Purification and characterization of ATM from human placenta. A manganese-dependent, wortmannin-sensitive serine/threonine protein kinase

ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC(50) of approximately 100 nM. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme.     

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites. In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.     

Identification of domains of ataxia-telangiectasia mutated required for nuclear localization and chromatin association

Ataxia-telangiectasia mutated (ATM) is essential for rapid induction of cellular responses to DNA double strand breaks (DSBs). In this study, we mapped a nuclear localization signal (NLS), 385KRKK388, within the amino terminus of ATM and demonstrate its recognition by the conventional nuclear import receptor, the importin alpha1/beta1 heterodimer. Although mutation of this NLS resulted in green fluorescent protein (GFP) x ATM(NLSm) localizing predominantly within the cytoplasm, small amounts of nuclear GFP x ATM(NLSm) were still sufficient to elicit a DNA damage response. Insertion of an heterologous nuclear export signal between GFP and ATM(NLSm) resulted in complete cytoplasmic localization of ATM, concomitantly reducing the level of substrate phosphorylation and increasing radiosensitivity, which indicates a functional requirement for ATM nuclear localization. Interestingly, the carboxyl-terminal half of ATM, containing the kinase domain, which localizes to the cytoplasm, could not autophosphorylate itself or phosphorylate substrates, nor could it correct radiosensitivity in response to DSBs even when targeted to the nucleus by insertion of an exogenous NLS, demonstrating that the ATM amino terminus is required for optimal ATM function. Moreover, we have shown that the recruitment/retention of ATM at DSBs requires its kinase activity because a kinase-dead mutant of GFP x ATM failed to form damage-induced foci. Using deletion mutation analysis we mapped a domain in ATM (amino acids 5-224) required for its association with chromatin, which may target ATM to sites of DNA damage. Combined, these data indicate that the amino terminus of ATM is crucial not only for nuclear localization but also for chromatin association, thereby facilitating the kinase activity of ATM in vivo.     

Substrate specificities and identification of putative substrates of ATM kinase family members

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.     

ATM: the product of the gene mutated in ataxia-telangiectasia.

Ataxia-telangiectasia mutated (ATM) is the product of the gene mutated in the human genetic disorder ataxia-telangeictasia (A-T). It is a 370 kDa protein that is a member of the phosphatidyl inositol 3-kinases superfamily. A-T cells and those derived from Atm-/- mice are characterized by hypersensitivity to ionizing radiation and defective cell cycle checkpoints. Defects are observed at all cell cycle checkpoints in A-T cells post-irradiation including the G1/S interface where ATM plays an important role in the activation of the tumour suppressor gene product p53. Activation leads to the induction of p21/WAF1, inhibition of cyclin-dependent kinase activity, failure to phosphorylate key substrates such as the retinoblastoma protein and consequently G1 arrest. ATM also plays an important role in the regulation and surveillance of meiotic progression. Absence of ATM gives rise to a spectrum of defects including immunodeficiency, neurodegeneration, radiosensitivity and cancer predisposition. It is clear that a better definition of the role of ATM in DNA damage recognition, cell cycle control and cell signalling may assist in the treatment of the progressive neurodegeneration in this syndrome.     

Recruitment of ATM protein to double strand DNA irradiated with ionizing radiation.

The product of the ATM gene, which is mutated in ataxia telangiectasia, is a nuclear phosphoprotein, and it involves the activation of the p53 pathway after ionizing radiation. Here we show that the ATM protein is constitutively associated with double strand DNA and that the interaction increases when the DNA is exposed to ionizing radiation. The ATM protein also had affinity to restriction endonuclease PvuII-digested DNA, but not to UV-irradiated DNA nor X-irradiated single-stranded DNA. The immunoprecipitation experiment detected very weak association between ATM and DNA-PK proteins, and immunodepletion of DNA-PK showed little or no effect on the interaction of the ATM protein with damaged DNA, indicating that an interaction with DNA-PK might not be required for the recruitment of the ATM protein to damaged DNA. Furthermore, the association was also confirmed in xrs-5 and xrs-6e cells, which are Chinese hamster ovary mutant cell lines defective in Ku80 function. These results indicate that the ATM protein is recruited to the site of DNA damage and it recognizes double strand breaks by itself or through an association with other DNA-binding protein other than DNA-PK and Ku80 proteins.     

S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and γH2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.     

Fragments of ATM which have dominant-negative or complementing activity.

The ATM protein has been implicated in pathways controlling cell cycle checkpoints, radiosensitivity, genetic instability, and aging. Expression of ATM fragments containing a leucine zipper motif in a human tumor cell line abrogated the S-phase checkpoint after ionizing irradiation and enhanced radiosensitivity and chromosomal breakage. These fragments did not abrogate irradiation-induced G1 or G2 checkpoints, suggesting that cell cycle checkpoint defects alone cannot account for chromosomal instability in ataxia telangiectasia (AT) cells. Expression of the carboxy-terminal portion of ATM, which contains the PI-3 kinase domain, complemented radiosensitivity and the S-phase checkpoint and reduced chromosomal breakage after irradiation in AT cells. These observations suggest that ATM function is dependent on interactions with itself or other proteins through the leucine zipper region and that the PI-3 kinase domain contains much of the significant activity of ATM.     

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.     

Sensing of ionizing radiation-induced DNA damage by ATM through interaction with histone deacetylase.

