Activation of p53 transcriptional activity requires ATM's kinase ... - Nature

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Oncogene (2001) 20, 5100 ± 5110 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Activation of p53 transcriptional activity requires ATM's kinase domain and multiple N-terminal serine residues of p53 Gaetan A Turenne1, Proma Paul1, Lareina La¯air1 and Brendan D Price*,1 Department of Radiation Oncology, D810A, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts, MA 02115, USA

The ATM protein kinase regulates the cell's response to DNA damage by regulating cell cycle checkpoints and DNA repair. ATM phosphorylates several proteins involved in the DNA-damage response, including p53. We have examined the mechanism by which ATM regulates p53's transcriptional activity. Here, we demonstrate that reintroduction of ATM into AT cells restores the activation of p53 by the radio-mimetic agent bleomycin. Further, p53 activation is lost when a kinase inactive ATM is used, or if the N-terminal of ATM is deleted. In addition, AT cells stably expressing ATM showed decreased sensitivity to Ionizing Radiationinduced cell killing, whereas cells expressing kinase inactive ATM or N-terminally deleted ATM were indistinguishable from AT cells. Finally, single pointmutations of serines 15, 20, 33 or 37 did not individually block the ATM-dependent activation of p53 transcriptional activity by bleomycin. However, double mutations of either serines 15 and 20 or serines 33 and 37 blocked the ability of ATM to activate p53. Our results indicate that the N-terminal of ATM and ATM's kinase activity are required for activation of p53's transcriptional activity and restoration of normal sensitivity to DNA damage. In addition, activation of p53 by ATM requires multiple serine residues in p53's transactivation domain. Oncogene (2001) 20, 5100 ± 5110. Keywords: ATM; p53; chk2; phosphorylation; bleomycin Introduction In response to DNA damage, mammalian cells activate cell cycle checkpoints (Canman and Lim, 1998a; Weinert, 1998). These checkpoints block DNA synthesis or chromosome segregation if the DNA is damaged (Jeggo et al., 1998; Meyn, 1999; Weinert, 1998), which may allow cells extra time to repair DNA damage. The ATM protein is a key regulator of the DNA-damage response (Savitsky et al., 1995). Muta-

*Correspondence: BD Price; E-mail: [email protected] Received 13 February 2001; revised 17 May 2001; accepted 24 May 2001

tions in the ATM protein give rise to the inherited disease Ataxia Telangiectasia (AT). AT is characterized by immunode®ciency, cerebellar ataxia, increased incidence of cancer and extreme sensitivity to Ionizing Radiation (Beamish and Lavin, 1994; Beamish et al., 1994; Luo et al., 1996; Meyn, 1999; Taylor et al., 1975). AT cells exhibit increased sensitivity to IR, radioresistant DNA synthesis and increased genetic instability (Cornforth and Bedford, 1985; Meyn, 1999; Taylor et al., 1975). The kinetics of DNA doublestrand break repair is grossly normal in AT cells, although the cells may contain residual levels of double-strand breaks (Jeggo et al., 1998). AT cells also exhibit high rates of spontaneous DNA recombination, poor ®delity during repair (Luo et al., 1996), and do not undergo potentially lethal damage repair (Weichselbaum et al., 1977). Further, AT cells have elevated levels of chromosomal abnormalities, including shortened telomeres and chromosome end joining (Metcalfe et al., 1996; Pandita et al., 1995). The ATM protein is a 3056 amino-acid protein containing a C-terminal kinase domain (Savitsky et al., 1995). ATM can phosphorylate several proteins involved in DNA repair, including p95/nibrin, (which may regulate the S phase check point; Lim et al., 2000; Zhao et al., 2000) and Brca1, a protein involved in DNA repair (Cortez et al., 1999). ATM can also directly phosphorylate two key regulators of cell cycle checkpoints ± the p53 tumor suppressor gene (Banin et al., 1998; Canman et al., 1998; Kastan et al., 1991; 1992) and the checkpoint kinase chk2 (Blasina et al., 1999a; Brown et al., 1999; Matsuoka et al., 1998). chk2 is involved in the activation of the G2checkpoint (Blasina et al., 1999b; Brown et al., 1999; Matsuoka et al., 1999). Exit from G2 requires activation of cyclin B/cdc2 through the tyrosine dephosphorylation of cdc2 by cdc25 phosphatase (reviewed in Rhind and Russell, 1998). In cells exposed to Ionizing Radiation, ATM phosphorylates chk2, activating chk2's kinase activity (Blasina et al., 1999a; Brown et al., 1999; Matsuoka et al., 1998). Activated chk2 then phosphorylates the cdc25 tyrosine phosphatase, inhibiting its activity (Blasina et al., 1999b; Furnari et al., 1999). Cyclin B/cdc2 therefore remains inactive, and the cells are arrested in G2 (Blasina et al., 1999b; Furnari et al., 1999). If AT cells are irradiated in G2-M, they exhibit a much shorter G2 arrest than

