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1 Department of Molecular Pathology, Cancer Research Institute, Kanazawa University, Kanazawa, 13-1 Takaramachi, Ishikawa 920-0934, Japan
2 Department of Environmental Science, Faculty of Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 310-8512, Japan
3 Radiation Biology Center, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan
4 Graduate School of Bioagricultural Science, Nagoya University, Nagoya 464-8601, Japan
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Abstract |
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12,14-prostaglandin J2 (15d-PGJ2), in NBS1- or MSH6-deficient cells. We further found that ATM is activated by treating chromatin-free immunoprecipitated ATM with MNNG or 15d-PGJ2, which modifies free sulfhydryl (SH) groups, and that 15d-PGJ2 binds covalently to ATM. Interestingly, 15d-PGJ2-induced ATM activation leads to p53 activation and apoptosis, but not to Chk2 or H2AX phosphorylation. These results indicate that ATM is activated through the direct modification of its SH groups, independent of DNA damage, and this activation leads, downstream, to apoptosis. |
Introduction |
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The results of several recent studies indicate that ATM is activated by a variety of noxious agents, such as alkylating agents, oxidative stress, poly-Q aggregates and UVA (Shackelford et al. 2001; Adamson et al. 2002; Zhang et al. 2002; Giuliano et al. 2003), which may not directly generate DSB. Furthermore, it is now clear that the loss of ATM results in an anomalous cellular response to oxidative stress. This includes decreased levels of catalase activity, increased levels of lipid hydroperoxides, reduced levels of cyclic nucleotides such as NADH and NADPH, enhanced generation of reactive oxygen species, enhanced heme oxygenase mRNA expression and increased levels of oxidant-modified proteins and lipids in ATM-deficient cells lines or tissues, including the brain of ATM knockout mice (Barlow et al. 1999; Takao et al. 2000; Kamsler et al. 2001; Stern et al. 2002). More recently, impaired self-renewal of hematopoietic stem cells in ATM knockout mice has been attributed to the defective response to oxidative stress (Ito et al. 2004). Thus, the defective response to oxidative stress may underlie the pathogenesis of some of the clinical phenotypes of A-T, such as cerebellar ataxia, premature aging, growth retardation and cancer predisposition. Nonetheless, it remains to be established how ATM is activated by oxidative stress or other noxious agents that may not directly induce DSB. Furthermore, the molecular mechanisms underlying the apoptosis of postmitotic cerebellar Purkinje cells, which is responsible for cerebellar ataxia, have remained elusive.
A reactive cyclopentenone-type prostaglandin 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) is a prostaglandin D2 metabolite generated during inflammatory processes that is, due to the presence of a reactive 
,ß-unsaturated ketone in the cyclopentenone ring, a potent inducer of intracellular oxidative stress (Gilroy et al. 1999; Kondo et al. 2001; Shibata et al. 2002). Furthermore, 15d-PGJ2 has been shown to directly modulate the activity of several biologically important molecules, such as NF-
B, IB kinase, H-Ras and thioredoxin, through the specific alkylation of free sulfhydryl (SH) groups on cysteine residues of the target proteins (Rossi et al. 2000; Cernuda-Morollon et al. 2001; Oliva et al. 2003; Shibata et al. 2003). An alkylating agent, N-methyl-N'-nitro-nitrosoguanidine (MNNG), is another potential oxidative stress inducer (Reitman et al. 1988) that reacts with free SH groups (Sugimura et al. 1968; Nagao et al. 1971; Johnson & Greenberg 1973), although MNNG is well known to methylate DNA (e.g. O6-methylguanine (O6-MeG)) (Stojic et al. 2004a), and effectively induces checkpoint activation through the activation of both ATM and ATR with the subsequent activation of Chk1 and Chk2 (Wang & Qin 2003; Stojic et al. 2004b; Beardsley et al. 2005).
