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.     

Determination of substrate motifs for human Chk1 and hCds1/Chk2 by the oriented peptide library approach

Mammalian Chk1 and Chk2 are two Ser/Thr effector kinases that play critical roles in DNA damage-activated cell cycle checkpoint signaling pathways downstream of ataxia telangiectasia-mutated and ataxia telangiectasia-related. Endogenous substrates have been identified for human hCds1/Chk2 and Chk1; however, the sequences surrounding the substrate residues appear unrelated, and consensus substrate motifs for the two Ser/Thr kinases remain unknown. We have utilized peptide library analyses to develop specific, highly preferred substrate motifs for hCds1/Chk2 and Chk1. The optimal motifs are similar for both kinases and most closely resemble the previously identified Chk1 and hCds1/Chk2 substrate target sequences in Cdc25C and Cdc25A, the regulation of which plays an important role in S and G(2)M arrest. Essential residues required for the definition of the optimal motifs were also identified. Utilization of the peptides to assay the substrate specificities and catalytic activities of Chk1 and hCds1/Chk2 revealed substantial differences between the two Ser/Thr kinases. Structural modeling analyses of the peptides into the Chk1 catalytic cleft were consistent with Chk1 kinase assays defining substrate suitability. The library-derived substrate preferences were applied in a genome-wide search program, revealing novel targets that might serve as substrates for hCds1/Chk2 or Chk1 kinase activity.     

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.     

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.     

MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation

The cellular response to DNA damage is mediated by evolutionarily conserved Ser/Thr kinases, phosphorylation of Cdc25 protein phosphatases, binding to 14-3-3 proteins, and exit from the cell cycle. To investigate DNA damage responses mediated by the p38/stress-activated protein kinase (SAPK) axis of signaling, the optimal phosphorylation motifs of mammalian p38alpha SAPK and MAPKAP kinase-2 were determined. The optimal substrate motif for MAPKAP kinase-2, but not for p38 SAPK, closely matches the 14-3-3 binding site on Cdc25B/C. We show that MAPKAP kinase-2 is directly responsible for Cdc25B/C phosphorylation and 14-3-3 binding in vitro and in response to UV-induced DNA damage within mammalian cells. Downregulation of MAPKAP kinase-2 eliminates DNA damage-induced G2/M, G1, and intra S phase checkpoints. We propose that MAPKAP kinase-2 is a new member of the DNA damage checkpoint kinase family that functions in parallel with Chk1 and Chk2 to integrate DNA damage signaling responses and cell cycle arrest in mammalian 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.     

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.     

Questioning the role of checkpoint kinase 2 in the p53 DNA damage response

Cdc25C and p53 have been reported to be physiological targets of checkpoint kinase 2 (Chk2). Surprisingly, although Chk2 purified from DNA damage sustaining cells has dramatically increased ability to phosphorylate Cdc25C when compared with untreated cells, its ability to phosphorylate p53 is weak before treatment, and there is no increase in its activity toward p53 after DNA damage by gamma irradiation or the radiomimetic agent neocarzinostatin. Furthermore, introduction of Chk2 short interfering RNA into three different human tumor cell lines leads to marked reduction of Chk2 protein, but p53 is still stabilized and active after DNA damage. The results with Chk1 short interfering RNA indicate as well that Chk1 does not play a role in human p53 stabilization after DNA damage. Thus, Chk1 and Chk2 are unlikely to be regulators of p53 in at least some human tumor cells. We discuss our results in the context of previous findings demonstrating a requirement for Chk2 in p53 stabilization and activity.     

Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding

The order and fidelity of cell cycle events in mammals is intimately linked to the integrity of the Chk1 kinase-Cdc25A phosphatase pathway. Chk1 phosphorylation targets Cdc25A for destruction and, as shown here, inhibits interactions between Cdc25A and its mitotic substrate cyclin B1-Cdk1. Phosphorylation of Cdc25A on serine 178 and threonine 507 facilitates 14-3-3 binding, and Chk1 phosphorylates both residues in vitro. Mutation of T507 to alanine (T507A) enhanced the biological activity of Cdc25A. Cdc25A(T507A) was more efficient in binding to cyclin B1, activating cyclin B1-Cdk1, and promoting premature entry into mitosis. We propose that the Chk1/Cdc25A/14-3-3 pathway functions to prevent cells from entering into mitosis prior to replicating their genomes to ensure the fidelity of the cell division process.     

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.     

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.     

MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p

We show that MAD3 encodes a novel 58-kD nuclear protein which is not essential for viability, but is an integral component of the spindle checkpoint in budding yeast. Sequence analysis reveals two regions of Mad3p that are 46 and 47% identical to sequences in the NH(2)-terminal region of the budding yeast Bub1 protein kinase. Bub1p is known to bind Bub3p (Roberts et al. 1994) and we use two-hybrid assays and coimmunoprecipitation experiments to show that Mad3p can also bind to Bub3p. In addition, we find that Mad3p interacts with Mad2p and the cell cycle regulator Cdc20p. We show that the two regions of homology between Mad3p and Bub1p are crucial for these interactions and identify loss of function mutations within each domain of Mad3p. We discuss roles for Mad3p and its interactions with other spindle checkpoint proteins and with Cdc20p, the target of the checkpoint.     

Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage

The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.     

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.     

Redox regulation of Cdc25C

The Cdc25 family of dual specific phosphatases are critical components of cell cycle progression and checkpoint control. Certain stresses such as ultraviolet light stimulate the rapid and selective destruction of Cdc25A protein through a Chk1 protein kinase-dependent pathway. We demonstrate that in contrast to cellular stresses previously examined, hydrogen peroxide exposure affects Cdc25C but not Cdc25A levels. Pharmacological inhibition of Chk1 activity or a mutant of Cdc25C that lacks the Chk1 phosphorylation site still undergoes degradation in response to oxidants. We also demonstrate that in vitro hydrogen peroxide stimulates an intramolecular disulfide bond between the active site cysteine at position 377 and another invariant cysteine at position 330. The in vivo stability of Cdc25C is substantially reduced by the mutation of either of these two cysteine residues. In contrast, a double (C2) mutant of both cysteine 330 and cysteine 377 results in a protein that is more stable than wild type Cdc25C and is resistant to oxidative stress-induced degradation. In addition, the C2 mutant, which is unable to form an intramolecular disulfide bond, has reduced binding to 14-3-3 in vitro and in vivo. These results suggest that oxidative stress may induce cell cycle arrest in part through the degradation of Cdc25C.     

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.     

Cdc25: mechanisms of checkpoint inhibition and recovery

Members of the eukaryotic Cdc25 phosphatase family are key targets of the Chk1 and Chk2 checkpoint kinases, which inactivate Cdc25 to halt cell cycle progression when DNA is damaged or incompletely replicated. Now, new kinases that phosphorylate and inactivate Cdc25 are being discovered, including MAPKAP kinase-2, a component of the p38 stress-activated MAP kinase pathway. The roles of other kinases, such as cyclin-dependent kinase, Polo and Aurora A kinase, in controlling the localization or the activation of Cdc25, are controversial. Here, we discuss new data that suggests that different Cdc25 isoforms and regulators of Cdc25 are differentially required for normal cell cycle progression and recovery from checkpoint arrest.     

The DNA damage effector Chk1 kinase regulates Cdc14B nucleolar shuttling during cell cycle progression

Chk1 is a critical effector of DNA damage checkpoints necessary for the maintenance of chromosome integrity during cell cycle progression. Here we report, that Chk1 co-localized with the nucleolar marker, fibrillarin in response to radiation-induced DNA damage in human cells. Interestingly, in vitro studies using GST pull down assays identified the dual-specificity serine/threonine nucleolar phosphatase Cdc14B as a Chk1 substrate. Furthermore, Chk1, but not a kinase-dead Chk1 control, was shown to phosphorylate Cdc14B using an in vitro kinase assay. Co-immunoprecipitation experiments using exogenous Cdc14B transfected into human cells confirmed the interaction of Cdc14B and Chk1 during cell cycle. In addition, reduction of Chk1 levels via siRNA or UCN-01 treatment demonstrated that Chk1 activation following DNA damage was required for Cdc14B export from the nucleolus. These studies have revealed a novel interplay between Chk1 kinase and Cdc14B phosphatase involving radiation-induced nucleolar shuttling to facilitate error-free cell cycle progression and prevent genomic instability.     

Cdc7-dependent and -independent phosphorylation of Claspin in the induction of the DNA replication checkpoint

Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. In this study we use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. We demonstrate that inhibition of Cdc7 kinase delays HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. We find that while chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. We demonstrate that Claspin is an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, we identify multiple phosphorylation sites that help to define a Cdc7 phosphorylation motif. Finally, we show that the interaction between Claspin and Cdc7 is not dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. We propose Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin’s function in the DNA replication checkpoint.