Checkpoint Kinase 2

Description

CHK2, a protein kinase that is activated in response to DNA damage, is involved in cell cycle arrest.

Cloning and Expression

In response to DNA damage and replication blocks, cells prevent cell cycle progression through the control of critical cell cycle regulators. To investigate checkpoint conservation, Matsuoka et al. (1998) used PCR and database analysis to identify CHK2, the mammalian homolog of Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe cds1+, protein kinases required for DNA damage and replication checkpoints. The longest human cDNA encoded a 543-amino acid protein with 83% identity to mouse Chk2 and 34% identity to Drosophila Dmnk, a protein highly expressed in ovaries for which a function in meiosis had been suggested. Human CHK2 protein is 26% identical to Rad53 and 26% identical to cds1+. Sequence analysis revealed a single forkhead-associated (FHA) domain, a 60-amino acid protein interaction domain essential for activation in response to DNA damage that is conserved in the Rad53/cds1+ family of kinases. CHK2 has a potential regulatory region rich in SQ and TQ amino acid pairs. Northern blot analysis revealed wide expression of small amounts of CHK2 mRNA with larger amounts in human testis, spleen, colon, and peripheral blood leukocytes. CHK2 complemented the lethality of a Rad53 deletion.

Blasina et al. (1999) and Chaturvedi et al. (1999) independently identified CHK2.

Gene Function

Matsuoka et al. (1998) demonstrated that CHK2 was rapidly phosphorylated and activated in response to replication blocks and DNA damage. The response to DNA damage occurred in an ATM (see 607585)-dependent manner. In vitro, CHK2 phosphorylated CDC25C (157680) 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 (603078), which suggests that, in response to DNA damage and DNA replicational stress, CHK1 and CHK2 may phosphorylate CDC25C to prevent entry into mitosis.

Brown et al. (1999) referred to CHK2 as human CDS1. Affinity-purified antibodies to CHK2 recognized an endogenous 65-kD protein in 293 cells and 65-kD protein in cells transfected with a plasmid encoding untagged CHK2. When several human tissues were analyzed by immunoblotting, CHK2 protein was detected only in testis. Brown et al. (1999) found that CHK2 was modified by phosphorylation and activated in response to ionizing radiation, and was also modified in response to hydroxyurea treatment. Functional ATM protein was required for CHK2 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, CHK2 phosphorylated CDC25C to promote the binding of 14-3-3 proteins (see 113508). These findings suggest that the checkpoint function of CHK2 is conserved in yeast and mammals.

Chehab et al. (2000) expressed CHK2 in human cells and analyzed its cell cycle profile. Wildtype, but not catalytically inactive, CHK2 led to G1 arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 (TP53; 191170) mutant, indicating that CHK2 acted upstream of p53. In vitro, CHK2 phosphorylated p53 on serine-20 and dissociated preformed complexes of p53 with MDM2 (164785), a protein that targets p53 for degradation. In vivo, ectopic expression of wildtype CHK2 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative CHK2 mutant abrogated both phosphorylation of p53 on serine-20 and p53 stabilization. Thus, in response to DNA damage, CHK2 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G1.

Lee et al. (2000) reported that CHK2 regulates BRCA1 (113705) function after DNA damage by phosphorylating serine-988 of BRCA1. Lee et al. (2000) demonstrated that CHK2 and BRCA1 interact and colocalize within discrete nuclear foci but separate after gamma irradiation. Phosphorylation of BRCA1 at serine-988 is required for the release of BRCA1 from CHK2. This phosphorylation is also important for the ability of BRCA1 to restore survival after DNA damage in the BRCA1-mutated cell line HCC1937.

