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:2.7.11.22 (cdc2)
8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cell cycle checkpoints that are engaged in response to damaged and unreplicated DNA may serve additional, constitutive functions. In the developing Xenopus laevis embryo, the checkpoint kinase Chk1 is transiently activated at the midblastula transition (MBT), a period of extensive cell cycle remodeling including the acquisition of cell cycle checkpoints. The timing of many cell cycle remodeling events at the MBT, such as the lengthening of cell cycles, depends upon a critical nucleocytoplasmic (N/C) ratio. However, other events, including the degradation of maternal cyclin E, do not depend upon the N/C ratio, and are regulated by an autonomous developmental timer. To better understand what regulates Chk1 activation at the MBT, embryos were treated with aphidicolin, at different developmental times and for different lengths of time, to reduce the DNA content at the MBT. Chk1 was activated at the MBT in these embryos establishing that Chk1 activation occurs independently of the N/C ratio. Cdc25A is normally phosphorylated by Chk1 at the MBT and then degraded. The degradation of Cdc25A demonstrated partial dependence on DNA content, suggesting that factors other than Chk1 regulate its degradation. When the cyclin E developmental timer was disrupted with the Cdk2 inhibitor delta34-Xic1, Chk1 was still activated at the MBT, indicating that activation of Chk1 at the MBT was not directly linked to the cyclin E timer. Conversely, unreplicated or damaged DNA, delayed the degradation of cyclin E at the MBT, indicating that the cyclin E/Cdk2 timer is sensitive to engagement of cell cycle checkpoints.
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PMID:Chk1 is activated at the midblastula transition in Xenopus laevis embryos independently of DNA content and the cyclin E/Cdk2 developmental timer. 1841 41

When cells traversing G(1) are irradiated with UV light, two parallel damage checkpoint pathways are activated: Chk1-Cdc25A and p53-p21(WAF1/CIP1), both targeting Cdk2, but the latter inducing a long lasting arrest. In similarly treated S phase-progressing cells, however, only the Cdc25A-dependent checkpoint is active. We have recently found that the p21-dependent checkpoint can be activated and induce a prolonged arrest if S phase cells are damaged with a base-modifying agent, such as methyl methanesulfonate (MMS) and cisplatin. But the mechanistic basis for the differential activation of the p21-dependent checkpoint by different DNA damaging agents is not understood. Here we report that treatment of S phase cells with MMS but not a comparable dose of UV light elicits proteasome-mediated degradation of Cdc6, the assembler of pre-replicative complexes, which allows induced p21 to bind Cdk2, thereby extending inactivation of Cdk2 and S phase arrest. Consistently, enforced expression of Cdc6 largely eliminates the prolonged S phase arrest and Cdk2 inactivation induced with MMS, whereas RNA interference-mediated Cdc6 knockdown not only prolongs such arrest and inactivation but also effectively activates the p21-dependent checkpoint in the UV-irradiated S phase cells.
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PMID:Cdc6 determines utilization of p21(WAF1/CIP1)-dependent damage checkpoint in S phase cells. 1845 79

Checkpoint pathways inhibit mitotic progression by inducing the phosphorylation of serine 216 in cdc25C resulting in the generation of a 14-3-3 binding site on cdc25C. Two 14-3-3 isoforms, 14-3-3epsilon and 14-3-3gamma form a complex with cdc25C and inhibit cdc25C function. To examine the contribution of 14-3-3gamma to checkpoint regulation, the expression of 14-3-3gamma was inhibited in HCT116 cells using vector based RNA interference. A transient reduction in the expression of 14-3-3gamma in HCT116 cells resulted in an override of both the incomplete S phase and the G(2) DNA damage checkpoint. A 14-3-3gamma knockdown clone also showed an override of both checkpoint pathways. These phenotypes were reversed upon expression of a shRNA resistant 14-3-3gamma cDNA. Override of the G(2) DNA damage checkpoint pathway was accompanied by a decrease in the levels of inhibitory phosphorylation on cdc25C and cdk1. However, there was no difference in the gamma-H2AX foci formation and levels of phospho-chk1 and phospho-chk2, suggesting that activation of the DNA damage checkpoint response and subsequent activation of the checkpoint kinases Chk1 and Chk2 was not perturbed. These results suggest that the override of checkpoint observed in 14-3-3gamma knockdown cells is due to failure to inhibit cdc25C function.
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PMID:14-3-3 Gamma is required to enforce both the incomplete S phase and G2 DNA damage checkpoints. 1884 1

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.
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PMID:Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation. 1898 24

Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.
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PMID:Transient treatment with CDK inhibitors eliminates proliferative potential even when their abilities to evoke apoptosis and DNA damage are blocked. 1906 55

The intrinsic damage response is activated by DNA damage that arises during the cell division process. The ability of the cell to repair this damage during proliferation is important for normal cell growth and, when disrupted, may lead to increased mutagenesis and tumorigenesis. The atypical CDK activator, Spy1, was previously shown to promote cell survival, prevent apoptosis and inhibit checkpoint activation in response to DNA damage. Prior studies have shown that Spy1 is upregulated in breast carcinomas and accelerates mammary tumorigenesis in vivo. In this report, first, we demonstrate that the ability of Spy1 to inhibit apoptosis and bypass UV-induced checkpoint activation is dependent on the presence of the gene regulatory protein p53 and the CKI p21. Second, we demonstrate that Spy1 expression has the following effects: prevents repair of cyclobutane pyrimidine dimers through bypass of nucleotide excision repair; increases the cellular mutation frequency; and reduces the formation of cyclin E induced gammaH2A.X foci. Lastly, we show that knockdown of endogenous Spy1 leads to gammaH2A.X foci formation, Chk1 phosphorylation and proliferation defects, demonstrating a functional role for Spy1 in the intrinsic DNA damage response. These results also demonstrate that Spy1 fulfills a novel regulatory role in the intrinsic DNA damage response and maintains the balance between checkpoint activation, apoptosis, repair and cell cycle progression in response to exogenous or intrinsic damage. Furthermore, the overexpression of Spy1 as a contributing factor in cancer progression will most likely be confined to p53-positive cells.
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PMID:The atypical CDK activator Spy1 regulates the intrinsic DNA damage response and is dependent upon p53 to inhibit apoptosis. 1910 3

