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.     

ATM is activated in response to N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA alkylation

p53 plays an important role in response to ionizing radiation by regulating cell cycle progression and triggering apoptosis. These activities are controlled, in part, by the phosphorylation of p53 by the protein kinase ATM. Recent evidence indicates that the monofunctional DNA alkylating agent N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) also triggers up-regulation and phosphorylation of p53; however, the mechanism(s) responsible for this are unknown. We observed that in MNNG-treated normal human fibroblasts, up-regulation and phosphorylation of p53 was sensitive to the ATM kinase inhibitor wortmannin. ATM-deficient fibroblasts exhibited a delay in p53 up-regulation indicating a role for ATM in triggering the MNNG-induced response. Likewise, a mismatch repair (MMR)-deficient colorectal tumor line failed to show rapid up-regulation of p53. However, unlike ATM-deficient cells, these MMR-deficient cells displayed rapid phosphorylation of the p53 residue serine 15 after MNNG. In vitro kinase assays indicate that ATM is rapidly activated in both normal and MMR-deficient cells in response to MNNG. Using a number of morphological and biochemical approaches, we failed to observe MNNG-induced apoptosis in normal human fibroblasts, suggesting that apoptosis-induced DNA strand breaks are not required for the activation of ATM in response to MNNG. Comet assays indicated that strand breaks accumulated, and p53 up-regulation/phosphorylation occurred quite rapidly (within 30 min) after MNNG treatment, suggesting that DNA strand breaks that arise during the repair process activate ATM. These findings indicate that ATM activation is not limited to the ionizing radiation-induced response and potentially plays an important role in response to DNA alkylation.     

Chromatin modifying protein 1A (Chmp1A) of the endosomal sorting complex required for transport (ESCRT)-III family activates ataxia telangiectasia mutated (ATM) for PanC-1 cell growth inhibition

Chromatin modifying protein 1A (Chmp1A) is a member of the endosormal sorting complex required for transport (ESCRT)-III family whose overexpression induces growth inhibition, chromatin condensation and p53 phosphorylation. p53 is a substrate for ataxia telangiectasia mutated (ATM), which can be activated upon chromatin condensation. Thus, we propose that Chmp1A regulates ATM, and the nuclear localization signal (NLS) is required for ATM activation. Our data demonstrated that overexpression of full-length Chmp1A induced an increase in active, phosphorylated ATM in the nucleus, where they co-localized. It also induced an increase in phospho-p53 in the nucleus, and in vitro ATM kinase and p53 reporter activities. The intensity of phospho-p53 closely followed that of ectopically induced full-length Chmp1A, suggesting a tight correlation between Chmp1A overexpression and p53 phosphorylation. On the other hand, Chmp1A depletion (reported to promote cell growth) had minor effects on phospho-ATM and p53 expression compared with control, which had very little expression of these proteins. NLS-deleted cells showed uniform cytoplasmic-Chmp1A expression and acted like shRNA-expressing cells (cell growth promotion and minimal effect on ATM), demonstrating the significance of NLS on ATM activation and growth inhibition. C-deleted Chmp1A, detected in the cytoplasm at the enlarged vesicles, increased phospho-ATM and p53, and inhibited growth; yet it had no effect on in vitro ATM kinase or p53 reporter activities, suggesting that the C-domain is not required for ATM activation. Finally, ATM inactivation considerably reduced Chmp1A mediated growth inhibition and phosphorylation of p53, showing that Chmp1A regulates tumor growth partly through ATM signaling.     

Chromatin modifying protein 1A (Chmp1A) of the endosomal sorting complex required for transport (ESCRT)-III family activates ataxia telangiectasia mutated (ATM) for PanC-1 cell growth inhibition