Here, we used these two agents to study whether ATM responds to the oxidative or methylation damage of DNA or, rather, to a change in the intracellular redox state, independent of DNA damage. To address this issue specifically, we used NBS1- and MSH6-deficient cells that were generated by targeted disruption. Nbs1 forms an evolutionarily conserved complex with Mre11 and Rad50, in which Nbs1 interacts with ATM and recruits it to DSB sites. Nbs1 is essential for DSB-induced ATM activation and the subsequent phosphorylation of ATM target proteins, such as Nbs1 itself, Chk2, and SMC1, in vertebrate cells (Uziel et al. 2003; Kitagawa et al. 2004; Lee & Paull 2004, 2005; Falck et al. 2005). Msh6 and Msh2 form an evolutionarily conserved mismatch repair complex (MutS). They recognize methylated DNAs such as O6-MeG (Stojic et al. 2004a) and are required for MNNG-induced ATR activation and Chk1 phosphorylation by ATR (Wang & Qin 2003; Stojic et al. 2004b). In the present study, we found that ATM is activated by MNNG and 15d-PGJ2 in an Nbs1- and Msh6-independent manner. We further found that ATM is activated by the direct treatment of chromatin-free immunoprecipitated ATM with MNNG or 15d-PGJ2, through the modification of SH groups on ATM, and that 15d-PGJ2 covalently binds to ATM. Interestingly, 15d-PGJ2-induced ATM activation leads to the phosphorylation and accumulation of p53 and to apoptosis, but not to Chk2 or H2AX phosphorylation in human tumor cells. These results indicate that ATM is directly activated by free SH-group-modifying agents and thereafter induces a distinct downstream response.
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Results |
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To study the mechanisms by which MNNG activates the ATM kinase, we developed an IP kinase assay for ATM in chicken DT40 cells, using anti-chicken ATM antibodies. The rational for using DT40 cells was the availability of a panel of stable isogenic knockout cell lines, including ATM–/–, NBS1–/–/–, RAD17–/–, RAD9–/–, and MSH6–/– lines (Takao et al. 1999; Tauchi et al. 2002; Kobayashi et al. 2004) (Supplementary Fig. S1). However, since the human ATM autophosphorylation site (Ser-1981) (Bakkenist & Kastan 2003) is not conserved in chicken ATM (Supplementary Fig. S2), and since some agents such as okadaic acid (OA) induce ATM autophosphorylation on Ser-1981 but not ATM kinase activation (Goodarzi et al. 2004), we adopted the IP ATM kinase assay to measure ATM activation. In this kinase assay, a GST-chicken Rad9 fusion protein rather than a PHAS-1 artificial substrate was used as the substrate for the chicken ATM kinase, because a region containing an ATM phosphorylation site corresponding to Ser-272 in human Rad9 (Chen et al. 2001) is well conserved in chicken Rad9 (Kobayashi et al. 2004) (Supplementary Fig. S3) and because this fusion protein gives more reliable results than PHAS-1, with low background. Using this ATM IP kinase assay, we confirmed that Nbs1 is required for effective IR-induced ATM activation (Supplementary Fig. S4) (Uziel et al. 2003; Kitagawa et al. 2004; Falck et al. 2005). Having established the ATM kinase assay in DT40 cells, we then analyzed the activation of ATM kinase in wild-type DT40 cells treated with various concentrations of MNNG. As shown in Fig. 1A, the ATM kinase was effectively activated with MNNG; the maximum activation of ATM was observed with 200 µM MNNG for 1 h, though 10 µM MNNG was sufficient to kill 99% DT40 cells (data not shown). However, higher concentrations of MNNG were somewhat inhibitory for ATM kinase activity, as we found that excess SH modification of ATM is inhibitory (Fig. 2).
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MutS, which consists of Msh2 and Msh6, is known to bind the O6-MeG generated by MNNG and is involved in the ATR-mediated Chk1 phosphorylation induced by MNNG (Stojic et al. 2004b), through direct interactions with ATR (Wang & Qin 2003). As shown in Fig. 1D, we confirmed the requirement for Msh6 for MNNG-induced and ATR-mediated Chk1 phosphorylation. However, we observed that the ATM kinase was effectively activated in MSH6–/– cells in response to MNNG (Fig. 1E), in agreement with previous findings that MNNG induces the activation of ATM kinase in human MLH1-deficient HCT116 cells (Adamson et al. 2002). In addition, O6-benzyl guanine, a specific inhibitor for methylguanine methyltransferase, neither inhibited nor enhanced MNNG-induced ATM activation (data not shown).
