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PubMed2000

Activation of Xenopus Chk1 by mutagenesis of threonine-377.

Xenopus Chk1 (Xchk1) is required for the checkpoint-associated delay of the cell cycle in frog egg extracts containing unreplicated or UV-damaged DNA. Phosphorylation of Xchk1 at multiple sites in the SQ/TQ domain (residues 314-366) in response to unreplicated or UV-damaged DNA results in elevation of its kinase activity. We have found that mutagenesis of Thr-377 in the conserved Thr-Arg-Phe (TRF) motif of Xchk1 also leads to a substantial increase in kinase activity. Thr-377 does not appear to be a site of phosphorylation in Xchk1. These findings suggest that Thr-377 may play a role in suppressing the activity of Xchk1.

Amino Acid Sequence↗
PubMed2000

Substrate specificity determinants of the checkpoint protein kinase Chk1.

The Chk1 protein kinase plays a critical role in a DNA damage checkpoint pathway conserved between fission yeast and animals. We have developed a quantitative assay for Chk1 activity, using a peptide derived from a region of Xenopus Cdc25C containing Ser-287, a known target of Chk1. Variants of this peptide were used to determine the residues involved in substrate recognition by Chk1, revealing the phosphorylation motif Phi-X-beta-X-X-(S/T)*, where * indicates the phosphorylated residue, Phi is a hydrophobic residue (M>I>L>V), beta is a basic residue (R>K) and X is any amino acid. This motif suggests that Chk1 is a member of a group of stress-response protein kinases which phosphorylate target proteins with related specificities.

Amino Acid Motifs↗
PubMed2000

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.

Catalytic Domain↗
PubMed1997

A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair.

Mutations in the S. cerevisiae RAD27 (also called RTH1 or YKL510) gene result in a strong mutator phenotype. In this study we show that the majority of the resulting mutations have a structure in which sequences ranging from 5-108 bp flanked by direct repeats of 3-12 bp are duplicated. Such mutations have not been previously detected at high frequency in the mutation spectra of mutator strains. Epistasis analysis indicates that RAD27 does not play a major role in MSH2-dependent mismatch repair. Mutations in RAD27 cause increased rates of mitotic crossing over and are lethal in combination with mutations in RAD51 and RAD52. These observations suggest that the majority of replication errors that accumulate in rad27 strains are processed by double-strand break repair, while a smaller percentage are processed by a mutagenic repair pathway. The duplication mutations seen in rad27 mutants occur both in human tumors and as germline mutations in inherited human diseases.

Base Sequence↗
PubMed2003

Nitrogen-containing bisphosphonates induce S-phase cell cycle arrest and apoptosis of myeloma cells by activating MAPK pathway and inhibiting mevalonate pathway.

Bisphosphonates have been used for the treatment of hypercalcemia associated with malignancies and osteoporosis. It was previously reported that the mevalonate pathway is involved in nitrogen-containing bisphosphonate-induced apoptosis in osteoclasts and myeloma cells. The aim of this study was to determine the effects of two bisphosphonates, incadronate, and newly developed bisphosphonate YM529 on human myeloma cells, U266, RPMI-8226, and HS-Sultan. Both incadronate and YM529 induced S-phase cell cycle arrest and apoptosis in these myeloma cells. Treatment of the myeloma cells with cell-permeable substrates for mevalonate pathways, geranylgeraniol, and farnesol prevented bisphosphonate-mediated growth suppression. Checkpoint kinases, Chk1/2, and MAPK became phosphorylated after stimulation with bisphosphonates in the myeloma cells. Bisphosphonate-induced apoptosis was partially prevented by the pretreatment with MAPK inhibitor. These results demonstrate that incadronate and YM529 suppress the proliferation of myeloma cells through mevalonate pathway and MAPK pathway.

Apoptosis↗
PubMed2003

Loss of XChk1 function triggers apoptosis after the midblastula transition in Xenopus laevis embryos.

