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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Members of the phosphoinositide-3-kinase-related kinase (PIKK) family, which includes mTOR, ATM, ATR, and hSMG-1, play important roles in regulating the cellular response to environmental stimuli. Despite the similarity of their catalytic domain to that of phosphoinositide-3-kinase, these extremely large (>250 kDa) polypeptides function as serine/threonine protein kinases. The catalytic activities of these PIKK family members can now be measured in immune-complex kinase assays. This assay involves isolation of the kinase by immunoprecipitation and the in vitro phosphorylation of a specific substrate in the presence of radio-labeled ATP. Here we describe, in detail, the determination of PIKK catalytic activity with a standardized immune-complex kinase assay protocol.
Methods Mol Biol 2004
PMID:Determination of the catalytic activities of mTOR and other members of the phosphoinositide-3-kinase-related kinase family. 1522 May 25

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.
Methods Mol Biol 2004
PMID:CHK1 kinase activity assay. 1522 May 26

Mammalian ATR and ATM checkpoint kinases modulate chromatin structures near DNA breaks by phosphorylating a serine residue in the carboxy-terminal tail SQE motif of histone H2AX. Histone H2A is similarly regulated in Saccharomyces cerevisiae. The phosphorylated forms of H2AX and H2A, known as gamma-H2AX and gamma-H2A, are thought to be important for DNA repair, although their evolutionarily conserved roles are unknown. Here, we investigate gamma-H2A in the fission yeast Schizosaccharomyces pombe. We show that formation of gamma-H2A redundantly requires the ATR/ATM-related kinases Rad3 and Tel1. Mutation of the SQE motif to AQE (H2A-AQE) in the two histone H2A genes caused sensitivity to a wide range of genotoxic agents, increased spontaneous DNA damage, and impaired checkpoint maintenance. The H2A-AQE mutations displayed a striking synergistic interaction with rad22Delta (Rad52 homolog) in ionizing radiation (IR) survival. These phenotypes correlated with defective phosphorylation of the checkpoint proteins Crb2 and Chk1 and a failure to recruit large amounts of Crb2 to damaged DNA. Surprisingly, the H2A-AQE mutations substantially suppressed the IR hypersensitivity of crb2Delta cells by a mechanism that required the RecQ-like DNA helicase Rqh1. We propose that gamma-H2A modulates checkpoint and DNA repair through large-scale recruitment of Crb2 to damaged DNA. This function correlates with evidence that gamma-H2AX regulates recruitment of several BRCA1 carboxyl terminus domain-containing proteins (NBS1, 53BP1, MDC1/NFBD1, and BRCA1) in mammals.
Mol Cell Biol 2004 Jul
PMID:Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast. 1522 25

To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.
Mol Cell Biol 2004 Jul
PMID:Intra-S-phase checkpoint activation by direct CDK2 inhibition. 1522 29

The interaction of cyclic peptides with surface-bound model peptides was investigated by ATR-FTIR spectroscopy, principal components analysis and force field calculations. Information about the interacting functional COOH, COO-, and NH3+ groups and the peptide backbone was gained through a set of cyclohexapeptides (seven of the type c(X1KX2KX3K) (K = L-lysine) and one of the type c(X1KX2KX3k) (k = D-lysine), which are interacting with L-arginine- or tripeptide-coated Si-ATR crystals. All measurements were performed in aqueous solutions. Spectra evaluation in the range 1800-1500 cm(-1) was done by band and principal components analysis (PCA). Only adsorbed molecules were present in these spectra. The coatings were investigated by ATR-FTIR spectroscopy too in order to characterise their functional groups. Based on this knowledge, the spectra of the interacting partners could be evaluated in relation to cyclohexapeptides and coatings. As a result, it was possible to identify the distinct differences in the bonding behaviour of the various peptides.
Spectrochim Acta A Mol Biomol Spectrosc 2004 Jul
PMID:Small interacting peptides. Part II: Interaction of cyclohexapeptides with immobilised model peptides. Comparison of infrared investigations, principal components analysis and force field calculations. 1524 46