ATM's kinase activity is required to activate p53 GA Turenne et al

normal cells and exit rapidly into G1 (Beamish et al., 1994; Nagasawa et al., 1994). Thus the defective G2 checkpoint seen in AT cells may be seen, at least in part, as the failure of ATM to activate chk2 following DNA damage. AT cells also fail to activate the p53-dependent G1 checkpoint (Kastan et al., 1991; 1992). In normal cells, exposure to Ionizing Radiation increases p53 protein levels by decreasing the proteolytic degradation of p53 (Haupt et al., 1997; Kastan et al., 1991; Kubbutata et al., 1998; Tishler et al., 1993). Degradation of p53 is mediated by the mdm2 oncogene (Haupt et al., 1997; Kubbutata et al., 1998), such that mdm2-p53 complexes are targeted for ubiquitin dependent degradation. The full activation of p53 by Ionizing Radiation requires the ATM protein (Kastan et al., 1992), and ATM protein can phosphorylate serine 15 of the p53 protein in vitro (Banin et al., 1998; Canman et al., 1998). Further, serines 15 and 20 of p53 are phosphorylated in vivo following exposure of normal, but not AT cells, to Ionizing Radiation (Chebab et al., 1999; Sakaguchi et al., 1998; Shieh et al., 1997, 1999; Siliciano et al., 1997; Unger et al., 1999a). Since phosphorylation of serine 20 of p53 is carried out by chk2 kinase (Chebab et al., 2000; Hirao et al., 2000; Tominaga et al., 1999), ATM therefore mediates both the direct phosphorylation of serine 15 and indirectly regulates serine 20 phosphorylation by controlling activation of chk2. There are additional sites for DNA damage induced phosphorylation at serines 33 and 37 of p53 (Sakaguchi et al., 1998; Shieh et al., 1997; 1999; Siliciano et al., 1997), which may be regulated by Atr and other kinases (Hall-Jackson et al., 1999; Tibbets et al., 1999). The phosphorylation of p53's N-terminal has been shown to block binding of mdm2, and therefore increase the stability of the p53 protein. More detailed work has shown that phosphorylation of serine-20 is the key-regulator of mdm2-p53 interaction, with other residues, including serines 15, 33 and 37 playing a lesser role (Chebab et al., 1999; Shieh et al., 1997; Unger et al., 1999a). mdm2 is also phosphorylated by ATM, and this may further contribute to the stabilization of p53 following exposure to Ionizing Radiation (Khosravi et al., 1999). Thus the control of p53 activation by ATM is complex, involving direct phosphorylation of serine 15 by ATM, phosphorylation of serine 20 by chk2 (which is activated by ATM) and further by direct phosphorylation of mdm2 by ATM. In addition, DNA damage increases the phosphorylation of serines 33 and 37 of p53 through an ATM-independent mechanism (Sakaguchi et al., 1998). These phosphorylations are thought to prevent mdm2 binding and leads to accumulation of p53 protein in the cell. Although accumulation of p53 protein is the initial step in the ATM-dependent activation of p53, subsequent steps, including activation of p53's DNA binding and changes in p53's transcriptional regulatory activity are also involved. For example, p53's DNA binding activity is increased by the Ionizing Radiation-induced acetylation of the Cterminal of p53, and this acetylation requires the prior

phosphorylation of the N-terminal of p53 (Sakaguchi et al., 1998). Serines 15, 20, 33 and 37 are all within the N-terminal transactivation domain of p53, and phosphorylation of some or all of these serine residues may be required to stimulate transcriptional activation of p53 target genes. However, the role of serines 15, 20, 33 and 37 in regulating the ATM-dependent activation of p53 transcriptional activity is not known. In this study, we have determined how ATM's kinase activity regulates activation of p53's transcriptional activity, and identi®ed the serine residues in the N-terminal of p53 which mediate this eect. Using a kinase inactive ATM construct, we demonstrate that the kinase activity of ATM is required for activation of p53 and chk2 kinase, and for the restoration of normal radiosensitivity in AT cells. Further, we have identi®ed an N-terminal region of ATM which is required for correct ATM functioning. In addition, we demonstrate that double-mutations of either serines 15 and 20 or serines 33 and 37 abolishes the ATM-dependent activation of p53 by ATM.

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Results To examine how ATM activates p53, we generated several deletion constructs of the ATM protein (Figure 1a). ATM constructs were inserted into the pcDNA3.1/ HisA expression vector, which adds the Omni tag to the N-terminal of ATM. The construction of full-

Figure 1 Construction and expression of ATM constructs. (a) Map of ATM constructs showing location of key domains and size of fragments. FL-AT encodes amino-acids 1-3056, FL-ATkd encodes the triple mutation D2879A/N2884K/D2898A, AT-DN encodes amino-acids 769-3056 and ATK encodes amino-acids =Omni Tag, =Leucine zipper, =kinase 2138-3056. domain. (b) Left panel. GM5849 cells were transiently transfected with vector (C) or FL-AT (AT), immunoprecipitated with goat IgG (lanes 1 and 2) or Omni-tag antibody (lanes 3 and 4) and ATM detected by Western blotting with anti-Omni antibody. Center and right panels. GM5849 AT cells expressing vector (pcDNA3.1/HisA), FL-AT, FL-ATkd, AT-DN, ATK or ATKkd were immunoprecipitated with anti-Omni antibody and analysed by Western blotting using Omni antibody Oncogene


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length ATM (FL-AT) and kinase inactive ATM (FLATkd) and the demonstration that FL-ATkd does not display kinase activity have been previously reported by us (Blasina et al., 1999a). Two further constructs were prepared, described in Figure 1. The N-terminal region of ATM has been reported to contain a site for interaction with both p53 (Khanna et al., 1998) and histone deacetylase (Kim et al., 1999). In AT-DN, the N-terminal of ATM (amino-acids 1-768) was deleted to examine the role of this region in p53 activation. Further reports have indicated that the kinase domain of ATM may be sucient to restore normal radiosensitivity to AT cells (Morgan et al., 1997). To explore this possibility, ATK, which encodes amino-acids 2138 ± 3056 of ATM, including the kinase domain, was constructed. The speci®city of the Omni antibody was examined by transiently expressing either vector or FL-AT in the Ataxia Telangiectasia cell line GM5849. ATM was only detected in cells transfected with FL-AT and was speci®cally immunoprecipitated by the Omni antibody but not by IgG (Figure 1b, left panel). In Figure 1b, AT cells stably expressing the indicated construct were immunoprecipitated with Omni antibody and examined by Western blotting. Vector transformed cells did not display any immunoreactive protein (Figure 1b, center panel), whereas ATM was clearly detected in cells expressing FL-AT, FL-ATkd and AT-DN. Expression of the kinase domain of ATM, ATK, and the kinase inactive version, ATkd, was also seen. Note that ATkd had a slightly increased mobility on SDS ± PAGE, presumably due to the triple mutation in the kinase domain which results in the net loss of two negatively charged amino-acids. Prior to examining the ability of these ATM constructs to activate p53, we examined the ability of each to complement the increased sensitivity of AT cells to IR. Cell lines stably expressing these ATM constructs were exposed to increasing amounts of Ionizing Radiation and clonogenic cell survival measured. GM5849 AT cells containing only the selection marker were very sensitive to radiation (Figure 2a,*). Cells expressing FL-AT showed a statistically signi®cant increase in cell survival following exposure to Ionizing Radiation (Figure 2a,*) whereas AT cells expressing the kinase inactive ATM construct were not statistically dierent from control cells (Figure 2a,D). AT cells typically display a 3 ± 8fold increase in sensitivity to DNA damage (Beamish and Lavin, 1994; Beamish et al., 1994; Taylor et al., 1975). In our complementation studies, we were only able to achieve about a twofold increase in cell survival following exposure to Ionizing Radiation (Figure 2a). This is similar to other published results, where stable transfection of ATM into AT cells increased cell survival by only 2 ± 3-fold (Zhang et al., 1997; Ziv et al., 1997). This low level of complementation may derive from our use of pooled clonal populations, which may express heterogeneous levels of ATM protein and therefore of radiosensitivity. In addition, AT cells are genetically unstable (Meyn, 1999) and will