Prior to the midblastula transition (MBT), Xenopus laevis embryos do not engage cell cycle checkpoints, although overexpression of the kinase XChk1 arrests cell divisions. At the MBT, XChk1 transiently activates and promotes cell cycle lengthening. In this study, endogenous XChk1 was inhibited by the expression of dominant-negative XChk1 (DN-XChk1). Development appeared normal until the early gastrula stage, when cells lost attachments and chromatin condensed. TUNEL and caspase assays indicated these embryos died by apoptosis during gastrulation. Embryos with unreplicated DNA likewise died by apoptosis. Embryos expressing DN-XChk1 proceeded through additional rapid rounds of DNA replication but initiated zygotic transcription on schedule. Therefore, XChk1 is essential in the early Xenopus embryo for cell cycle remodeling and for survival after the MBT.

Animals↗
PubMed2001

DNA damage: Chk1 and Cdc25, more than meets the eye.

Control of mitotic entry is a component of the checkpoint response that contributes to cell survival following DNA damage. In some eukaryotic cells, mitotic entry relies heavily on regulation of the state of tyrosine phosphorylation of the cyclin-dependent kinase Cdc2. Evidence that checkpoint regulation of cell-cycle progression operates through controlling the state of Cdc2 tyrosine phosphorylation exists. Whether other targets of the checkpoint pathway could play important roles in the response to DNA damage is a subject of ongoing investigations.

Animals↗
PubMed2002

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.

Animals↗
PubMed2000

The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP.

Small GTPase proteins such as Ras are key regulators of cellular proliferation and are activated by guanine nucleotide exchange/releasing factors (GEFs/GRFs). Three classes of Ras GRFs have been identified to date, represented by Sos1/2, Ras-GRF1/2 and Ras-GRP. Here, we describe a novel candidate Ras activator, cyclic nucleotide rasGEF (CNrasGEF), which contains CDC25, Ras exchange motif (REM), Ras-association (RA), PDZ and cNMP (cAMP/cGMP) binding (cNMP-BD) domains, two PY motifs and a carboxy-terminal SxV sequence. CNrasGEF can activate Ras in vitro, and it binds cAMP directly via its cNMP-BD. In cells, CNrasGEF activates Ras in response to elevation of intracellular cAMP or cGMP, or treatment with their analogues 8-Br-cAMP or 8-Br-cGMP, independently of protein kinases A and G (PKA and PKG). This activation is prevented in CNrasGEF lacking its CDC25 domain or cNMP-BD. CNrasGEF can also activate the small GTPase Rap1 in cells, but this activation is constitutive and independent of cAMP. CNrasGEF is expressed mainly in the brain and is localized at the plasma membrane, a localization dependent on the presence of intact PDZ domain but not the SxV sequence. These results suggest that CNrasGEF may directly connect cAMP-generating pathways or cGMP-generating pathways to Ras.

Animals↗
PubMedDate not supplied

Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint.

BACKGROUND: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available. RESULTS: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation. CONCLUSIONS: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.

Amino Acid Sequence↗
PubMed2002

Hus1 acts upstream of chk1 in a mammalian DNA damage response pathway.

The evolutionarily conserved Hus1 proteins function in DNA damage response pathways that serve to maintain genomic stability. Cells lacking mouse Hus1 are hypersensitive to certain genotoxins, and we have explored the molecular basis for this defect by examining how Hus1 inactivation affects genotoxin-induced signaling events. p53 accumulation and activation in response to DNA damage appeared normal in Hus1 null cells. Likewise, Hus1 was dispensable for genotoxin-induced Chk2 phosphorylation. In contrast, Chk1 phosphorylation after genotoxic stress was greatly reduced in the absence of Hus1, but was restored in Hus1 null fibroblasts complemented by infection with a Hus1-expressing retrovirus. These results demonstrate that mouse Hus1 is required for a specific subset of DNA damage signaling events and functions to promote genotoxin-induced Chk1 phosphorylation.

Animals↗
PubMed2003

Evolution of eukaryotic cell cycle regulation: stepwise addition of regulatory kinases and late advent of the CDKs.