Chromatin modifying protein 1A (Chmp1A) is a member of the Endosormal sorting complex required for transport (ESCRT)-III family whose over-expression induces growth inhibition, chromatin condensation, and p53 phosphorylation. p53 is a substrate for Ataxia telangiectasia mutated (ATM), which can be activated upon chromatin condensation. Thus, we propose that Chmp1A regulates ATM, and the nuclear localization signal (NLS) is required for ATM activation. Our data demonstrated that over-expression of full-length Chmp1A induced an increase in active, phosphorylated ATM in the nucleus, where they co-localized. It also induced an increase in phospho-p53 in the nucleus, and in vitro ATM kinase and p53 reporter activities. The intensity of phospho-p53 closely followed that of ectopically induced full-length Chmp1A, suggesting a tight correlation between Chmp1A over-expression and p53 phosphorylation. On the other hand, Chmp1A depletion (reported to promote cell growth) had minor effects on phospho-ATM and p53 expression compared to control, which had very little expression of these proteins. NLS-deleted cells showed uniform cytoplasmic-Chmp1A expression and acted like shRNA-expressing cells (cell growth promotion and minimal effect on ATM), demonstrating the significance of NLS on ATM activation and growth inhibition. C-deleted Chmp1A, detected in the cytoplasm at the enlarged vesicles, increased phospho-ATM and p53, and inhibited growth; yet it had no effect on in vitro ATM kinase or p53 reporter activities, suggesting that the C-domain is not required for ATM activation. Finally, ATM inactivation considerably reduced Chmp1A mediated growth inhibition and phosphorylation of p53, showing that Chmp1A regulates tumor growth partly through ATM signaling.     

TGFβ1-Induced Activation of ATM and p53 Mediates Apoptosis in a Smad7-Dependent Manner

ATM, a DNA-damage sensitive kinase and p53, are frequently inactivated in a variety of cancers as they together with γH2AX are critical guardians against DNA damage. Here, we report of a functional cross-talk between the cytokine TGFβ and p53, leading to apoptosis of epithelial cells, involving Smad7, a TGF-β target gene, p38 MAP kinase, and ATM. Using ectopic expression of p53, siRNA for Smad7, p38α -/- deficient cells and specific inhibitors, we show that TGF-β induces apoptosis via ATM and p53 in epithelial cells. Intriguingly, Smad7 act as a scaffold protein to promote functional interactions between p38, ATM and p53 upon TGFβ treatment, facilitating their activation. Smad7 colocalizes with γH2AX in DNA damage foci and was required for proper cell cycle checkpoints to prevent genetic instability. Our data imply that Smad7 plays a crucial role upstream of ATM and p53 to protect the genome from insults evoked by extracellular stress.     

Requirement of ATM in UVA-induced signaling and apoptosis.

Solar UVA, but not UVC, reaches the earth's surface and therefore is an important etiological factor for the induction of human skin cancer. ATM kinase is an important regulator of cell survival and cell cycle checkpoints. Here, we observe that UVA, unlike UVC, triggers ATM kinase activity, and the activation may occur through reactive oxygen species produced after irradiation of cells with UVA. We also show that ATM activation is involved in the apoptotic response to UVA but not UVC. Furthermore, we provide evidence that ATM-dependent p53 and c-Jun N-terminal kinase (JNK) pathways are linked to UVA-induced apoptosis. On the other hand, UVC-induced apoptosis occurs through ATR-dependent p53 phosphorylation as well as the JNK pathway. Therefore, these results suggest that ATM, like p53, is involved in the UVA-induced apoptosis to suppress carcinogenesis.     

Radiation-induced apoptosis in developing mouse retina exhibits dose-dependent requirement for ATM phosphorylation of p53

Ionizing radiation (IR) induces DNA breakage to activate cell cycle checkpoints, DNA repair, premature senescence or cell death. A master regulator of cellular responses to IR is the ATM kinase, which phosphorylates a number of downstream effectors, including p53, to inhibit cell cycle progression or to induce apoptosis. ATM phosphorylates p53 directly at Ser15 (Ser18 of mouse p53) and indirectly through other kinases. In this study, we examined the role of ATM and p53 Ser18 phosphorylation in IR-induced retinal apoptosis of neonatal mice. Whole-body irradiation with 2 Gy IR induces apoptosis of postmitotic and proliferating cells in the neonatal retinas. This apoptotic response requires ATM, exhibits p53-haploid insufficiency and is defective in mice with the p53S18A allele. At a higher dose of 14 Gy, retinal apoptosis still requires ATM and p53 but can proceed without Ser18 phosphorylation. These results suggest that ATM activates the apoptotic function of p53 in vivo through alternative pathways depending on IR dose.     