have accumulated numerous genetic changes during growth in culture. These changes may aect the inherent radiosensitivity of the AT cells, and reintroduction of ATM may not completely correct this. To examine if the kinase domain alone was sucient to complement the increased sensitivity of AT cells, GM5849 AT cells expressing ATK or AT-DN were exposed to increasing doses of Ionizing Radiation and clonogenic cell survival measured. Neither ATK, containing approximately 900 amino-acids from the C-terminal, or AT-DN increased the ability of AT cells to survive radiation induced cell death (Figure 2B). Figure 2 therefore demonstrates that the kinase domain of ATM as well sequences in the N-terminal are required for ATM to mediate cell survival following DNA damage. The induction of transcriptionally active p53 by DNA damage involves at least two steps. First, p53's stability is increased by inhibiting the interaction between mdm2 and p53 (Sakaguchi et al., 1998; Shieh et al., 1999; Siliciano et al., 1997; Unger et al., 1999a) and second, there is activation of p53's DNA binding activity and increased transcription of p53 target genes (Tishler et al., 1993). In AT cells, activation of p53 is defective, and there is little or no increase in transcriptional activity (Kastan et al., 1991, 1992). To analyse the mechanism by which ATM activates p53's transcriptional activity, we employed the SV40 transformed AT ®broblast cell line GM5849. Although GM5849 AT cells express wtp53 (Jung et al., 1997), they also express the SV40Tag, which binds to the DNA binding domain of p53 and inactivates p53 (Hess and Brandner, 1997; Kohli and Jorgensen, 1999; O'Neil et al., 1996). However, several reports demonstrate that the p53 response to DNA damage is intact in SV40 transformed human ®broblast cells, including the activation of G1 arrest (Kohli and Jorgensen, 1999; O'Neil et al., 1996), phosphorylation of serines 15 and 37 of p53 (Tibbets et al., 1999), activation of p53-DNA binding activity and increased transcriptional activity (Hess and Brandner, 1997; Kohli and Jorgensen, 1999; O'Neil et al., 1996; Sheppard and Liu, 1999). Further, a signi®cant proportion of the p53 in these cells is not associated with the SV40Tag (O'Neil et al., 1996), indicating that these cells contain a pool of free wtp53 which can potentially be regulated in response to DNA damage. In Figure 3a, we ®rst determined if GM5849 AT cells contained p53 which was not bound to SV40Tag. AT cell extracts were immunodepleted of SV40Tag by three sequential immunoprecipitations with anti-SV40Tag antibody (Figure 3a, lanes 1 ± 3), followed by immunoprecipitation with anti-p53 antibody (Figure 3a, lane 4). The immunoprecipitated protein was then examined by Western blot for both SV40Tag and p53 protein (Figure 3a). The ®rst immunoprecipitation with SV40Tag antibody pulled down both Tag and p53, indicating that both proteins exist as a complex, as previously described (O'Neil et al., 1996). Two further immunoprecipitations with SV40Tag antibody did not yield any additional p53 or SV40Tag. When the SV40Tag depleted extracts were


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Figure 2 Radiosensitivity of AT cells expressing ATM constructs. GM5849 cells stably expressing the indicated construct were irradiated and the number of surviving colonies counted 14 days later. (a) *=Vector, ~=FL-ATkd, *=FL-AT. (b) *=vector, &=AT-DN, ~=ATK. Results+s.e.m., errors shown where larger than symbol size

Figure 3 Activation of p53 transcriptional activity by Bleomycin. (a) GM5849 cell lysates were immuno-depleted of SV40 Tag by three consecutive immunoprecipitations with anti-Tag antibody (lanes 1 ± 3, S), followed by immunoprecipitation of p53 with antip53 antibody (lane 4, P) or immnuo-depleted of p53 by three consecutive immunoprecipitations with anti-p53 antibody (lanes 5 ± 7, P), followed by immunoprecipitation of Tag with anti-Tag antibody (lane 8, S). At each step, the amount of p53 and Tag present was examined by Western blotting. (b+c) GM5849 AT cells were transiently transfected with either the pGL3(T+) or p50-Luc reporter constructs. Transfections contained wtp53 (150 ng), mtp53 (300 ng) or full-length ATM (1.8 mg) as indicated. All transfections contained reporter (0.6 mg), pCMV-Gal (0.8 mg) and total DNA was maintained at 4 mg using the promoterless ATM vector AT-Sure and/or pcDNA3.1/HisA. Cells were incubated for 18 h post-transfection with either bleomycin (3 mM) or solvent (PBS). Luciferase activities were measured and transfection eciency monitored using pCMV-gal activity. All experiments were carried out in triplicate, results+s.e.m.

immunoprecipitated with anti-p53 antibody, signi®cant amounts of p53 were seen, but no co-precipitating SV40Tag was detected (Figure 3a, lane 4). This indicates that the AT cells contain both p53-SV40Tag

complexes as well as free p53. The reciprocal experiment was also carried out. AT cell extracts were subject to three consecutive immunoprecipitations with anti-p53 antibody. Both p53 and SV40Tag were Oncogene


