The Interaction between Checkpoint Kinase 1 (Chk1) and the Minichromosome Maintenance (MCM) Complex Is Required for DNA Damage-induced Chk1 Phosphorylation

Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage.     

Revealing targeted therapeutic opportunities in triple-negative breast cancers: A new strategy

Nek6 is a recently identified NIMA-related kinase that is required for mitotic cell cycle progression. In the present study, we examined the role of Nek6 in the DNA damage response. We found that Nek6 is phosphorylated upon IR and UV irradiation through the DNA damage checkpoint in vivo. Nek6 is also directly phosphorylated by the checkpoint kinases Chk1 and Chk2 in vitro. Notably, Nek6 activation during mitosis is completely abolished by IR and UV irradiation. Moreover, the ectopic expression of Nek6 overrides DNA damage-induced G2/M arrest. These results suggest that Nek6 is a novel target of the DNA damage checkpoint and that the inhibition of Nek6 activity is required for proper cell cycle arrest in the G2/M phase upon DNA damage.     

Polo-like kinase 1 and Chk2 interact and co-localize to centrosomes and the midbody

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 (1). We report here that 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, we 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.     

14‐3‐3γ mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage

14‐3‐3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14‐3‐3 (β and ζ isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345‐phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14‐3‐3γ forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296‐phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14‐3‐3γ mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.     

Replication stress activates DNA polymerase alpha-associated Chk1

Chk1 contributes to both intra-S and DNA damage checkpoint responses. Here, we show that depletion of DNA Polα and not Polε or Polδ by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure. Combinatorial ablation of DNA Polα and Chk1 causes an accumulation of γ-H2A.X, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Co-depletion of DNA Polα with ATR yields similar phenotypes, suggesting that ATR and Chk1 are epistatic and required for maintenance of genomic integrity following replication stress. Significantly, Chk1 and DNA Polα can be co-immunoprecipated from native cell extracts. Moreover, following replication stress, Polα-associated Chk1 becomes rapidly phosphorylated on Ser345 in a TopBP1 and ATR-dependent manner. Hence, the ability to efficiently phosphorylate Chk1 in the context of DNA Polα complexes is correlated with suppression of DNA damage following replication stress. These findings identify DNA Polα as an important component of the signal transduction cascade that activates the intra-S checkpoint.     

Chk2 activation and phosphorylation-dependent oligomerization

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. We show here that 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.     

Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345

Checkpoints are biochemical pathways that provide the cell with mechanisms to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The conserved checkpoint kinase Chk1 regulates mitotic progression in response to DNA damage and replication interference by blocking the activation of Cdk1/cyclin B. Chk1 is phosphorylated on Ser-317 and Ser-345 following a checkpoint signal, a process that is regulated by Atr, and by the sensor complexes containing Rad17 and Hus1. We show that Chk1 is associated with chromatin in cycling cells and that the chromatin-associated Chk1 is phosphorylated in the absence of exogenous DNA damage. The UV-induced Ser-345-phosphorylated forms of Chk1 that appear minutes after treatment are predominantly associated with chromatin. The Ser-345 site is in a 14-3-3 consensus binding motif and is required for nuclear retention of Chk1 following an hydroxyurea-induced checkpoint signal; nonetheless, Ser-345 or Ser-317 are not required for the chromatin association of Chk1. Hus1, a member of the proliferating cell nuclear antigen-like damage recognition complex plays a role in the phosphorylation of Chk1 on Ser-345, however, Hus1 is not required for phosphorylation on Ser-317 or for Chk1 localization to chromatin. These results indicate that there is more than one step in Chk1 activation and that the regulation of this checkpoint signaling is achieved at least in part through phosphorylation of Ser-345, which serves to localize Chk1 in the nucleus presumably by blocking Crm1-dependent nuclear export.     

