Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mammalian Chk1 and Chk2 protein kinases are two important components of the G(2) DNA damage checkpoint. They are activated by upstream kinases (ataxia telangectasia mutated gene and ATM and Rad 3 related gene) and interfere with the activity of the cdc2/cyclinB1 complex, necessary for the G(2)-M transition, through the inactivation of the cdc25 phosphatases (cdc25A and cdc25C). To understand the role of Chk1 and Chk2 in the cellular response to different anticancer agents, we knocked down the expression of each protein or simultaneously of both proteins by using the small interfering RNA technique in the HCT-116 colon carcinoma cell line and in its isogenic systems in which p53 and p21 have been inactivated by targeted homologous recombination. We here show that inhibition of Chk1 but not of Chk2 in p21(-/-) and p53(-/-) cells caused a greater abrogation of G(2) block induced by ionizing radiation and cis-diamine-dichloroplatinum treatments and a greater sensitization to the same treatments than in the parental cell line with p53 and p21 wild type proteins. These data further emphasise the role of Chk1 as a molecular target to inhibit in tumors with a defect in the G(1) checkpoint with the aim of increasing the selectivity and specificity of anticancer drug treatments.
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PMID:Chk1, but not Chk2, is involved in the cellular response to DNA damaging agents: differential activity in cells expressing or not p53. 1532 76

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.
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PMID:A role for the Fanconi anemia C protein in maintaining the DNA damage-induced G2 checkpoint. 1537 54

Response to DNA damage and cell-cycle regulation differ markedly between embryonic stem (ES) cells and somatic cells. ES cells require exquisitely sensitive mechanisms to maintain genomic integrity and do so, in part, by suppressing spontaneous mutation. Spontaneous mutation frequency in somatic cells is approximately 10(-4) compared with 10(-6) for ES cells. ES cells also lack a G(1) checkpoint and are hypersensitive to IR and other DNA-damaging agents. These characteristics facilitate apoptosis and the removal of cells with a mutational burden from the population, thereby keeping the population free of damaged cells. Here, we identify signaling pathways that are compromised and lead to a natural absence of aG(1) arrest in ES cells after DNA damage. The affected pathways are those mediated by p53 and p21 and by ATM, Chk2, Cdc25A, and Cdk2. In ES cells, Chk2 kinase is not intranuclear as in somatic cells but is sequestered at centrosomes and is unavailable to phosphorylate Cdc25A phosphatase and cause its degradation. Although ectopic expression of Chk2 does not rescue the p53/p21 pathway, its expression is sufficient to allow it to phosphorylate Cdc25A, activate downstream targets, restore a G(1) arrest, and protect the cell from apoptosis.
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PMID:Restoration of an absent G1 arrest and protection from apoptosis in embryonic stem cells after ionizing radiation. 1545 51

Initiation of DNA replication occurs at origins of replication, traditionally defined by specific sequence elements. Sequence-dependent initiation of replication is the rule in prokaryotes and in the yeast Saccharomyces cereviseae. However, sequence-dependent initiation does not appear to be absolutely required in metazoan eukaryotes. Origin firing is instead likely dependent on stochastic initiation from chromatin-defined loci, despite the demonstration of some specific origins. Based on some recent observations in Xenopus laevis egg extracts and in mammalian cell culture, we propose that timing of origin firing is dependent on feedback from active replicons. This dynamic regulation of replication is mediated by sensing of ongoing replication by the DNA-damage checkpoint kinases ATM and ATR, which in turn downregulate neighboring and distal origins and replicons by inhibition of the S-phase kinases Cdk2 and Cdc7 and by inhibition of the replicative Mcm helicase. Origin selection, activation, and replicon progression are therefore constrained in both space and time via feedback from the cell cycle and ongoing replication.
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PMID:ATM and ATR check in on origins: a dynamic model for origin selection and activation. 1565 72

Cyclin-dependent kinases (Cdk) promote cell proliferation, are often deregulated in human cancers, and are targets of ongoing cancer chemotherapy trials. We show here that Cdk activity is also required in human cells to maintain function of the Chk1 pathway, a key component of the response to DNA damage or stalled replication. Chk1 expression was markedly reduced in primary fibroblasts and U2OS osteogenic sarcoma cells by treatment with small molecule Cdk inhibitors or induction of a dominant-negative mutant of Cdk2. The findings of decreased Chk1 activity and accumulation of Cdc25A, a protein targeted for degradation by Chk1, confirmed that Chk1 function was impaired. Furthermore, Cdk inhibition triggered a DNA damage response, characterized by the accumulation of activated forms of ATM and Chk2 as well as nuclear foci containing phosphorylated substrates of ATM/ATR, including histone H2AX (gammaH2AX). Time course experiments showed that the bulk of ATM activation followed Chk1 down-regulation. Chk1 RNA interference combined with partial inhibition of DNA replication was sufficient to evoke the DNA damage response. Conversely, ectopic expression of Chk1 blunted induction of gammaH2AX foci by Cdk inhibitors, indicating that Chk1 down-regulation was necessary to elicit the full phenotype. Finally, both Cdk and Chk1 inhibitors enhanced the cytotoxity of etoposide, a DNA-damaging agent. These results define a pathway through which Cdk inhibition can mediate DNA damage and potentially enhance the efficacy of extant cancer chemotherapies.
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PMID:Cdk inhibition in human cells compromises chk1 function and activates a DNA damage response. 1570 74

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.
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PMID:Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. 1583 61