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detected after the ®rst immunoprecipitation with p53 antibody, and subsequent immunoprecipitations revealed only small amounts of p53 (Figure 3a, lanes 5 ± 7). When the extracts which were immunodepleted of p53 were further immunoprecipitated with SV40Tag antibody, free SV40tAG was detected but no coprecipitating p53 was seen (Figure 3a, lane 8). This interaction between p53 and SV40Tag was unaected by either reintroduction of the ATM gene or exposure of the cells to the DNA-damaging agent bleomycin, indicating that ATM and DNA damage do not measurably alter the association between p53 and SV40 Tag (B Price, unpublished results). Therefore GM5849 AT cells contain a pool of free wtp53 with the potential to be activated by DNA-damaging agents. p53's transcriptional activity was monitored in a transient transfection experiment using the p53 luciferase reporter construct p50-Luc (Youmell et al., 1998). p50-Luc contains two copies of a p53-consensus binding site linked to the TATA box and minimal promoter of the pGL3(T+) luciferase reporter construct. AT cells transfected with pGL3(T+) alone displayed negligible luciferase activity, and cotransfection with a wtp53 expression vector did not increase this activity (Figure 3b). GM5849 AT cells transfected with p50-Luc had increased basal activity, and displayed strong activation of the reporter following cotransfection with a wtp53 expression vector (Figure 3b). Since the only dierence between pGL3(T+) and p50-Luc is the presence of the p53-consensus binding site in p50-Luc, Figure 3b demonstrates that p50-Luc can be activated by wtp53. In Figure 3c, AT cells were transfected with p50-Luc and exposed to the radiomimetic agent bleomycin. Bleomycin induces strand breaks in DNA that are similar to those caused by Ionizing Radiation, and AT cells show increased sensitivity to bleomycin compared to normal cells (Beamish and Lavin, 1994; Meyn, 1999). Bleomycin caused a small increase in transcription from the p50Luc reporter which was abolished by mtp53, which functions as a dominant negative inhibitor of wtp53 function. When AT cells were cotransfected with FL-AT, there was an increase in basal activity from the promoter which was increased further by bleomycin treatment. When cells were cotransfected with ATM plus the dominant negative mtp53, both basal and bleomycin induced transcription were decreased (Figure 3c). The results in Figure 3 demonstrate that p50-Luc is speci®cally responsive to wild type, but not mutant p53. Further, bleomycin activates transcription from p50-Luc, and this activation is increased in the presence of ATM, but inhibited by mtp53. These results are consistent with bleomycin increasing wtp53 transcriptional activity through an ATM-dependent mechanism. In Figure 4, GM5849 AT cells were transiently transfected with either FL-AT, FL-ATkd or wtp53 in the indicated combinations and the ability of bleomycin to increase the transcriptional activity of p50-Luc measured. Control cells (Figure 4, *) showed an increase in activity from the reporter as the bleomycin

Figure 4 ATM's kinase activity is required to activate p53 transcription. GM5849 AT cells were transiently transfected with p50-Luc reporter (0.6 mg), pCMV-Gal (0.8 mg) and either the promoterless AT-Sure vector (Con, *), pFL-AT (FL-AT, ~), pFL-ATkd (ATkd, &), in the absence (open symbols) or presence (closed symbols) of pcDwtp53 (p53; 150 ng). Cells were exposed to bleomycin for 18 h, and extracts assayed for luciferase activity. b-galactosidase activity from the constitutive pCMV-Gal vector was used to control for transfection eciencies. All data points were carried out in triplicate. Error bars indicated where greater than symbol size+s.e.m.

concentration was increased. When FL-AT was transfected into the GM5849 AT cells, bleomycin caused a signi®cant increase in activity from the reporter, with maximal activation between 1 ± 5 mM bleomycin (Figure 4, ~). In contrast, the full-length kinase inactive ATM construct was indistinguishable from control cells (Figure 4, &). Because activation of the reporter was only seen in the presence of both FLAT and a DNA-damage signal from bleomycin, this indicates that there is speci®c activation of the p53 reporter by the ATM protein, presumably through activation of endogenous p53. To determine if this activation was indeed the result of p53 activation rather than through some alternative mechanism, GM5849 cells were transiently transfected with an expression vector for wtp53. In the absence of bleomycin, the exogenous p53 induced a fourfold increase in basal transcriptional activity, and, when combined with the DNA-damage signal from bleomycin, gave a further twofold increase in activity (Figure 4, *). Since GM5849 cells do not express ATM protein, this increase in p53 activity by bleomycin treatment may re¯ect activation of other regulators of p53, such as Atr or DNA-PK. If cells were cotransfected with FL-ATkd and p53, no further increase in transcriptional activity was seen compared to cells transfected with p53 alone (Figure 4, &). However, when cells were transfected with both wtp53 and FL-AT, strong activation of the p53 reporter construct was seen. We interpret this to mean that ATM can activate the transcriptional activity of p53 in these cells when exposed to an appropriate DNA-


damage signal. Further, Figure 4 indicates that activation of p53's transcriptional activity by ATM requires an intact kinase domain. In Figure 5a, we further examined the role of ATM's kinase domain in activating p53's transcriptional activity. GM5849 AT cells were transfected with wtp53 in the presence of the indicated construct. The per cent increase in reporter activity following bleomycin exposure was then calculated. When GM5849 AT cells transfected with p53 were exposed to bleomycin, the activity of the p50-Luc reporter was increased (Figure 5a: Vector), similar to that seen in Figure 4. When GM5849 cells were transfected with both p53 and FL-AT (Figure 5a: FL-AT), strong activation of the p53 reporter construct by bleomycin was observed. However, cells transfected with p53 and either AT-DN or the kinase domain of ATM, ATK, failed to stimulate the transcriptional activity of wtp53 when exposed to bleomycin. The results in Figures 4 and 5a indicate that both the kinase activity and sequences in the N-terminal of the ATM protein are required for the ecient activation of p53 by ATM following exposure to a DNA-damaging signal. ATM activation of p53 is thought to occur through two distinct phosphorylation steps. First, ATM can phosphorylate serine 15 of p53 (Banin et al., 1998; Canman et al., 1998). Second, ATM activates chk2 kinase which can phosphorylate serine 20 of p53