Constitutive phosphorylation of ATM in lymphoblastoid cell lines from patients with ICF syndrome without downstream kinase activity

Double strand DNA breaks in the genome lead to the activation of the ataxia-telangiectasia mutated (ATM) kinase in a process that requires ATM autophosphorylation at serine-1981. ATM autophosphorylation only occurs if ATM is previously acetylated by Tip60. The activated ATM kinase phosphorylates proteins involved in arresting the cell cycle, including p53, and in repairing the DNA breaks. Chloroquine treatment and other manipulations that produce chromatin defects in the absence of detectable double strand breaks also trigger ATM phosphorylation and the phosphorylation of p53 in primary human fibroblasts, while other downstream substrates of ATM that are involved in the repair of DNA double strand breaks remain unphosphorylated. This raises the issue of whether ATM is constitutively activated in patients with genetic diseases that display chromatin defects. We examined lymphoblastoid cell lines (LCLs) generated from patients with different types of chromatin disorders: Immunodeficiency, Centromeric instability, Facial anomalies (ICF) syndrome, Coffin Lowry syndrome, Rubinstein Taybi syndrome and Fascioscapulohumeral Muscular Dystrophy. We show that ATM is phosphorylated on serine-1981 in LCLs derived from ICF patients but not from the other syndromes. The phosphorylated ATM in ICF cells did not phosphorylate the downstream targets NBS1, SMC1 and H2AX, all of which require the presence of double strand breaks. We demonstrate that ICF cells respond normally to ionizing radiation, ruling out the possibility that genetic deficiency in ICF cells renders activated ATM incapable of phosphorylating its downstream substrates. Surprisingly, p53 was also not phosphorylated in ICF cells or in chloroquine-treated wild type LCLs. In this regard the response to chromatin-altering agents differs between primary fibroblasts and LCLs. Our findings indicate that although phosphorylation at serine-1981 is essential in the activation of the ATM kinase, serine-1981 phosphorylation is insufficient to render ATM an active kinase towards downstream substrates, including p53.     

Functional interaction of H2AX, NBS1, and p53 in ATM-dependent DNA damage responses and tumor suppression

Ataxia-telangiectasia (A-T) mutated (ATM) kinase signals all three cell cycle checkpoints after DNA double-stranded break (DSB) damage. H2AX, NBS1, and p53 are substrates of ATM kinase and are involved in ATM-dependent DNA damage responses. We show here that H2AX is dispensable for the activation of ATM and p53 responses after DNA DSB damage. Therefore, H2AX functions primarily as a downstream mediator of ATM functions in the parallel pathway of p53. NBS1 appears to function both as an activator of ATM and as an adapter to mediate ATM activities after DNA DSB damage. Phosphorylation of ATM and H2AX induced by DNA DSB damage is normal in NBS1 mutant/mutant (NBS1m/m) mice that express an N-terminally truncated NBS1 at lower levels. Therefore, the pleiotropic A-T-related systemic and cellular defects observed in NBS1m/m mice are due to the disruption of the adapter function of NBS1 in mediating ATM activities. While H2AX is required for the irradiation-induced focus formation of NBS1, our findings indicate that NBS1 and H2AX have distinct roles in DNA damage responses. ATM-dependent phosphorylation of p53 and p53 responses are largely normal in NBS1m/m mice after DNA DSB damage, and p53 deficiency greatly facilitates tumorigenesis in NBS1m/m mice. Therefore, NBS1, H2AX, and p53 play synergistic roles in ATM-dependent DNA damage responses and tumor suppression.     

Cellular response to DNA damage. Link between p53 and DNA-PK

Cells which lack DNA-activated protein kinase (DNA-PK) are very susceptible to ionizing radiation and display an inability to repair double strand DNA breaks. DNA-PK is a member of a protein kinase family that includes ATR and ATM which have strong homology in their carboxy-terminal kinase domain with PL-3 kinase. ATM has been proposed to act upstream of p53 in cellular response to ionizing radiation. DNA-PK may similarly interact with p53 in cellular growth control and in mediation of the response to ionizing radiation.     

The isoflavonoids genistein and quercetin activate different stress signaling pathways as shown by analysis of site-specific phosphorylation of ATM, p53 and histone H2AX

The ataxia-telangiectasia mutated (ATM) protein kinase is activated in response to ionizing radiation (IR) and activates downstream DNA-damage signaling pathways. Although the role of ATM in the cellular response to ionizing radiation has been well characterized, its role in response to other DNA-damaging agents is less well defined. We previously showed that genistein, a naturally occurring isoflavonoid, induced increased ATM protein kinase activity, ATM-dependent phosphorylation of p53 on serine 15 and activation of the DNA-binding properties of p53. Here, we show that genistein also induces phosphorylation of p53 at serines 6, 9, 20, 46, and 392, and that genistein-induced accumulation and phosphorylation of p53 is reduced in two ATM-deficient human cell lines. Also, we show that genistein induces phosphorylation of ATM on serine 1981 and phosphorylation of histone H2AX on serine 139. The related bioflavonoids, daidzein and biochanin A, did not induce either phosphorylation of p53 or ATM at these sites. Like genistein, quercetin induced phosphorylation of ATM on serine 1981, and ATM-dependent phosphorylation of histone H2AX on serine 139; however, p53 accumulation and phosphorylation on serines 6, 9, 15, 20, 46, and 392 occurred in ATM-deficient cells, indicating that ATM is not required for quercetin-induced phosphorylation of p53. Our data suggest that genistein and quercetin induce different DNA-damage induced signaling pathways that, in the case of genistein, are highly ATM-dependent but, in the case of quercetin, may be ATM-dependent only for some downstream targets.     

