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.     

ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1

Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.     

BRCA1-dependent Chk1 phosphorylation triggers partial chromatin disassociation of phosphorylated Chk1 and facilitates S-phase cell cycle arrest

Chk1 phosphorylation by the PI3-like kinases ATR and ATM is critical for its activation and its role in prevention of premature mitotic entry in response to DNA damage or stalled replication. The breast and ovarian tumor suppressor, BRCA1, is among several checkpoint mediators that are required for Chk1 activation by ATM and ATR. Previously we showed that BRCA1 is necessary for Chk1 phosphorylation and activation following ionizing radiation. BRCA1 has been implicated in S-phase checkpoint control yet its mechanism of action is not well characterized. Here we report that BRCA1 is critical for Chk1 phosphorylation in response to inhibition of replication by either cisplatin or hydroxyurea. While Chk1 phosphorylation of S317 is fully dependent on BRCA1, additional proteins may mediate S345 phosphorylation at later time points. In addition, we show that a subset of phosphorylated Chk1 is released from the chromatin in a BRCA1-dependent manner which may lead to the phosphorylation of Chk1 substrate, Cdc25C, on S216 and to S-phase checkpoint activation. Inhibition of Chk1 kinase by UCN-01 or expression of Chk1 phosphorylation mutants in which the serine residues were substituted with alanine residues abrogates BRCA1-dependent cell cycle arrest in response replication inhibition. These data reveal that BRCA1 facilitates Chk1 phosphorylation and its partial chromatin dissociation following replication inhibition that is likely to be required for S-phase checkpoint signaling.     

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.     

Specific role of Chk1 phosphorylations in cell survival and checkpoint activation

Chk1 is a multifunctional protein kinase that plays essential roles in cell survival and cell cycle checkpoints. Chk1 is phosphorylated at multiple sites by several protein kinases, but the precise effects of these phosphorylations are largely unknown. Using a knockout-knockin system, we examined the abilities of Chk1 mutants to reverse the defects of Chk1-null cells. Wild-type Chk1 could rescue all the defects of Chk1-null cells. Like endogenous Chk1, wild-type Chk1 localized in both the cytoplasm and the nucleus, and its centrosomal association was enhanced by DNA damage. The mutation at S345 resulted in mitotic catastrophe, impaired checkpoints, and loss of the ability to localize in the cytoplasm, but the mutant retained the ability to be released from chromatin upon encountering genotoxic stressors. In contrast, the mutation at S317 resulted in impaired checkpoints and loss of chromatin release upon encountering genotoxic stressors, but its mutant retained the abilities to prevent mitotic catastrophes and to localize in the cytoplasm, suggesting the distinct effects of these phosphorylations. The forced immobilization of S317A/S345A in centrosomes resulted in the prevention of apoptosis in the presence or absence of DNA damage. Thus, two-step phosphorylation of Chk1 at S317 and S345 appeared to be required for proper localization of Chk1 to centrosomes.     

P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

The ataxia telangiectasia mutated- and rad3-related kinase (ATR)/Chk1 pathway is a sentinel of cell cycle progression. On the other hand, the Ras/mitogen-activated protein kinase/90-kDa ribosomal S6 kinase (p90 RSK) pathway is a central node in cell signaling downstream of growth factors. These pathways are closely correlated in cell proliferation, but their interaction is largely unknown. Here we show that Chk1 is phosphorylated predominantly at Ser-280 and translocated from cytoplasm to nucleus in response to serum stimulation. Nonphosphorylated Chk1-Ser-280 mutation attenuates nuclear Chk1 accumulation, whereas the phosphomimic mutation has a reverse effect on the localization. Treatment with p90 RSK inhibitor impairs Chk1 phosphorylation at Ser-280 and accumulation at the nucleus after serum stimulation, whereas these two phenomena are induced by the expression of the constitutively active mutant of p90 RSK in serum-starved cells. In vitro analyses indicate that p90 RSK stoichiometrically phosphorylates Ser-280 on Chk1. Together with Chk1 phosphorylation at Ser-345 by ATR and its autophosphorylation at Ser-296, which are critical for checkpoint signaling, Chk1-Ser-280 phosphorylation is elevated in a p90 RSK-dependent manner after UV irradiation. In addition, Chk1 phosphorylation at Ser-345 and Ser-296 after UV irradiation is also attenuated by the treatment with p90 RSK inhibitor or by Ser-280 mutation to Ala. These results suggest that p90 RSK facilitates nuclear Chk1 accumulation through Chk1-Ser-280 phosphorylation and that this pathway plays an important role in the preparation for monitoring genetic stability during cell proliferation.     

Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.     

