Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.     

p53-dependent Chk1 phosphorylation is required for maintenance of prolonged G2 Arrest

Targeting checkpoint kinases has been shown to have a potential chemosensitizing effect in cancer treatment. However, inhibitors of such kinases preferentially abrogate the DNA damage-induced G2 checkpoint in p53-/- as opposed to p53+/+ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G2 checkpoint are unclear. Using normal human diploid p53+/+ and p53-/- fibroblasts as model systems, we have compared the effects of three checkpoint inhibitors, caffeine, staurosporine and UCN-01, on gamma-radiation-induced G2 arrest. The G2 arrest in p53+/+ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G2 arrest in p53-/- cells was sensitive to all three inhibitors. Chk2 (CHEK1) phosphorylation was maintained in the presence of all three inhibitors in both p53+/+ and p53-/- cells. Chk1 phosphorylation was maintained only in the presence of staurosporine and UCN-01 in p53+/+ cells. In the presence of caffeine Chk1 phosphorylation was inhibited regardless of p53 status. The pathway of Chk1 phosphorylation --> Cdc25A degradation --> inhibition of cyclin B1/Cdk1 activity --> G2 arrest is accordingly resistant to staurosporine and UCN-01 in p53+/+ cells. Moreover, sustained phosphorylation of Chk1 in the presence of staurosporine and UCN-01 is strongly related to phosphorylation of p53. The present study suggests the unique role of Chk1 in preventing abrogation of the G2 checkpoint in p53+/+ cells.     

Cdc7-Dbf4 and the human S checkpoint response to UVC

The S checkpoint response to ultraviolet radiation (UVC) that inhibits replicon initiation is dependent on the ATR and Chk1 kinases. Downstream effectors of this response, however, are not well characterized. Data reported here eliminated Cdc25A degradation and inhibition of Cdk2-cyclin E as intrinsic components of the UVC-induced pathway of inhibition of replicon initiation in human cells. A sublethal dose of UVC (1 J/m(2)), which selectively inhibits replicon initiation by 50%, failed to reduce the amount of Cdc25A protein or decrease Cdk2-cyclin E kinase activity. Cdc25A degradation was observed after irradiation with cytotoxic fluences of UVC, suggesting that severe inhibition of DNA chain elongation and activation of the replication checkpoint might be responsible for the UVC-induced degradation of Cdc25A. Another proposed effector of the S checkpoint is the Cdc7-Dbf4 complex. Dbf4 interacted weakly with Chk1 in vivo but was recognized as a substrate for Chk1-dependent phosphorylation in vitro. FLAG-Dbf4 formed complexes with endogenous Cdc7, and this interaction was stable in UVC-irradiated HeLa cells. Overexpression of FLAG- or Myc-tagged Dbf4 abrogated the S checkpoint response to UVC but not ionizing radiation. These findings implicate a Dbf4-dependent kinase as a possible target of the ATR- and Chk1-dependent S checkpoint response to UVC.     

Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition

In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo-cytoplasmic ratio. Here we show that the checkpoint kinase Chk1, but not Cds1 (Chk2), is activated transiently at the MBT in a maternal/zygotic gene product-regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre-MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1-independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development.     

The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway

Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G(2) checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G(2) checkpoint. Here we report that A1-5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G(2) arrest response when compared with its counterpart, B4, although A1-5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1-5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1-5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G(2) arrest and at the same time reduces the phosphorylation of Cdc2 in A1-5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1-5 cells to radiation-induced killing and reduces the G(2) arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G(2) arrest in A1-5 cells.     

Differential roles for checkpoint kinases in DNA damage-dependent degradation of the Cdc25A protein phosphatase