(Chebab et al., 2000; Hirao et al., 2000; Tominaga et al., 1999). Thus ATM activation leads to phosphorylation of p53 at serines 15 and 20. The inability of FLATkd, AT-DN or ATK to activate p53 might therefore stem from their inability to phoshorylate serine 15 or to activate chk2. To examine the relative importance of these two events in activating p53, we examined the ability of ATM constructs to activate chk2 kinase following exposure to bleomycin. chk2 activation was examined by transfecting cells with expression vectors for chk2 or a kinase inactive chk2 termed chk2kd. Both contain a HA-epitope tag at the N-terminal, which increases their apparent mass by approximately 2 kd. These constructs were then transiently expressed in GM637 cells, an SV40 transformed ®broblast cell line which expresses normal ATM protein. In Figure 5b (top panel), both endogenous (lower band) and transfected chk2 constructs (upper band) were readily detected by Western blotting. GM637 cells were then transfected with chk2 or chk2kd and immunokinase assays carried out to monitor chk2 autophosphorylation after exposure bleomycin. chk2 anti-sera immunoprecipitated a 64 kd protein whose autophosphorylation was stimulated by bleomycin (Figure 5b, lanes 3 and 4). This activity was absent from immunoprecipitates using IgG (Figure 5b, lanes 1 and 2), and was not detected in GM5849 AT cells (lanes 9 and 10). Cells transfected

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Figure 5 Activation of chk2 requires the ATM protein. (a) p53 activation requires the N-terminal of ATM. GM5849 cells were transiently transfected with p50-Luc reporter (0.6 mg), pCMVGal (0.8 mg), wtp53 (100 ng) and either vector, FL-AT, AT-DN or ATK. Cells were exposed to bleomycin (2 mM) for 16 h, and extracts assayed for luciferase activity. b-galactosidase activity from the constitutive pCMV-Gal vector was used to control for transfection eciencies. Results are expressed as the % increase in reporter activity by bleomycin in the presence of the indicated ATM construct. Results+s.e.m. (b) Chk2 expression. Upper panel. Normal GM637 ®broblasts were transiently transfected with HA tagged chk2 (chk2), HA tagged kinase inactive chk2 (chk2kd) or vector and examined by Western blotting using an anti-chk2 antibody. Middle panel. GM637 cells (lanes 1 ± 8) were transiently transfected with chk2 (C), chk2kd (kd) or vector (V). Non-transfected GM5849 AT cells were used as controls (lanes 9 and 10). Cells were exposed to bleomycin (2 mM,+) or solvent (PBS, 7) for 10 min and extracts immunoprecipitated with anti-chk2 anti-sera (lanes 3 ± 10) or IgG (lanes 1 and 2) and chk2 immunokinase assays carried out as described in Materials and methods. Bottom panel. GM5849 AT cells were transfected with chk2 (lanes 11 ± 18) and either vector (V, lanes 11 and 12), pFL-AT (A, lanes 13 and 14), pFL-ATkd (Ak, lanes 15 and 16) or AT-DN (DN, lanes 17 and 18). Cells were treated with bleomycin (2 mM,+) or solvent (PBS, 7) for 10 min and extracts immunoprecipitated with anti-chk2 anti-sera (lanes 11 ± 18). Chk2 was immunoprecipitated and immunokinase assays were carried out as described in Materials and methods Oncogene


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with exogenous chk2 (which has a 2 kd HA tag) displayed activation of both endogenous and transfected chk2 after bleomycin (lanes 7 and 8), whereas cells transfected with chk2kd exhibited only endogenous kinase activity (lanes 5 and 6). Figure 5 demonstrates that chk2 is activated in an ATM dependent manner, as previously shown (Blasina et al., 1999a; Brown et al., 1999; Matsuoka et al., 1998). GM5489 AT cells were then transfected with chk2 in the presence of the indicated ATM construct. Transfection of GM5849 AT cells with chk2 alone did not restore bleomycin induced activation of chk2 (Figure 5b, lanes 11 and 12). However, cotransfection of FLAT with chk2 restored bleomycin dependent activation (lanes 13 and 14), whereas both FL-ATkd and AT-DN were inactive (lanes 15 ± 18). Interestingly, the unstimulated chk2 kinase activity in FL-AT transfected cells was slightly increased (Figure 5, lane 13). The data in Figure 5b is consistent with Figures 4 and 5a, indicating that both the kinase activity and the Nterminal of the ATM protein are required for the correct functioning of the ATM protein. Further, they demonstrate that chk2 is activated by FL-AT, but not by kinase inactive versions or by the AT-DN construct. Figures 4 and 5 demonstrate that the kinase activity of ATM is required for the activation of p53 and chk2 by bleomycin. In vitro studies demonstrate that ATM phosphorylates both serine 15 of p53 and threonine 68 of chk2 kinase (Banin et al., 1998; Canman et al., 1998b; Melchionna et al., 2000). Phosphorylation of chk2 by ATM activates its kinase activity, and chk2 then phosphorylates serine 20 of p53. In vivo, the DNA-damage induced phosphorylation of serines 15 and 20 of p53 requires functional ATM protein (Chebab et al., 1999; Sakaguchi et al., 1998; Shieh et al., 1997, 1999; Siliciano et al., 1997; Unger et al., 1999a). These observations imply that ATM regulates p53 transcriptional activity through the phosphorylation of serines 15 and 20 of p53. However, serines 33 and 37 of p53 are also phosphorylated, in vivo, following exposure to DNA damage, although these phosphorylations are not known to be ATM-dependent (Sakaguchi et al., 1998; Shieh et al., 1997, 1999; Siliciano et al., 1997). Thus activation of p53's transcriptional activity is likely to involve both the ATM-dependent phosphorylation of serines 15 and 20 as well as the phosphorylation of serines 33 and 37 by other kinases. In the following experiments, we set out to determine which of the N-terminal phosphorylation sites in p53 are required for the ATM-dependent activation of p53 by bleomycin. Single serine to alanine mutations in serines 15, 20, 33 and 37, as well as double mutations in serines 15 and 20 (directly regulated by ATM) and serines 33 and 37 (not directly regulated by ATM). Each of the constructs had similar basal transcriptional activity, indicating that the amino-acid substitutions introduced did not signi®cantly alter the transcriptional activity of these mutant p53 proteins (B Price, unpublished results). This is similar to data reported by others (Ashcroft et al., 1999; Blattner et al., 1999; Unger et

