FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage

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1 Reeive 19 Jan 212 Aepte 11 Jul 212 Pulishe 14 Aug 212 DOI: 1.138/nomms28 signalling links to the p apoptoti pathway following DNA amage Young Min Chung 1, *, See-Hyoung Park 1, *, Wen-Bin Tsai 2, Shih-Ya Wang 3, Masa-Aki Ikea 4, Jonathan S. Berek 1, Davi J. Chen 3 & Mikey C.-T. Hu 1 DNA amage as a result of environmental stress is reognize y sensor proteins that trigger repair mehanisms, or, if repair is unsuessful, initiate apoptosis. Defets in DNA amageinue apoptosis promote genomi instaility an tumourigenesis. The protein ataxiatelangietasia mutate () is ativate y DNA oule-stran reaks an regulates apoptosis via p. Here we show that interats with the p omplex, augments phosphorylation of the omplex an inues the formation of nulear foi in ells on DNA amage. is essential for DNA amage-inue apoptosis an onversely requires, an phosphorylate p isoforms to trigger apoptosis as a result of DNA amage. Uner these onitions may also have a role in regulating hromatin retention of phosphorylate p. These results suggest an essential link etween an the p-meiate apoptoti programme following DNA amage. 1 Division of Gyneologi Onology, Department of Ostetris an Gyneology, Stanfor University Shool of Meiine, Stanfor, California 9435, USA. 2 Department of Moleular Pathology, University of Texas M.D. Anerson Caner Center, Houston, Texas 773, USA. 3 Division of Moleular Raiation Biology, Department of Raiation Onology, University of Texas Southwestern Meial Center, Dallas, Texas 7, USA. 4 Setion of Moleular Emryology, Grauate Shool of Meial an Dental Sienes, Tokyo Meial an Dental University, Tokyo , Japan. *These authors ontriute equally to this work. Corresponene an requests for materials shoul e aresse to M.C.-T.H. ( mhu1@stanfor.eu).

2 nature ommuniations DOI: 1.138/nomms28 All living organisms are inexoraly expose to environmental stress that ause DNA oule-stran reaks (DSBs), whih pose the greatest hallenge to the maintenane of genomi staility in ells. Many antianer agents also inue DSBs in aner ells that promote ellular apoptosis. DSBs are generally reognize y sensors an transuers, ontaining signalling kinases, whih an signal repair mehanisms or, if this fails, they trigger apoptosis. One of the most important proteins is ataxia-telangietasia mutate (), a ruial tumour suppressor, whih is ativate y DSBs though autophosphorylation at Serine-1981 () 1. One ativate, phosphorylates various ownstream sustrates suh as histone H2AX (H2AX), nirin (Ns1), BRCA1, ellyle hekpoint kinases Chk1 an, an p that exert vital DNA-amage funtions inluing regulation of ell-yle hekpoints or inuing repair of amage DNA or promoting apoptosis through p (refs 2,3). The p tumour suppressor protein has a entral role in the eision of a ell to unergo either ell-yle arrest or apoptosis after iverse stresses, inluing DNA amage, hypoxia an the ativation of onogenes 4 6. The amount an transriptional ativity of p is regulate y post-translational moifiation, suh as phosphorylation, sumoylation, neation an aetylation 7. Uner normal onitions, p protein levels are low owing to Hm2 (human Mm2)-meiate uiquitylation an egraation through the proteasome pathway. On DNA amage, DSBs-ativate phosphorylates p at Serine- (p-ps) 8 that may inhiit the interation of p with Hm2 (ref. 9), resulting in p stailization. In aition, an ativate an homeoomain-interating protein kinase 2 (HK2) to phosphorylate p at Serine-2 (p- ps2) 1 an Serine-46 (p-ps46) 11,12, respetively, in its transativation omain, whih in turn leas to upregulation of the expression of pro-apoptoti genes suh as Bax (Bl2-assoiate X protein) an Puma (p-upregulate moulator of apoptosis) 13. HK2 has a ritial role in regulating the apoptoti programme inue y DNA amage 11,12,14. Notaly, it has een shown that mutations in Serine- an Serine-2 of human p impair its apoptoti ativity, an Serine-46 phosphorylation is ruial for p-meiate apoptosis after DNA amage 16. Thus, the regulation of -p ativation is ertainly important in DNA amage-triggere apoptosis. However, the upstream signalling mehanisms that regulate p stailization an ativity y -meiate phosphorylation are largely unlear. Interestingly, moleular interations etween p an (or a) have een isovere is a pivotal transription fator that regulates the transription of a numer of genes important for moulating ell-yle ontrol 21, DNA amage response 22,23, oxiative an nutritional stress 17,24, aging an longevity 27, ellular apoptosis 28 3 an suppression of aner in animal an human ells. Gene knokout finings support FOXOs essential funtions in tumour suppression 34 an the maintenane of the hematopoieti stem ell pool 35. Although multiple mehanisms have een shown to regulate ativity, phosphorylation inhiits nulear transloation that is essential for its regulation an funtion. This nulear exlusion an transloation of into the ytoplasm inhiits -epenent transription. Loss of funtion of through phosphorylation has een linke to tumourigenesis an poor patient survival in reast aner 31,32, suggesting that is a key tumour suppressor. Notaly, an p share a numer of similarities in their funtions an regulations. Not only o they interat with eah other ut also they regulate eah other s ativity or staility. For instane, it has een reporte that ativation of p an suppress transriptional ativity through serum/gluoortioi-inuile kinase 1 (SGK1) 36 or inue egraation through Mm2 (ref. 37). Uner oxiative stress, p an inhiit transriptional ativity 2. Conversely, it has een shown that ativate an stailize p an promote p-epenent apoptosis 18. However, uring