Antagonism of Chk1 signaling in the G2 DNA damage checkpoint by dominant alleles of Cdr1

Activation of the Chk1 protein kinase by DNA damage enforces a checkpoint that maintains Cdc2 in its inactive, tyrosine-15 (Y15) phosphorylated state. Chk1 downregulates the Cdc25 phosphatases and concomitantly upregulates the Wee1 kinases that control the phosphorylation of Cdc2. Overproduction of Chk1 causes G(2) arrest/delay independently of DNA damage and upstream checkpoint genes. We utilized this to screen fission yeast for mutations that alter sensitivity to Chk1 signaling. We describe three dominant-negative alleles of cdr1, which render cells supersensitive to Chk1 levels, and suppress the checkpoint defects of chk1Delta cells. Cdr1 encodes a protein kinase previously identified as a negative regulator of Wee1 activity in response to limited nutrition, but Cdr1 has not previously been linked to checkpoint signaling. Overproduction of Cdr1 promotes checkpoint defects and exacerbates the defective response to DNA damage of cells lacking Chk1. We conclude that regulation of Wee1 by Cdr1 and possibly by related kinases is an important antagonist of Chk1 signaling and represents a novel negative regulation of cell cycle arrest promoted by this checkpoint.     

Requirement of MTA1 in ATR-mediated DNA Damage Checkpoint Function

MTA1 (metastasis-associated protein 1), an integral component of the nucleosome remodeling and deacetylase complex, has recently been implicated in the ionizing radiation-induced DNA damage response. However, whether MTA1 also participates in the UV-induced DNA damage checkpoint pathway remains unknown. In response to UV radiation, ATR (ataxia teleangiectasia- and Rad3-related) is the major kinase activated that orchestrates cell cycle progression with DNA repair machinery by phosphorylating and activating a number of downstream substrates, such as Chk1 (checkpoint kinase 1) and H2AX (histone 2A variant X). Here, we report that UV radiation stabilizes MTA1 in an ATR-dependent manner and increases MTA1 binding to ATR. On the other hand, depletion of MTA1 compromises the ATR-mediated Chk1 activation following UV treatment, accompanied by a marked down-regulation of Chk1 and its interacting partner Claspin, an adaptor protein that is required for the phosphorylation and activation of Chk1 by ATR. Furthermore, MTA1 deficiency decreases the induction of phosphorylated H2AX (referred to as γ-H2AX) and γ-H2AX focus formation after UV treatment. Consequently, depletion of MTA1 results in a defect in the G₂-M checkpoint and increases cellular sensitivity to UV-induced DNA damage. Thus, MTA1 is required for the activation of the ATR-Claspin-Chk1 and ATR-H2AX pathways following UV treatment, and the noted abrogation of the DNA damage checkpoint in the MTA1-depleted cells may be, at least in part, a consequence of dysregulation of the expression of these two pathways. These findings suggest that, in addition to its role in the repair of double strand breaks caused by ionizing radiation, MTA1 also participates in the UV-induced ATR-mediated DNA damage checkpoint pathway.     

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.     

Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint.

DNA damage induces cell cycle arrest (called the damage checkpoint), during which cells carry out actions for repair. A fission yeast protein, Crb2/Rhp9, which resembles budding yeast Rad9p and human BRCA1, promotes checkpoint by activating Chk1 kinase, which restrains Cdc2 activation. We show here that phosphorylation of the T215 Cdc2 site of Crb2 is required for reentering the cell cycle after the damage-induced checkpoint arrest. If this site is nonphosphorylatable, irradiated cells remain arrested, though damage is repaired, and maintain the phosphorylated state of Chk1 kinase. The T215 site is in vitro phosphorylated by purified Cdc2 kinase. Phosphorylation of T215 occurs intensely in response to DNA damage at a late stage, suggesting an antagonistic role of Cdc2 phosphorylation toward checkpoint.     

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.     

Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.     

Cdc25 Inhibited In Vivo and In Vitro by Checkpoint Kinases Cds1 and Chk1

In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S-M replication checkpoint that prevents mitosis when DNA is incompletely replicated. Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2. Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2. In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S-M replication checkpoint. Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro. Chk1, a protein kinase that is required for the G2-M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay. In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99. The Cdc25 alanine-99 mutation partially impaired the S-M replication and G2-M damage checkpoints in vivo. Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.     

Repeated phosphopeptide motifs in human Claspin are phosphorylated by Chk1 and mediate Claspin function

Claspin is a checkpoint protein involved in ATR (ataxia telangiectasia mutated- and Rad3-related)-dependent Chk1 activation in Xenopus and human cells. In Xenopus, Claspin interacts with Chk1 after DNA damage through a region containing two highly conserved repeats, which becomes phosphorylated during the checkpoint response. Because this region is also conserved in human Claspin, we investigated the regulation and function of these potential phosphorylation sites in human Claspin. We found that Claspin is phosphorylated in vivo at Thr-916 in response to replication stress and UV damage. Mutation of these phosphorylation sites on Claspin inhibited Claspin-Chk1 interaction in vivo, impaired Chk1 activation, and induced premature chromatin condensation in cells, indicating a defect in replication checkpoint. In addition, we found that Thr-916 on Claspin is phosphorylated by Chk1, suggesting that Chk1 regulates Claspin during checkpoint response. These results together indicate that phosphorylation of Claspin repeats in human Claspin is important for Claspin function and the regulation of Claspin-Chk1 interaction in human cells.     

Autophosphorylated residues involved in the regulation of human chk2 in vitro

Human checkpoint kinase 2 is a major actor in checkpoint activation through phosphorylation by ataxia telangiectasia mutated in response to DNA double-strand breaks. In the absence of de novo DNA damage, its autoactivation, reported in the event of increased Cds1/checkpoint kinase 2 (Chk2) expression, has been attributed to oligomerization. Here we report a study performed on autoactivated recombinant Chk2 proteins that aims to correlate kinase activity and phosphorylation status. Using a fluorescence-based technique to assay human checkpoint kinase 2 catalytic activity, slight differences in the ability to phosphorylate Cdc25C were observed, depending on the recombinant system used. Using mass spectrometry, the phosphorylation sites were mapped to identify sites potentially involved in the kinase activity. Five phosphorylated positions, at Ser120, Ser260, Thr225, Ser379 and Ser435, were found to be common to bacteria and insect cells expression systems. They were present in addition to the six known phosphorylation sites induced by ionizing radiation (Thr68, Thr432, Thr387, Ser516, Ser33/35 and Ser19) detected by immunoblotting. After phosphatase treatment, Chk2 regained activity via autorephosphorylation. The determination of the five common sites and ionizing-radiation-inducible positions as rephosphorylated confirms that they are potential positive regulators of Chk2 kinase activity. For Escherichia coli's most highly phosphorylated 6His-Chk2, 13 additional phosphorylation sites were assigned, including 7 novel sites on top of recently reported phosphorylation sites.     

DNA Damage-Induced Accumulation of Centrosomal Chk1 Contributes to its Checkpoint Function

The checkpoint kinase Chk1 is an established transducer of ATR- and ATM-dependent signalling in response to DNA damage. In addition to its nuclear localization, Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B-Cdk1 during unperturbed cell cycles. Here, we demonstrate that DNA damage caused by ultraviolet irradiation or hydroxyurea treatment leads to centrosomal accumulation of endogenous Chk1 in normal human BJ fibroblasts and in ATR- or ATM-deficient fibroblasts. Chemical inhibition of ATR/ATM by caffeine led to enhanced centrosomal Chk1 deposition associated with nuclear Chk1 depletion. In contrast to normal or ATM-deficient fibroblasts, genetically ATR-deficient Seckel-fibroblasts showed detectable constitutive centrosomal accumulation of Chk1 even in the absence of exogenous insults. After DNA damage, the centrosomal fraction of Chk1 was found to be phosphorylated at ATR/ATM phosphorylation sites. Forced immobilization of kinase-inactive but not wild-type Chk1 to centrosomes resulted in a G2/M checkpoint defect. Finally, both DNA damage, and forced centrosomal expression of Chk1 in the absence of genotoxic treatments, induced centrosome amplification in a subset of cells, a phenomenon which could be suppressed by inhibition of ATM/ATR-mediated signaling. Taken together, our results suggest that accumulation of phosphorylated Chk1 at centrosomes constitutes an additional element in the DNA damage response. Centrosomal Chk1 induces G2/M cell cycle arrest and may evoke centrosome amplification, the latter possibly providing a backup mechanism for elimination of cells with impaired DNA damage checkpoints operating earlier during the cell cycle.