Chk1-Mad2 interaction

Chk1 is implicated in several checkpoints of the cell cycle acting as a key player in the signal transduction pathway activated in response to DNA damage and crucial for the maintenance of genomic stability. Chk1 also plays a role in the mitotic spindle checkpoint, which ensures the fidelity of mitotic segregation during mitosis, preventing chromosomal instability and aneuploidy. Mad2 is one of the main mitotic checkpoint components and also exerts a role in the cellular response to DNA damage. To investigate a possible crosslink existing between Chk1 and Mad2, we studied Mad2 protein levels after Chk1 inhibition either by specific siRNAs or by a specific and selective Chk1 inhibitor (PF-00477736), and we found that after Chk1 inhibition, Mad2 protein levels decrease only in tumor cells sensitive to Chk1 depletion. We then mapped six Chk1’s phosphorylatable sites on Mad2 protein, and found that Chk1 is able to phosphorylate Mad2 in vitro on more than one site, while it is incapable of phoshorylating the Mad2 form mutated on all six phosphorylatable sites. Moreover our studies demonstrate that Chk1 co-localizes and physically associates with Mad2 in cells both under unstressed conditions and after DNA damage, thus providing new and interesting evidence on Chk1 and Mad2 crosstalk in the DNA damage checkpoint and in the mitotic spindle checkpoint.     

Chk2-dependent phosphorylation of XRCC1 in the DNA damage response promotes base excision repair

The DNA damage response (DDR) has an essential function in maintaining genomic stability. Ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2) and ATM- and Rad3-related (ATR)-Chk1, triggered, respectively, by DNA double-strand breaks and blocked replication forks, are two major DDRs processing structurally complicated DNA damage. In contrast, damage repaired by base excision repair (BER) is structurally simple, but whether, and how, the DDR is involved in repairing this damage is unclear. Here, we demonstrated that ATM-Chk2 was activated in the early response to oxidative and alkylation damage, known to be repaired by BER. Furthermore, Chk2 formed a complex with XRCC1, the BER scaffold protein, and phosphorylated XRCC1 in vivo and in vitro at Thr(284). A mutated XRCC1 lacking Thr(284) phosphorylation was linked to increased accumulation of unrepaired BER intermediate, reduced DNA repair capacity, and higher sensitivity to alkylation damage. In addition, a phosphorylation-mimic form of XRCC1 showed increased interaction with glycosylases, but not other BER proteins. Our results are consistent with the phosphorylation of XRCC1 by ATM-Chk2 facilitating recruitment of downstream BER proteins to the initial damage recognition/excision step to promote BER.     

USP7 controls Chk1 protein stability by direct deubiquitination

Chk1, an essential checkpoint kinase in the DNA damage response pathway (DDR), is tightly regulated by both ATR-dependent phosphorylation and proteasome-mediated degradation. Here we identify ubiquitin hydrolase USP7 as a novel regulator of Chk1 protein stability. USP7 was shown before to regulate other DDR proteins such as p53, Hdm2 and Claspin, an adaptor protein in the ATR-Chk1 pathway required for Chk1 activation. Depletion or inhibition of USP7 leads to lower Chk1 levels. The decreased Chk1 protein after USP7 knock down cannot be rescued by simultaneously elevating Claspin levels, demonstrating that the effect of USP7 on Chk1 is independent of its known effect on Claspin. Conversely, overexpression of USP7 wild type, but not a catalytic mutant version, elevates Chk1 levels and increases the half-life of Chk1 protein. Importantly, wild type, but not catalytic mutant USP7 can deubiquitinate Chk1 in vivo and in vitro, confirming that USP7 directly regulates Chk1 protein levels. Finally we show that USP7 catalytic mutant is (mono-)ubiquitinated, which suggests auto-deubiquitination by this ubiquitin hydrolase, possibly important for its regulation.     

Chk1 is essential for the development of murine epidermal melanocytes

Embryonic deletion of mouse Chk1 is lethal; however, whether Chk1 is essential in all individual tissues is unknown. By breeding C57Bl/ 6 mice homozygous for a conditional allele of Chk1 (Chk1fl/fl) and bearing melanocyte‐specific Tyr::Cre and DCT:: LacZ transgenes, we investigated the consequences of Chk1 deletion in the melanocytic lineage. We show that adult Tyr::Cre; Chk1fl/fl mice lack coat pigmentation and epidermal melanocytes in the hair follicles, but retain eye pigmentation in the retinal pigmented epithelium (RPE). Melanoblasts formed normally during embryogenesis in Tyr::Cre; Chk1fl/fl mice at early times (embryonic day 10.5; E10.5) but were completely absent by stage E13.5, most probably as a consequence of spontaneous DNA damage and apoptosis. By contrast, melanoblast numbers were only slightly reduced in heterozygous Tyr::Cre; Chk1fl/ + embryos, and these mice exhibited normal coat pigmentation as adults. Thus, Chk1 is essential for the developmental formation of murine epidermal melanocytes but hemizygosity has little, if any, permanent developmental consequence in this cell type.     

