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.     

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

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

Methylator-induced, mismatch repair-dependent G2 arrest is activated through Chk1 and Chk2

SN1 DNA methylating agents such as the nitrosourea N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) elicit a G2/M checkpoint response via a mismatch repair (MMR) system-dependent mechanism; however, the exact nature of the mechanism governing MNNG-induced G2/M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G2/M checkpoint response. We document that MNNG induces a robust, dose-dependent G2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMR-dependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases.     

Recovery from DNA damage checkpoint arrest by PP1-mediated inhibition of Chk1

The G2 DNA damage checkpoint delays mitotic entry via the upregulation of Wee1 kinase and the downregulation of Cdc25 phosphatase by Chk1 kinase, and resultant inhibitory phosphorylation of Cdc2. While checkpoint activation is well understood, little is known about how the checkpoint is switched off to allow cell cycle re-entry. To identify proteins required for checkpoint release, we screened for genes in Schizosaccharomyces pombe that, when overexpressed, result in precocious mitotic entry in the presence of DNA damage. We show that overexpression of the type I protein phosphatase Dis2 sensitises S. pombe cells to DNA damage, causing aberrant mitoses. Dis2 abrogates Chk1 phosphorylation and activation in vivo, and dephosphorylates Chk1 and a phospho-S345 Chk1 peptide in vitro. dis2Delta cells have a prolonged chk1-dependent arrest and a compromised ability to downregulate Chk1 activity for checkpoint release. These effects are specific for the DNA damage checkpoint, because Dis2 has no effect on the chk1-independent response to stalled replication forks. We propose that inactivation of Chk1 by Dis2 allows mitotic entry following repair of DNA damage in the G2-phase.     

Characterization of a Saccharomyces cerevisiae homologue of Schizosaccharomyces pombe Chk1 involved in DNA-damage-induced M-phase arrest

Chk1 is an evolutionarily conserved protein kinase that plays an essential role in mediating G2 arrest in response to DNA damage in Schizosaccharomyces pombe and human cells. It functions by maintaining the inhibition (by phosphorylation of a specific tyrosine residue) of the cyclin-dependent kinase Cdc2 that initiates the G2/M transition. Here, we characterize a structural homologue of Chk1 in the budding yeast Saccharomyces cerevisiae. In this organism, G2/M arrest following DNA damage is considered to be independent of tyrosine phosphorylation of the Cdc2 homologue Cdc28. Nevertheless, a partial defect in G2/M-phase arrest following treatment with ionizing radiation, but not UV radiation, is associated with deletion of CHK1. The fact that such an effect remains detectable in cells synchronized with the microtubule inhibitor nocodazole prior to γ irradiation implies the existence of a CHK1-dependent checkpoint in M phase. We conclude from epistasis analysis that Chk1 participates in the Pds1-dependent subpathway of M-phase arrest. In spite of the partial checkpoint defect of the chk1 mutant, the survival of colony-forming cells is not notably decreased following UV and γ irradiation. In two-hybrid screens, we identified a heme-binding stress protein (encoded by the yeast ORF YNL234W), a protein involved in genomic silencing (Sas3) and Chk1 itself as interacting partners of Chk1. Received: 7 July 1999 / Accepted: 29 October 1999     

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.     

Unleashing Chk1 in cancer therapy

The checkpoint kinase 1 (Chk1) is one of the major players in the signal transduction pathway set in motion in response to DNA damage which activates different cell cycle checkpoints including the G₁/S, the intra-S, G₂-M and the mitotic spindle checkpoint, contributing to the maintenance of genomic stability. Chk1 is considered a good molecular target to inhibit, in combination with other anticancer agents, to increase the sensitivity of treatment, especially in tumors with a defective G₁ checkpoint. Experimental evidence highlights the essential role of Chk1 in normal and cancer cells even under unstressed conditions, especially in controlling DNA replication and cell division. This review looks at the main functions of Chk1 and the data on Chk1 inhibitors at their preclinical and clinical development are reported. This information may suggest novel approaches for new treatments with Chk1 inhibitors in combination with anticancer agents or as single agents. The emergent synthetic lethality approach may help define the genetic background features where Chk1 inhibitors alone could be very effective.     