ATM is directly activated by MNNG through the modification of free SH groups
The above results, showing that MNNG-induced ATM kinase activation requires neither Nbs1 nor Msh6 (Fig. 1), raise the possibility that MNNG acts directly on ATM, rather than through the generation of methylated DNA. To explore this possibility, immunoprecipitated ATM, prepared from untreated DT40 cells with antibodies specific for chicken ATM, was treated with MNNG and the ATM kinase activity was measured. As shown in Fig. 2A, ATM was significantly activated by MNNG in this chromatin-free experiment, and much lower concentrations of MNNG (0.2 µM) were sufficient for the maximum activation of ATM activity. These results further support the possibility that MNNG acts directly on ATM rather than through the generation of methylated DNA. Since, in addition to methylating DNA, MNNG modifies SH groups on cysteine residues in some proteins (Sugimura et al. 1968; Nagao et al. 1971; Johnson & Greenberg 1973), we investigated whether the MNNG-mediated modification of ATM's SH groups is involved in the cell-free activation of ATM by MNNG. Immunoprecipitated ATM was therefore pretreated with a SH-specific agent, N-ethylmaleimide (NEM), before MNNG treatment. As shown in Fig. 2B, the activation of ATM by 0.5 µM MNNG was completely inhibited by the pretreatment with excess NEM (1 mM). Furthermore, the basal activity of ATM decreased after pretreatment with high concentrations of NEM (4 mM), indicating that excess SH modification is inhibitory for the ATM kinase activity. These results suggest that ATM is activated by MNNG through the modification of critical SH groups in ATM, independent of DNA modification, and that excess SH modification is rather inhibitory for the ATM kinase activity.
15d-PGJ2 covalently binds and activates ATM
Because SH groups seemed to be important for the direct activation of the ATM kinase by MNNG (Fig. 2B), we investigated whether another specific SH-reactive oxidative stress inducer, 15d-PGJ2, would induce ATM kinase activation. DT40 cells were treated with various concentrations of 15d-PGJ2, for 1 h, and the ATM kinase activity was measured. As shown in Fig. 3A, ATM was effectively activated with 20 µM of 15d-PGJ2, and this 15d-PGJ2-induced ATM activation was observed in NBS1–/–/– DT40 cells (data not shown). However, ATM activation was not observed with 9,10-dihydro-15d-PGJ2 (CAY, Fig. 3B). This 15d-PGJ2 analog, unlike 15d-PGJ2, lacks an electrophilic ,ß-unsaturated ketone, and therefore can neither function as an oxidative stress inducer nor react with the SH groups of target proteins (Shibata et al. 2003). Interestingly, we did not detect significant Chk1, Chk2 and H2AX phosphorylation in cells treated with 15d-PGJ2 (data not shown, see also Fig. 5A and 5C), although MNNG effectively induced Chk1, Chk2 and H2AX phosphorylation together with ATM kinase activation (Figs 1 and 5). We then treated immunoprecipitated ATM, prepared from untreated cells, with 15d-PGJ2, and measured the ATM kinase activity. The results, shown in Fig. 3C, indicate that, similar to the MNNG treatment, the ATM kinase was also effectively activated by the direct treatment of immunoprecipitated ATM with low concentrations of 15d-PGJ2 (0.2 nM).