The checkpoint mediator protein Claspin facilitates the phosphorylation and activation of Chk1 by ATR and thus is required for efficient DNA replication. However, the physical association of Claspin homologues with replication factors and forks suggests that it might have additional functions in controlling DNA replication. DNA combing was used to examine the functions of Chk1 and Claspin at individual forks and to determine whether Claspin functions independently of Chk1. We find that Claspin, like Chk1, regulates fork stability and density in unperturbed cells. As expected, Chk1 regulates origin firing predominantly by controlling Cdk2-Cdc25 function. By contrast, Claspin functions independently of the Cdc25-Cdk2 pathway in mammalian cells. The findings support a model in which Claspin plays a role regulating replication fork stability that is independent of its function in mediating Chk1 phosphorylation.
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PMID:Claspin and Chk1 regulate replication fork stability by different mechanisms. 1927 May 16

When replication is blocked by a template lesion or polymerase inhibitor while helicase continues unwinding the DNA, single stranded DNA (ssDNA) accumulates and becomes coated with RPA, which then initiates signals via PCNA mono-ubiquitination to activate trans-lesion polymerases and via ATR and Chk1 to inhibit Cdk2-dependent cell cycle progression. The signals are conveyed by way of a complex network of molecular interactions. To clarify those complexities, we have constructed a molecular interaction map (MIM) using a novel hierarchical assembly procedure. Molecules were arranged on the map in hierarchical levels according to interaction step distance from the DNA region of stalled replication. The hierarchical MIM allows us to disentangle the network's interlocking pathways and loops and to suggest functionally significant features of network architecture. The MIM shows how parallel pathways and multiple feedback loops can provide failsafe and robust switch-like responses to replication stress. Within the central level of hierarchy ATR and Claspin together appear to function as a nexus that conveys signals from many sources to many destinations. We noted a division of labor between those two molecules, separating enzymatic and structural roles. In addition, the network architecture disclosed by the hierarchical map, suggested a speculative model for how molecular crowding and the granular localization of network components in the cell nucleus can facilitate function.
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PMID:Network architecture of signaling from uncoupled helicase-polymerase to cell cycle checkpoints and trans-lesion DNA synthesis. 1955 79

The role of the mismatch repair (MMR) system in correcting base-base mismatches is well established; its involvement in the response to DNA double strand breaks, however, is less clear. We investigated the influence of the essential component of MMR, the hMLH1 protein, on the cellular response to DNA-double strand breaks induced by treatment with SN-38, the active metabolite of topoisomerase I inhibitor irinotecan, in a strictly isogenic cell system (p53(wt), hMLH1(+)/p53(wt), hMLH1(-)). By using hMLH1 expressing clones or cells transduced with the hMLH1-expressing adenovirus as well as siRNA technology, we show that in response to SN-38-induced DNA damage the MMR proficient (MMR(+)) cells make: (i) a stronger G2/M arrest, (ii) a subsequent longer tetraploid G1 arrest, (iii) a stronger activation of Chk1 and Chk2 kinases than the MMR deficient (MMR(-)) counterparts. Both Cdk2 and Cdk4 kinases contribute to the basal tetraploid G1 arrest in MMR(+) and MMR(-) cells. Although the Chk1 kinase is involved in the G2/M arrest, neither Chk1 nor Chk2 are involved in the enhancement of the tetraploid G1 arrest. The long-lasting tetraploid G1 arrest of MMR(+) cells is associated with their lower clonogenic survival after SN-38 treatment, the abrogation of the tetraploid G1 arrest resulted in their better clonogenic survival. These data show that the stabilization of the tetraploid G1 arrest in response to double strand breaks is a novel function of the MMR system that contributes to the lesser survival of MMR(+) cells.
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PMID:Mismatch repair system decreases cell survival by stabilizing the tetraploid G1 arrest in response to SN-38. 1973 70

Cyclin E is a regulator of cyclin-dependent protein kinases (Cdks) and is involved in mediating the cell cycle transition from G(1) to S phase. Here, we describe a novel function for cyclin E in the long term maintenance of checkpoint arrest in response to replication barriers. Exposure of cells to mitomycin C or UV irradiation, but not ionizing radiation, induces stabilization of cyclin E. Stabilization of cyclin E reduces the activity of Cdk2-cyclin A, resulting in a slowing of S phase progression and arrest. In addition, cyclin E is shown to be required for stabilization of Cdc6, which is required for activation of Chk1 and the replication checkpoint pathway. Furthermore, the stabilization of cyclin E in response to replication fork barriers depends on ATR, but not Nbs1 or Chk1. These results indicate that in addition to its well studied role in promoting cell cycle progression, cyclin E also has a role in regulating cell cycle arrest in response to DNA damage.
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PMID:Cyclin E is stabilized in response to replication fork barriers leading to prolonged S phase arrest. 1981 34


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