Protein kinases regulate a number of critical events in mitosis and meiosis. A study of the evolution of kinases involved in cell cycle control (CCC) might shed light on the evolution of the eukaryotic cell cycle. In particular, applying quantitative phylogenetic methods to key CCC kinases could provide information on the relative timing of gene duplication events. To investigate the evolution of CCC kinases, we constructed phylogenetic trees for the CDC28 family and performed statistical tests of the tree topology. This family includes the cyclin-dependent kinases (CDKs), which are key regulators of the eukaryotic cell cycle, as well as other CCC kinases. We found that CDKs and, in particular, the principal cell cycle regulator Cdc28p, branch off the phylogenetic tree at a late stage, after several other kinases involved in either mitosis or meiosis regulation. On the basis of this tree topology, it is proposed that, at early stages of evolution, the eukaryotic cell cycle was not controlled by CDKs and that only a subset of extant kinases, notably the DNA damage checkpoint kinase Chk1p, were in place. During subsequent evolution, a series of duplications of kinase genes occurred, gradually adding more kinases to the CCC system, the CDKs being among the last major additions.

Animals↗
PubMed1997

The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity.

BACKGROUND: Cell cycle checkpoints maintain the fidelity of the somatic cell cycle by ensuring that one step in the cell cycle is not initiated until a previous step has been completed. The extent to which cell cycle checkpoints play a role in the initial rapid embryonic divisions of higher eukaryotes is unclear. The initial syncytial divisions of Drosophila embryogenesis provide an excellent opportunity to address this issue as they are amenable to both genetic and cellular analysis. In order to study the relevance of cell cycle checkpoints in early Drosophila embryogenesis, we have characterized the maternal-effect grapes (grp) mutation, which may affect feedback control during early syncytial divisions. RESULTS: The Drosophila grp gene encodes a predicted serine/threonine kinase and has significant homology to chk1/rad27, a gene required for a DNA damage checkpoint in Schizosaccharomyces pombe. Relative to normal embryos, embryos derived from grp-mutant mothers exhibit elevated levels of DNA damage. During nuclear cycles 12 and 13, alignment of the chromosomes on the metaphase plate was disrupted in grp-derived embryos, and the embryos underwent a progression of cytological events that were indistinguishable from those observed in normal syncytial embryos exposed to X-irradiation. The mutant embryos also failed to progress through a regulatory transition in Cdc2 activity that normally occurs during interphase of nuclear cycle 14. CONCLUSION: We propose that the primary defect in grp-derived embryos is a failure to replicate or repair DNA completely before mitotic entry during the late syncytial divisions. This suggests that wild-type grp functions in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions. These results are discussed with respect to the proposed function of the chk1/rad27 gene.

Amino Acid Sequence↗
PubMed1997

Cell-cycle signaling: Atm displays its many talents.

The discovery of multiple signaling cascades downstream of Atm may lead to a clearer understanding of the diverse defects seen in ataxia-telangiectasia. These pathways - which include evolutionarily conserved Chk1 and Atr, and non-conserved p21, p53 and AbI - guard genomic integrity after DNA damage.

Animals↗
PubMed1997

Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes.

BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest. RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors. CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.

Amino Acid Sequence↗
PubMed1995

The chk1 pathway is required to prevent mitosis following cell-cycle arrest at 'start'.

BACKGROUND: The G2-M-phase transition is controlled by cell-cycle checkpoint pathways which inhibit mitosis if previous events are incomplete or if the DNA is damaged. Genetic analyses in yeast have defined two related, but distinct, pathways which prevent mitosis--one which acts when S phase is inhibited, and one which acts when the DNA is damaged. In the fission yeast Schizosaccharomyces pombe, many of the gene products involved have been identified. Six 'radiation checkpoint' (rad) gene products are required for both the S-M and DNA-damage checkpoints, whereas Chk1, a putative protein kinase, is required only for the DNA-damage checkpoint and not for the S-M checkpoint following the inhibition of DNA synthesis. RESULTS: We have genetically defined a third mitotic control checkpoint pathway in fission yeast which prevents mitosis when passage through 'start' (the commitment point in G1) is compromized. In cycling cells arrested at start, mitosis is prevented by a Chk1-dependent pathway. In the absence of Chk1, G1 cells attempt an abortive mitosis with a 1C DNA content without entering S phase. Similar results are seen in the absence of Rad17, a typical example of a rad gene product. CONCLUSIONS: Genetic dissection of checkpoints in logarithmically growing fission yeast has identified a pathway that couples mitosis to correct passage through start. This pathway is related to the DNA-structure check-points which ensure that mitosis is dependent on the completion of replication and the integrity of the DNA. We propose that all three mitotic control checkpoints monitor distinct DNA or protein structures at different stages in the cell cycle.

Cell Cycle↗