The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ATM dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. ATM phosphorylates in vitro the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduces the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocks all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD (Matsuoka, 2000).
The Chk2 Ser/Thr kinase plays crucial, evolutionarily conserved roles in cellular responses to DNA damage. Identification of two pro-oncogenic mutations within the Chk2 FHA domain has highlighted its importance for Chk2 function in checkpoint activation. The X-ray structure of the Chk2 FHA domain in complex with an in vitro selected phosphopeptide motif reveals the determinants of binding specificity and shows that both mutations are remote from the peptide binding site. The Chk2 FHA domain mediates ATM-dependent Chk2 phosphorylation and targeting of Chk2 to in vivo binding partners such as BRCA1 through either or both of two structurally distinct mechanisms. Although phospho-dependent binding is important for Chk2 activity, previously uncharacterized phospho-independent FHA domain interactions appear to be the primary target of oncogenic lesions (Li, 2002).
The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. Autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways (Xu, 2002).
Mutants of the Saccharomyces cerevisiae ataxia telangiectasia mutated (ATM) homolog MEC1/SAD3/ESR1 were identified that could live only if the RAD53/SAD1 checkpoint kinase was overproduced. MEC1 and a structurally related gene, TEL1, have overlapping functions in response to DNA damage and replication blocks that in mutants can be provided by overproduction of RAD53. Both MEC1 and TEL1 were found to control phosphorylation of Rad53p in response to DNA damage. These results indicate that RAD53 is a signal transducer in the DNA damage and replication checkpoint pathways and functions downstream of two members of the ATM lipid kinase family. Because several members of this pathway are conserved among eukaryotes, it is likely that a RAD53-related kinase will function downstream of the human ATM gene product and play an important role in the mammalian response to DNA damage (Sanchez, 1996).
Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases. Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks. It is shown here that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1). This phenotype is highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro. Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain results in an increased DNA damage sensitivity in vivo. Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1) (Pike, 2001).
The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53. Rad52, in turn, inhibit spindle elongation and late origin firing and this leads to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. The restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. These data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks (K. Shimada, 2002).
During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. This study investigates the mechanism that controls the meiotic progression in Schizosaccharomyces pombe cells that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis has revealed that meiotic DSB repair in meu13Delta is retarded. The delay in entering nuclear division is dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. It is proposed that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression (M. Shimada, 2002).
During meiosis, genetic recombination between homologous chromosomes is essential for proper chromosome segregation at the first meiotic division. In response to incomplete recombination, the pachytene checkpoint (also known as the meiotic recombination checkpoint) arrests or delays meiotic cell cycle progression, thus preventing the formation of defective gametes. A role is described for a meiosis-specific kinase, Mek1, in the meiotic recombination checkpoint in fission yeast. Mek1 belongs to the Cds1/Rad53/Chk2 family of kinases containing forkhead-associated domains, which participate in a number of checkpoint responses from yeast to mammals. Defects in meiotic recombination generated by the lack of the fission yeast Meu13 protein lead to a delay in entry into meiosis I owing to inhibitory phosphorylation of the cyclin-dependent kinase Cdc2 on tyrosine 15. Mutation of mek1(+) alleviates this checkpoint-induced delay, resulting in the formation of largely inviable meiotic products. Experiments involving ectopic overexpression of the mek1(+) gene indicate that Mek1 inhibits the Cdc25 phosphatase, which is responsible for dephosphorylation of Cdc2 on tyrosine 15. Furthermore, the meiotic recombination checkpoint is impaired in a cdc25 phosphorylation site mutant. Thus, the first evidence is provided of a connection between an effector kinase of the meiotic recombination checkpoint and a crucial cell cycle regulator and present a model for the operation of this meiotic checkpoint in fission yeast (Perez-Hidalgo, 2003).
A Chk2-like gene was found to be essential for meiosis in Caenorhabditis elegans. Ce-chk-2 consists of 450 amino acid residues shows good homology with the Chk2/Cds1 gene family. The results of RNA-mediated interference (RNAi) indicates that the F1 generation from dsRNA injected animals grow to adulthood, but approximately 95% of their eggs (F2) die during early embryogenesis. Among the few surviving progeny, males (XO animals) arise at an abnormally high frequency (30%). In addition, 12 univalents are observed in full grown oocytes of the F1, while six bivalents are normally observed in wild-type oocytes. Ce-chk-2 gene expression increases in the adult stage, and the expression level decreases in the glp-4 mutant, which is defective in germ line proliferation. The radiation sensitivity of F1 embryos carrying Ce-chk-2 RNAi was not significantly affected (Higashitani, 2000).