Rotavirus activates a noncanonical ATM‐Chk2 branch of DNA damage response during infection to positively regulate viroplasm dynamics

Surveillance for maintaining genomic pristineness, a protective safeguard of great onco‐preventive significance, has been dedicated in eukaryotic cells to a highly conserved and synchronised signalling cascade called DNA damage response (DDR). Not surprisingly, foreign genetic elements like those of viruses are often potential targets of DDR. Viruses have evolved novel ways to subvert this genome vigilance by twisting canonical DDR to a skewed, noncanonical response through selective hijacking of some DDR components while antagonising the others. Though reported for many DNA and a few RNA viruses, potential implications of DDR have not been addressed yet in case of infection with rotavirus (RV), a double‐stranded RNA virus. In the present study, we aimed at the modulation of ataxia telangiectasia mutated (ATM)‐checkpoint kinase 2 (Chk2) branch of DDR in response to RV infection in vitro. We found activation of the transducer kinase ATM and its downstream effector Chk2 in RV‐SA11‐infected cells, the activation response being maximal at 6‐hr post infection. Moreover, ATM activation was found to be dependent on induction of the upstream sensor Mre11‐Rad50‐Nbs1 (MRN) complex. Interestingly, RV‐SA11‐mediated maximal induction of ATM‐Chk2 pathway was revealed to be neither preceded by occurrence of nuclear DNA damage nor transduced to formation of damage‐induced canonical nuclear foci. Subsequent investigations affirmed sequestration of MRN components as well as ATM‐Chk2 proteins away from nucleus into cytosolic RV replication factories (viroplasms). Chemical intervention targeting ATM and Chk2 significantly inhibited fusion and maturation of viroplasms leading to attenuated viral propagation. Cumulatively, the current study describes RV‐mediated activation of a noncanonical ATM‐Chk2 branch of DDR skewed in favour of facilitated viroplasm fusion and productive viral perpetuation.     

Function and regulation mechanism of Chk1 during meiotic maturation in porcine oocytes

Checkpoint 1 (Chk1), as an important member of DNA replication checkpoint and DNA damage response, has an important role during the G2/M stage of mitosis. In this study, we used porcine oocyte as a model to investigate the function of Chk1 during porcine oocyte maturation. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages, mainly localized in the cytoplasm at GV stage and moved to the spindle after germinal vesicle breakdown (GVBD). Chk1 depletion not only induced oocytes to be arrested at MI stage with abnormal chromosomes arrangement, but also inhibited the degradation of Cyclin B1 and decreased the expression of Mitotic Arrest Deficient 2-Like 1 (Mad2L1), one of spindle assembly checkpoint (SAC) proteins, and cadherin 1 (Cdh1), one of coactivation for anaphase-promoting complex/cyclosome (APC/C). Moreover, Chk1 overexpression delayed GVBD. These results demonstrated that Chk1 facilitated the timely degradation of Cyclin B1 at anaphase I (AI) and maintained the expression of Mad2L1 and Cdh1, which ensured that all chromosomes were accurately located in a line, and then oocytes passed metaphase I (MI) and AI and exited from the first meiotic division successfully. In addition, we proved that Chk1 had not function on GVBD of porcine oocytes, which suggested that maturation of porcine oocytes did not need the DNA damage checkpoint, which was different from the mouse oocyte maturation.     

Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1^+/− nor Chk2^−/− mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1^+/−Chk2^−/− and Chk1^+/−Chk2^+/− mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.     