DNA amage-inue apoptosis, the regulation of funtional interation etween an p to oorinate with ruial DNA amage response proteins suh as an remains largely unknown. Although several important proteins responsile for ativation of the p-meiate apoptosis have een ientifie, the mehanism y whih ativates p apoptoti signalling has remaine elusive. Our reent finings suggest that is essential for ativation of the -BRCA1-Ra17 omplex that signals the -meiate repair mehanism at early stages of DNA amage 23. In aition, it has een foun that is neessary for the regulation of normal expression to maintain homoeostasis of hematopoieti stem ells 38. The fat that prolonge ativation of y antianer agents an trigger apoptosis 29,3 suggests that may e involve in regulating the -meiate p apoptoti signalling pathway at late stages of DNA amage. Beause amptothein (CPT), a topoisomerase-1 inhiitor, an inue DNA amage an ativate the - apoptoti pathway in aner ells 39,4, we set out to etermine whether has a key role in linking an to the p-meiate apoptosis after CPT-inue DNA amage, an if so, how the -p apoptoti mahinery is regulate. Here we show that is funtionally assoiate with the p omplex, an regulates phosphorylation an the formation of p omplexes at sites of DNA amage that inue apoptosis. Colletively, we suggest a novel network etween an the p-meiate apoptoti programme after DNA amage. Results interats with the p omplex. Beause p-ps is phosphorylate iretly y an our an other reent stuies suggest that may e neessary for the regulation of ativation on DNA amage 23,38, we investigate whether has an essential role in the regulation of the meiate -p apoptoti signalling pathway after DNA amage inue y CPT. To assess the effet of CPT on enogenous expression an the levels of p-ps/ps2/ps46,, -pt68 an phosphorylate H2AX (), we treate ells with CPT or ontrol (imethylsulphoxie (DMSO)) an analyse the levels of these proteins in ell lysates y immunolotting (). We foun that the levels of, p-ps/ps2/ps46, ps1981, -pt68 an were inrease signifiantly 1 4 h post treatment with CPT, whereas total expressions of, an H2AX remaine unhange an the ontrol vehile i not alter the levels of the teste proteins (Supplementary Fig. S1). In aition, other DNA-amaging agents suh as etoposie (VP16), ionizing raiation an ultraviolet showe similar results to those of CPT (Supplementary Fig. S1). To valiate the interation etween an or p after DNA amage, we ompare the levels of, an p using o-immunopreipitation (o-) an assays with ell lysates otaine from ells treate for 2 4 h with CPT. We foun that was assoiate with not only an p ut also p-ps speifially, a iret phosphorylation target of ativate, suggesting that may interat with ps1981 also. Inee, we showe that was physially assoiate with p-ps, p-ps2, p-ps46 (p-ps/ps2/ps46),,, HK2 an in ells treate with CPT (Fig. 1). In aition, the expression of HK2 was inue after CPT treatment. Colletively, these results suggest that may have a role in regulating the p pathway after ritial DNA amage. To onfirm the su-ellular loations of, p-ps/ps2/ ps46,, -pt68 an in the CPT-treate

3 nature ommuniations DOI: 1.138/nomms28 ARTICLE a (h) p-ps p-ps2 p-ps46 p HK (h) p-ps p-ps2 p-ps46 (L) 4 p-ps p (L) p-ps 2 4 (h) (L) 4 p-ps2 2 4 (h) e 4 HK2 p-ps (h) 13 f 4 p-ps2 2 4 (h) p-ps2 p p-ps46 p p-ps46 (H) (H) g (h) p-ps p-ps2 p-ps46 h HK (h) p-ps46 HK2 (H) 13 (L) (L) Figure 1 interats with the p H2AX omplex. (a) Whole-ell lysates of treate with CPT (1 µm) for 2 or 4 h or DMSO (, ontrol) were sujete to immunopreipitation () with an anti- antioy (A) or an isotype (ontrol) followe y immunolotting () with As as iniate, or an anti- ( ontrol) or a ontrol A against light hain (L). ( h) Similarly, ell lysates were sujete to with an A against (), p-ps (),p-ps2 (), p-ps46 (e), (f), (g), HK2 (h) or an isotype followe y with the iniate As or a ontrol A against (L) or heavy hain (H). ells, we performe analysis with ytoplami an nulear extrats from ells treate with CPT an showe that the levels of, p-ps/ps2/ps46, an were elevate in the nuleus (Supplementary Fig. S2). These results suggest that DNA amaging agents suh as CPT an sustantially inue expression an promote an p ativation in the nuleus onurrently. is o-loalize with p H2AX nulear foi. An important question in this stuy is the ellular loation of DNA amage-inue. In light of the signifiane of DNA amage-inue interation with p-ps/ps2/ps46,, an -pt68, it was plausile to examine whether was reruite to the amage DNA sites after DNA amage. Thus, we inue spatially loalize DSBs in ells y using fouse laser miro-irraiation as esrie previously 41,42. This tehnique allowe us to etet the reruitment of a unique fration of an an/or to the laser-inue DNA amage traks (Fig. 2a). In aition, to etermine the physial assoiations etween an p-ps/ ps2/ps46 or or or -pt68 at the sites of DNA lesions, we assesse whether loalize to the potential sites of DSBs y o-loalization with p-ps/ps2/ps46,, or -pt68 following CPT-inue DNA amage. ells were treate with CPT or ontrol an staine with speifi antioies. We foun that approximately 7 % of loate in the nuleus an o-loalize with p-ps/ps2/ps46,, an -pt68 to form nulear foi after exposure to a low ose of CPT for 2 h, whereas primarily resie in the ytoplasm of ells treate with ontrol (Fig. 2 f; Supplementary Fig. S3). To generalize these results, we performe the same experiments in another p wil-type (WT) ell line A549 an showe that o-loalize with p-ps/ps2/ps46,, an -pt68 to form nulear foi in A549 ells after CPT treatment (Supplementary Figs S3 an S4), suggesting that may o-loalize with p-ps/ps2/ps46,, an -pt68 to sites of DNA reaks in response to DNA amage.

