71 (1998) 49–63. J. Xu, S. Xin, W. Du, Drosophila Chk2 is required for DNA damage-mediated cell cycle arrest and apoptosis, FEBS Lett 508 (2001) 394–398. J. Xu, W. Du, Drosophila chk2 plays an important role in a mitotic checkpoint in syncytial embryos, FEBS Lett 545 (2003) 209– 212. S. Takada, A. Kelkar, W.E. Theurkauf, Drosophila checkpoint kinase 2 couples centrosome function and spindle assembly to genomic integrity, Cell 113 (2003) 87–99. M. Peters, C. DeLuca, A. Hirao, V. Stambolic, J. Potter, L. Zhou, J. Liepa, B. Snow, S. Arya, J. Wong, D. Bouchard, R. Binari, A.S. Manoukian, T.W. Mak, Chk2 regulates irradiation-induced,
[110]
[111]
[112]
[113]
[114]
1047
p53-mediated apoptosis in Drosophila, Proc Natl Acad Sci USA 99 (2002) 11305–11310. M.H. Brodsky, B.T. Weinert, G. Tsang, Y.S. Rong, N.M. McGinnis, K.G. Golic, D.C. Rio, G.M. Rubin, Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage, Mol Cell Biol 24 (2004) 1219– 1231. I. Oishi, K. Iwai, Y. Kagohashi, H. Fujimoto, K. Kariya, T. Kataoka, H. Sawa, H. Okano, H. Otani, H. Yamamura, Y. Minami, Critical role of Caenorhabditis elegans homologs of Cds1 (Chk2)-related kinases in meiotic recombination, Mol Cell Biol 21 (2001) 1329– 1335. A. Higashitani, H. Aoki, A. Mori, Y. Sasagawa, T. Takanami, H. Takahashi, Caenorhabditis elegans Chk2-like gene is essential for meiosis but dispensable for DNA repair, FEBS Lett 485 (2000) 35–39. A.J. MacQueen, A.M. Villeneuve, Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2, Genes Dev 15 (2001) 1674–1687. U. Abdu, M. Brodsky, T. Schupbach, Activation of a meiotic checkpoint during Drosophila oogenesis regulates the translation of Gurken through Chk2/Mnk, Curr Biol 12 (2002) 1645–1651.