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B, IB kinase, H-Ras, and thioredoxin through covalent binding to specific cysteine residues (Rossi et al. 2000; Cernuda-Morollon et al. 2001; Oliva et al. 2003; Shibata et al. 2003), we used biotinylated 15d-PGJ2 to investigate whether 15d-PGJ2 binds to ATM. Immunoprecipitates prepared from wild-type or ATM–/– DT40 cells with antibodies specific to chicken ATM were treated with 50 µM biotinylated 15d-PGJ2 and then immunoblotted with HRP-conjugated NeutrAvidin. As shown in Fig. 4A, biotinylated 15d-PGJ2 bound to ATM was detected in the immunoprecipitates prepared from wild-type but not ATM–/– DT40 cells. In addition, we analyzed 15d-PGJ2 binding to HA-tagged human ATM (HA-hATM). HA-hATM was induced with doxycycline in ATM–/– DT40 cells stably expressing inducible HA-hATM (ATM–/–/HA-hATM), and was immunoprecipitated with anti-HA antibodies. The immunoprecipitated HA-hATM was then treated with biotinylated 15d-PGJ2, and the 15d-PGJ2 binding to HA-hATM was determined by immunoblot analysis with HRP-conjugated NeutrAvidin. Similar to chicken ATM, we detected biotinylated 15d-PGJ2 bound to HA-hATM only when the immunoprecipitates were prepared from cells in which the HA-hATM expression was induced by doxycycline (Fig. 4B). In another experiment, we treated ATM–/–/HA-hATM cells with biotinylated 15d-PGJ2 after HA-hATM induction. Biotinylated 15d-PGJ2-binding proteins were then precipitated with NeutrAvidin Plus beads, and both the precipitates ("Bound") and the supernatants ("Unbound") were immunoblotted with anti-HA. As shown in Fig. 4C, while HA-hATM was found exclusively in the "Unbound" fraction in the absence of treatment with biotinylated 15d-PGJ2, all the HA-hATM was detected in the "Bound" fraction after treatment with biotinylated 15d-PGJ2. The results of these experiments demonstrate that 15d-PGJ2 binds to ATM, presumably through the alkylation of ATM's cysteine residues.
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Since 15d-PGJ2 effectively activated the ATM kinase, it was important to investigate the possible functional consequences of ATM activation by 15d-PGJ2. However, we could not study one of the important ATM target molecules, p53 (Shiloh & Kastan 2001), in DT40 cells, because p53 expression is extremely low in these cells (Takao et al. 1999), and the ATM phosphorylation site in the N-terminal region of p53 is not conserved in chicken p53 (Soussi et al. 1988). In addition, the phosphorylation of Chk1, Chk2 and H2AX was not observed in DT40 cells in response to 15d-PGJ2 (data not shown). We therefore analyzed the phosphorylation of p53 (Ser-15) and Chk2 (Thr-68) in the YZ5 (ATM+) and EBS (ATM–) human cell lines (Ziv et al. 1997) following treatment with 15d-PGJ2. As shown in Fig. 5A, 15d-PGJ2 induced p53 phosphorylation in the YZ5 cells but not the EBS cells, establishing that ATM-dependent phosphorylation of p53 is induced by 15d-PGJ2. However, the phosphorylation of Chk2 (Fig. 5A) was not detected in either cell line, as in the DT40 cells. On the other hand, IR effectively induced the phosphorylation of both Chk2 and p53 in the YZ5 cells, and the degree of phosphorylation of both proteins was much lower in the EBS cells (Fig. 5B), confirming the induction of ATM-dependent p53 and Chk2 phosphorylation by IR. However, MNNG effectively induced the phosphorylation of Chk2 and p53 in both the YZ5 and EBS cells, indicating that MNNG-induced p53 or Chk2 phosphorylation is only partially dependent on ATM. Since we could not detect ATM autophosphorylation on Ser-1981 in YZ5 cells following IR treatment, due to the extremely low ATM expression in the YZ5 line, we studied ATM autophosphorylation and H2AX phosphorylation (Ser-139) along with p53 phosphorylation in MCF7 cells. As shown in Fig. 5C, 15d-PGJ2 (40 µM) induced only a marginal degree of ATM autophosphorylation, though 15d-PGJ2 was effective in p53 phosphorylation and accumulation. In addition, we could not detect significant H2AX phosphorylation following the 15d-PGJ2 treatment, even though MNNG was very effective in phosphorylating H2AX (Fig. 5C). This result is consistent with previous findings that agents that alter chromatin structure without introducing detectable DSB induce ATM autophosphorylation but not H2AX phosphorylation (Bakkenist & Kastan 2003).
Because ATM activation and p53 phosphorylation were observed in response to 15d-PGJ2, we investigated whether 15d-PGJ2 induces ATM-dependent apoptosis in human cells. YZ5 and EBS cells were treated with 40 µM of 15d-PGJ2 for 24 h, and apoptotic cells were quantified using annexin V FACS analysis. The results shown in Fig. 5D indicate that 15d-PGJ2 induces ATM-dependent apoptosis in human tumor cells.