4 nature ommuniations DOI: 1.138/nomms28 a Merge DAPI e DAPI Merge -ps 1981 Merge DAPI CPT DMSO Sale ar, 6 µm f -ps 1981 DAPI Merge -ps 1981 Merge DAPI Sale ar, 6 µm CPT DMSO p-ps DAPI Merge Sale ar, 6 µm CPT DMSO g % p-ps nulear foi 4 P=.4 j % nulear foi 4 P=.1 p-ps2 DAPI Merge Sale ar, 6 µm shrna: + + Control shrna: + + Control CPT DMSO p-ps46 DAPI Merge Sale ar, 6 µm h % p-ps2 nulear foi 4 shrna: P= Control k % nulear foi 4 shrna: P= Control CPT DMSO i % p-ps46 nulear foi 4 P=.6 l % -pt68 nulear foi 4 P=.2 Sale ar, 6 µm shrna: Control shrna: Control Figure 2 is essential for the formation of p H2AX nulear foi. (a) Aumulation of a fration of at laser-inue amage was etete in ells 3 min after fouse laser miro-irraiation. The ells were staine with antioies (As) against an or, followe y fluoresene mirosopy as esrie in the setion of Immunofluoresene uner Methos. DAPI was use to show the nulei, an o-loalizations of with were shown as the merge images. Sale ar, 6 µm. ( f) ells were treate with CPT (1 µm) or DMSO ontrol for 2 h, an o-loalizations etween an p-ps (), p-ps2 (), p-ps46 (), (e) or ps1981 (f) were etete using As as iniate an followe y fluoresene mirosopy. Sale ar, 6 µm. (g l) is neessary for the formation of p-ps, p-ps2 an p-ps46,, an -pt68 nulear foi on CPT-inue DNA amage. stale ell lines transfete with -shrna or ontrol-shrna were treate with CPT (1 µm) or DMSO for 2 h, an then the su-ellular loalizations an oloalization of an p-ps (g), p-ps2 (h), p-ps46 (i), (j), (k) or -pt68 (l) were etete using speifi As as iniate. An average of 2 ells with speifi nulear foi in eah omparison was etermine an shown. The images of nulear foi of these proteins were shown in Supplementary Fig. S5. The error ars represent stanar eviation, an the statistial test is the paire t-test.

5 nature ommuniations DOI: 1.138/nomms28 ARTICLE a shrna: Control H2AX p p-ps p-ps2 p-ps46 -pt68 p-ps6 p-ps9 p-ps37 43 shrna: Control H2AX p p-ps p-ps2 p-ps46 -pt68 p-ps6 p-ps9 p-ps37 A PARP-1 p116 p85 shrna: Control H2AX -pt68 H1299 Control-shRNA shrna (h) Figure 3 is require for p phosphorylation an PARP-1 leavage. (a) stale ell lines transfete with -shrna or ontrol-shrna were treate with CPT (1 µm) or DMSO (negative ontrol) for 4 h, an whole-ell lysates were prepare an the levels of proteins were analyse y with speifi antioies (As) as highlighte or an anti-β-atin (loaing ontrol). (,) A549 () an H1299 () stale ell lines transfete with -shrna or ontrol-shrna were treate with CPT (1 µm) or DMSO (negative ontrol) for 4 h, an whole-ell lysates were analyse y with speifi As as iniate or an anti-β-atin. () Cell lysates from stale ell lines (-shrna an ontrol-shrna) treate with CPT (1 µm) for an iniate time ourse were sujete to analysis using As against PARP-1 an β-atin (loaing ontrol). is essential for forming p nulear foi. Although nulear foi of p-ps, p-ps2, p-ps46,, an -pt68 were forme signifiantly (aroun 65 75%) in the presene of in stale ell line transfete with ontrol-short hairpin RNA (shrna) after exposure to a low ose (1 µm) of CPT for 2 h, the formations of p-ps, p-ps2, p-ps46,, an - pt68 nulear foi were greatly reue in stale ell line transfete with -shrna following the same CPT treatment (Fig. 2g l; Supplementary Fig. S5). To show the generality of this effet, we performe the same experiments in A549 ells y treating A549 (ontrol-shrna) an A549 (-shrna) ells with CPT an showe that knokown of expression in A549 ells arogate the formations of p-ps, p- ps2, p-ps46,, an -pt68 nulear foi in A549 ells following CPT treatment (Supplementary Figs S6 an S7). These results suggest that may e ruial for the formation of p-ps, p-ps2, p-ps46,, ps1981 an -pt68 nulear foi after DNA amage inue y CPT. p H2AX phosphorylation requires. Next, we etermine whether silening of alters the expression or phosphorylation of,, p an H2AX following DNA amage y iohemial approahes. We treate (ontrolshrna) an (-shrna) ells with a low ose of CPT for 4 h an performe analysis. Interestingly, silening of i not hange the expression of,, H2AX an p total proteins ut, signifiantly, i reue the levels of phosphorylation (or ativation) of,, p an H2AX (Fig. 3a). To emonstrate the generality of this fining, we performe the same experiments in A549 ells y treating A549 (ontrol-shrna) an A549 (- shrna) ells with CPT an performing analysis. In agreement with our results in ells, silening of expression in A549 ells signifiantly reue phosphorylation of,, p an H2AX in A549 ells after CPT treatment (Fig. 3), suggesting that inee is require for p H2AX ativation