Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein kinase Chk1 is essential for the DNA damage checkpoint. Cells lacking Chk1 are hypersensitive to DNA-damaging agents such as UV light and gamma-irradiation because they fail to arrest the cell cycle when DNA damage is generated. Phosphorylation of Chk1 occurs after DNA damage and is dependent on the integrity of the DNA damage checkpoint pathway. We have tested whether a topoisomerase I inhibitor, camptothecin (CPT), generates DNA damage in the fission yeast Schizosaccharomyces pombe that results in Chk1 phosphorylation. We demonstrate that Chk1 is phosphorylated in response to CPT treatment in a time- and dose-dependent manner and that phosphorylation is dependent on an intact DNA damage checkpoint pathway. Furthermore, we show that cells must be actively dividing in order for CPT to generate a Chk1-responsive DNA damage signal. This observation is consistent with a model whereby the cytotoxic event caused by CPT treatment is the production of a DNA double-strand break resulting from the collision of a DNA replication fork with a trapped CPT-topoisomerase I cleavable complex. Cells lacking Chk1 are hypersensitive to CPT treatment, suggesting that the DNA damage checkpoint pathway can be an important determinant for CPT sensitivity or resistance. Finally, as a well-characterized, soluble agent that specifically causes DNA damage, CPT will allow a biochemical analysis of the checkpoint pathway that responds to DNA damage.
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PMID:The topoisomerase I poison camptothecin generates a Chk1-dependent DNA damage checkpoint signal in fission yeast. 1040 62

In fission yeast, the Hsk1 protein kinase is essential for the initiation of DNA replication. We have shown previously that Hsk1 forms a heterodimeric complex with the regulatory subunit, Dfp1. In this report we describe the further characterization of Dfp1. Reconstitution experiments with purified proteins indicate that Dfp1 is necessary and sufficient to activate Hsk1 phosphorylation of exogenous substrates, such as the Schizosaccharomyces pombe minichromosome maintenance protein Cdc19. The dfp1(+) gene is essential for viability of S. pombe, and depletion of the Dfp1 protein significantly delays the onset of S phase. Dfp1 is a phosphoprotein in vivo and becomes hyperphosphorylated when cells are blocked in S phase by treatment with the DNA synthesis inhibitor hydroxyurea. Hyperphosphorylation in S phase depends on the checkpoint kinase Cds1. The abundance of Dfp1 varies during progression through the cell cycle. The protein is absent when cells are arrested in G(1) phase. When cells are released into the cell cycle, Dfp1 appears suddenly at the G(1)/S transition, coincident with the initiation of DNA replication. The absence of Dfp1 before S phase is due largely, but not exclusively, to posttranscriptional regulation. We propose that cell cycle-regulated activation of Dfp1 expression at the G(1)/S transition results in activation of the Hsk1 protein kinase, which, in turn, leads to the initiation of DNA replication.
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PMID:Cell cycle regulation of Dfp1, an activator of the Hsk1 protein kinase. 1041 94

The Schizosaccharomyces pombe checkpoint gene named rad3(+) encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. Here we describe a novel role for Rad3 in the control of telomere integrity. Mutations in the rad3(+) gene alleviated telomeric silencing and produced shortened lengths in the telomere repeat tracts. Genetic analysis revealed that the other checkpoint rad mutations rad1, rad17, and rad26 belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening. tel1(+), another ATM homologue in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1(+) caused no obvious changes in the telomeric DNA structure. Our results demonstrate a novel role of the S. pombe ATM homologues that is independent of chk1(+) and cds1(+).
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PMID:Genetic control of telomere integrity in Schizosaccharomyces pombe: rad3(+) and tel1(+) are parts of two regulatory networks independent of the downstream protein kinases chk1(+) and cds1(+). 1043 May 79

Caffeine exposure sensitizes tumor cells to ionizing radiation and other genotoxic agents. The radiosensitizing effects of caffeine are associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints. The similarity of these checkpoint defects to those seen in ataxia-telangiectasia (A-T) suggested that caffeine might inhibit one or more components in an A-T mutated (ATM)-dependent checkpoint pathway in DNA-damaged cells. We now show that caffeine inhibits the catalytic activity of both ATM and the related kinase, ATM and Rad3-related (ATR), at drug concentrations similar to those that induce radiosensitization. Moreover, like ATM-deficient cells, caffeine-treated A549 lung carcinoma cells irradiated in G2 fail to arrest progression into mitosis, and S-phase-irradiated cells exhibit radioresistant DNA synthesis. Similar concentrations of caffeine also inhibit gamma- and UV radiation-induced phosphorylation of p53 on Ser15, a modification that may be directly mediated by the ATM and ATR kinases. DNA-dependent protein kinase, another ATM-related protein involved in DNA damage repair, was resistant to the inhibitory effects of caffeine. Likewise, the catalytic activity of the G2 checkpoint kinase, hChk1, was only marginally suppressed by caffeine but was inhibited potently by the structurally distinct radiosensitizer, UCN-01. These data suggest that the radiosensitizing effects of caffeine are related to inhibition of the protein kinase activities of ATM and ATR and that both proteins are relevant targets for the development of novel anticancer agents.
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PMID:Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. 1048 86

The Brca1 (breast cancer gene 1) tumor suppressor protein is phosphorylated in response to DNA damage. Results from this study indicate that the checkpoint protein kinase ATM (mutated in ataxia telangiectasia) was required for phosphorylation of Brca1 in response to ionizing radiation. ATM resides in a complex with Brca1 and phosphorylated Brca1 in vivo and in vitro in a region that contains clusters of serine-glutamine residues. Phosphorylation of this domain appears to be functionally important because a mutated Brca1 protein lacking two phosphorylation sites failed to rescue the radiation hypersensitivity of a Brca1-deficient cell line. Thus, phosphorylation of Brca1 by the checkpoint kinase ATM may be critical for proper responses to DNA double-strand breaks and may provide a molecular explanation for the role of ATM in breast cancer.
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PMID:Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. 1061 May 23

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 Deltamik1 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.
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PMID:Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint. 1063 86

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.
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PMID:Substrate specificity determinants of the checkpoint protein kinase Chk1. 1064 19

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 gamma 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 gamma 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.
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PMID:Characterization of a Saccharomyces cerevisiae homologue of Schizosaccharomyces pombe Chk1 involved in DNA-damage-induced M-phase arrest. 1066 74

Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.
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PMID:The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. 1067 1

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.
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PMID:Rapid destruction of human Cdc25A in response to DNA damage. 1082 53


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