When exposed to ionizing radiation, eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the ionizing radiation-induced S-phase checkpoint cause 'radioresistant DNA synthesis,' a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia. The CDC25A phosphatase (116947) activates the cyclin-dependent kinase 2 (CDK2; 116953) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. Falck et al. (2001) reported a functional link between ATM, checkpoint signaling kinase CHK2, and CDC25A, and implicated this mechanism in controlling the S-phase checkpoint. Falck et al. (2001) showed that ionizing radiation-induced destruction of CDC25A requires both ATM and the CHK2-mediated phosphorylation of CDC25A on serine-123. An ionizing radiation-induced loss of CDC25A protein prevents dephosphorylation of CDK2 and leads to a transient blockade of DNA replication. Falck et al. (2001) also showed that tumor-associated CHK2 alleles cannot bind or phosphorylate CDC25A, and that cells expressing these CHK2 alleles, elevated CDC25A, or a CDK2 mutant unable to undergo inhibitory phosphorylation (CDK2AF) fail to inhibit DNA synthesis when irradiated. Falck et al. (2001) concluded that their 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 et al. (2002) demonstrated that experimental blockade of either the NBS1 (602667)-MRE11 (600814) function or the CHK2-triggered events leads to a partial radioresistant DNA synthesis phenotype in human cells. In contrast, concomitant interference with NBS1-MRE11 and the CHK2-CDC25A-CDK2 pathways entirely abolishes inhibition of DNA synthesis induced by ionizing radiation, resulting in complete radioresistant DNA synthesis analogous to that caused by defective ATM. In addition, CDK2-dependent loading of CDC45 (603465) onto replication origins, a prerequisite for recruitment of DNA polymerase, was prevented upon irradiation of normal or NBS1/MRE11-defective cells but not cells with defective ATM. Falck et al. (2002) concluded that in response to ionizing radiation, phosphorylation of NBS1 and CHK2 by ATM triggers 2 parallel branches of the DNA damage-dependent S-phase checkpoint that cooperate by inhibiting distinct steps of DNA replication.

By transfection of human embryonic kidney and adenocarcinoma cells with CHEK2 carrying various domain or point mutations, Ahn et al. (2002) demonstrated that the phosphorylation of thr68 by ATM promotes oligomerization of CHEK2 via binding of the thr68-phosphorylated region in 1 CHEK2 molecule to the unphosphorylated FHA domain of another. CHEK2 also phosphorylates its own FHA domain, and this modification reduces its affinity for thr68-phosphorylated CHEK2. Ahn et al. (2002) concluded that oligomerization of CHEK2 increases the efficiency of transautophosphorylation, resulting in the release of active CHEK2 monomers that proceed to enforce checkpoint control in irradiated cells.

Lopes et al. (2001) used the 2-dimensional gel type technique to examine replication intermediates in response to hydroxyurea-induced replication blocks in S. cerevisiae. They showed that hydroxyurea-treated Rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. Lopes et al. (2001) proposed that Rad53 prevents collapse of the fork when replication pauses.

To characterize the mechanisms controlling replication fork integrity in S. cerevisiae, Sogo et al. (2002) analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wildtype cells accumulated short single-stranded regions, which likely causes checkpoint activation, whereas Rad53 mutants exhibited extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Furthermore, Rad53 mutant cells accumulated Holliday junctions through fork reversal. Sogo et al. (2002) speculated that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

Yang et al. (2002) determined that PML (102578) and CHEK2 mediated p53-independent apoptosis following gamma irradiation of several human cell lines. Endogenous CHEK2 bound PML within PML nuclear bodies. Following gamma irradiation, CHEK2 phosphorylated PML on ser117, causing dissociation of the 2 proteins. Yang et al. (2002) concluded that this pathway to gamma irradiation-induced apoptosis utilizes ATM, CHEK2, and PML.

Bartkova et al. (2005) showed that in clinical specimens from different stages of human tumors of the urinary bladder, breast, lung, and colon, the early precursor lesions, but not normal tissues, commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM (607585) and CHK2 and phosphorylated histone H2AX (601772) and p53 (191170). Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genomewide assessment of allelic imbalances, Bartkova et al. (2005) concluded that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM-CHK2-p53 pathway, might allow cell proliferation, survival, increased genomic instability, and tumor progression.