in A549 ells on DNA amage. In aition, we have arrie out similar experiments in a p-efiient ell line H1299 y treating H1299 (ontrol-shrna) an H1299 (- shrna) ells with CPT as esrie aove. We showe that knokown of expression in H1299 ells iminishe phosphorylation of, an H2AX in H1299 ells after CPT treatment (Fig. 3), suggesting that is neessary for - -H2AX ativation in H1299 ells following DNA amage. Colletively, these results suggest that is essential for phosphorylation or ativation of,, p an H2AX when DNA amage ours. is neessary for inuing DNA amage-inue apoptosis. To examine the role of in DNA amage-inue apoptosis, we treate ells with a low ose of CPT an analyse apoptosis y the stanar poly-adp-riose polymerase (PARP) egraation, an iniator of apoptosis 43 an terminal eoxynuleotiyl transferase UTP nik en laelling (TUNEL) apoptosis assays. We showe that CPT treatment inue signifiant PARP-1 egraation an TUNEL-positive ells ~8 h post rug treatment, respetively (Fig. 3). Furthermore, we showe that silening of expression in these ells signifiantly attenuate this CPT-inue apoptosis (Figs 3 an 4a,), suggesting that may e neessary for promoting apoptosis after DNA amage. To etermine whether knokown also ontriutes to survival, we treate (ontrolshrna) an (-shrna) ells with various oses of CPT or DMSO (ontrol) for 24 h or with. µm CPT for a time ourse, an performe ell survival assays y ell ounting or the 3-(4,5-imethylthiazol-2-yl)-2,5-iphenyltetrazolium romie (MTT)

6 nature ommuniations DOI: 1.138/nomms28 a TUNEL DAPI Merge (Control-shRNA) (-shrna) CPT DMSO CPT DMSO P=.9 % TUNEL-positive ells shrna: Control Sale ar, 2 µm Relative ell viaility (% of ontrol) (Control-shRNA) (-shrna) P=.4 P=.6 P=.5 Relative ell viaility (% of ontrol) (Control-shRNA) (-shrna) P=.7 P=.5 P=.3 Dose (µm): h Time (h): µm Figure 4 is neessary for inuing DNA amage-inue apoptosis. (a) stale ell lines (-shrna an ontrol-shrna) were treate with CPT (1 µm) or DMSO (ontrol) for 48 h, an the ells were fixe on the slies for etermining ellular apoptosis y TUNEL assays (Promega). Nulei were staine with DAPI (olour-inverte to re), an merge images (yellow) were onsiere as apoptoti ells. Sale ar, 2 µm. () An average (%) of apoptoti (TUNEL-positive) ells was etermine an shown in the iagram; the samples inlue three iologial repliates, the error ars represent stanar eviation, an the statistial test is the paire t-test. The signifiant P-values etween the -shrna group versus the ontrol group treate with CPT are highlighte. (,) (ontrol-shrna) an (-shrna) ells were treate with various oses of CPT or DMSO (ontrol) for 24 h () or with a low ose CPT (. µm) for a time ourse as iniate (), an performe ell survival assays y the MTT assays () or ell ounting (). The signifiant P-values etween the -shrna group versus the ontrol group treate with CPT are iniate. The numer of iologial repliates is three, the error ars represent stanar eviation, an the statistial test is the paire t-test. assays. We foun that knokown in these ells signifiantly promote survival after CPT treatment (Fig. 4,). To generalize these results, we ompare the survival rates in A549 (ontrol-shrna) versus A549 (-shrna) ells an H1299 (ontrol-shrna) versus H1299 (-shrna) ells following CPT treatment. We showe that silening of in these ells signifiantly enhane survival after CPT treatment (Supplementary Fig. S8). Taken together, these results suggest that is essential for regulating survival an apoptosis when DNA amage ourre. may nee p ativation to lea to apoptosis. Further, to etermine the roles of phosphorylate p in the regulation of -meiate apoptosis following DNA amage inue y CPT, we transfete ells with ontrolshort interfering RNA (sirna) or sirna-targeting, or p, an treate these ells with a low ose of CPT. Markely, silening the expression of, or p with speifi sirnas in ells aolishe CPT-inue PARP-1 egraation (Fig. 5a), suggesting that p proteins are neessary for the -meiate apoptosis on CPT-inue DNA amage. In aition, we treate ells with speifi inhiitors of, an p or ontrol for 6 h efore exposure to a low ose of CPT or ontrol, an we showe that inhiiting the ativity of, or p signifiantly iminishe the levels of, -pt68 or p phosphorylate isoforms (p-ps/ps2/ps46) an arogate CPT-inue PARP-1 egraation (Fig. 5). As ontrols, we showe that these inhiitor treatments i not alter expression signifiantly; thus, the lak of PARP-1 egraation oul not e attriute to attenuation of expression in these ells. These results suggest that the ativities of -, -pt68- an p-phosphorylate isoforms (p-ps/ps2/ps46) may e essential for -meiate apoptosis. Next, we transfete the p-knokown, (p- sirna) (Fig. 5), ells with pdna3 (ontrol) or p-wt or