Claspin is phosphorylated in the Chk1-binding domain by a kinase distinct from Chk1

Chk1 protein kinase plays a critical role in checkpoints that restrict progression through the cell cycle if DNA replication has not been completed or DNA damage has been sustained. ATR-dependent activation of Chk1 is mediated by Claspin. Phosphorylation of Claspin at two sites (Thr916 and Ser945 in humans) in response to DNA replication arrest or DNA damage recruits Chk1 to Claspin. Chk1 is subsequently phosphorylated by ATR and fully activated to control cell cycle progression. We show that ablation of Chk1 by siRNA in human cells or its genetic deletion in chicken DT40 cells does not prevent phosphorylation of Claspin at Thr916 (Ser911 in chicken). Chk1, however, does play other roles, possibly indirect, in the phosphorylation of Claspin and its induction. These results demonstrate that phosphorylation of Claspin within the Chk1-binding domain is catalysed by an ATR-dependent kinase distinct from Chk1.     

Chk1 inhibition in p53-deficient cell lines drives rapid chromosome fragmentation followed by caspase-independent cell death

Activation of Checkpoint kinase 1 (Chk1) following DNA damage mediates cell cycle arrest to prevent cells with damaged DNA from entering mitosis. Here we provide a high-resolution analysis of cells as they undergo S- and G₂-checkpoint bypass in response to Chk1 inhibition with the selective Chk1 inhibitor GNE-783. Within 4–8 h of Chk1 inhibition following gemcitabine induced DNA damage, cells with both sub-4N and 4N DNA content prematurely enter mitosis. Coincident with premature transition into mitosis, levels of DNA damage dramatically increase and chromosomes condense and attempt to align along the metaphase plate. Despite an attempt to congress at the metaphase plate, chromosomes rapidly fragment and lose connection to the spindle microtubules. Gemcitabine mediated DNA damage promotes the formation of Rad51 foci; however, while Chk1 inhibition does not disrupt Rad51 foci that are formed in response to gemcitabine, these foci are lost as cells progress into mitosis. Premature entry into mitosis requires the Aurora, Cdk1/2 and Plk1 kinases and even though caspase-2 and -3 are activated upon mitotic exit, they are not required for cell death. Interestingly, p53, but not p21, deficiency enables checkpoint bypass and chemo-potentiation. Finally, we uncover a differential role for the Wee-1 checkpoint kinase in response to DNA damage, as Wee-1, but not Chk1, plays a more prominent role in the maintenance of S- and G₂-checkpoints in p53 proficient cells.     

Regulation of checkpoint kinases through dynamic interaction with Crb2

ATR/Rad3-like kinases promote the DNA damage checkpoint through regulating Chk1 that restrains the activation of cyclin-dependent kinases. In fission yeast, Crb2, a BRCT-domain protein that is similar to vertebrate 53BP1, plays a crucial role in establishing this checkpoint. We report here that Crb2 regulates DNA damage checkpoint through temporal and dynamic interactions with Rad3, Chk1 and replication factor Cut5. The active complex formation between Chk1 and Crb2 is regulated by Rad3 and became maximal during the checkpoint arrest. Chk1 activation seems to need two steps of interaction changes: the loss of Rad3-Chk1 and Rad3-Crb2 interactions, and the association between hyperphosphorylated forms of Chk1 and Crb2. Chk1 is the major checkpoint kinase for the arrest of DNA polymerase mutants. The in vitro assay of Chk1 showed that its activation requires the presence of Crb2 BRCT. Hyperphosphorylation of Crb2 is also dependent on its intact BRCT. Finally, we show direct interaction between Rad3 and Crb2, which is inhibitory to Rad3 activity. Hence, Crb2 is the first to interact with both Rad3 and Chk1 kinases.     

DNA damage-induced S phase arrest in human breast cancer depends on Chk1, but G2 arrest can occur independently of Chk1, Chk2 or MAPKAPK2

Checkpoint kinases Chk1 and Chk2 are two key components in the DNA damage-activated checkpoint signaling pathways. To distinguish the roles of Chk1 and Chk2 in S and G₂ checkpoints after DNA damage, derivatives of the human breast cancer cell line MDA-MB-231 were established that express short hairpin RNAs to selectively suppress Chk1 or Chk2 expression. DNA damage was induced with the topoisomerase I inhibitor SN38 which arrests cells in S or G₂ phase depending on concentration. Depletion of Chk1 resulted in loss of S phase arrest upon incubation with SN38, but the cells still arrested in G₂. Suppression of Chk2 had no impact on cell cycle arrest, while cells concurrently suppressed for both Chk1 and Chk2 still arrested primarily in G₂ suggesting the presence of an alternate checkpoint regulator. One critical target for Chk1 is Cdc25A which is phosphorylated and degraded to prevent cell cycle progression. Cells arrested in G₂ in the absence of Chk1/Chk2 still showed regulation of Cdc25A consistent with the action of an alternate kinase. One candidate for an alternate checkpoint kinase is MAPKAPK2 (MK2), yet this kinase was minimally activated by DNA damage and its inhibition did not facilitate either S or G₂ progression. Furthermore, we were unable to substantiate the recent observation that the Chk1 inhibitor UCN-01 inhibits MK2. These results show that Chk1, but neither Chk2 nor MK2, is an important regulator of S phase arrest, and suggest that an additional kinase can contribute to the G2 arrest.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