Gorgoulis et al. (2005) analyzed a panel of human lung hyperplasias, all of which retained wildtype p53 genes and had no signs of gross chromosomal instability, and found signs of a DNA damage response, including histone H2AX and CHK2 phosphorylation, p53 accumulation, focal staining of p53 binding protein-1 (53BP1; 605230), and apoptosis. Progression to carcinoma was associated with p53 or 53BP1 inactivation and decreased apoptosis. A DNA damage response was also observed in dysplastic nevi and in human skin xenografts, in which hyperplasia was induced by overexpression of growth factors. Both lung and experimentally-induced skin hyperplasias showed allelic imbalance at loci that are prone to DNA double-strand break formation when DNA replication is compromised (common fragile sites). Gorgoulis et al. (2005) proposed that, from its earliest stages, cancer development is associated with DNA replication stress, which leads to DNA double-strand breaks, genomic instability, and selective pressure for p53 mutations.

Pregueiro et al. (2006) found that the Neurospora checkpoint kinase-2, called Prd4, is regulated by the circadian clock and that, reciprocally, Prd4 physically interacts with the clock component Frq, promoting its phosphorylation. DNA-damaging agents can reset the clock in a manner that depends on time of day, and this resetting is dependent on Prd4. Thus, Pregueiro et al. (2006) concluded that Prd4, the Neurospora Chk2, identifies a molecular link that feeds back conditionally from circadian output to input and the cell cycle.

Janssen et al. (2011) demonstrated that chromosome segregation errors can also result in structural chromosome aberrations. Chromosomes that missegregate are frequently damaged during cytokinesis, triggering a DNA double-strand break response in the respective daughter cells involving ATM (607585), CHK2, and p53. Janssen et al. (2011) showed that these double-strand breaks can lead to unbalanced translocations in the daughter cells. Janssen et al. (2011) concluded that their data showed that segregation errors can cause translocations and provided insights into the role of whole-chromosome instability in tumorigenesis.

Bolcun-Filas et al. (2014) reported that Chek2 is essential for culling mouse oocytes bearing unrepaired meiotic or induced DNA double-strand breaks. Female infertility caused by a meiotic recombination mutation or irradiation was reversed by mutation of Chek2. Both meiotically programmed and induced double-strand breaks trigger Chek2-dependent activation of Tp53 (191170) and Tp63 (603273), effecting oocyte elimination. Bolcun-Filas et al. (2014) concluded that these data established CHEK2 as essential for DNA damage surveillance in female meiosis and indicated that the oocyte double-strand break damage response primarily involves a pathway hierarchy in which ATR (601215) signals to CHEK2, which then activates TP53 and TP63.

Molecular Genetics

Bell et al. (1999) identified heterozygous germline mutations in CHK2 in patients with Li-Fraumeni syndrome-2 (609265). Bell et al. (1999) suggested that CHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and that their observations provided a link between the central role of p53 (191170) inactivation in human cancer and the well-defined G2 checkpoint in yeast.

Vahteristo et al. (2001) analyzed the CHK1 (603078), CHK2, and p53 genes for mutations in 44 Finnish families with Li-Fraumeni syndrome, Li-Fraumeni-like syndrome (see 151623), or a phenotype suggestive of Li-Fraumeni syndrome. Five different disease-causing mutations were observed in 7 families: 4 in the p53 gene and 1 in the CHK2 gene. The CHK2 mutation occurred in 2 families and was the same as that reported by Bell et al. (1999): 1100delC (604373.0001). The families originated from different parts of Finland, were not known to be related, and segregated different chromosome 22 haplotypes. Thus, 1100delC is clearly a disease-causing mutation and represents a mutation hotspot in the CHK2 gene. The phenotypes of the 2 families were considered atypical because of the lack of sarcomas or childhood cancers. In contrast, the family with this mutation reported by Bell et al. (1999) had classic LFS.

Ino et al. (2000) concluded that the CHK2 gene is not the target of somatic inactivation in malignant gliomas.

Bell et al. (1999) identified a C-to-T transition at nucleotide 433 of the CHK2 gene resulting in an arginine-to-tryptophan substitution at codon 145 (R145W) in a colon cancer (114500) cell line.

Lee et al. (2001) demonstrated inactivating mutations of both alleles of CHEK2 in a sporadic colon carcinoma cell line; the 2 mutations were A247D and R145W (604373.0003).