al., 1999b), who showed that single or double mutations in N-terminal serine residues did not signi®cantly aect the basal transcriptional activity of these p53 constructs. In Figure 6, each of the p53 constructs was examined for the ability of bleomycin to activate p53's transcriptional activity in the absence or presence of ATM. In the absence of ATM, wtp53 and p53 with single mutations in serines 15, 20, 33 or 37 all activated the p53 reporter to similar levels when exposed to bleomycin, with p53S33A showing the highest activation (Figure 6, ± ). When each of these p53 mutants were cotransfected with FLAT and exposed to bleomycin, bleomycin increased the detected p53 transcriptional activity to similar levels. Mutation of residues 15, 20, 33 and 37 therefore did not aect the ATM dependent activation of p53 by bleomycin. Next, we examined double mutations in the ATM regulated phosphorylation sites (serines 15 and 30), or in the ATM-independent sites (serines 33 and 37). Exposure of cells transfected with only p53S15/ 20A to bleomycin increased p53's transcriptional activity to a higher level than that seen with wtp53. However, when p53S15/20A was co-transfected with ATM, bleomycin did not induce any further increase in transcriptional activity (Figure 6). Further, when cells were transfected with p53S33/37A and exposed to bleomycin, no signi®cant activation of the p53-reporter construct was seen. This is in contrast to wtp53, which showed a 20% increase in p53 activity following exposure to bleomycin. When p53S3/37A was cotransfected with ATM, bleomycin was unable to further activate p53's transcriptional activity seen. Figure 6 demonstrates that mutation of either serines 15 and 20 or 33 and 37 of p53 inhibits the ATM-dependent activation of p53 by bleomycin.

Figure 6 Activation of p53 by ATM requires N-terminal serine residues. GM5849 cells were transiently transfected with the promoterless ATM vector AT-Sure (7) or FL-AT (+) in the presence of either wtp53 expression vector or the indicated serine to alanine mutation (150 ng each). p50-Luc reporter (0.6 mg) and pCMV-Gal (0.8 mg) were also added. Cells were to exposed to bleomycin (3 mM:+) for 18 h, and extracts assayed for luciferase activity. b-galactosidase activity from the constitutive pCMVGal vector was used to control for transfection eciencies. Results are expressed as the % increase in reporter activity caused by bleomycin relative to solvent exposed cells. Results+s.e.m.


Discussion We have shown that the kinase activity of the ATM protein is required for activation of p53 and chk2 by DNA damage, and is also required for the restoration of normal cellular radiosensitivity by the ATM protein. This implies that the ability of ATM to activate appropriate downstream signal transduction pathways in response to DNA damage is dependent on the kinase activity of ATM. We also expressed an ATM fragment containing only the kinase domain plus sequences N-terminal to this (amino-acids 2138 ± 3056) in AT cells; this fragment was inactive and did not activate p53, chk2 or alter the radiosensitivity of AT cells. In contrast, others have reported that a smaller, approximately 400 amino-acid fragment containing the kinase domain could complement the radiosensitivity of AT cells (Morgan et al., 1997), suggesting that their fragment retained some intrinsic kinase activity. The reason for the discrepancy between these observations is unclear, but may stem from the size dierences in the fragments employed. However, our studies demonstrate that an intact kinase domain is required for restoration of normal sensitivity to DNA damage and for activation of p53 and chk2. Previous studies have shown that ATM's kinase activity is increased in cells exposed to DNA damage (Banin et al., 1998; Blasina et al, 1999a; Canman et al., 1998), and that ATM can phosphorylate multiple proteins involved in the DNA damage response, including p53 (Banin et al., 1998; Canman et al., 1998b), chk2 kinase (Chebab et al, 2000; Hirao et al., 2000; Tominaga et al., 1999), p95/nibrin (Lim et al., 2000; Zhao et al., 2000) and Brca1 (Cortez et al., 1999). The diverse phenotype expressed by AT cells, including aberrant checkpoint activation, genomic instability and defective repair may therefore derive from the inability of AT cells to correctly phosphorylate and upregulate key proteins involved in all aspects of DNA repair. We also examined the role of the N-terminal of ATM. Using an ATM construct lacking the Nterminal, we did not observe any activation of either the p53 or chk2 proteins, or any increase in cell survival following exposure to lonizing Radiation, indicating that an intact N-terminal is essential for correct ATM functioning. The N-terminal of ATM can associate with both p53 (Khanna et al., 1998) and histone deacetylase in vitro (Kim et al., 1999), and both of these proteins co-precipitate with ATM in vivo (Khanna et al., 1998; Kim et al., 1999). ATM has also been shown to bind to dsDNA (Smith et al., 1999a; Suzuki et al., 1999), and this may involve sequences in the N-terminal of ATM (Smith et al., 1999b). Whether ATM binds directly to DNA or if this interaction is mediated through a speci®c DNA-binding sub-unit is not known. However, we speculate that the N-terminal of ATM may contain sites for interaction with DNA as well as for association with p53 and other ATM eectors. Deletion of this region would be predicted to inactivate the ATM protein. An alternative explanation is that deletion of the N-terminal may disrupt the