Differential roles of ATM- and Chk2-mediated phosphorylations of Hdmx in response to DNA damage

The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.     

Mode of ATM-dependent suppression of chromosome translocation

It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.     

ATM activates p53 by regulating MDM2 oligomerization and E3 processivity

Rapid activation of p53 by ionizing irradiation is a classic DNA damage response mediated by the ATM kinase. However, the major signalling target and mechanism that lead to p53 stabilization are unknown. We show in this report that ATM induces p53 accumulation by phosphorylating the ubiquitin E3 ligase MDM2. Multiple ATM target sites near the MDM2 RING domain function in a redundant manner to provide robust DNA damage signalling. In the absence of DNA damage, the MDM2 RING domain forms oligomers that mediate p53 poly ubiquitination and proteasomal degradation. Phosphorylation by ATM inhibits RING domain oligomerization, specifically suppressing p53 poly ubiquitination. Blocking MDM2 phosphorylation by alanine substitution of all six phosphorylation sites results in constitutive degradation of p53 after DNA damage. These observations show that ATM controls p53 stability by regulating MDM2 RING domain oligomerization and E3 ligase processivity. Promoting or disrupting E3 oligomerization may be a general mechanism by which signalling kinases regulate ubiquitination reactions, and a potential target for therapeutic intervention.     

CKIP-1 recruits nuclear ATM partially to the plasma membrane through interaction with ATM

CKIP-1 (casein kinase-2 interacting protein-1) is implicated in muscle differentiation, regulation of cell morphology and actin cytoskeleton. More recently, we showed that CKIP-1 regulated AP-1 activity and promoted apoptosis via caspase-3-dependent cleavage and translocation. Here, we report that overexpression of CKIP-1 in SK-BR-3 breast cancer cells prevents p53 degradation induced by cycloheximide treatment through increase of p53 N-terminal Ser-15 phosphorylation level. CKIP-1 could interact with ATM, which is an upstream kinase of p53, thereby enhance the stability of p53. Interestingly, CKIP-1 is localized both at the plasma membrane and in the nucleus dependent on the cell types, and only the plasma membrane-localized CKIP-1 could form a complex with ATM. Importantly, CKIP-1 recruits nuclear ATM proteins partially to the plasma membrane. Our data provide the first evidence that ATM, a predominantly nuclear kinase, could be relocalized to the plasma membrane by CKIP-1 and shed new light on the multi-functional CKIP-1.     

Down-regulation of Fer induces ROS levels accompanied by ATM and p53 activation in colon carcinoma cells

Fer is an intracellular tyrosine kinase which resides in both the cytoplasm and nucleus of mammalian cells. This kinase was also found in all malignant cell-lines analyzed and was shown to support cell-cycle progression in cancer cells. Herein we show that knock-down of Fer, both, impairs cell-cycle progression and imposes programmed cell death in colon carcinoma (CC) cells. The cell-cycle arrest and apoptotic death invoked by the depletion of Fer were found to depend on the activity of p53. Accordingly, down regulation of Fer led to the activation of the Ataxia Telangiectasia Mutated protein (ATM) and its down-stream effector-p53. Knock-down of Fer also increased the level of Reactive-Oxygen Species (ROS) in CC cells, and subjection of Fer depleted cells to ROS neutralizing scavengers significantly decreased the induced phosphorylation and activation of ATM and p53. Notably, over-expression of Fer opposed the Doxorubicin driven activation of ATM and p53, which can be mediated by ROS. Collectively, our findings imply that Fer sustains low ROS levels in CC cells, thereby restraining the activation of ATM and p53 in these cells.     

The Cellular Ataxia Telangiectasia-Mutated Kinase Promotes Epstein-Barr Virus Lytic Reactivation in Response to Multiple Different Types of Lytic Reactivation-Inducing Stimuli

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor β (TGF-β) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53- and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect.