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)

Mammalian CHK1 is a Ser/Thr effector kinase that plays critical roles in the DNA damage-activated cell cycle checkpoint signaling pathway downstream of ATR (ATM and Rad3-related protein kinase). This chapter is focused on describing an assay to measure CHK1 activity in vitro. The basic mechanism of this assay is to observe the phosphorylated levels of a fragment of CDC25C containing the site that can be phosphorylated by CHK1 in vitro. This assay includes five major steps: (1) preparing extracts from the control or treated cells; (2) preparing substrate; (3) immunoprecipitating CHK1 protein from the cells; (4) assembling the kinase assay; and (5) analyzing the phosphorylated level of the substrates by CHK1. Besides CHK1, CHK2 is another important checkpoint regulator that responds to DNA damage. Because CHK1 and CHK2 share some substrates such as CDC25C in vitro, this assay could also be used for CHK2 activity assay, except that the CHK2 antibody will replace the CHK1 antibody.
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PMID:CHK1 kinase activity assay. 1522 May 26

Recent studies have identified, hSMG-1 as the newest member of the phosphoinositide 3-kinase(PI3-kinase)-related kinase (PIKK) family. The protein kinase activity of hSMG-1 resembles that of the related PIKK, ATM, both in terms of substrate specificity and its sensitivity to inhibition by the fungal metabolite wortmannin. hSMG-1 is the ortholog of a Caenorhabditis elegans protein, CeSMG-1, which has been genetically linked to a critical mRNA surveillance pathway termed nonsense-mediated decay (NMD). The function of NMD is to mark for rapid degradation mRNAs that bear a premature termination codon. Compelling evidence now indicates that hSMG-1 is also a central player in the NMD pathway in human cells. In addition, hSMG-1, like ATM, appears to be involved in the recognition and/or repair of damaged DNA in these cells. In this review, we introduce a model in which hSMG-1 teams with ATM and ATR to insure the overall quality of the transcriptome in human cells.
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PMID:The ATM-related kinase, hSMG-1, bridges genome and RNA surveillance pathways. 1527 77

Genotoxic stress activates checkpoint signaling pathways that block cell cycle progression, trigger apoptosis, and regulate DNA repair. Studies in yeast and humans have shown that Rad9, Hus1, Rad1, and Rad17 play key roles in checkpoint activation. Three of these proteins-Rad9, Hus1, and Rad1-interact in a heterotrimeric complex (dubbed the 9-1-1 complex), which resembles a PCNA-like sliding clamp, whereas Rad17 is part of a clamp-loading complex that is related to the PCNA clamp loader, replication factor-C (RFC). In response to genotoxic damage, the 9-1-1 complex is loaded around DNA by the Rad17-containing clamp loader. The DNA-bound 9-1-1 complex then facilitates ATR-mediated phosphorylation and activation of Chk1, a protein kinase that regulates S-phase progression, G2/M arrest, and replication fork stabilization. In addition to its role in checkpoint activation, accumulating evidence suggests that the 9-1-1 complex also participates in DNA repair. Taken together, these findings suggest that the 9-1-1 clamp is a multifunctional complex that is loaded onto DNA at sites of damage, where it coordinates checkpoint activation and DNA repair.
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PMID:Dial 9-1-1 for DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex. 1527 87

Eukaryotic cells slow their progression through S phase upon DNA damage. The mechanism that leads to this slowing is called the intra-S-phase checkpoint. Previous studies demonstrated that in the fission yeast Schizosaccharomyces pombe this checkpoint is mediated by a pathway that includes Rad3 (similar to human ATR and ATM) and Cds1 (similar to human Chk1 and Chk2). Here we present evidence that a major downstream target of this pathway is the cyclin-dependent kinase, Cdc2. We also present evidence suggesting that the intra-S-phase checkpoint makes a relatively minor contribution to the survival of cells with damaged DNA.
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PMID:On the slowing of S phase in response to DNA damage in fission yeast. 1529 57