The ATM gene is mutated in individuals with ataxia telangiectasia, a human genetic disease characterized by extreme sensitivity to radiation. The ATM protein acts as a sensor of radiation-induced cellular damage and contributes to cell cycle regulation, signal transduction, and DNA repair; however, the mechanisms underlying these functions of ATM remain largely unknown. Binding and immunoprecipitation assays have now shown that ATM interacts with the histone deacetylase HDAC1 both in vitro and in vivo, and that the extent of this association is increased after exposure of MRC5CV1 human fibroblasts to ionizing radiation. Histone deacetylase activity was also detected in immunoprecipitates prepared from these cells with antibodies to ATM, and this activity was blocked by the histone deacetylase inhibitor trichostatin A. These results suggest a previously unanticipated role for ATM in the modification of chromatin components in response to ionizing radiation.     

Rapid activation of ATR by ionizing radiation requires ATM and Mre11

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.     

14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response

The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phsophorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G₂/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.     

DNA损伤生物学反应中ATM对p21~(WAF1/CIP1)蛋白的直接磷酸化

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1 CIP1蛋白的快速活化过程     

Human cytomegalovirus disrupts both ataxia telangiectasia mutated protein (ATM)- and ATM-Rad3-related kinase-mediated DNA damage responses during lytic infection

Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.     

Human PTIP facilitates ATM-mediated activation of p53 and promotes cellular resistance to ionizing radiation

Mus musculus Pax2 transactivation domain-interacting protein (Ptip) is an essential gene required for the maintenance of genome stability, although its precise molecular role is unclear. Human PTIP (hPTIP) was recently isolated in a screen for proteins, translated from cDNA pools, capable of interacting with peptides phosphorylated by the ATM (ataxia telangiectasia-mutated)/ATR (ataxia telangiectasia-related) protein kinases. hPTIP was described as a 757-amino acid protein bearing four BRCT domains. Here we report that instead full-length endogenous hPTIP contains 1069 amino acids and six BRCT domains. hPTIP shows increased association with 53BP1 in response to ionizing radiation (IR) but not in response to other DNA-damaging agents. Whereas translocation of both 53BP1 and hPTIP to sites of IR-induced DNA damage occurs independently of ATM, IR-induced association of PTIP and 53BP1 requires ATM. Deletion analysis identified the domains of 53BP1 and hPTIP required for protein-protein interaction and focus formation. Data characterizing the cellular roles of hPTIP are also presented. Small interfering RNA was used to show that hPTIP is required for ATM-mediated phosphorylation of p53 at Ser(15) and for IR-induced up-regulation of the cyclin-dependent kinase inhibitor p21. Lowering hPTIP levels also increased cellular sensitivity to IR, suggesting that this protein plays a critical role in maintaining genome stability.     

DNA damage-induced acetylation of lysine 3016 of ATM activates ATM kinase activity

The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-lysine 3016 demonstrate rapid (within 5 min) in vivo acetylation of ATM following exposure to bleomycin. Furthermore, lysine 3016 of ATM is a substrate in vitro for the Tip60 histone acetyltransferase. Mutation of lysine 3016 does not affect unstimulated ATM kinase activity but does abolish upregulation of ATM's kinase activity by DNA damage, inhibits the conversion of inactive ATM dimers to active ATM monomers, and prevents the ATM-dependent phosphorylation of the p53 and chk2 proteins. These results are consistent with a model in which acetylation of lysine 3016 in the FATC domain of ATM activates the kinase activity of ATM. The acetylation of ATM on lysine 3016 by Tip60 is therefore a key step linking the detection of DNA damage and the activation of ATM kinase activity.     

Vaccinia-related kinase 1 (VRK1) is an upstream nucleosomal kinase required for the assembly of 53BP1 foci in response to ionizing radiation-induced DNA damage

Cellular responses to DNA damage require the formation of protein complexes in a highly organized fashion. The complete molecular components that participate in the sequential signaling response to DNA damage remain unknown. Here we demonstrate that vaccinia-related kinase 1 (VRK1) in resting cells plays an important role in the formation of ionizing radiation-induced foci that assemble on the 53BP1 scaffold protein during the DNA damage response. The kinase VRK1 is activated by DNA double strand breaks induced by ionizing radiation (IR) and specifically phosphorylates 53BP1 in serum-starved cells. VRK1 knockdown resulted in the defective formation of 53BP1 foci in response to IR both in number and size. This observed effect on 53BP1 foci is p53- and ataxia-telangiectasia mutated (ATM)-independent and can be rescued with VRK1 mutants resistant to siRNA. VRK1 knockdown also prevented the activating phosphorylation of ATM, CHK2, and DNA-dependent protein kinase in response to IR. VRK1 activation in response to DNA damage is a novel and early step in the signaling of mammalian DNA damage responses.     

Checkpoint signaling: epigenetic events sound the DNA strand-breaks alarm to the ATM protein kinase

The ATM protein kinase is centrally involved in the cellular response to ionizing radiation (IR) and other DNA double-strand-break-inducing insults. Although it has been well established that IR exposure activates the ATM kinase domain, the actual mechanism by which ATM responds to damaged DNA has remained enigmatic. Now, a landmark paper provides strong evidence that DNA-strand breaks trigger widespread activation of ATM through changes in chromatin structure (1). This review discusses a checkpoint activation model in which chromatin perturbations lead to the conversion of inactive ATM domains to phosphorylated, active ATM monomers. The new findings underscore the critical importance of epigenetic events in genome function and surveillance in mammalian cells.