Common fragile sites are loci that form chromosome gaps or breaks when DNA synthesis is partially inhibited. Fragile sites are prone to deletions, translocations, and other rearrangements that can cause the inactivation of associated tumor suppressor genes in cancer cells. It was previously shown that ATR is critical to fragile-site stability and that ATR-deficient cells have greatly elevated fragile-site expression (A. M. Casper, P. Nghiem, M. F. Arlt, and T. W. Glover, Cell 111:779-789, 2002). Here we demonstrate that mouse and human cells deficient for BRCA1, due to mutation or knockdown by RNA interference, also have elevated fragile-site expression. We further show that BRCA1 functions in the induction of the G(2)/M checkpoint after aphidicolin-induced replication stalling and that this checkpoint function is involved in fragile-site stability. These data indicate that BRCA1 is important in fragile-site stability and that fragile sites are recognized by the G(2)/M checkpoint pathway, in which BRCA1 plays a key role. Furthermore, they suggest that mutations in BRCA1 or interacting proteins could lead to rearrangements at fragile sites in cancer cells.
Mol Cell Biol 2004 Aug
PMID:BRCA1 is required for common-fragile-site stability via its G2/M checkpoint function. 1525 37

The mammalian mismatch repair (MMR) system has been implicated in activation of the G(2) checkpoint induced by methylating agents. In an attempt to identify the signaling events accompanying this phenomenon, we studied the response of MMR-proficient and -deficient cells to treatment with the methylating agent temozolomide (TMZ). At low TMZ concentrations, MMR-proficient cells were growth-inhibited, arrested in G(2)/M, and proceeded to apoptosis after the second post-treatment cell cycle. These events were accompanied by activation of the ATM and ATR kinases, and phosphorylation of Chk1, Chk2, and p53. ATM was activated later than ATR and was dispensable for phosphorylation of Chk1, Chk2, and p53 on Ser15 and for triggering of the G(2)/M arrest. However, it conferred protection against cell growth inhibition induced by TMZ. ATR was activated earlier than ATM and was required for an efficient phosphorylation of Chk1 and p53 on Ser15. Moreover, abrogation of ATR function attenuated the TMZ-induced G(2)/M arrest and increased drug-induced cytotoxicity. Treatment of MMR-deficient cells with low TMZ concentrations failed to activate ATM and ATR and to cause phosphorylation of Chk1, Chk2, and p53, as well as G(2)/M arrest and apoptosis. However, all these events occurred in MMR-deficient cells exposed to high TMZ concentrations, albeit with faster kinetics. These results demonstrate that TMZ treatment activates ATM- and ATR-dependent signaling pathways and that this process is absolutely dependent on functional MMR only at low drug concentrations.
Mol Pharmacol 2004 Sep
PMID:DNA damage induced by temozolomide signals to both ATM and ATR: role of the mismatch repair system. 1532 39

YCa4O(BO3)3-(YCOB) is a non-linear optical (NLO) material grown by Czochralski technique. Polarised IR, ATR-IR, polarised Raman and optical transmission spectral measurements were made. A series of absorption bands have been observed with intensities depending on the functional groups of the crystals. The observed bands were assigned and discussed.
Spectrochim Acta A Mol Biomol Spectrosc 2004 Oct
PMID:Polarised infrared and Raman studies of YCa4O(BO3)3 a non-linear optical single crystal. 1535 Sep 7

Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemis-depleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.
Mol Cell Biol 2004 Oct
PMID:Artemis is a phosphorylation target of ATM and ATR and is involved in the G2/M DNA damage checkpoint response. 1545 91

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.
Hum Mol Genet 2004 Dec 01
PMID:ATM is required for the cellular response to thymidine induced replication fork stress. 1545 81


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