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Discussion |
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, is required for the checkpoint response to methylated DNA (Wang & Qin 2003; Stojic et al. 2004b). The results of a recent study indicate that Nbs1, in addition to its role in DSB-induced ATM activation, is required for the ATR-mediated Chk1 phosphorylation induced by HU or UV (Stiff et al. 2005). Consistent with the results of these studies, we found that MNNG-induced and ATR-mediated Chk1 phosphorylation is Nbs1- and Msh6-dependent (Fig. 1B,D). These findings establish that the Chk1 and Chk2 phosphorylation that is linked to methylated DNA damage requires the DNA damage sensors, Msh6 and Nbs1, in addition to early acting checkpoint proteins such as Rad17 and Rad9. In contrast, we found that ATM is activated by MNNG in an Nbs1- and Msh6-independent manner (Fig. 1C,E), suggesting that, in addition to strict DNA damage-dependent ATR activation, MNNG activates ATM by mechanisms that do not require methylated DNA damage or DSB, which are potentially generated by damage to methylated DNA during DNA repair or replication. This result is consistent with the previous findings that agents that alter chromatin structure without introducing detectable DSB induce ATM autophosphorylation on Ser-1981 (Bakkenist & Kastan 2003). However, this hypothesis does not necessarily rule out the possibility that the damage to methylated DNA is converted to DSB (Fig. 5C), the accumulation of which might result in abnormalities in the cellular redox state under conditions of ATM deficiency.
The results of our experiments using cells deficient for NBS1 or MSH6 suggest that ATM is directly activated by MNNG in the absence of DNA damage. Indeed, we found that ATM is activated by the direct treatment of chromatin-free immunoprecipitated ATM with MNNG, through the modification of critical SH groups (Fig. 2). However, it is possible that catalytic SH groups of ATM-associated phosphatase-5 or phosphatase 2A (Ali et al. 2004; Goodarzi et al. 2004) are the actual targets of MNNG. To further explore the possible involvement of SH groups on ATM, we investigated whether 15d-PGJ2 activates ATM. 15d-PGJ2 is a potent inducer of intracellular oxidative stress (Kondo et al. 2001) and directly modulates the activity of several biologically important molecules through the specific alkylation of free SH groups on cysteine residues of target proteins (Rossi et al. 2000; Cernuda-Morollon et al. 2001; Oliva et al. 2003; Shibata et al. 2003). We found that 15d-PGJ2 effectively activated the kinase activity of ATM in both intact cells and immunoprecipitates (Fig. 3). Importantly, ATM activation was not induced with a 15d-PGJ2 analog, 9,10-dihydro-15d-PGJ2 (CAY, Fig. 3B), which lacks the reactive electrophilic ,ß-unsaturated ketone that is involved in oxidative stress induction and SH-group reactivity (Shibata et al. 2003). Using biotinylated 15d-PGJ2, we further demonstrated that 15d-PGJ2 covalently binds to ATM through the alkylation of ATM's cysteine residues (Fig. 4). These results indicate that ATM is a direct target for 15d-PGJ2, raising the interesting possibility that critical SH groups on ATM function as a direct oxidative stress sensor, independent of ATM's role in the cellular response to DSB.
Alternatively, it is plausible that free SH groups, together with Ser-1981, are involved in dormant dimer formation (Bakkenist & Kastan 2003), and the modification of the SH groups and Ser-1981 autophosphorylation results in ATM dissociation and activation. This idea is consistent with the results of recent studies that suggest that ATM autophosphorylation is not sufficient for dimer dissociation and subsequent ATM activation (Goodarzi et al. 2004; Lee & Paull 2005). We also found that, while 15d-PGJ2 effectively activates ATM kinase activity (Fig. 3) and subsequent p53 phosphorylation and accumulation (Fig. 5A,C), 15d-PGJ2 is not effective in ATM autophosphorylation (Fig. 5C). However, the formal establishment of these hypotheses requires the identification of the cysteine residues involved.