overall structure of the ATM protein and inactivate it. A more detailed deletion and mutagenesis approach will be needed to address this issue. We have also determined which of several serine phosphorylation sites in the N-terminal of p53 were required for activation of p53's transcriptional activity by ATM. We used a minimal p53 reporter construct containing only a p53 consensus binding site linked to a TATA box and basal promoter (Youmell et al., 1998). This minimal reporter was speci®cally responsive to p53. Although the promotor regions of p53 regulated genes contain binding sites for multiple transcription factors, this minimal promoter was used since it is more likely to be regulated by p53 alone, rather than in concert with other DNA-damage activated transcription factors. The role of N-terminal serine residues in the activation of p53 has been the subject of intense investigation, but these experiments have generally examined only the basal (unstimulated) transcriptional activity of the p53 protein. This approach has lead to con¯icting results, with most experiments showing no alteration in p53 transcriptional activity (Ashcroft et al., 1999; Blattner et al., 1999). Here, we have addressed this issue by examining the interaction between ATM, a DNA damage signal and p53 transcriptional activity. Using this system, we have shown that bleomycin can increase p53's transcriptional activity by a process which depends on both ATM's kinase activity and on multiple serine residues in the N-terminal of p53. A potential confounding factor in these experiments is the presence of the SV40 Tag protein in the AT cells used. It is possible that ATM regulates the interaction between p53 and SV40Tag, such that activation of ATM causes release of p53 bound to SV40Tag, which in turn increases transcription from the p53 reporter construct. This is unlikely for several reasons. First, neither bleomycin nor ATM status alters the amount of p53 bound to SV40Tag in GM5849 AT cell (B Price, unpublished results). Second, a recent report demonstrates that the interaction between p53 and SV40Tag is unaected by phosphorylation of serines 15 and 37 of p53 (Sheppard and Liu, 1999). Further, previously published data has shown that the p53-DNA damage response is intact in many SV40 transformed ®broblast cells, including the phosphorylation of p53, increased p53-DNA binding and transcription of the p21 gene (Hess and Brandner, 1997; Kohli and Jorgensen, 1999; O'Neil et al., 1996; Tibbets et al., 1999). We have shown that approximately 50% of the p53 in GM5849 cells is not bound to SV40Tag (Figure 3) and the transient expression of p53 into these cells will further increase the levels of free p53. Thus we feel it is unlikely that ATM is regulating p53-SV40Tag interactions, but is more likely to be aecting the transcriptional activity of the p53 protein directly. We have attempted to replicate the results in non-transformed AT ®broblasts or in transformed AT lymphoblasts, but for technical reasons we are unable to obtain high enough transfection eciencies to perform these experiments. We interpret our data to mean that

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ATM can directly aect the transcriptional activity of the p53 protein. In our experimental system, single mutations of either serines 15, 20, 33 or 37 had minimal eect on the ability of ATM to increase p53 transcriptional activity. This indicates that there is some redundancy in the role of these serine residues, such that loss of a single serine does not impair p53 function. However, double mutation of either serines 15 and 20 or serines 33 and 37 blocked the ability of ATM to activate p53. This is consistent with our observation that ATM's kinase activity is required for p53 activation, and implies that phosphorylation of p53 is one of the key steps in activation of p53. Serines 15 and 20 have been implicated in regulating the p53-mdm2 interaction (Chebab et al., 1999; Kubbutata et al, 1998; Unger et al., 1999a), with serine 20 being the main regulator of p53-mdm2 interaction (Unger et al., 1999a). Phosphorylation of serines 15 and 20 is proposed to block the interaction between mdm2 and p53, leading to accumulation of the p53 protein. Since phosphorylation of serines 15 and 20 is ATM-dependent (Chebab et al., 1999; Sakaguchi et al., 1998; Shieh et al., 1997; 1999; Siliciano et al., 1997), our results are consistent with the hypothesis that serines 15 and 20 of p53 are required for the ATM-dependent activation of the p53 protein. Surprisingly, mutation of serines 33 and 37 also blocked the ability of ATM to activate p53. Phosphorylation of serines 33 and 37 is not known to be dependent on the ATM protein, indicating that additional DNA-damage activated kinases are required for the activation of p53, speci®cally kinases which phosphorylate serines 33 and 37. Potential protein kinases which might phosphorylate serines 33 and 37 include the Jun and Atr kinases respectively (Tibbets et al., 1999). There are also other phosphorylation sites within p53 N-terminal which can be regulated by DNA damage, including serines 6 and 9 (Higashimoto et al., 2000) and serine 46 (Oda et al., 2000), which may contribute to the overall activation of p53. These observations demonstrate that a complex pattern of phosphorylations within the N-terminal of p53 are required for the full activation of p53's transcriptional activity by DNA damage. In addition, whilst ATM and serines 15 and 20 of p53 are essential, they are not sucient to elicit full activation of p53's transcriptional activity. The requirement for serines 33 and 37 of p53 strongly suggests a role for additional, DNA-damage activated protein kinases in the activation of p53. The activation of p53's transcriptional activity by DNA damage involves several steps. These include decreased degradation of the p53 protein (Sakaguchi et al., 1998; Shieh et al., 1997; 1999; Siliciano et al., 1997), increases in p53 DNA-binding activity (Tishler et al., 1993) and changes in the actual transcriptional activity of the p53 protein. The activation of p53 by ATM in our study re¯ects the overall relative contributions of each of these steps to the formation of transcriptionally active p53 complexes. However, in our system, the presence of stable SV40 Tag-p53 complexes as well as

