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Query: UMLS:C0004135 (ATM)
 13,001 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of the cyclin kinase inhibitor, p21, is regulated both transcriptionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p21 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppressed the ubiquitination of p21. In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cells resulting from 15 Gy gamma-irradiation was diminished by Wortmannin or LY294002 pretreatment of cells. However, the levels of p21 mRNA were not affected by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after gamma-irradiation to a lesser degree than that of p21. In addition, we examined the involvement of DNA-PK, whose activity is inhibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibroblast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by gamma-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY294002, was evaluated. ATM deficient cells induced p21 after gamma-irradiation, gamma-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclude that the p21 stabilization mechanism functions after gamma-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor ATM gene product for activity.
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PMID:Phosphatidylinositol 3-kinase inhibitors, Wortmannin or LY294002, inhibited accumulation of p21 protein after gamma-irradiation by stabilization of the protein. 1077 Oct 89

Activation of the p53-stress response pathway has been implicated in excitotoxic neuronal cell death. Recent studies have demonstrated an age-dependent induction of both p53 mRNA and protein in the rat brain following lithium-pilocarpine-mediated status epilepticus (LPSE). We investigated whether other proteins that have been shown to participate in the p53 cascade are induced by LPSE. We used immunohistochemistry to examine the expression of Mdm2, Bax, CD95/Fas/APO-1, ATM, Ref-1 and ubiquitin. A significant increase in nuclear Mdm2 immunoreactivity, which colocalized with p53, was observed in cells within hippocampal pyramidal cell layers, dentate gyrus, piriform cortex, amygdala and thalamus. Dual immunofluorescence microscopy revealed a reduction in free ubiquitin expression in cells with p53 and Mdm2 accumulation. Increased immunoreactivity for CD95/Fas/APO-1 and Bax was also detected in the same p53-positive cells. Moreover, expression of Ref-1 and ATM, which are involved in the response to oxidative stress-induced DNA damage and regulation of p53 function, were increased. Colocalization of Ref-1 and p53 suggests that Ref-1 might activate p53 function in LPSE-induced neurodegeneration. In contrast, ATM immunoreactivity was predominantly cytoplasmic suggesting that ATM may not directly modulate p53 activity in injured neurons. These results extend our previous observations with regard to activation and stabilization of p53 in injured central nervous system neurons. The data indicate that p53 induction following LPSE may activate downstream pro-apoptotic genes leading to neurodegeneration.
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PMID:Immunohistochemical study of p53-associated proteins in rat brain following lithium-pilocarpine status epilepticus. 1185 39

The human genetic disorder ataxia-telangiectasia (A-T) is due to lack of functional ATM, a protein kinase which is involved in cellular responses to DNA double strand breaks (DSBs) and possibly other oxidative stresses, as well as in regulation of several fundamental cellular functions. Studies regarding responses in A-T cells to the induction of DSBs utilize ionizing radiation or radiomimetic chemicals, such as neocarzinostatin (NCS), which induce DNA DSBs. This critical DNA lesion activates many defense systems, such as the cell cycle checkpoints. The cell cycle is also regulated through a timed and coordinated degradation of regulatory proteins via the ubiquitin pathway. Our recent studies indicate that the ubiquitin pathway is influenced by the cellular redox status and that it is the major cellular pathway for removal of oxidized proteins. Accordingly, we hypothesized that the absence of a functional ATM protein might involve perturbations to the ubiquitin pathway as well. We show here that upon treatment with NCS, there was a transient 50-70% increase in endogenous ubiquitin conjugates in A-T and wt lymphoblastoid cells. Ubiquitin conjugation capabilities per se and levels of substrates for conjugation were also similarly enhanced in wt and A-T cells upon NCS treatment. We also compared the ubiquitination response in A-T and wt cells using H(2)O(2) as the stress, in view of preexisting evidence of the effects of H(2)O(2) on ubiquitination capabilities in other types of cells. As with NCS treatment, there was an approximately 45% increase in endogenous ubiquitin conjugates by 2-4 h after exposure to H(2)O(2). Both cell types showed a rapid 50-150% increase in de novo formed 125I-ubiquitin conjugates. As compared with wt cells, unexposed A-T cells had higher endogenous levels of conjugates and enhanced conjugation capability. However, A-T cells mounted a more muted ubiquitination response to the stress. The enhanced ubiquitin conjugation in unstressed A-T cells and attenuated ability of these cells to respond to stress are consistent with the A-T cells being under oxidative stress and with their having an 'aged' phenotype. The indication that ubiquitin conjugate levels and ubiquitin conjugation capabilities are enhanced upon oxidative stress without significant changes in GSSG/GSH ratios indicates that assays of ubiquitination provide a sensitive measure of cellular stress. The data also add support to the impression that potentiated ubiquitination response to mild oxidative stress is a generalizable phenomenon.
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PMID:Ubiquitination capabilities in response to neocarzinostatin and H(2)O(2) stress in cell lines from patients with ataxia-telangiectasia. 1208 Apr 67

Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. Here, we show that CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. We show that radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. Our study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage.
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PMID:Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. 1457 10

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.
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PMID:Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage. 1460 23