Analysis of mutants defective in meiotic chromosome pairing has uncovered a role for Caenorhabditis elegans chk-2 in initial establishment of pairing between homologous chromosomes during early meiotic prophase. chk-2 is also required for the major spatial reorganization of nuclei that normally accompanies the onset of pairing, suggesting a mechanistic coupling of these two events. Despite failures in pairing, nuclear reorganization, and crossover recombination, chk-2 mutants undergo many other aspects of meiotic chromosome morphogenesis and complete gametogenesis. Although chk-2 encodes a C. elegans ortholog of the Cds1/Chk2 checkpoint protein kinases, germ-line nuclei in chk-2 mutants are competent to arrest proliferation in response to replication inhibition and to trigger DNA damage checkpoint responses to ionizing radiation. However, chk-2 mutants are defective in triggering the pachytene DNA damage checkpoint in response to an intermediate block in the meiotic recombination pathway, suggesting that chk-2 is required either for initiation of meiotic recombination or for monitoring a specific subset of DNA damage lesions. It is proposed that chk-2 functions during premeiotic S phase to enable chromosomes to become competent for subsequent meiotic prophase events and/or to coordinate replication with entry into prophase (MacQueen, 2001).
Although chromosomal segregation at meiosis I is the critical process for genetic reassortment and inheritance, little is known about molecules involved in this process in metazoa. Utilizing double-stranded RNA (dsRNA)-mediated genetic interference it has been shown that novel protein kinases (Ce-CDS-1 and Ce-CDS-2) related to Cds1 (Chk2) play an essential role in meiotic recombination in Caenorhabditis elegans. Injection of dsRNA into adult animals results in the inhibition of meiotic crossing over and induces the loss of chiasmata at diakinesis in oocytes of F(1) animals. However, electron microscopic analysis reveals that synaptonemal complex formation in pachytene nuclei of the same progeny of injected animals appears to be normal. Thus, Ce-CDS-1 and Ce-CDS-2 are the first examples of Cds1-related kinases that are required for meiotic recombination in multicellular organisms (Oishi, 2001).
Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. The ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells (Ahn, 2000).
Phosphorylation of Thr-68 by the ATM protein is necessary for efficient activation of Chk2 when cells are exposed to ionizing radiation. By an unknown mechanism, this initial event promotes additional autophosphorylation events including modifications of Thr-383 and Thr-387, two amino acid residues located within the activation loop segment within the Chk2 catalytic domain. Chk2 and related kinases possess one or more Forkhead-associated (FHA) domains that are phosphopeptide-binding modules believed to be crucial for their checkpoint control activities. The Chk2 FHA domain is dispensable for Thr-68 phosphorylation but necessary for efficient autophosphorylation in response to ionizing radiation. Phosphorylation of Thr-68 promotes oligomerization of Chk2 by serving as a specific ligand for the FHA domain of another Chk2 molecule. In addition, Chk2 phosphorylates its own FHA domain, and this modification reduces its affinity for Thr-68-phosphorylated Chk2. Thus, activation of Chk2 in irradiated cells may occur through oligomerization of Chk2 via binding of the Thr-68-phosphorylated region of one Chk2 to the FHA domain of another. Oligomerization of Chk2 may therefore increase the efficiency of trans-autophosphorylation resulting in the release of active Chk2 monomers that proceed to enforce checkpoint control in irradiated cells (Ahn, 2002a).
ATM is necessary for phosphorylation and activation of Cds1/Chk2 in vivo and can phosphorylate Cds1 in vitro, although evidence is lacking that the sites phosphorylated by ATM are required for activation. This study shows that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionizing radiation (Melchionna, 2000).