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.     

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.     

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.     

Oxidative stress induces cell cycle-dependent Mre11 recruitment,ATM and Chk2 activation and histone H2AX phosphorylation

DNA damage response recruits complex molecular machinery involved in DNA repair, arrest of cell cycle progression, and potentially in activation of apoptotic pathway. Among the first responders is the Mre11- (MRN) complex of proteins (Mre11, Rad50, Nbs1), essential for activation of ATM; the latter activates checkpoint kinase 2 (Chk2) and phosphorylates histone H2AX. In the present study the recruitment of Mre11 and phosphorylation of ATM, Chk2 and H2AX (γH2AX) detected immunocytochemically were measured by laser scanning cytometry to assess kinetics of these events in A549 cells treated with H2O2. Recruitment of Mre11 was rapid, peaked at 10 min of exposure to the oxidant, and was of similar extent in all phases of the cell cycle. ATM and Chk2 activation as well as H2AX phosphorylation reached maximum levels after 30 min of treatment with H2O2; the extent of phosphorylation of each was most prominent in S-, less in G₁-, and the least in G₂M- phase cells. A strong correlation between activation of ATM and Chk2, measured in the same cells, was seen in all phases of the cycle. In untreated cells activated Chk2 and Mre11 were distinctly present in centrosomes while in interphase cells they had characteristic punctate nuclear localization. The punctate expression of activated Chk2 both in untreated and H₂O₂ treated cells was accentuated when measured as maximal pixel rather than integrated value of immunofluorescence (IF) per nucleus, and was most pronounced in G₁ cells, likely reflecting the function of Chk2 in activating Cdc25A. Subpopulations of G₁ and G₂M cells with strong maximal pixel of Chk2-Thr68P IF in association with centrosomes were present in untreated cultures. Cytometric multiparameter assessment of the DNA damage response utilizing quantitative image analysis that allows one to measure inhomogeneity of fluorochrome distribution (e.g. maximal pixel) offers unique advantage in studies of the response of different cell constituents in relation to cell cycle position.     

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.     

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.     

Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

In yeasts, the replication protein Cdc6/Cdc18 is required for the initiation of DNA replication and also for coupling S phase with the following mitosis. In metazoans a role for Cdc6 has only been shown in S phase entry. Here we provide evidence that human Cdc6 (HuCdc6) also regulates the onset of mitosis, as overexpression of HuCdc6 in G(2) phase cells prevents entry into mitosis. This block is abolished when HuCdc6 is expressed together with a constitutively active Cyclin B/CDK1 complex or with Cdc25B or Cdc25C. An inhibitor of Chk1 kinase activity, UCN-01, overcomes the HuCdc6 mediated G(2) arrest indicating that HuCdc6 blocks cells in G(2) phase via a checkpoint pathway involving Chk1. When HuCdc6 is overexpressed in G(2), we detected phosphorylation of Chk1. Thus, HuCdc6 can trigger a checkpoint response, which could ensure that all DNA is replicated before mitotic entry. We also present evidence that the ability of HuCdc6 to block mitosis may be regulated by its phosphorylation.     

Hyperactive Cdc2 kinase interferes with the response to broken replication forks by trapping S.pombe Crb2 in its mitotic T215 phosphorylated state

Although it is well established that Cdc2 kinase phosphorylates the DNA damage checkpoint protein Crb2^53BP1 in mitosis, the full impact of this modification is still unclear. The Tudor-BRCT domain protein Crb2 binds to modified histones at DNA lesions to mediate the activation of Chk1 by Rad3^ATR kinase. We demonstrate here that fission yeast cells harbouring a hyperactive Cdc2^CDK1 mutation (cdc2.1w) are specifically sensitive to the topoisomerase 1 inhibitor camptothecin (CPT) which breaks DNA replication forks. Unlike wild-type cells, which delay only briefly in CPT medium by activating Chk1 kinase, cdc2.1w cells bypass Chk1 to enter an extended cell-cycle arrest which depends on Cds1 kinase. Intriguingly, the ability to bypass Chk1 requires the mitotic Cdc2 phosphorylation site Crb2-T215. This implies that the presence of the mitotic phosphorylation at Crb2-T215 channels Rad3 activity towards Cds1 instead of Chk1 when forks break in S phase. We also provide evidence that hyperactive Cdc2.1w locks cells in a G1-like DNA repair mode which favours non-homologous end joining over interchromosomal recombination. Taken together, our data support a model such that elevated Cdc2 activity delays the transition of Crb2 from its G1 to its G2 mode by blocking Srs2 DNA helicase and Casein Kinase 1 (Hhp1).     