15d-PGJ2 is a prostaglandin D2 metabolite generated during inflammatory processes and is characterized by the presence of a reactive ,ß-unsaturated ketone in the cyclopentenone ring, which functions as a potent inducer of intracellular oxidative stress (Gilroy et al. 1999; Kondo et al. 2001; Shibata et al. 2002). The results of immunohistochemical analysis indicate that 15d-PGJ2 is detected in foamy macrophages in human atherosclerotic plaques, lipopolysaccharide-stimulated macrophages, inflammation-induced pleural exudates or spinal cord tissues from patients with amyotrophic lateral sclerosis (Gilroy et al. 1999; Kondo et al. 2002; Shibata et al. 2002). Furthermore, 15d-PGJ2 directly modulates the activity of several biologically important molecules, such as NF-B, IB kinase, H-Ras, and thioredoxin, through the specific alkylation of free SH groups on cysteine residues in target proteins (Rossi et al. 2000; Cernuda-Morollon et al. 2001; Oliva et al. 2003; Shibata et al. 2003). In the present study, we demonstrated that ATM is another direct target of 15d-PGJ2 (Figs 3 and 4). However, in contrast to DSB-induced ATM activation, this 15d-PGJ2-induced ATM activation is independent of Nbs1 and results in the phosphorylation and accumulation of p53 and in apoptosis, but does not cause the phosphorylation of Chk2 or H2AX (Fig. 5). This pattern of ATM activation by 15d-PGJ2 is consistent with previous findings; although p53 phosphorylation and ATM autophosphorylation are Nbs1-independent, Chk2 phosphorylation is Nbs1-dependent and strictly linked to DNA damage (Kitagawa et al. 2004; Falck et al. 2005); agents that alter chromatin structure without introducing detectable DSB, induce ATM autophosphorylation but not H2AX phosphorylation (Bakkenist & Kastan 2003). This pattern of ATM activation by 15d-PGJ2 may be physiologically significant, considering the anti-inflammatory and pro-apoptotic roles that 15d-PGJ2 plays in various disease states (Castrillo et al. 2000; Straus & Glass 2001; Kondo et al. 2002; Lee et al. 2003). In particular, 15d-PGJ2 has been implicated in p53-dependent neuronal apoptosis (Kondo et al. 2002). This pattern is also consistent with the observation that a portion of the extranuclear ATM localizes to peroxisomes (Watters et al. 1999). In addition, we recently showed that 4-oxo-2-nonenal, a major lipid peroxidation product, induces ATM autophosphorylation, p53 phosphorylation, and apoptosis in SH-SY5Y human neuroblastoma cells (Shibata et al. 2006). ATM, therefore, may function as an early monitor of various endogenously produced electrophilic metabolites and as a regulator of the cellular oxidative stress response to protect cells from the toxic effects of these metabolites, or to induce apoptosis. It is conceivable that the impairment of the ATM functions proposed here can eventually result in the clinical manifestations of A-T, such as cerebellar ataxia, premature aging, growth retardation or cancer.
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Experimental procedures |
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DT40 cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 1% chicken serum, 2 mM L-glutamine, 50 µg/mL penicillin, 50 units/mL streptomycin, and 10–5 M ß-mercaptoethanol at 39.5 °C in a humidified 5% CO2 incubator. The 293T, YZ5, EBS, and MCF7 cells were cultured in Dulbecco's modified Eagle medium (Nissui) supplemented with 15% FCS, 2 mM L-glutamine, 50 µg/mL penicillin, and 50 units/mL streptomycin. MNNG and NEM were obtained from Nacalai tesque and Sigma, respectively. 15d-PGJ2 and its analog, 9,10-dihydro-15d-PGJ2 (CAY10410), were from Cayman Chemical. Biotinylated 15d-PGJ2 was prepared as previously described (Shibata et al. 2003). X-ray irradiation was performed using an MBR-1520R radiator (Hitachi) set at 150-kVp, 20 mA, 0.5 mm aluminum, and 0.5 mm copper filtration with a dose rate of 1 Gy/min.
Gene targeting
A chicken EST clone (AI981357 [GenBank] ) containing the amino acid sequence homologous to human Msh6 was obtained at the NCBI web site. Oligonucleotide PCR primers were designed based on the EST clone, and partial chicken MSH6 cDNA was amplified by RT-PCR using RNA extracted from DT40 cells. Using the cDNA fragment as a probe, chicken MSH6 genomic clones were isolated from a spleen lambda genomic library (Stratagene). The identity of this clone was confirmed by DNA sequencing. MSH6 targeting vectors were constructed by replacing exons 2–4 of the MSH6 gene with his- and bsr-selection marker gene cassettes. For gene targeting of the chicken MSH6 locus, wild-type DT40 cells were sequentially transfected with pMSH6-his and pMSH6-bsr (Supplementary Fig. S1), as previously described (Takao et al. 1999).