Human Chk2 has been characterized and purified both from baculovirus-infected insect cells and from either untreated or DNA damage-stressed human HCT116 cells. Chk2 from unstressed human cells is largely monomeric and inactive in phosphorylating its substrate, Cdc25C. It is also unphosphorylated at Thr-68, a site that is the target of the ataxia telangiectasia-mutated protein kinase. After treatment of HCT116 cells with a radiomimetic compound neocarzinostatin, active Chk2 exists as stable Thr-68-phosphorylated dimers as well as interconvertable Thr-68-unphosphorylated monomers and dimers. Interestingly, Chk2 from insect cells behaves by all criteria tested like active Chk2 from neocarzinostatin-treated HCT116 cells. Based on Stokes radius and sedimentation coefficient values, Chk2 monomers and dimers have asymmetric rather than globular shapes. Both Thr-68-phosphorylated and Thr-68-unphosphorylated forms of active Chk2 are capable of phosphorylating Cdc25C. Thus, although phosphorylation of Thr-68 may be required for initial oligomerization and activation of Chk2, it is not needed for maintenance of dimerization or kinase activity (Ahn, 2002b).
In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ATM gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, gene-targeted Chk2-deficient mice were generated. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis is restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis (Hirao, 2002).
Signaling pathways in response to DNA double strand breaks involve molecular cascades consisting of sensors, transducers, and effector proteins that activate cell cycle checkpoints and recruit repair machinery proteins. NFBD1 (a nuclear factor with BRCT domains protein 1) contains FHA (forkhead-associated), BRCT (breast cancer susceptibility gene 1 carboxyl terminus) domains, and internal repeats and is an early participant in nuclear foci in response to IR. To elucidate its role in the response pathways, small interfering RNA (siRNA) directed against NFDB1 in human cells demonstrated that its absence is associated with increased radio-sensitivity and delayed G(2)/M transition, but not G(1) to S. NFBD1 associates with nuclear foci within minutes following IR, a property similar to histone H2AX, 53BP1, and Chk2, which are all early participants in the DNA damage signaling cascade. Temporal studies show that H2AX is required for the foci positive for NFBD1, but NFBD1 is not needed for 53BP1- and H2AX-positive foci. NFBD1, together with 53BP1, plays a partially redundant role in regulating phosphorylation of the downstream effector protein, Chk2, since abrogation of both diminishes phosphorylated Chk2 in IR-induced foci. These results place NFBD1 parallel to 53BP1 in regulating Chk2 and downstream of H2AX in the recruitment of repair and signaling proteins to sites of DNA damage (Peng, 2003).
The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. This study shows that the ATM-dependent activation of Chk2 by gamma-radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein (Drosophila homolog Nbs), Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2 (Buscemi, 2001).
The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. MRN deficiency occurs in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, checkpoint responses were examined to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation is independent of MRN. Chk2 activation is ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restores Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibits Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restores Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. It is proposed that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers (Takemura, 2006).
In response to DNA damage and replication blocks, cells prevent cell cycle progression through the control of critical cell cycle regulators. This study identifies Chk2, the mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 protein kinases required for the DNA damage and replication checkpoints. Chk2 was rapidly phosphorylated and activated in response to replication blocks and DNA damage; the response to DNA damage occurs in an ATM-dependent manner. In vitro, Chk2 phosphorylates Cdc25C on serine-216, a site known to be involved in negative regulation of Cdc25C. This is the same site phosphorylated by the protein kinase Chk1: this suggests that, in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis (Matsuoka, 1998).
In human cells, the mitosis-inducing kinase Cdc2 is inhibited by phosphorylation on Thr14 and Tyr15. Disruption of these phosphorylation sites abrogates checkpoint-mediated regulation of Cdc2 and renders cells highly sensitive to agents that damage DNA. Phosphorylation of these sites is controlled by the opposing activities of the Wee1/Myt1 kinases and the Cdc25 phosphatase. The regulation of these enzymes is therefore likely to be crucial for the operation of the G2-M DNA-damage checkpoint. The activity of Cdc25 decreases following exposure to ionizing radiation. The irradiation-induced decrease in Cdc25 activity is suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinases, and is dependent on the function of the gene that is mutated in ataxia telangiectasia. Two human kinases have been identified that phosphorylate and inactivate Cdc25 in vitro. One is the previously characterized Chk1 kinase. The second is novel and is homologous to the Cds1/Rad53 family of checkpoint kinases in yeast. Human Cds1 is activated in response to DNA damage. These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1 (Blasina, 1999).
When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. A functional link is reported between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and this mechanism is implicated in controlling the S-phase checkpoint. IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. Tumor-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and cells expressing these Chk2 alleles or elevated Cdc25A, or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) all fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumor suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis (Falck, 2001).
Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. A S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). Recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with the observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage (Shieh, 2000).