Genetically distinct checkpoints, activated as a consequence of either DNA replication arrest or ionizing radiation-induced DNA damage, integrate DNA repair responses into the cell cycle programme. The ataxia-telangiectasia mutated (ATM) protein kinase blocks cell cycle progression in response to DNA double strand breaks, whereas the related ATR is important in maintaining the integrity of the DNA replication apparatus. Here, we show that thymidine, which slows the progression of replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, depends on both ATM and ATR. Thymidine induces ATM-mediated phosphorylation of Chk2 and NBS1 and an ATM-independent phosphorylation of Chk1 and SMC1. AT cells exposed to thymidine showed decreased viability and failed to induce homologous recombination repair (HRR). Taken together, our results implicate ATM in the HRR-mediated rescue of replication forks impaired by thymidine treatment.
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PMID:ATM is required for the cellular response to thymidine induced replication fork stress. 1545 81

Eukaryotic cells regulate progression through the cell cycle in response to DNA damage. Cell cycle checkpoints are the signal transduction pathways that couple the detection of DNA damage to the proteins that control transitions in the cell cycle. The protein kinase Chk1, originally discovered in fission yeast, but conserved in humans, is essential for preventing mitotic entry in the presence of DNA damage or blocks to DNA replication that cannot be reconciled. Chk1 is phosphorylated in response to DNA damage. Phosphorylation depends on the activity of conserved components of the checkpoint pathway including Rad3, a member of the ATM/ATR family of kinases. Phosphorylation leads to activation of Chk1 kinase activity. In this chapter, we describe an assay for monitoring the activity of Chk1 isolated.
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PMID:Assaying cell cycle checkpoints: activity of the protein kinase Chk1. 1557 43

Both the incidence and severity of asthma in women are influenced by fluctuations in estrogen (E(2)) levels, raising the possibility that E(2)s may reduce the hyperresponsiveness that is characteristic of asthma. We examined the effect of E(2) and its downstream signaling pathways in isolated mouse bronchial and tracheal rings passively sensitized either with serum from patients with atopic asthma (ATR) or with serum from control subjects (CTR). ATR exhibited significantly higher sensitivity to carbachol than CTR. Pretreatment of ATR with E(2) shifted the carbachol concentration-response curve (CCRC) toward that of CTR. The E(2) effect was abolished by the nitric oxide synthase inhibitor, L-nitroarginine methyl ester, the soluble guanyl cyclase inhibitor, quinoxalin-1, or the protein kinase G inhibitor, KT5823. Inhibition of the large-conductance, calcium-activated potassium (BK(Ca)) channel activity with iberiotoxin also attenuated the E(2) effect on ATR. In patch-clamp studies, E(2) increased by 50-fold the BK(Ca) channel activity in freshly isolated airway smooth muscle cells. This increase was completely blocked by KT5823. These studies suggest that, at physiologic concentrations, E(2) can prevent cholinergic-induced constriction of asthmatic tracheal rings by activating the nitric oxide-cGMP-protein kinase G pathway to increase BK(Ca) channel activity.
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PMID:Estrogen reduces carbachol-induced constriction of asthmatic airways by stimulating large-conductance voltage and calcium-dependent potassium channels. 1562 73

Double-stranded DNA breaks (DSBs) are a particularly dangerous form of DNA damage because they can lead to chromosome loss, translocations or truncations. When DSBs occur, many proteins are recruited to the break site; these proteins serve to both initiate DNA repair and to activate a checkpoint response. Repair occurs via one of two pathways: non-homologous end-joining (NHEJ), in which broken DNA ends are directly ligated; or homologous recombination (HR), in which a homologous chromosome is used as a template in a replicative repair process. The checkpoint response is mediated by the phosphatidyl inositol 3-kinase-like kinases, Mec1 and Tel1 (ATR and ATM in humans, respectively). Two recent studies in yeast have significantly increased our understanding of when each of the proteins involved in these processes is localized to a break and, in addition, how their sequential localization is achieved. Specifically, these studies support and expand upon a model in which Tel1 and the NHEJ proteins are the first proteins to localize to the break to initiate signaling and attempt repair, but are subsequently replaced by Mec1 and the HR proteins. This transition is mediated by a cyclin-dependent kinase-dependent initiation of 5'-->3' processing (resection) of the DSB. Thus, the cell-cycle stage at which DSBs occur affects the way in which the DSBs are processed and recognized.
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PMID:Damage in transition. 1569 49