Because inherited CHK2 mutations are found in some Li-Fraumeni cancer syndrome families, Miller et al. (2002) examined the role of CHK2 mutations in sporadic cancer. They found missense mutations affecting the forkhead and kinase domains in 4 of 57 osteosarcomas (259500), 1 of 20 ovarian cancers, and 1 of 35 nonsmall cell lung cancers. The finding of CHK2 gene mutations were consistent with osteosarcoma being a defining tumor of Li-Fraumeni syndrome. The occurrence of CHK2 mutations in sporadic cancers emphasized the importance of the stress pathway, which includes TP53.

Mutations in BRCA1 (113705) and BRCA2 (600185) 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, Meijers-Heijboer et al. (2002) analyzed the CHEK2 gene, which was considered a plausible candidate gene because it encodes a cell-cycle checkpoint kinase that is implicated in DNA repair processes involving BRCA1 and p53. They found that CHEK2*1100delC (604373.0001), a truncating variant that abrogates the kinase activity, has a frequency of 1.1% in healthy individuals and 5.1% in individuals with breast cancer derived from 718 families that did not carry mutations in BRCA1 or BRCA2, including 13.5% of individuals from families with male breast cancer. They estimated that the CHEK2*1100delC variant results in an approximately 2-fold increase of breast cancer risk in women and a 10-fold increase of risk in men. By contrast, the variant conferred no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggested that the biologic 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.

Among 578 men with prostate cancer (176807), Dong et al. (2003) found 28 (4.8%) germline CHEK2 mutations, 16 of which were unique. Additional screening for CHEK2 mutations in 149 families with familial prostate cancer revealed 11 mutations (5 unique) in 9 families, including 2 frameshift and 3 missense mutations. Sixteen of 18 unique CHEK2 mutations identified in both sporadic and familial cases were not detected among 423 unaffected men, suggesting a pathologic effect of CHEK2 mutations in prostate cancer development. Analysis of 2 frameshift mutations revealed abnormal splicing in one and a dramatic reduction of CHEK2 protein levels in both.

Wu et al. (2006) presented evidence that both germline and somatic CHEK2 mutations identified in prostate cancer may contribute to the development of prostate cancer through the reduction of CHEK2 activation in response to DNA damage and/or oncogenic stress.

To investigate whether CHEK2 variants confer susceptibility to breast cancer, Schutte et al. (2003) screened the full CHEK2 coding sequence in BRCA1/BRCA2-negative breast cancer cases from 89 pedigrees with 3 or more cases of breast cancer. One novel germline variant and 2 other mutations were identified, but none occurred at significantly elevated frequency in familial breast cancer cases compared with controls. Schutte et al. (2003) concluded that the 1100delC may be the only CHEK2 allele that makes an appreciable contribution to breast cancer susceptibility.

Meijers-Heijboer et al. (2003) identified the 1100delC variant in affected members of families segregating a breast and colorectal cancer phenotype. The 1100delC mutation was not, however, the major predisposing factor for the phenotype, but appeared to act in synergy with at least 1 unknown susceptibility gene.

In Poland, there are 3 polymorphic variants of CHEK2, which, in aggregate, are present in 5.5% of the population. Two of these, 1100delC (604373.0001) and IVS2+1G-A (604373.0013), are rare and result in premature protein termination; a third is a common missense variant, I157T (604373.0002). Cybulski et al. (2004) found that all 3 variants are associated with an increased risk of prostate cancer in Poland. Cybulski et al. (2004) ascertained the prevalence of each of these alleles in 4,008 cancer cases and 4,000 controls, all from Poland. The majority of the common cancer sites were represented. Positive associations with protein-truncating alleles were seen for cancer of the thyroid, breast, and prostate. The missense variant I157T was associated with an increased risk of breast cancer, colon cancer, kidney cancer, prostate cancer, and thyroid cancer. The range of cancers associated with mutations of the CHEK2 gene may be much greater than previously thought.