DNA damage regulates Chk2 association with chromatin

DNA damage triggers cellular signaling pathways that control the cell cycle and DNA repair. Chk2 is a critical mediator of diverse responses to DNA damage. Chk2 transmits signals from upstream phosphatidylinositol 3'-kinase-like kinases to effector substrates including p53, Brca1, Cdc25A, and Cdc25C. Using chromatin fractionation as well as immunostaining combined with detergent pre-extraction, we have found that a small pool of Chk2 is associated with chromatin prior to DNA damage. Recovery of chromatin-bound Chk2 is reduced in an ATM-dependent manner by exposure to ionizing radiation. Camptothecin and adriamycin also reduce the amount of chromatin-associated Chk2. The Thr(68)-phosphorylated forms of Chk2 induced by DNA damage are found in soluble fractions, but not in the chromatin-enriched fraction. Functional serine/threonine glutamine cluster domain, forkhead-associated domain, and kinase activity are all required for efficient reduction of chromatin-bound Chk2 in response to DNA damage. Artificial induction of Chk2 oligomerization concomitant with exposure to low dose ionizing radiation reduces chromatin-bound Chk2. When Chk2 is incubated with chromatin-enriched fractions in vitro in the presence of ATP, hyperphosphorylated forms of Chk2 bind more weakly to chromatin than hypophosphorylated forms. Taken together, our data suggest that DNA damage induces activation of chromatin-bound Chk2 by a chromatin-derived signal, and that this results in dissociation of activated Chk2 from chromatin, facilitating further signal amplification and transmission to soluble substrates.     

Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell cycle progression. In vertebrates, Chk1 and Chk2 phosphorylate Cdc25A at multiple N-terminal sites and target it for rapid degradation in response to genotoxic stress. Here we show that Chk1, but not Chk2, phosphorylates Xenopus Cdc25A at a novel C-terminal site (Thr504) and inhibits it from C-terminally interacting with various Cdk-cyclin complexes, including Cdk1-cyclin A, Cdk1-cyclin B, and Cdk2-cyclin E. Strikingly, this inhibition, rather than degradation itself, of Cdc25A is essential for the Chk1-induced cell cycle arrest and the DNA replication checkpoint in early embryos. 14-3-3 proteins bind to Chk1-phosphorylated Thr504, but this binding is not required for the inhibitory effect of Thr504 phosphorylation. A C-terminal site presumably equivalent to Thr504 exists in all known Cdc25 family members from yeast to humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibit all examined Cdc25 family members from C-terminally interacting with their Cdk-cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtually all Cdc25 phosphatases by a novel common mechanism.     

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.     

Death receptor-induced activation of the Chk2- and histone H2AX-associated DNA damage response pathways

TRAIL is an endogenous death receptor ligand also used therapeutically because of its selective proapoptotic activity in cancer cells. In the present study, we examined chromatin alterations induced by TRAIL and show that TRAIL induces a rapid activation of DNA damage response (DDR) pathways with histone H2AX, Chk2, ATM, and DNA-PK phosphorylations. Within 1 h of TRAIL exposure, immunofluorescence confocal microscopy revealed γ-H2AX peripheral nuclear staining (γ-H2AX ring) colocalizing with phosphorylated/activated Chk2, ATM, and DNA-PK inside heterochromatin regions. The marginal distribution of DDR proteins in early apoptotic cells is remarkably different from the focal staining seen after DNA damage. TRAIL-induced DDR was suppressed upon caspase inhibition or Bax inactivation, demonstrating that the DDR activated by TRAIL is downstream from the mitochondrial death pathway. H2AX phosphorylation was dependent on DNA-PK, while Chk2 phosphorylation was dependent on both ATM and DNA-PK. Downregulation of Chk2 decreased TRAIL-induced cell detachment; delayed the activation of caspases 2, 3, 8, and 9; and reduced TRAIL-induced cell killing. Together, our findings suggest that nuclear activation of Chk2 by TRAIL acts as a positive feedback loop involving the mitochondrion-dependent activation of caspases, independently of p53.