The transcription factor NF-kappaB is critical for setting the cellular sensitivities to apoptotic stimuli, including DNA damaging anticancer agents. Central to NF-kappaB signaling pathways is NEMO/IKKgamma, the regulatory subunit of the cytoplasmic IkappaB kinase (IKK) complex. While NF-kappaB activation by genotoxic stress provides an attractive paradigm for nuclear-to-cytoplasmic signaling pathways, the mechanism by which nuclear DNA damage modulates NEMO to activate cytoplasmic IKK remains unknown. Here, we show that genotoxic stress causes nuclear localization of IKK-unbound NEMO via site-specific SUMO-1 attachment. Surprisingly, this sumoylation step is ATM-independent, but nuclear localization allows subsequent ATM-dependent ubiquitylation of NEMO to ultimately activate IKK in the cytoplasm. Thus, genotoxic stress induces two independent signaling pathways, SUMO-1 modification and ATM activation, which work in concert to sequentially cause nuclear targeting and ubiquitylation of free NEMO to permit the NF-kappaB survival pathway. These SUMO and ubiquitin modification pathways may serve as anticancer drug targets.
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PMID:Sequential modification of NEMO/IKKgamma by SUMO-1 and ubiquitin mediates NF-kappaB activation by genotoxic stress. 1465 48

Eukaryotic cells respond to DNA damage and stalled replication forks by activating protein kinase-mediated signaling pathways that promote cell cycle arrest and DNA repair. A central target of the cell cycle arrest program is the Cdc25A protein phosphatase. Cdc25A is required for S-phase entry and dephosphorylates tyrosine-15 phosphorylated Cdk1 (Cdc2) and Cdk2, positive regulators of cell division. Cdc25A is unstable during S-phase and is degraded through the ubiquitin-proteasome pathway, but its turnover is enhanced in response to DNA damage. Although basal and DNA-damage-induced turnover depends on the ATM-Chk2 and ATR-Chk1 pathways, how these kinases engage the ubiquitin ligase machinery is unknown. Here, we demonstrate a requirement for SCFbeta-TRCP in Cdc25A turnover during an unperturbed cell cycle and in response to DNA damage. Depletion of beta-TRCP stabilizes Cdc25A, leading to hyperactive Cdk2 activity. SCFbeta-TRCP promotes Chk1-dependent Cdc25A ubiquitination in vitro, and this involves serine 76, a known Chk1 phosphorylation site. However, recognition of Cdc25A by beta-TRCP occurs via a noncanonical phosphodegron in Cdc25A containing phosphoserine 79 and phosphoserine 82, sites that are not targeted by Chk1. These data indicate that Cdc25A turnover is more complex than previously appreciated and suggest roles for an additional kinase(s) in Chk1-dependent Cdc25A turnover.
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PMID:SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase. 1468 Dec 6

In eukaryotic cells, control mechanisms of cell-cycle progression have evolved to accurately monitor the integrity of genetic information to be transferred to the progeny. Cdc25A phosphatase is an essential activator of cell-cycle progression and is targeted by checkpoint signals. Ubiquitylation regulates Cdc25A activity through fine tuning of its protein levels. Two different ubiquitin ligases (APC/C and SCF complex) are involved in Cdc25A turnover. While APC/C is involved in regulating Cdc25A at the exit of mitosis, SCF regulates the abundance of Cdc25A in S phase and G2. In response to DNA damage or to stalled replication, the activation of the ATM and ATR protein kinases leads to Chk1 and Chk2 activation and to Cdc25A hyperphosphorylation. These events stimulate SCF-mediated ubiquitylation of Cdc25A and its proteolysis. This contributes to delaying cell-cycle progression, thereby preventing genomic instability. Based on recent findings, we discuss the role of Cdc25A ubiquitylation and degradation in cell-cycle progression and in response to DNA damage. Moreover, we discuss the role of phosphorylation at multiple sites in triggering ubiquitylation signals.
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PMID:Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis. 1502 92

Checkpoint kinase 2 (Chk2) is one of the critical kinases governing the cell cycle checkpoint, DNA damage repair, and cell apoptosis in response to DNA damaging signals. In the present report, we demonstrate that Chk2 kinase is degraded at the protein level in response to cisplatin through ubiquitin-proteasome pathway. This degradation was independent of the Thr68 phosphorylation, ATM kinase, and BRCA1 tumor suppressor. Examination of Chk2 protein revealed a decreased expression of Chk2 protein in cisplatin-resistant ovarian cancer cell lines, suggesting that degradation or decreased expression of Chk2 is partially responsible for chemo-resistance. Site-directed mutation of the putative destruction box in the Chk2 protein did not affect the Chk2 degradation induced by cisplatin. Therefore, these results are the first to indicate a novel mechanism of regulating Chk2 in cisplatin-induced resistance of cancer cells.
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PMID:Inducible degradation of checkpoint kinase 2 links to cisplatin-induced resistance in ovarian cancer cells. 1569 85

Drosophila telomeres are maintained by transposition of specialized retrotransposons rather than by telomerase activity, and their stability is independent of the sequence of DNA termini. Recent studies have identified several proteins that protect Drosophila telomeres from fusion events. These proteins include the telomere capping factors HP1/ORC-associated protein (HOAP) and heterochromatin protein 1 (HP1), the Rad50 and Mre11 DNA repair proteins that are required for HOAP and HP1 localization at telomeres, and the ATM kinase. Another telomere-protecting factor identified in Drosophila is UbcD1, a polypeptide highly homologous to class I ubiquitin-conjugating E2 enzymes. In addition, it has been shown that HP1 and both components of the Drosophila Ku70/80 heterodimer act as negative regulators of telomere length. Except for HOAP, all these proteins are conserved in humans and are associated with human telomeres. Collectively, these results indicate that Drosophila is an excellent model system for the analysis of the mechanisms of telomere maintenance. In past and current studies, 15 Drosophila genes have been identified that prevent telomeric fusion, and it has been estimated that the Drosophila genome contains at least 40 genes required for telomere protection. We believe that the molecular characterization of these genes will lead to identification of many novel human genes with roles in telomere maintenance.
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PMID:The mechanism of telomere protection: a comparison between Drosophila and humans. 1601 58


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