Chk2/hcds1, the human homolog of the Saccharomyces cerevisiae RAD53/SPK1 and Schizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G(2) in response to DNA damage. Chk2/hCds1 was expressed in human cells and their cell cycle profile was analyzed. Wild-type, but not catalytically inactive, Chk2/hCds1 leads to G(1) arrest after DNA damage. The arrest is inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylates p53 on Ser-20 and dissociates preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 leads to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogates both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G(1) (Chehab, 2000).
Chk2 is a protein kinase that is activated in response to DNA damage and may regulate cell cycle arrest. Chk2-deficient mouse cells were generated by gene targeting. Chk2-/- embryonic stem cells fail to maintain gamma-irradiation-induced arrest in the G2 phase of the cell cycle. Chk2-/- thymocytes are resistant to DNA damage-induced apoptosis. Chk2-/- cells are defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to gamma irradiation. Reintroduction of the Chk2 gene restores p53-dependent transcription in response to gamma irradiation. Chk2 directly phosphorylates p53 on serine 20, which is known to interfere with Mdm2 binding. This provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage (Hirao, 2000).
The mammalian Chk2 kinase is thought to mediate ATM-dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2-deficient mice. Although Chk2(-/-) mice appeared normal, they are resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain are also resistant to IR-induced apoptosis. The IR-induced G(1)/S cell cycle checkpoint, but not the G(2)/M or S phase checkpoints, is impaired in embryonic fibroblasts derived from Chk2(-/-) mice. IR-induced stabilization of p53 in Chk2(-/-) cells was 50%-70% of that in wild-type cells. Caffeine further reduces p53 accumulation, suggesting the existence of an ATM/ATR-dependent but Chk2-independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53-dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2(-/-) cells. These results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability (Takai, 1992).
In response to genotoxic stress, mammalian cells can activate cell cycle checkpoint pathways to arrest the cell for repair of DNA damage or induce apoptosis to eliminate damaged cells. The checkpoint kinase, Chk2, has been implicated in both of these responses and is believed to function in an ataxia telangiectasia (Atm)-dependent manner. Chk2-/- mouse embryo fibroblasts (MEFs), unlike Atm-/- or p53-/- MEFs, behaved like normal MEFs in manifesting p21 induction and G(1) arrest upon exposure to gamma-irradiation. Therefore, Chk2 is not involved in p53-mediated G(1) arrest. To examine the role of Chk2 in p53-dependent apoptotic response, adenovirus E1A-expressing MEFs were used. Chk2-/- cells, like p53-/- cells, do not undergo DNA damage-induced apoptosis, whereas Atm-/- cells behave like normal cells in invoking an apoptotic response. Furthermore, this apoptosis can occur in the absence of protein synthesis, suggesting that it is preexisting, or 'latent', p53 that mediates this response. It is concluded that Chk2 is not involved in Atm- and p53-dependent G(1) arrest, but is involved in the activation of latent p53, independently of Atm, in triggering DNA damage-induced apoptosis (Jack, 2002).
The checkpoint kinases Chk1 and Chk2 are central to the induction of cell cycle arrest, DNA repair, and apoptosis as elements in the DNA-damage checkpoint. In several human tumor cell lines, Chk1 and Chk2 control the induction of the p53 related transcription factor p73 in response to DNA damage. Multiple experimental systems were used to show that interference with or augmentation of Chk1 or Chk2 signaling strongly impacts p73 accumulation. Furthermore, Chk1 and Chk2 control p73 mRNA accumulation after DNA damage. E2F1 directs p73 expression in the presence and absence of DNA damage. Chk1 and Chk2, in turn, are vital to E2F1 stabilization and activity after genotoxic stress. Thus, Chk1, Chk2, E2F1, and p73 function in a pathway mediating p53-independent cell death produced by cytotoxic drugs. Since p53 is often obviated through mutation as a cellular port for anticancer intervention, this pathway controlling p53 autonomous pro-apoptotic signaling is of potential therapeutic importance (Urist, 2004).