7 nature ommuniations DOI: 1.138/nomms28 ARTICLE a sirna: Control PARP PARP-1 Cleave PARP1 85 Cleave PARP1 p p-ps p-ps2 p-ps46 Bax p 2 42 Inhiitor: -pt68 p-ps p-ps2 p-ps46 p Control p p e (p-sirna) % TUNEL-positive ells Vetor: Cont-siRNA p-sirna pdna3 ** p-wt ** p-sa ** ** p-s2a p-s46a Vetor: PARP-1 leave PARP1 p p-ps p -ps2 p-ps46 pdna3 (p-sirna) p-wt p-sa p-s2a p-s46a Figure 5 Phosphorylate p may e neee for -meiate apoptosis. (a) ells were transfete with ontrol-sirna (ontrol) or sirna (.3 µm) targeting, or p an inuate for 48 h, an then these ells were treate with CPT (1 µm) ( + ) or DMSO ( ) for 48 h. Whole-ell lysates were prepare from these treate ells an the levels of PARP-1 an its egrae proteins,,, p an their phosphorylate proteins or a p target (Bax) were analyse y with speifi antioies (As) as iniate or an anti-β-atin (loaing ontrol). () ells were first treate with inhiitor (Mirin) or inhiitor (NSC19) or p inhiitor (Pifithrin), 2 µm of eah, or DMSO for 6 h, an then treate with CPT (1 µm) or DMSO for 16 h. Whole-ell lysates were prepare from these treate ells an the levels of PARP-1 an its egrae proteins, an the iniate proteins an β-atin were analyse y analysis with speifi As. () ells were transfete with p-sirna or Cont-siRNA (Control-siRNA) for 48 h. Whole-ell lysates were prepare from these transfete ells an the expressions of p an (ontrol) were etermine y analysis with speifi As. () (p-sirna) ells were transfete with pdna3 (ontrol) or p wil-type (WT) or mutant (p-sa or p-s2a or p-46a) expression vetors (2 µg DNA for eah) for 48 h, an then treate with CPT (1 µm) ( + ) or DMSO ( ) for 48 h. Whole-ell lysates were prepare from these treate ells an the levels of PARP-1 an its egrae proteins, an the iniate proteins were analyse y analysis with speifi As. (e) (p-sirna) ells were transfete with ontrol an expression vetors for 24 h, the transfete ells were treate with CPT (1 µm) or DMSO ontrol for 36 h, an ellular apoptosis was etermine y TUNEL assays (Promega). Images of apoptoti ells were shown in Supplementary Fig. S9. An average (%) of TUNEL-positive (apoptoti) ells was etermine an shown in the iagram. **P =.1 (paire t-test). mutant (p-sa or p-s2a or p-46a) expression vetors for 48 h, an then treate them with a low ose (1 µm) of CPT or ontrol for 48 h. Interestingly, over-expression of p-wt-promote CPT-inue PARP-1 egraation an apoptosis, whereas overexpression of p-sa, p-s2a or p-s46a mutant faile to inue PARP-1 egraation an TUNEL apoptosis after CPT treatment (Fig. 5,e; Supplementary Fig. S9). As ontrols, we showe that over-expression of these p mutants i not signifiantly hange expression, suggesting that the oserve negative effet on PARP-1 egraation or TUNEL apoptosis annot e asrie to eilitation of in these ells. In aition, we repeate the same experiments in another p-null ell line, H1299, an otaine the same reprouile results (Supplementary Fig. S1), onfirming that these p phosphorylate

8 nature ommuniations DOI: 1.138/nomms28 a Fration: I III IV I III IV p-sirna + pdna3 (Control-shRNA) (-shrna) Fration: I III IV I III IV pt68 p-ps p-ps2 p-ps46 β-tuulin Lamin A/C HMG14 p β-tuulin Lamin A/C HMG14 Fration: p β-tuulin Lamin A/C HMG14 (Control-shRNA) (-shrna) p-sirna + p-wt (Control-shRNA) (-shrna) I III IV I III IV Fration: I III IV I III IV p β-tuulin Lamin A/C HMG14 e Fration: p β-tuulin Lamin A/C HMG14 f p-sirna +p-sd (Control-shRNA) (-shrna) (Control-shRNA) (-shrna) I p-sirna +p-s2d III IV I III IV Fration: I III IV I III IV p β-tuulin Lamin A/C HMG14 p-sirna +p-s46d (Control-shRNA) (-shrna) Figure 6 has a role in regulating hromatin retention of phosphorylate p. (a) stale ell lines transfete with -shrna or ontrol-shrna were treate with CPT (1 µm) ( + ) or DMSO ( ) (ontrol) for 4 h, an ells were ollete an frationate with Noniet P-4 as esrie in the setion of Chromatin Retention Assay uner Methos. Equal amount (2 µg) of eah fration was analyse y analysis with the highlighte antioies as esrie aove. Proteins β-tuulin, lamin A/C an HMG14 (high-moility-group 14, a hromosome-ining protein) represent the frationation an loaing ontrols of the ytosol (fration I), the nuleoplasm (fration III) an the hromatin (fration IV), respetively. ( f) (ontrol-shrna) an (-shrna) ells were transfete with p sirna for 48 h, then transfete with pdna3 (negative ontrol) () or the p-wt vetor () or the speifi vetors expressing p-sd (), p-s2d (e) an p-s46d (f) for 36 h. The transfete ells were treate with CPT (1 µm) or DMSO for 4 h, then ells were ollete an sujete to hromatin frationation an analysis as esrie aove isoforms (p-ps/ps2/ps46) are neessary for -meiate apoptosis following DNA amage inue y CPT. Colletively, these results suggest that may require p phosphorylate isoforms to lea to apoptosis after DNA amage. regulates hromatin retention of phosphorylate p. An important question in mehanism is how ontriutes to the ativation of, -pt68 an p-ps/ps2/ps46 in ells on DNA amage. Hene, we sought to etermine whether is require for ining an retention of, - pt68 an p-ps/ps2/ps46 proteins to the hromatin following DNA amage. We treate (ontrol-shrna) an (-shrna) ells with a low ose of CPT for 4 h an performe hromatin retention assays. Remarkaly, silening of expression in ells signifiantly iminishe hromatin retention of, -pt68, p-ps/ps2/ps46 an (Fig. 6a), suggesting that may have a role in regulating hromatin retention of phosphorylate p H2AX. To emonstrate the important role of in regulating hromatin retention of the ativate p proteins in a phosphorylation-inepenent manner, we silene enogenous p in (ontrol-shrna) an (-shrna) ells with p sirna as esrie aove. We then transfete these p- knokown ells with pdna3 (negative ontrol) or the p-wt vetor or the speifi vetors expressing p-sd, p-s2d an p-s46d, whih are the onstitutively ativate mutant p proteins with the speifi Serine (S) resiue eing mutate to Asparti ai (D) that mimis the phosphorylate-s resiue 16,44,45, for 36 h. We treate these transfete ells with a low ose of CPT for 4 h, then harveste ells an performe hromatin frationation as esrie aove. Remarkaly, silening signifiantly iminishe hromatin retention of these p-sd, p-s2d an

9 nature ommuniations DOI: 1.138/nomms28 ARTICLE p-s46d proteins (Fig. 6 f), suggesting that may have a neessary role in hromatin retention of the ativate p proteins inepenent of their phosphorylation. Disussion is a key DNA amage sensor that has a entral role in ontrolling the DNA amage response. A great eal of information has een otaine on the DNA-amage sensor mehanisms eteting DSBs in reent years; however, it is still unlear how speifi DNA lesions are reognize an how signalling is elivere ownstream to the apoptoti mahinery 1 3. It also remains largely unknown when the sensor proteins shoul signal repair mehanisms to repair amage DNA an when they woul trigger apoptosis. Although ativation of the p-meiate apoptosis has a ritial role in tumour suppression, the ontrol of ativation of p apoptoti signalling is unlear In this stuy, we show a ritial role of in regulating the formation of --p- ps/ps2/ps46 omplexes at sites of DNA reaks, an its role in promoting the p apoptoti-signalling pathway after DNA amage. Our finings suggest that may e important for regulating hromatin retention of these phosphorylate (an/or ativate) p proteins in the nuleus when DNA amage ours. Although it is possile that the general aunane of the proteins examine may regulate the levels of protein protein interation an hromatin retention oserve here, our ata show speifi interations etween an p-ps or p an the extent of these interations in o- is signifiantly lower at 4 h than at 2 h after CPT treatment (Fig. 1a,), whereas the levels of an p-ps are onsieraly higher at 4 h than at 2 h post CPT treatment (Supplementary Fig. S1a). Taken together, these results learly iniate that the formation of speifi /p-ps, /p, - an other omplexes (as shown y o-) is not solely epenent on the aunane of these proteins examine. Interestingly, it has een shown that p ativation an ownregulate through inution of SGK1 or Hm2 (refs 36,37), whih in turn promotes uiquitin-epenent egraation of p (ref. 46). These intriguing finings suggest the possile roles of p an Hm2 (refs 37,47) in ownregulation of as negative feeak loops. On the other han, it has een reporte that ativation of an enhane p stailization y iret phosphorylation of Hm2 (refs 48,49) an inhiition of its RING omain oligomerization an E3 ligase 5. Colletively, we have propose a shemati representation of the -epenent phosphorylation or ativation of ps1981/p-ps, -pt68/p-ps2 an HK2/p-pS46 together with their negative feeak loops an the ownstream p apoptoti-signalling pathways after DNA amage (Fig. 7). At the early stages of DNA amage with low levels of DSBs, it is possile that only a minor fration of p is ativate that is suffiient to rive the transription of the p21cip1 gene, resulting in ell-yle arrest. However, with high levels of DSBs, p is ativate an aumulate