Geminin deficiency causes a Chk1-dependent G2 arrest in Xenopus

Geminin is an unstable inhibitor of DNA replication that gets destroyed at the metaphase/anaphase transition. The biological function of geminin has been difficult to determine because it is not homologous to a characterized protein and has pleiotropic effects when overexpressed. Geminin is thought to prevent a second round of initiation during S or G2 phase. In some assays, geminin induces uncommitted embryonic cells to differentiate as neurons. In this study, geminin was eliminated from developing Xenopus embryos by using antisense techniques. Geminin-deficient embryos show a novel and unusual phenotype: they complete the early cleavage divisions normally but arrest in G2 phase immediately after the midblastula transition. The arrest requires Chk1, the effector kinase of the DNA replication/DNA damage checkpoint pathway. The results indicate that geminin has an essential function and that loss of this function prevents entry into mitosis by a Chk1-dependent mechanism. Geminin may be required to maintain the structural integrity of the genome or it may directly down-regulate Chk1 activity. The data also show that during the embryonic cell cycles, rereplication is almost entirely prevented by geminin-independent mechanisms.     

Human Immunodeficiency Virus Type 1 Vpr Induces Cell Cycle G2 Arrest through Srk1/MK2-Mediated Phosphorylation of Cdc25

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G₂ arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G₂ arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G₂/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G₂ arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G₂ delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G₂/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G₂ arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G₂ arrest at least in part through a Srk1/MK2-mediated mechanism.     

Regulation of Cdc2/cyclin B activation in Xenopus egg extracts via inhibitory phosphorylation of Cdc25C phosphatase by Ca(2+)/calmodulin-dependent protein [corrected] kinase II

Activation of Cdc2/cyclin B kinase and entry into mitosis requires dephosphorylation of inhibitory sites on Cdc2 by Cdc25 phosphatase. In vertebrates, Cdc25C is inhibited by phosphorylation at a single site targeted by the checkpoint kinases Chk1 and Cds1/Chk2 in response to DNA damage or replication arrest. In Xenopus early embryos, the inhibitory site on Cdc25C (S287) is also phosphorylated by a distinct protein kinase that may determine the intrinsic timing of the cell cycle. We show that S287-kinase activity is repressed in extracts of unfertilized Xenopus eggs arrested in M phase but is rapidly stimulated upon release into interphase by addition of Ca2+, which mimics fertilization. S287-kinase activity is not dependent on cyclin B degradation or inactivation of Cdc2/cyclin B kinase, indicating a direct mechanism of activation by Ca2+. Indeed, inhibitor studies identify the predominant S287-kinase as Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates Cdc25C efficiently on S287 in vitro and, like Chk1, is inhibited by 7-hydroxystaurosporine (UCN-01) and debromohymenialdisine, compounds that abrogate G2 arrest in somatic cells. CaMKII delays Cdc2/cyclin B activation via phosphorylation of Cdc25C at S287 in egg extracts, indicating that this pathway regulates the timing of mitosis during the early embryonic cell cycle.     

The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation

The checkpoint kinase Chk1 is an important mediator of cell cycle arrest following DNA damage. The 1.7 A resolution crystal structures of the human Chk1 kinase domain and its binary complex with an ATP analog has revealed an identical open kinase conformation. The secondary structure and side chain interactions stabilize the activation loop of Chk1 and enable kinase activity without phosphorylation of the catalytic domain. Molecular modeling of the interaction of a Cdc25C peptide with Chk1 has uncovered several conserved residues that are important for substrate selectivity. In addition, we found that the less conserved C-terminal region negatively impacts Chk1 kinase activity.     

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