Antibodies
The 1.8 kbp DNA fragment was amplified by PCR using a partial chicken ATM cDNA fragment (Takao et al. 1999) as a template. The PCR fragment was treated with BamHI and KpnI, and the purified 600-bp fragment was then ligated to the BamHI- and KpnI-treated pQE31 (Qiagen), yielding His-tag-fused partial chicken ATM expression plasmids. This plasmid contains the chicken ATM amino acid sequence corresponding to the 1943–2124 amino acids of human ATM (Supplementary Fig. S2). The His-tag ATM was purified with an Ni-NTA column (Qiagen) and was used to immunize white rabbits, and anti-ATM antisera were obtained from these rabbits. Antibodies against phospho-Chk1 (Ser-345), phospho-Chk2 (Thr-68), phospho-p53 (Ser-15), and phospho-ATM (ser-1981) were from Cell Signaling Technology. Antibodies against phospho-H2AX (Ser-139), Chk1, p53, ATM, and actin were from Upstate, Santa Cruz Biotechnology, Calbiochem, GeneTex, Inc., and Sigma, respectively.
GST-Rad9 fusion proteins
The expression vector for the GST-fused chicken Rad9 fragment containing the ATM phosphorylation site (Supplementary Fig. S3) was constructed as follows. A chicken RAD9 DNA fragment was amplified by PCR using chicken RAD9 cDNA (Kobayashi et al. 2004). The PCR fragment was treated with BamHI and XhoI, and the fragment was ligated into BamHI- and XhoI-treated pGEX4T-3 (Pharmacia Biotech), yielding a GST-Rad9 fusion protein expression plasmid. The GST-Rad9 fusion protein was purified with Glutathione Sepharose 4B (Pharmacia Biotech).
Immunoblot analysis
Cells were harvested, washed, and lyzed in lysis buffer A (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 µg/mL aprotinin, 1 µg/mL leupeptin). After centrifugation at 16 000 g for 15 min, the protein concentrations of the cleared lysates were determined by the Bio-Rad protein quantification assay. The same amounts of protein were separated by SDS-PAGE (8 or 15%), followed by blotting to nitrocellulose or to a Hybond-P (Amersham Biosciences) membrane.
Immunoprecipitation (IP) kinase assay for ATM
Cells were treated with IR, MNNG, or 15d-PGJ2 in a CO2 incubator, then collected, washed with ice-cold PBS, and lyzed in NETN150 (20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, 1 mM PMSF, 10 mM
ß–glycerophosphate, 1 mM NaF, 0.1 mM Na3VO4, 0.2 U/mL aprotinin, 10 µg/mL leupeptin, 5 µg/mL pepstatin). After clarifying the cell lysates by centrifugation, antibodies to chicken ATM were added to the extracts, and the reaction mixtures were rotated for 1 h at 4 °C, followed by incubation with protein G-Sepharose 4 Fast Flow (Amersham Biosciences) for 1 h at 4 °C. The immunoprecipitates were washed twice with lysis buffer and twice with kinase buffer (20 mM HEPES/KOH pH 7.5, 10 mM MgCl2, 10 mM MnCl2). The immunoprecipitates thus prepared were not contaminated with chromatin components, as we could not detect Histone H3 in the immunoprecipates by immunoblot analysis (data not shown). After centrifugation, the immunoprecipitates were resuspended in 20 µL of kinase buffer containing 25 µM ATP, 1 µg GST-Rad9-fusion proteins, and 10 µCi of [
-32P]ATP. The reaction mixtures were incubated for 30 min at 30 °C, then boiled in SDS sample buffer for SDS-PAGE (13%). Gels were dried on to a filter paper and analyzed by autoradiography. Radioactivity was quantified with a Bio-image Analyzer BAS1000 (Fujix). In other experiments, immunoprecipitated ATM, prepared as described above, was mixed with 10 µL of MNNG or 15d-PGJ2 diluted in kinase buffer and 10 µL of kinase buffer containing 50 µM ATP, 1 µg GST-Rad9 protein, and 10 µCi [-32P]ATP. The reaction mixtures were incubated for 30 min at 30 °C, and the ATM kinase activity was measured as described above. For NEM treatment, immunoprecipitated ATM was resuspended in kinase buffer containing NEM and incubated for 15 min at 25 °C. The immunoprecipitates were washed once with kinase buffer, then used for the kinase assay.