The E2F-1 transcription factor is regulated during cell cycle progression and induced by cellular stress, such as DNA damage. Checkpoint kinase 2 (Chk2) regulates E2F-1 activity in response to the DNA-damaging agent etoposide. A Chk2 consensus phosphorylation site in E2F-1 is phosphorylated in response to DNA damage, resulting in protein stabilization, increased half-life, transcriptional activation and localization of phosphorylated E2F-1 to discrete nuclear structures. Expression of a dominant-negative Chk2 mutant blocks induction of E2F-1 and prevents E2F-1-dependent apoptosis. Moreover, E2F-1 is resistant to induction by etoposide in tumor cells expressing mutant chk2. Therefore, Chk2 phosphorylates and activates E2F-1 in response to DNA damage, resulting in apoptosis. These results suggest a role for E2F-1 in checkpoint control and provide a plausible explanation for the tumor suppressor activity of E2F-1 (Stevens, 2003).
Chk2 is a protein kinase intermediary in DNA damage checkpoint pathways. DNA damage induces phosphorylation of Chk2 at multiple sites concomitant with activation. Chk2 phosphorylated at Thr-68 is found in nuclear foci at sites of DNA damage. Chk2 phosphorylated at Thr-68 and Thr-26 or Ser-28 is localized to centrosomes and midbodies in the absence of DNA damage. In a search for interactions between Chk2 and proteins with similar subcellular localization patterns, it was found that Chk2 coimmunoprecipitates with Polo-like kinase 1, a regulator of chromosome segregation, mitotic entry, and mitotic exit. Plk1 overexpression enhances phosphorylation of Chk2 at Thr-68. Plk1 phosphorylates recombinant Chk2 in vitro. Indirect immunofluorescence (IF) microscopy revealed the co-localization of Chk2 and Plk1 to centrosomes in early mitosis and to the midbody in late mitosis. These findings suggest lateral communication between the DNA damage and mitotic checkpoints (Tsvetkov, 2003).
The promyelocytic leukaemia (PML) gene is translocated in most acute promyelocytic leukaemias and encodes a tumor suppressor protein. PML is involved in multiple apoptotic pathways and is thought to be pivotal in gamma irradiation-induced apoptosis. The DNA damage checkpoint kinase hCds1/Chk2 is necessary for p53-dependent apoptosis after gamma irradiation. In addition, gamma irradiation-induced apoptosis also occurs through p53-independent mechanisms, although the molecular mechanism remains largely unknown. hCds1/Chk2 is shown to mediates gamma irradiation-induced apoptosis in a p53-independent manner through an ataxia telangiectasia-mutated (ATM)-hCds1/Chk2-PML pathway. These results provide the first evidence of a functional relationship between PML and a checkpoint kinase in gamma irradiation-induced apoptosis (Yang, 2002).
Genetic errors in meiosis can lead to birth defects and spontaneous abortions. Checkpoint mechanisms of hitherto unknown nature eliminate oocytes with unrepaired DNA damage, causing recombination-defective mutant mice to be sterile. This study reports that checkpoint kinase 2 (Chk2 or Chek2: Loki in Drosophila), is essential for culling mouse oocytes bearing unrepaired meiotic or induced DNA double-strand breaks (DSBs). Female infertility caused by a meiotic recombination mutation or irradiation was reversed by mutation of Chk2. Both meiotically programmed and induced DSBs trigger CHK2-dependent activation of TRP53 (p53) and TRP63 (p63), effecting oocyte elimination. These data establish CHK2 as essential for DNA damage surveillance in female meiosis and indicate that the oocyte DSB damage response primarily involves a pathway hierarchy in which ataxia telangiectasia and Rad3-related (ATR) signals to CHK2, which then activates p53 and p63 (Bolcun-Filas, 2014).
UCN-01 (7-hydroxystaurosporine) is a cell-cycle checkpoint abrogator that sensitizes cells to ionizing radiation (IR) and chemotherapeutic agents. UCN-01 abrogates DNA-damage-induced G(2) checkpoint most selectively in p53-defective cells, by primarily targeting Chk1. UCN-01 prevents IR-induced p53 up-regulation and p53 phosphorylation on serine 20, a site identified for Chk2 (or/and Chk1) kinase. In human colon carcinoma HCT116 cells, IR treatment enhances Chk2 kinase activity, whereas Chk1 activity remains unchanged, which suggests that UCN-01 may interrupt IR-induced p53 response by inhibiting Chk2 kinase. This conclusion is supported by in vitro kinase assays, showing that UCN-01 inhibits Chk2 immunoprecipitated from HCT116 cells. In addition, UCN-01 efficiently abrogated both the initiation and maintenance of IR-induced G(2) arrest in HCT116 cells and their isogenic p53 (-/-) derivative, indicating that G(2) checkpoint abrogation by UCN-01 is p53 independent. In the p53 (-/-) cells, there was neither p21(Waf1/Cip1) induction nor UCN-01-induced apoptosis. Taken together, these observations indicate that UCN-01 can modulate both Chk1 and Chk2 in intact cells and enhance IR-induced apoptosis in p53-deficient, and consequently p21-deficient, cells (Yu, 2002).