The regulation of a pre-replicative complex (pre-RC) at origins ensures that the genome is replicated only once per cell cycle. Cdt1 is an essential component of the pre-RC that is rapidly degraded at G1-S and also inhibited by Geminin (Gem) protein to prevent re-replication. We have previously shown that destruction of the Drosophila homolog of Cdt1, Double-parked (Dup), at G1-S is dependent upon cyclin-E/CDK2 and important to prevent re-replication and cell death. Dup is phosphorylated by cyclin-E/Cdk2, but this direct phosphorylation was not sufficient to explain the rapid destruction of Dup at G1-S. Here, we present evidence that it is DNA replication itself that triggers rapid Dup destruction. We find that a range of defects in DNA replication stabilize Dup protein and that this stabilization is not dependent on ATM/ATR checkpoint kinases. This response to replication stress was cell-type specific, with neuroblast stem cells of the larval brain having the largest increase in Dup protein. Defects at different steps in replication also increased Dup protein during an S-phase-like amplification cell cycle in the ovary, suggesting that Dup stabilization is sensitive to DNA replication and not an indirect consequence of a cell-cycle arrest. Finally, we find that cells with high levels of Dup also have elevated levels of Gem protein. We propose that, in cycling cells, Dup destruction is coupled to DNA replication and that increased levels of Gem balance elevated Dup levels to prevent pre-RC reformation when Dup degradation fails.
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PMID:Levels of the origin-binding protein Double parked and its inhibitor Geminin increase in response to replication stress. 1614 Dec 38

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins and effector proteins, the cyclomodulins, that deregulate the host cell cycle. Upon injection into HeLa cells by the enteropathogenic Escherichia coli (EPEC) type III secretion system, Cif induces a cytopathic effect characterized by the recruitment of focal adhesion plates and the formation of stress fibres, an irreversible cell cycle arrest at the G(2)/M transition, and sustained inhibitory phosphorylation of mitosis inducer, CDK1. Here, we report that the reference typical EPEC strain B171 produces a functional Cif and that lipid-mediated delivery of purified Cif into HeLa cells induces cell cycle arrest and actin stress fibres, implying that Cif is necessary and sufficient for these effects. EPEC infection of intestinal epithelial cells (Caco-2, IEC-6) also induces cell cycle arrest and CDK1 inhibition. The effect of Cif is strikingly similar to that of cytolethal distending toxin (CDT), which inhibits the G(2)/M transition by activating the DNA-damage checkpoint pathway. However, in contrast to CDT, Cif does not cause phosphorylation of histone H2AX, which is associated with DNA double-stranded breaks. Following EPEC infection, the checkpoint effectors ATM/ATR, Chk1 and Chk2 are not activated, the levels of the CDK-activating phosphatases Cdc25B and Cdc25C are not affected, and Cdc25C is not sequestered in host cell cytoplasm. Hence, Cif activates a DNA damage-independent signalling pathway that leads to inhibition of the G(2)/M transition.
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PMID:Escherichia coli cyclomodulin Cif induces G2 arrest of the host cell cycle without activation of the DNA-damage checkpoint-signalling pathway. 1684 90

Bloom syndrome (BS) is a rare human autosomal recessive disorder characterized by marked genetic instability associated with greatly increased predisposition to a wide range of cancers affecting the general population. BS arises through mutations in both copies of the BLM gene which encodes a 3'-5' DNA helicase identified as a member of the RecQ family. Several studies support a major role for BLM in the cellular response to DNA damage and stalled replication forks. However, the specific function(s) of BLM remain(s) unclear. The BLM protein is strongly expressed and phosphorylated during mitosis, but very little information is available about the origin and the significance of this phosphorylation. We show here that ATM kinase provides only a limited contribution to the mitotic phosphorylation of BLM. We also demonstrate that BLM is directly phosphorylated at multiple sites in vitro by the mitotic cdc2 kinase, and identify two new sites of mitotic BLM phosphorylation: Ser-714 and Thr-766. Our results identify BLM helicase as a new substrate for cdc2, which may have potential physiological implications for the role of BLM in mitosis.
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PMID:The Bloom syndrome helicase is a substrate of the mitotic Cdc2 kinase. 1688 Jul 35

Among the three mammalian genes encoding DNA ligases, only the LIG3 gene does not have a homolog in lower eukaryotes. In somatic mammalian cells, the nuclear form of DNA ligase IIIalpha forms a stable complex with the DNA repair protein XRCC1 that is also found only in higher eukaryotes. Recent studies have shown that XRCC1 participates in S phase-specific DNA repair pathways independently of DNA ligase IIIalpha and is constitutively phosphorylated by casein kinase II. In this study we demonstrate that DNA ligase IIIalpha, unlike XRCC1, is phosphorylated in a cell cycle-dependent manner. Specifically, DNA ligase IIIalpha is phosphorylated on Ser123 by the cell division cycle kinase Cdk2 beginning early in S phase and continuing into M phase. Interestingly, treatment of S phase cells with agents that cause oxygen free radicals induces the dephosphorylation of DNA ligase IIIalpha. This oxidative stress-induced dephosphorylation of DNA ligase IIIalpha is dependent upon the ATM (ataxia-telangiectasia mutated) kinase and appears to involve inhibition of Cdk2 and probably activation of a phosphatase.
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PMID:ATM mediates oxidative stress-induced dephosphorylation of DNA ligase IIIalpha. 1704 Aug 96


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