endogenous wtp53 prevents us from monitoring the half-life and DNA-binding activity of the p53 mutants we have examined. Thus we are unable to determine if the increased p53 transcriptional activity is due to changes in p53 stability, increased DNA binding or transcriptional activity. The most likely explanation is that all three contribute to this eect. In conclusion, we suggest that phosphorylation of p53 by ATM is the initiating event in a series of posttranslational modi®cations which leads to the formation of a transcriptionally active p53 complex. ATM directly regulates the phosphorylation of serines 15 and 20, inhibiting mdm2 binding. This leads to decreased p53 degradation and accumulation of p53. Phosphorylation of serines 33 and 37 (by unknown kinases) has been shown to increase the binding of Histone Acetytransferase to the N-terminal of p53 (Sakaguchi et al., 1998). Histone Acetyltransferase then acetylates lysines in the C-terminal of p53, which in turn activates the DNA-binding activity of the p53 protein (Sakaguchi et al., 1998). Loss of serines 15 and 20 or 33 and 37 would then impair the activation of the p53 protein by inhibiting either of the two key steps in p53 activation ± protein stabilization and increased DNA binding. This is the ®rst demonstration that the ATM protein can directly in¯uence the transcriptional activity of the p53 protein through a mechanism involving N-terminal phosphorylation site. Materials and methods Cell lines, plasmids and antibodies GM637 (normal) or GM5849 (AT) human ®broblast cell lines were obtained from the Coriel Institute, Camden, NJ, USA. Cells were maintained in Minimal Eagle's Media with Earles Balanced Salt Solution supplemented with 7% Fetal Calf Serum. Irradiation was carried out using a 250 kVp Xray machine under temperature controlled conditions. For clonogenic cell survival assays, cells were plated in triplicate 24 h prior to irradiation. Cells were allowed to recover for 14 days post-irradiation, and stained with crystal violet. Surviving colonies containing 450 cells were counted. Plating eciency was 30 ± 50%. ATM cDNA was provided by Y Shiloh, Tel Aviv, Israel. chk2 constructs and antibody were provided by C McGowan, Scripps, CA, USA. ATM antibodies and anti-Omni Tag were purchased from Santa Cruz Biotech, CA, USA and SV40 and p53 antibodies from Oncogene Science, NY, USA. pAT-DN was constructed by inserting an EcoRI fragment spanning bases 2309 ± 4310 of ATM, in frame, into pcDNA3.1/HisA to generate pAT-CD. pAT-CD was then digested with KpnI/XhoI (to remove bases 3909 ± 4310), and a KpnI/XhoI fragment containing bases 3901 ± 9197 of ATM inserted. pAT-DN contains amino-acids 769 ± 3056 of ATM. pATK was constructed by inserting a 2781bp EcoRI fragment of the ATM protein (amino-acids 2138 ± 3056), in frame, into the pcDNA3.1/HisA expression vector. The construction of pcDwtp53, p50-Luc, FL-AT, FLATkd have been previously described by us (Blasina et al., 1999a; Youmell et al., 1998). Single and double point mutations were generated using the p-Alter I mutagenesis system or ExSite PCR site-directed mutagenesis kit (Stratagene, CA, USA). All mutations were veri®ed by sequencing.


Transfection and Iuciferase reporter assays Stably transfected cell lines. 36106 GM5849 cells were cultured on 100 mm dishes for 18 h prior to the addition of a DNA-Lipofectin (Gibco ± BRL) mix (10 mg DNA/20 mg lipofectin in 1.62 ml Dulbecco's Modi®ed Eagle's Media; DMEM). Six hours later, cells were washed in DMEM and refed 48 h after transfection, cells were trypsinized, replated at 1 : 5 to 1 : 20 dilution, allowed to reattach for 8 h, and then exposed to G418 (400 mg/ml) for 12 ± 15 days, with medium changes every 3 days. Surviving colonies (approximately 100 ± 150) were trypsinized, pooled, grown up and tested for expression of the appropriate gene by Western blotting. Transient transfections. For chk2 kinase assays, GM5849 or GM637 cells were transfected as described above with chk2 (2 mg) or AT constructs (8 mg) and adjusted to 10 mg of DNA with a promoterless ATM vector. Cells were allowed to recover for 44 h before chk2 kinase assays were carried out. For luciferase reporter assays, 36105 GM5849 cells were transfected with pCMV-b-galatosidase (0.8 mg), p50-Luc (0.5 mg), AT construct (1.8 mg) or p53 (150 ng) and adjusted to 4 mg of DNA with pcDNA/HisA in a ®nal volume of 2 ml containing 6.5 mg of lipofectin. After 6 h exposure to the DNA-lipofectin mix, cells were washed, and then exposed to either solvent (PBS) or bleomycin for 16 h. Preparation of cell extracts and assays for luciferase and b-galactosidase activity are as described in (Youmell et al., 1998). Some variation between dierent bleomycin batches was noted, with stimulation of p53 transcriptional activity varying between 100 ± 300%. For this reason, each experiment was carried out with all appropriate controls and with each point performed in triplicate. Immunoprecipitation, Western blotting and kinase assays ATM Cells were lyzed directly in 1 ml of RIPA [50 mM Tris pH 7.4; 150 mM NaCl; 1% Triton X-100; 0.1% SDS; 0.5%

Deoxycholate; 1 mM PMSF; 1 mg/ml leupeptin; 1 mg/ml aprotinin; 500 mM Na-vanadate; 50 mM NaF] and centrifuged at (35 kg/15 min). Cell lysates are pre-cleared for 1 h with protein-A/G agarose beads, then immunoprecipitated with protein-A/G agarose beads (50% w/v) pre-coated with antiOmni Tag antibody (2 mg: Santa Cruz, CA, USA) for 4 h. Beads were washed four times in RIPA, and protein removed from the beads in 30 ml of sample buer (2% sodium dodecyl sulfate; 10% glycerol; 1% mercaptoethanol; 0.125 M Tris pH 6.8) at 928C. chk2 Cells were lyzed on the dish in 1 ml of RIPA buer. Extracts were centrifuged (15 kg/10 min), and the supernatant immunoprecipitated with anti-cds1 antibodies precoated onto protein-A/G agarose beads. Immunoprecipitates were washed in 461 ml of buer RIPA buer, twice in kinase buer (50 mM Tris, pH 7.4, 10 mM MgCl2) and kinase reactions carried out in 20 ml of kinase buer containing 10 mCi 32P-ATP. Reactions were terminated by the addition of 46SDS sample buer. Phosphorylated products were detected by SDS ± PAGE and autoradiography. For Western blotting, proteins were transferred to Immobilon PVDF membranes, blocked in 10% milk, and incubated in primary antibody for 2 ± 18 h. For analysis of ATM, proteins are separated by 6% SDS ± PAGE and transferred for 30 min at 12 V in methanol free buer to PVDF membranes. Membranes were incubated with Omni antibody, and antigens then detected with by a coupled biotin-avidin immuno-enzyme method (Vector Laboratories, Burlingame CA, USA) or by ECL (Amersham PLC, UK).

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Acknowledgments We thank Dr Y Shiloh for providing ATM cDNA and Dr C McGowan for providing chk2 plasmids and antibodies. This work was supported by grant number CA64585 from the NCl and by funds from the AJCRT foundation.

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