Forkhead-homology-associated (FHA) domains function as protein-protein modules that recognize phosphorylated serine/threonine motifs. Interactions between FHA domains and phosphorylated proteins are thought to have essential roles in the transduction of DNA damage signals; however, it is unclear how FHA-domain-containing proteins participate in mammalian DNA damage responses. A FHA-domain-containing protein-mediator of DNA damage checkpoint protein 1 (MDC1) is involved in DNA damage responses. MDC1 localizes to sites of DNA breaks and associates with CHK2 after DNA damage. This association is mediated by the MDC1 FHA domain and the phosphorylated Thr 68 of CHK2. Furthermore, MDC1 is phosphorylated in an ATM/CHK2-dependent manner after DNA damage, suggesting that MDC1 may function in the ATM-CHK2 pathway. Consistent with this hypothesis, suppression of MDC1 expression results in defective S-phase checkpoint and reduced apoptosis in response to DNA damage. These functions can be restored by the expression of wild-type MDC1 but not MDC1 with a deleted FHA domain. Suppression of MDC1 expression results in decreased p53 stabilization in response to DNA damage. These results suggest that MDC1 is recruited through its FHA domain to the activated CHK2, and has a critical role in CHK2-mediated DNA damage responses (Lou, 2003).
The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Heterozygous germ line mutations in hCHK2 occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in the p53 gene. These observations suggest that hCHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast (Bell, 1999).
Ionizing radiation damages chromosomal DNA and activates p53-dependent transcription in mammalian cells. The Chk2 protein kinase has been hypothesized to be the primary mediator of this response. This hypothesis has been rigorously tested in human cells by disrupting the CHK2 gene through homologous recombination. The p53 response was unexpectedly robust in such cells. Phosphorylation of p53 at serine 20, accumulation of p53 protein, transcriptional activation of p53 target genes, and cell cycle arrest and apoptotic death phenotypes were completely intact regardless of CHK2 status. These results indicate that Chk2 kinase is not required for p53 activation in human cells and explain why CHK2 and TP53 mutations can jointly occur in human tumors (Jallepalli, 2003).
Germ-line mutations in the p53 gene predispose individuals to Li-Fraumeni syndrome (LFS). The cell cycle checkpoint kinases CHK1 and CHK2 act upstream of p53 in DNA damage responses, and rare germ-line mutations in CHK2 have been reported in LFS families. CHK1, CHK2, and p53 genes were analyzed for mutations in 44 Finnish families with LFS, Li-Fraumeni-like syndrome, or families phenotypically suggestive of LFS with conformation-sensitive gel electrophoresis. Five different disease-causing mutations were observed in 7 families: 4 in the p53 gene and 1 in the CHK2 gene (2 of 44 families). Interestingly, the other CHK2-mutation carrier also has a mutation in the MSH6 gene. The cancer phenotype in the CHK2-families is not characteristic of LFS, and may indicate variable phenotypic expression in the rare families with CHK2 mutations. No mutations in the CHK1 gene were identified (Vahteristo, 2001).
CHEK2 (previously known as 'CHK2') is a cell-cycle-checkpoint kinase that phosphorylates p53 and BRCA1 in response to DNA damage. A protein-truncating mutation, 1100delC in exon 10, which abolishes the kinase function of CHEK2, has been found in families with Li-Fraumeni syndrome (LFS) and in those with a cancer phenotype that is suggestive of LFS, including breast cancer. In the present study, the frequency of 1100delC was found to be 2.0% among an unselected population-based cohort of 1,035 patients with breast cancer. This was slightly, but not significantly, higher than the 1.4% frequency found among 1,885 population control subjects. However, a significantly elevated frequency was found among those 358 patients with a positive family history (11/358). Furthermore, patients with bilateral breast cancer are sixfold more likely to be 1100delC carriers than are patients with unilateral cancer. Analysis of the 1100delC variant in an independent set of 507 patients with familial breast cancer with no BRCA1 and BRCA2 mutations confirms a significantly elevated frequency of 1100delC, with a high frequency also seen in patients with only a single affected first-degree relative. Finally, tissue microarray analysis indicates that breast tumors from patients with 1100delC mutations show reduced CHEK2 immunostaining. The results suggest that CHEK2 acts as a low-penetrance tumor-suppressor gene in breast cancer and that it makes a significant contribution to familial clustering of breast cancer-including families with only two affected relatives, which are more common than families that include larger numbers of affected women (Vahteristo, 2002).