In response to DNA damage, cells activate a signaling pathway that promotes cell cycle arrest and degradation of the cell cycle regulator Cdc25A. Cdc25A degradation occurs via the SCFbeta-TRCP pathway and phosphorylation of Ser-76. Previous work indicates that the checkpoint kinase Checkpoint kinase 1 (Chk1) is capable of phosphorylating Ser-76 in Cdc25A, thereby promoting its degradation. In contrast, other experiments involving overexpression of dominant Chk2 mutant proteins point to a role for Chk2 in Cdc25A degradation. However, loss-of-function studies that implicate Chk2 in Cdc25A turnover are lacking, and there is no evidence that Chk2 is capable of phosphorylating Ser-76 in Cdc25A despite the finding that Chk1 and Chk2 sometimes share overlapping primary specificity. We find that although Chk2 can phosphorylate many of the same sites in Cdc25A that Chk1 phosphorylates, albeit with reduced efficiency, Chk2 is unable to efficiently phosphorylate Ser-76. Consistent with this, Chk2, unlike Chk1, is unable to support SCFbeta-TRCP-mediated ubiquitination of Cdc25A in vitro. In CHK2(-/-) HCT116 cells, the kinetics of Cdc25A degradation in response to ionizing radiation is comparable with that seen in HCT116 cells containing Chk2, indicating that Chk2 is not generally required for timely DNA damage-dependent Cdc25A turnover. In contrast, depletion of Chk1 by RNA interference in CHK2(-/-) cells leads to Cdc25A stabilization in response to ionizing radiation. These data support the idea that Chk1 is the primary signal transducer linking activation of the ATM/ATR kinases to Cdc25A destruction in response to ionizing radiation.     

p38 and Chk1 kinases: different conductors for the G(2)/M checkpoint symphony.

The mechanism controlling G(2)/M checkpoint activation after DNA damage was thought to be mediated primarily by nuclear Chk1/Chk2 kinases. Recent evidence indicates that this checkpoint is more complex, involving at least two different biochemical systems that target the Cdc25B and Cdc25C phosphatases. Following genotoxic stress, different kinases integrate signaling from the damaged DNA and other damaged cellular components to regulate Cdc25 inactivation. Our current model for G(2)/M checkpoint activation after genotoxic stress is discussed emphasizing the roles for Chk1 and p38 kinases in checkpoint regulation.     

Cytoplasmic occurrence of the Chk1/Cdc25 pathway and regulation of Chk1 in Xenopus oocytes

Chk1, a nuclear DNA damage/replication G2 checkpoint kinase, phosphorylates Cdc25 and causes its nuclear exclusion in yeast and mammalian cells, thereby arresting the cell at the G2 phase until DNA repair/replication is completed. Chk1 is also involved, at least in part, in the natural G2 arrest of immature Xenopus oocytes, but it is unknown how Chk1 inhibits Cdc25 function and undergoes regulation during oocyte maturation. By using enucleated oocytes, we show here that Chk1 inhibits Cdc25 function in the cytoplasm of G2-arrested oocytes and that Cdc25 is activated exclusively in the cytoplasm of maturing oocytes. Moreover, we show that Chk1 activity is not appreciably altered during maturation, being maintained at basal levels, and that C-terminal truncation mutants of Chk1 have very high kinase activities, strong abilities to inhibit maturation, and altered subcellular localization in oocytes. These results, together with other results, suggest that the Chk1/Cdc25 pathway is involved cytoplasmically in G2 arrest of Xenopus oocytes, but moderately and independent of the G2 checkpoint, and that the C-terminal region of Chk1 negatively regulates its kinase activity and also determines its subcellular localization. Based on these results, we discuss the possibility that Chk1 (with the basal activity) may function as an ordinary regulator of Cdc25 in oocytes (and in other cell types) and that Chk1 might be hyperactivated in response to the G2 checkpoint via its dramatic conformational change.     

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.     

Regulation of Human Cdc25A Stability by Serine 75 Phosphorylation Is Not Sufficient to Activate a S-phase Checkpoint

Degradation of Cdc25A phosphatase is an ubiquitous feature of stress. There are some discrepancies in the reported roles for different phosphorylation sites in the regulation of Cdc25A stability. Using a panel of doxycycline-inducible phosphorylation mutants we show that the stability of human Cdc25A protein is dependent upon phosphorylation at S75. In non-stressed conditions and in non-mitotic cells, Cdc25A is unstable and its stability is regulated in a Chk1-dependent manner. During mitosis, Cdc25A becomes stable and does not undergo degradation after DNA damage. We further show that Chk1 kinase regulates Cdc25A stability after UV irradiation. Similar to Chk1 kinase, p38 MAPK controls Cdc25A protein level after osmotic stress. Using phospho-specific antibodies, we find that both kinases can phosphorylate S75 and S123 in vitro. Inactivation of either Chk1 after UV-irradiation or p38 MAPK after osmotic stress prevents activation of a S phase checkpoint and S75 and S123 phosphorylation. However, introduction of stable Cdc25A (S75A or S75/123A) proteins is not sufficient to overcome this checkpoint. We propose that regulation of human Cdc25A stability by its phosphorylation at S75 may contribute to S phase checkpoint activation only in cooperation with other regulatory mechanisms.     

Regulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint

Degradation of Cdc25A phosphatase is an ubiquitous feature of stress. There are some discrepancies in the reported roles for different phosphorylation sites in the regulation of Cdc25A stability. Using a panel of doxycycline-inducible phosphorylation mutants we show that the stability of human Cdc25A protein is dependent upon phosphorylation at S75. In non-stressed conditions and in non-mitotic cells, Cdc25A is unstable and its stability is regulated in a Chk1-dependent manner. During mitosis, Cdc25A becomes stable and does not undergo degradation after DNA damage. We further show that Chk1 kinase regulates Cdc25A stability after UV irradiation. Similar to Chk1 kinase, p38 MAPK controls Cdc25A protein level after osmotic stress. Using phospho-specific antibodies, we find that both kinases can phosphorylate S75 and S123 in vitro. Inactivation of either Chk1 after UV-irradiation or p38 MAPK after osmotic stress prevents activation of a S phase checkpoint and S75 and S123 phosphorylation. However, introduction of stable Cdc25A (S75A or S75/123A) proteins is not sufficient to overcome this checkpoint. We propose that regulation of human Cdc25A stability by its phosphorylation at S75 may contribute to S phase checkpoint activation only in cooperation with other regulatory mechanisms.     

Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes

Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes.     

The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.     

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.     

Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint

The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in Δmik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint.     

Degradation of Cdc25A phosphatase in HeLa cells under normal and stress conditions

Cdc25A phosphatase regulates cell cycle progression by removing the inhibitory phosphates from cyclin-dependent kinases. Activity of Cdc25A depends on its phosphorylation status. During normal cell cycle progression and after DNA damage phosphorylation by Chk1 (or Chk2) triggers Cdc25A degradation via ubiquitin-proteasome pathway. In this study we investigate the role of various phosphorylation sites (Ser123, Ser75, Ser17 and Ser115) in the regulation of Cdc25A stability. We have shown that only S75A mutation abrogates Cdc25A degradation both in normal and stress conditions. We also studied the influence of stable form of Cdc25A on checkpoint progression after DNA damage. We have found out that delay in DNA synthesis after UV and IR does not depend on Cdc25A activity. However, the presence of stable Cdc25A increases the number of mitotic cells after these stresses.     

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.     

Reoxygenation following hypoxia activates DNA-damage checkpoint signaling pathways that suppress cell-cycle progression in cultured human lymphocytes

Cellular responses to DNA damage after hypoxia and reoxygenation (H/R) were examined in human lymphocytes. Cultured lymphocytes exposed to H/R showed a lower cytokinesis block proliferation index and a higher frequency of micronuclei in comparison to control cells. Western blots showed that H/R exposure induced p53 expression; however, p21 and Bax expression did not increase, indicating that H/R did not affect p53 transactivational activity. Phosphorylation of p53 (Ser15), Chk1 (Ser345), and Chk2 (Thr68) was also observed, suggesting that H/R activates p53 through checkpoint signals. In addition, H/R exposure caused the phosphorylation and negative regulation of Cdc2 and Cdc25C, proteins that are involved in cell-cycle arrest at the G2/M checkpoint. The S-phase checkpoint, regulated by the ATM-p95/NBS1-SMC1 pathway, was also triggered in H/R-exposed lymphocytes. These results demonstrate that H/R exposure triggers checkpoint signaling and induces cell-cycle arrest in cultured human lymphocytes.     

Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast

Maintenance of genome integrity requires a checkpoint that restrains mitosis in response to DNA damage [1]. This checkpoint is enforced by Chk1, a protein kinase that targets Cdc25 [2--7]. Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS) and thereby inhibit Cdc25 nuclear import [6, 8--14]. Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking. We have tested this hypothesis in fission yeast. We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model. Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells. However, forced nuclear localization of Cdc25 fails to override the damage checkpoint. Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforcement. We propose that direct inhibition of Cdc25 phosphatase activity by Chk1, as demonstrated in vitro with fission yeast and human Chk1 [15, 16], is sufficient for proficient checkpoint regulation of Cdc25 and may be the primary mechanism of checkpoint enforcement in fission yeast.     

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.