Cell-cycle checkpoints induced by DNA damage or replication play critical roles in the maintenance of genomic integrity during cell proliferation. Biochemical analysis of checkpoint pathways has been greatly facilitated by the use of cell-free systems made from Xenopus eggs. In the present study, we describe a human cell-free system that reproduces a DNA-dependent checkpoint pathway acting on the Chk1 protein kinase. In this system, double-stranded DNA oligonucleotides induce the phosphorylation of Chk1 at activating sites targeted by ATR [ATM (ataxia telangiectasia mutated)- and Rad3-related] and ATM kinases. Phosphorylation of Chk1 is dependent on the interaction of Claspin, a protein first identified in Xenopus as a Chk1-binding protein. We show that the DNA-dependent binding of Chk1 to Claspin requires two phosphorylation sites, Thr916 and Ser945, which lie within the Chk1-binding domain of Claspin. Using a phosphopeptide derived from the consensus motif of these sites, we show that the interaction of Claspin with Chk1 is required for the ATR/ATM-dependent phosphorylation of Chk1. Using a panel of protein kinase inhibitors, we provide evidence that Chk1 is phosphorylated at an additional site in response to activation of the checkpoint response, probably by autophosphorylation. Claspin is phosphorylated in the Chk1-binding domain in an ATR/ATM-dependent manner and is also targeted by additional kinases in response to double-stranded DNA oligonucleotides. This cell-free system will facilitate further biochemical analysis of the Chk1 pathway in humans.
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PMID:DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system. 1570 91

In the present study, we employed a well established JB6 mouse epithelial cell model to define the molecular mechanism of efficacy of a naturally occurring flavonoid silibinin against ultraviolet B (UVB)-induced skin tumorigenesis. UVB exposure of cells caused a moderate phosphorylation of ERK1/2 and Akt and a stronger phosphorylation of p53 at Ser(15), which was enhanced markedly by silibinin pretreatment. Kinase activity of ERK1/2 for Elk-1 and Akt for glycogen synthase kinase-3beta was also potently enhanced by silibinin pretreatment. Furthermore, silibinin increased the UVB-induced level of cleaved caspase 3 as well as apoptotic cells. Based on these observations, next we investigated the role of upstream kinases, ATM/ATR and DNA-PK, which act as sensors for UVB-induced DNA damage and transduce signals leading to DNA repair or apoptosis. Whereas UVB strongly activated ATM as observed by Ser(1981) phosphorylation, it was not affected by silibinin pretreatment. However, pretreatment of cells with the DNA-protein kinase (PK) inhibitor LY294002 strongly reversed silibinin-enhanced Akt-Ser(473) and p53-Ser(15) as well as ERK1/2 phosphorylation together with a dose-dependent decrease in cleaved caspase 3 and apoptosis (p < 0.05). In addition, silibinin pretreatment strongly enhanced H2A.X-Ser(139) phosphorylation and DNA-PK-associated kinase activity as well as the physical interaction of p53 with DNA-PK; pretreatment of cells with LY294002 but not caffeine abolished the silibinin-caused increase in both DNA-PK activation and p53-Ser(15) phosphorylations. Together, these findings suggest that silibinin preferentially activates the DNA-PK-p53 pathway for apoptosis in response to UVB-induced DNA damage, and that this could be a predominant mechanism of silibinin efficacy against UVB-induced skin cancer.
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PMID:Silibinin up-regulates DNA-protein kinase-dependent p53 activation to enhance UVB-induced apoptosis in mouse epithelial JB6 cells. 1579 56


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