Mutations in BRCA1 and BRCA2 confer a high risk of breast and ovarian cancer, but account for only a small fraction of breast cancer susceptibility. To find additional genes conferring susceptibility to breast cancer, CHEK2 was examined. CHEK2(*)1100delC, a truncating variant that abrogates the kinase activity, has a frequency of 1.1% in healthy individuals. However, this variant is present in 5.1% of individuals with breast cancer from 718 families that do not carry mutations in BRCA1 or BRCA2, including 13.5% of individuals from families with male breast cancer. It is estimated that the CHEK2(*)1100delC variant results in an approximately twofold increase of breast cancer risk in women and a tenfold increase of risk in men. By contrast, the variant confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggests that the biological mechanisms underlying the elevated risk of breast cancer in CHEK2 mutation carriers are already subverted in carriers of BRCA1 or BRCA2 mutations, which is consistent with participation of the encoded proteins in the same pathway (Meijers-Heijboer, 2002).
Disruption of Brca1 results in cellular demise or tumorigenesis depending on cellular context. Inactivation of p53 contributes to Brca1-associated tumor susceptibility. However the activation of p53-dependent checkpoint/apoptotic signaling in the absence of Brca1 is poorly understood. Chk2 inactivation is shown to be partially equivalent to p53 inactivation, in that Chk2 deficiency facilitates the development, survival, and proliferation of Brca1-deficient T cells at the expense of genomic integrity. Brca1 deficiency results in Chk2 phosphorylation and the Chk2-dependent accumulation and activation of p53. Furthermore, inactivation of Chk2 and Brca1 is cooperative in breast cancer. These findings identify a critical role for Chk2 as a component of the DNA damage-signaling pathway activated in response to Brca1 deficiency (McPherson, 2004). Brca1-targeted disruption in mice results in embryonic lethality. To circumvent this limitation, conditional targeting strategies have been employed that facilitate the study of the consequences of Brca1 disruption in vivo and ex vivo. Mice that are tBrca1/ carry a targeted null mutation of Brca1 restricted to the T-cell lineage, thus bypassing the associated lethality of Brca1 germline disruption. Brca1 disruption in the T-cell compartment results in a drastic depletion of thymocytes and peripheral T cells, the accumulation of chromosomal abnormalities, and activation of p53. Thymocyte development of tBrca1/ mice is restored in the absence of p53 or the presence of overexpressed anti-apoptotic protein Bcl2 that antagonizes the p53 pathway. These findings suggest that genomic instability and p53 activation due to the ablation of Brca1 contribute to the observed defects in T-cell development, survival, and proliferation (McPherson, 2004).
Although several studies have linked the compromised viability and genomic instability of Brca1-deficient cells to the activation of p53 by genome surveillance, the molecular components that comprise this response are unclear. Recent studies of p53 have elucidated upstream signaling cascades that modulate its half-life, intracellular localization, and functional activity. DNA damage alters the phosphorylation status of p53 and other DNA damage response proteins by triggering molecular signaling cascades that include ATM, ATR, DNA-PK, Chk1, and Chk2 kinases. A component of these genome surveillance pathways, Chk2 (also known as Cds1) has been identified as a tumor suppressor; mutations in Chk2 have been implicated in familial cancer syndromes including Li-Fraumeni and low penetrance breast cancer susceptibility. Several studies have elucidated an ATM-Chk2-p53 DNA damage signaling cascade that is activated following DNA damage. Since Chk2 functions in a DNA damage response pathway that result in p53 activation, attempts were made to determine the contribution of this kinase to phenotypes associated with Brca1 deficiency. Deficiency in Chk2 partially mimics the loss of p53 and rescues the defective development, growth, and cellular demise of Brca1-deficient T cells at the expense of genomic instability and increased tumorigenicity. An important role for Chk2 has been identified in suppressing Brca1-associated breast cancer (McPherson, 2004).
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