Covalent binding of 15d-PGJ2 to ATM
We isolated several partial and overlapping human ATM cDNA clones by library screening and PCR based on the published human ATM cDNA sequence (Savitsky et al. 1995), and a full-length ATM cDNA was constructed from these cDNA clones. The full-length human ATM cDNA expression vector with an HA-tag (HA-hATM) was prepared step-wise by constructing an HA-tagged eukaryotic expression vector, pEF-3HA, and then cloning the entire ATM coding sequence in the correct orientation into the expression vector, pEF-HA-hATM (the HA-tag sequence is located upstream of the ATM coding sequence). To prepare ATM–/– DT40 cells stably expressing a doxycycline-inducible HA-hATM gene (ATM–/–/HA-hATM), we used pUHD10-1 and pUHD172–1neo plasmids (kindly provided by Dr M. Gossen) (Gossen et al. 1995). ATM–/– DT40 cells were transfected with both the PvuI-digested pUHD10-3-HA-hATM and XhoI-digested pUHD172–1neo and selected with neomycin, yielding ATM–/–/HA-hATM DT40 cells.
ATM–/–/HA-hATM cells were incubated with doxycycline (1 µg/mL) overnight, collected, washed with ice-cold PBS, and lyzed in lysis buffer B (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5% Tween20, 0.2% NP-40, 1 mM PMSF, 0.2 U/mL aprotinin, 10 µg/mL leupeptin, 5 µg/mL pepstatin). After clarifying the lysates by centrifugation, the extracts were reacted with an anti-HA antibody (Boehringer Mannheim) for 2 h at 4 °C. The reaction mixtures were then incubated with protein G-Sepharose 4 Fast Flow for 1 h at 4 °C. The immunoprecipitates were washed 4 times with lysis buffer B, and twice with reaction buffer (20 mM HEPES/KOH, pH 7.5). The immunoprecipitates were resuspended in 60 µL reaction buffer with or without 50 µM biotinylated 15d-PGJ2, incubated for 1 h at 30 °C, then boiled in SDS sample buffer. The reaction products were separated by SDS-PAGE (6%) and biotinylated proteins were detected with HRP-conjugated NeutrAvidin (Pierce Biotechnology). For chicken ATM, antisera to chicken ATM and NETN150 supplemented with protease inhibitors were used instead of the anti-HA antibodies and lysis buffer B, respectively.
ATM–/–/HA-hATM cells were incubated with doxycycline overnight, then incubated with or without 20 µM biotinylated 15d-PGJ2 for 1 h in a CO2 incubator. The cells were collected, washed with ice-cold PBS, and lyzed in NETN150 supplemented with protease inhibitors. After centrifugation, the cleared extracts were incubated with 50 µL NeutrAvidin-Plus beads (Pierce Biotechnology) for 3 h at 4 °C with rotation and shaking. After centrifugation, the supernatants were collected as the "Unbound" fractions. The beads were washed 4 times with the lysis buffer, and were boiled in SDS sample buffer to prepare the bound proteins ("Bound"). The "Unbound" and "Bound" proteins were separated by SDS-PAGE (6%), and HA-hATM was detected by using the anti-HA antibody as the primary antibody.
Assessment of apoptosis
YZ5 and EBS cells were untreated or treated with 40 µM of 15d-PGJ2. After a 24-h incubation, the cells were trypsinized and harvested. Apoptotic cells were stained with annexin V using the Annexin V apoptosis kit (Clontech), and the annexin V-positive cells were detected by a FACS Calibur (Becton Dickinson), following the manufacturer's instructions.
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Acknowledgements |
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* Correspondence: E-mail: kyamamot{at}kenroku.kanazawa-u.ac.jp
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References |
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Received: 19 February 2006
Accepted: 5 April 2006
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) or treated with 40 µm 15d-PGJ2 (
). After a 24-h incubation, the cells were trypsinized and harvested. Apoptotic cells were quantified by annexin V FACS analysis. Each value represents the mean ± SD for at least three experiments.