ATR,Claspin and the Rad9-Rad1-Hus1 Complex Regulate Chk1 and Cdc25A in the Absence of DNA Damage

The ATR and Chk1 kinases are essential to maintain genomicintegrity. ATR, with Claspin and the Rad9-Rad1-Hus1 complex,activates Chk1 after DNA damage. Chk1-mediated phosphorylation ofthe Cdc25A phosphatase is required for the mammalian S-phasecheckpoint. Here, we show that during physiological S phase theregulation of the Chk1-Cdc25A pathway depends on ATR, Claspin,Rad9, and Hus1. Human cells with chemically or genetically ablatedATR showed inhibition of Chk1-dependent phosphorylation of Cdc25A,and they accumulated Cdc25A without external DNA damage.Furthermore, siRNA-mediated depletion of Claspin, Rad9 and Hus1also stabilized Cdc25A. ATR ablation also inhibited the activatoryphosphorylation of Chk1 on serine 345. Thus, the ATR-Chk1-Cdc25Apathway represents an integral part of physiological S-phaseprogression, and interference with this mechanism underminesviability of somatic mammalian cells. DNA damage further activatesand switches this pathway from its constitutively operating“surveillance mode” compatible with DNA replication into an“emergency” checkpoint response.     

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.     

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3     

Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit

In higher eukaryotic organisms, the checkpoint kinase 1 (Chk1) contributes essential functions to both cell cycle and checkpoint control. Chk1 executes these functions, in part, by targeting the Cdc25A protein phosphatase for ubiquitin-mediated proteolysis. In response to genotoxic stress, Chk1 is phosphorylated on serines 317 (S317) and 345 (S345) by the ataxia-telangiectasia-related (ATR) protein kinase. Phosphorylation of Chk1 on these C-terminal serine residues is used as an indicator of Chk1 activation in vivo. Here, we report that inhibition of Chk1 kinase activity paradoxically leads to the accumulation of S317- and S345-phosphorylated Chk1 in vivo and that ATR catalyzes Chk1 phosphorylation under these conditions. We demonstrate that Chk1 phosphorylation by ATR is antagonized by protein phosphatase 2A (PP2A). Importantly, dephosphorylation of Chk1 by PP2A is regulated, in part, by the kinase activity of Chk1. We propose that the ATR-Chk1-PP2A regulatory circuit functions to keep Chk1 in a low-activity state during an unperturbed cell division cycle but at the same time keeps Chk1 primed to respond rapidly in the event that cells encounter genotoxic stress.     

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.     

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.     

Genotoxin-induced Rad9-Hus1-Rad1 (9-1-1) chromatin association is an early checkpoint signaling event

Rad17, Rad1, Hus1, and Rad9 are key participants in checkpoint signaling pathways that block cell cycle progression in response to genotoxins. Biochemical and molecular modeling data predict that Rad9, Hus1, and Rad1 form a heterotrimeric complex, dubbed 9-1-1, which is loaded onto chromatin by a complex of Rad17 and the four small replication factor C (RFC) subunits (Rad17-RFC) in response to DNA damage. It is unclear what checkpoint proteins or checkpoint signaling events regulate the association of the 9-1-1 complex with DNA. Here we show that genotoxin-induced chromatin binding of 9-1-1 does not require the Rad9-inducible phosphorylation site (Ser-272). Although we found that Rad9 undergoes an additional phosphatidylinositol 3-kinase-related kinase (PIKK)-dependent posttranslational modification, we also show that genotoxin-triggered 9-1-1 chromatin binding does not depend on the catalytic activity of the PIKKs ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), or DNA-PK. Additionally, 9-1-1 chromatin binding does not require DNA replication, suggesting that the complex can be loaded onto DNA in response to DNA structures other than stalled DNA replication forks. Collectively, these studies demonstrate that 9-1-1 chromatin binding is a proximal event in the checkpoint signaling cascade.     

Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response

BACKGROUND: Checkpoint signaling pathways are of crucial importance for the maintenance of genomic integrity. Within these pathways, the effector kinase Chk1 plays a central role in mediating cell-cycle arrest in response to DNA damage, and it does so by phosphorylating key cell-cycle regulators. RESULTS: By investigating the subcellular distribution of Chk1 by cell fractionation, we observed that around 20% of it localizes to chromatin during all phases of the cell cycle. Furthermore, we found that in response to DNA damage, Chk1 rapidly dissociates from the chromatin. Significantly, we observed a tight correlation between DNA-damage-induced Chk1 phosphorylation and chromatin dissociation, suggesting that phosphorylated Chk1 does not stably associate with chromatin. Consistent with these events being triggered by active checkpoint signaling, inhibition of the DNA-damage-activated kinases ATR and ATM, or siRNA-mediated downregulation of the DNA-damage mediator proteins Claspin and TopBP1, impaired DNA-damage-induced dissociation of Chk1 from chromatin. Finally, we established that Chk1 phosphorylation occurs at localized sites of DNA damage and that constitutive immobilization of Chk1 on chromatin results in a defective DNA-damage-induced checkpoint arrest. CONCLUSIONS: Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKK-dependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.     

Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of Claspin and the Rad9 carboxy terminus

The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.     

Autophosphorylation of checkpoint kinase 2 at serine 516 is required for radiation-induced apoptosis

In response to ionizing radiation, checkpoint kinase 2 (Chk2) is activated in an ataxia telangiectasia mutation-dependent manner and induces either cell cycle arrest or apoptosis. Chk2 is also autophosphorylated following DNA damage. It is proposed that autophosphorylation of Chk2 may contribute to Chk2 activation. To fully understand the regulation of Chk2, we mapped an in vitro Chk2 autophosphorylation site at C-terminal serine 516 site (Ser-516). Ser-516 of Chk2 is phosphorylated following radiation in vivo, and this phosphorylation depends on the kinase activity of Chk2. Mutation of this autophosphorylation site (S516A) results in reduced Chk2 kinase activity, suggesting that Chk2 autophosphorylation is required for full kinase activation following DNA damage. Moreover, the S516A mutant of Chk2 is defective in ionizing radiation-induced apoptosis, suggesting that Chk2 autophosphorylation is critical for Chk2 function following DNA damage.     

Regulatory interactions between the checkpoint kinase Chk1 and the proteins of the DNA-dependent protein kinase complex

Checkpoints are biochemical pathways that provide cells a mechanism 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 by blocking the activation of Cdk1/cyclin B. In this study, we investigate the regulatory interaction between Chk1 and members of the Atm family of kinases and the functional role of the C-terminal non-catalytic domains of Chk1. Chk1 stimulates the kinase activity of DNA-PK (protein kinase) complexes, which leads to increased phosphorylation of p53 on Ser-15 and Ser-37. In addition, Chk1 stimulates DNA-PK-dependent end-joining reactions in vitro. We also show that Chk1 protein complexes bind to single-stranded DNA and DNA ends. These results indicate a connection between components that regulate the checkpoint pathways and DNA-PK complex proteins, which have a role in the repair of double strand breaks.     

Inhibition of Chk1 by activated PKB/Akt

We have shown recently that DNA damage effector kinase Chk1 is phosphorylated in vitro by protein kinase B/Akt (PKB/Akt) on serine 280. Activation of Chk1 by DNA damage in vivo is suppressed in presence of activated PKB. In this study we show that Chk1 is phosphorylated by PKB in vivo, and that increased phosphorylation by PKB on serine 280 correlates with impairment of Chk1 activation by DNA damage. Our results indicate a likely mechanism for the negative effects that phosphorylation of serine 280 has on activation of Chk1. The Chk1 protein phosphorylated by PKB on serine 280 does not enter into protein complexes after replication arrest. Moreover, Chk1 phosphorylated by PKB fails to undergo activating phosphorylation on serine 345 by ATM/ATR. Phosphorylation by ATM/ATR and association with other checkpoint proteins are essential steps in activation of Chk1. Inhibition of these steps provides a plausible explanation for the observed attenuation of Chk1 activation by activated PKB after DNA damage.     

Regulation of intra-S phase checkpoint by ionizing radiation (IR)-dependent and IR-independent phosphorylation of SMC3

Structure maintenance of chromosome 1 (SMC1) is phosphorylated by ataxia telangiectasia-mutated (ATM) in response to ionizing radiation (IR) to activate intra-S phase checkpoint. A role of CK2 in DNA damage response has been implicated in many previous works, but the molecular mechanism for its activation is not clear. In the present work, we report that SMC3 is phosphorylated at Ser-1067 and Ser-1083 in vivo. Ser-1083 phosphorylation is IR-inducible, depends on ATM and Nijmegen breakage syndrome 1 (NBS1), and is required for intra-S phase checkpoint. Interestingly, Ser-1067 phosphorylation is constitutive and is not induced by IR but also affects intra-S phase checkpoint. Phosphorylation of Ser-1083 is weakened in cells expressing S1067A mutant, suggesting interplay between Ser-1067 and Ser-1083 phosphorylation in DNA damage response. Consistently, small interfering RNA knockdown of CK2 leads to attenuated phosphorylation of Ser-1067 as well as intra-S phase checkpoint defect. Our data provide evidence that phosphorylation of a core cohesin subunit SMC3 by ATM plays an important role in DNA damage response and suggest that a constitutive phosphorylation by CK2 may affect intra-S phase checkpoint by modulating SMC3 phosphorylation by ATM.