Shaag et al. (2005) identified 2 extended haplotypes at CHEK2 that cosegregated with breast cancer in high-risk families. Two amino acid substitutions were discovered: ser428-to-phe (S428F; 604373.0014) in the kinase domain and pro85-to-leu (P85L; 604373.0005) in the N-terminal region. The S428F allele failed to complement a Rad53 deletion in S. cerevisiae, reflecting abrogation of normal CHEK2 function, whereas the P85L allele complemented Rad53 as did wildtype CHEK2. Frequencies of S428F heterozygotes were 3% among 1,632 female breast cancer patients (not selected for family history or age at diagnosis) and 1.4% among 1,673 controls, whereas frequencies of P85L were 0.9% among cases and 0.8% among controls. On the basis of the experience of mothers, sisters, and daughters of probands, breast cancer risk due to S428F alleles was estimated as 0.17 (+/- 0.08) by age 60. Presence of an S428F allele increased breast cancer risk approximately 2-fold among Ashkenazi Jewish women, whereas P85L is a neutral allele. Shaag et al. (2005) suggested that selecting probands with extended haplotypes that cosegregate with disease may improve the efficiency of resequencing efforts, and that quantitative complementation tests in yeast may be used to evaluate variants in genes with highly conserved function.

Cybulski et al. (2006) identified 1 of 4 CHEK2 founder alleles (1100delC, IVS2+1G-A, I157T, and a 5.4-bp deletion; 604373.0012) in 184 (9.9%) of 1,864 Polish patients with prostate cancer. The odds ratio for disease development of patients with truncating mutations was higher than the odds ratio of those with missense mutations.

Animal Model

Hirao et al. (2000) generated Chk2-deficient mouse embryonic cells by gene targeting. Chk2 -/- embryonic stem cells failed to maintain gamma-irradiation-induced arrest in the G2 phase of the cell cycle. Chk2 -/- thymocytes were resistant to DNA damage-induced apoptosis. Chk2 -/- cells were defective for p53 stabilization and for induction of p53-dependent transcripts such as p21 in response to gamma irradiation. Reintroduction of the Chk2 gene restored p53-dependent transcription in response to gamma irradiation. Chk2 directly phosphorylated p53 on serine 20, which is known to interfere with Mdm2 binding. Hirao et al. (2000) concluded that this provides a mechanism for increased stability of p53 by prevention of ubiquitination in response to DNA damage. They further concluded, in light of the finding of 2 mutations in CHK2 in patients with Li-Fraumeni syndrome (Bell et al., 1999), that the results provided a mechanistic link between Chk2 and p53 to explain the phenotypic similarity of these 2 genetically distinct Li-Fraumeni syndrome families. Thus, like p53, Chk2 may contribute to a wide range of human cancers.

In an effort to clarify the roles of Chek2 and Atm in tumorigenesis, Hirao et al. (2002) compared the G1/S checkpoint, apoptosis, and expression of p53 proteins in thymocytes isolated from Chek2-null mice and Atm-null mice. They determined that Chek2 can regulate p53-dependent apoptosis in an Atm-independent manner. Radiation-induced apoptosis was restored in Chek2-null thymocytes by reintroduction of the wildtype Chek2 gene, but not by a Chek2 gene in which the sites phosphorylated by Atm or Atr (601215) were mutated to alanine.

Iijima-Ando et al. (2010) showed that Chk2 is a novel tau (MAPT; 157140) kinase. Overexpression of Drosophila Chk2 increased tau phosphorylation at ser262 and enhanced tau-induced neurodegeneration in transgenic flies expressing human tau. The nonphosphorylatable ser262-to-ala mutation abolished Chk2-induced enhancement of tau toxicity, suggesting that the ser262 phosphorylation site may be involved in the enhancement of tau toxicity by Chk2. In vitro kinase assays revealed that human CHK2 and CHK1 directly phosphorylated human tau at ser262. Drosophila Chk2 did not modulate the activity of the fly homolog of microtubule affinity regulating kinase (see MARK3, 602678), which has been shown to be a physiologic tau ser262 kinase. The authors suggested that CHK1 and CHK2 may be involved in tau phosphorylation and toxicity in the pathogenesis of Alzheimer disease (AD; 104300).