aove a partiular threshol that upregulates the expression of pro-apoptoti genes suh as Bax, Puma an pa1 (p-regulate apoptosis-inuing protein 1) 13,51. Although CPT an signifiantly upregulate the expression of these p ownstream target genes in the presene of, we have foun that the expression levels of Bax, Puma an pa1 were signifiantly reue in the -knokown ells after CPT treatment (ata not shown), suggesting that CPT inues these pro-apoptoti proteins in a -epenent manner. It has een shown that phosphorylation of p-ps46 is ativate y HK2 kinase. Although HK2 has een foun to e a ownstream target of 12, it is unlear how DNA amage inues the -HK2-p-pS46 signalling pathway. In this stuy, we show that CPT-inue DNA amage an inue the -epenent phosphorylation of an p-ps46 an the interations of -HK2-p-pS46, ps ps1981 p pt68 ps2 BAX, pa1, PUMA an so on. Apoptosis HK2 ps46 DNA amage HDM2 Figure 7 A link etween an the p-meiate apoptoti programme. A shemati shows the -epenent ativation of, -pt68, HK2, p-ps, p-ps2, p-ps46 an the possile roles of p an Hm2 in ownregulation of as negative feeak loops, an the ownstream p apoptotisignalling pathway after DNA amage inue y CPT. The ashe lines enote the inhiitory pathways aoring to the pulishe literature. suggesting that may have a ritial role in ativation of the -HK2-p-pS46 signalling pathway an provie an essential link etween an p-ps/ps2/ps46 simultaneously on DNA amage. In aition, we show that is neessary for inuing DNA amage-inue apoptosis. Taken together, these results suggest that has an essential role in the regulation of -p-meiate apoptosis after DNA amage. Moreover, we have foun that, -pt68 an p phosphorylate isoforms (p-ps/ps2/ps46) are essential for -meiate apoptosis following DNA amage. To the est of our knowlege, this is the first eviene that requires phosphorylation (or ativation) of p an its upstream kinases an together to lea to apoptosis after DNA amage. Our results are onsistent with a reent fining that p is neessary for transriptional upregulation an transativation in vivo 33. Beause there is no inrease in levels in ells after CPT treatment for 16 h (Fig. 5) an 48 h (Fig. 5), we performe analysis of an p proteins with full time-ourse of CPT treatment ( 48 h) in ells, an showe that the amount of protein remains at a similar level etween an 16 h an etween an 48 h after CPT treatment (Supplementary Fig. S11a), iniating that these results o not ontrait the inrease shown in Supplementary Fig. S1a an Fig. 1a. Together, our results suggest that ooperates with the p apoptoti programme onurrently, leaing to

10 nature ommuniations DOI: 1.138/nomms28 a high egree of ativation of the p-meiate apoptoti pathways following DNA amage. Although it is known that is physially assoiate with p (refs 17 2), the moleular mehanisms unerlying - meiate regulation of the p-inue apoptoti pathway on DNA amage an vie versa are largely unknown. Our finings suggest that is require for phosphorylation of p isoforms (p- ps/ps2/ps46). To further eluiate the mehanism y whih regulates p ativation in the DNA amage response, using the hromatin retention assays, we show that silening of expression in ells signifiantly iminishes hromatin retention of p-ps/ps2/ps46 on DNA amage (Fig. 6). Moreover, is triggere to migrate markely to the hromatin from the ytoplasm in parallel with phosphorylate p following DNA amage (Fig. 6a), suggesting that may ooperate with phosphorylate p an migrate to the DSB sites on the hromatin onurrently after DNA amage. To etermine whether is still loalize to the hromatin at late time treatment with CPT, we performe the same experiments of Fig. 6a at 24 h after CPT treatment an showe that the loalization of to the hromatin is signifiantly reue after 24 h CPT treatment ompare with 4 h (Supplementary Fig. S11). Colletively, these results suggest that ontriutes to the DNA amage response y ontrolling phosphorylation of p or regulating hromatin retention of these phosphorylate proteins in the nuleus or oth. In aition to, Mre11-Ra5-Ns1 (MRN) an other fators an interat with an regulate its kinase ativity that enhanes its aility to phosphorylate sustrates in vitro 52,. However, the fat that an e ativate y ionizing raiation treatment in ells laking Ns1 or BRCA1 ut annot e reruite to the DSB sites suggests that the MRN omplex may inrease the aumulation of at the DSB sites on the hromatin 54, ut other fators an ativate ativity in an MRN-inepenent manner. Thus, it is plausile that ativity may e regulate y two istint events, one eing the ativation of intermoleular autophosphorylation of 56 y multiple fators suh as an the MRN omplex, an the other, reruitment of the ativate to the sustrate sites. In this respet, the MRN omplex inues the DNA-amage response to failitate the repair of amage DNA at early stages of DNA amage 57, whereas if the amage DNA annot e repaire, triggers a ell eath programme y promoting the p apoptoti mehanism. This orhestrate genome maintenane programme in response to DNA amage stress meiates not only timely DNA repair to avert the most harmful lesions to the integrity of the genome ut also an apoptoti mehanism to eliminate ells with heavily amage genomes, therey the evelopment of anerous ells an e prevente 58. Methos Cell ulture an transfetion. All ell lines were grown at 37 C an 5% CO 2 in DMEM/F12 supplemente with L-glutamine, peniillin/streptomyin an 1% fetal ovine serum. All sirnas against (s-29761), CHK2 (s-29271), p (s-29435) an ontrol sirna (s-44231) were otaine from Santa Cruz Biotehnology (Santa Cruz, CA). Four HuSH 29mer shrna onstruts against human (NM_14) an ontrol HuSH shrna loning vetor (prs) using U6 promoter were purhase from OriGene Tehnologies, In. (Rokville, MD). The oligonuleotie sequenes in the shrna expression assettes are shown in Supplementary Tale S1. ells were transfete with a omination of four HuSH 29mer shrna onstruts onurrently or ontrol prs vetor y liposome using GenJet In Vitro DNA Tranfetion Reagent for Cell (SignaGen Laoratories, Gaithersurg, MD). After puromyin seletion (1 µg ml 1 ), the -knokown poole stale lones (esignate (- shrna)) an the vetor ontrol stale lones (esignate (ControlshRNA)) were selete. Similarly, the A549 or H1299 -knokown poole stale lones (esignate A549 (-shrna) or H1299 (-shrna)) an the vetor ontrol stale lones (esignate A549 (Control-shRNA) or H1299 (Control-shRNA)) were generate an selete as esrie aove. For transfetion with sirna, ells were transfete with speifi sirna or ontrol sirna as iniate y using DharmaFECT 1 transfetion reagent (Thermo Sientifi, Rokfor, IL) aoring to the manufaturer s instrutions. Whole-ell lysates were prepare 48 h after transfetion as esrie previously 31. Antioies an reagents. The DNA amaging agent CPT was purhase from Sigma (St Louis, MO). The rug was issolve in DMSO an store in aliquots at 2 C. Antioies speifi to (FKHRL1, H-144 (1:1, ilution) an N-16, 1:5 ilution), PARP (1:1, ilution), CHK2 (1:1, ilution), p (DO-1 an FL-393, 1:1, ilution), phospho-p (p-ps, p-ps2 an p-ps46, 1:1, ilution), Lamin A/C (1:2, ilution) an β-tuulin (1:2, ilution) were otaine from Santa Cruz Biotehnology. Antioies against H2AX (1:2, ilution), phospho-h2ax Serine-139 (, 1:1, ilution) an phospho- Serine-1981 (, 1:1, ilution) were purhase from Millipore (Billeria, MA). Antioies against phospho- Serine-1981 (, 1:1, ilution), phospho- (-pt68, 1:1, ilution), p (1:1, ilution), phospho-p (p-ps6, p-ps9, p-ps, p-ps2, p- ps37, an p-ps46, 1:1, ilution), Bax (1:5 ilution) an Puma (1:5 ilution) were purhase from Cell Signaling Tehnology (Danvers, MA). Antioies against (BL116G, 1:5 ilution) were purhase from Bethyl Laoratories (Montgomery, TX). Antioies against (32-1, 271-1, 1:1, ilution) an phospho- Serine-1981 (1:1, ilution) were otaine from Epitomis (Burlingame, CA), an an antioy against FKHRL1 (6-951) was otaine from Upstate Biotehnology (Lake Plai, NY). Antioies against p21cip1 (1:1, ilution) an p27kip1 (1:1, ilution) were purhase from Cell Signaling Tehnology an/or BD PharMingen (San Diego, CA). Antioy against β-atin (1:3, ilution) was purhase from Sigma. Antioy against HMG14 (highmoility-group 14, 1:2, ilution) was purhase from Aam (Camrige, MA). Alexa 488-, Alexa 594- an Alexa-647-onjugate seonary antioies (1:2 ilution) were otaine from Moleular Proes (Eugene, OR). inhiitor (Mirin) an inhiitor (NSC19) were purhase from Santa Cruz Biotehnology, an p inhiitor (Pifithrin) was otaine from Toris Biosiene. MTT was otaine from Invitrogen an DMSO was purhase from Sigma. Laser miro-irraiation an imaging. Live ell imaging omine with laser miro-irraiation was esrie previously 41,42. Briefly, fluoresene ata were aquire with an Axiovert 2M mirosope (Carl Zeiss MiroImaging, Thornwoo, NY). A 365-nm pulse nitrogen laser (Spetra-Physis, Mountain View, CA) was iretly ouple to the epifluoresene path of the mirosope. DSBs were introue in the nuleus y miro-irraiation with 365 nm laser. Cells were reovere for 3 min in the inuator, followe y immunofluoresent staining. Images were taken with an AxioCam HRm amera an fluoresene intensities of miro-irraiate areas were etermine y using Axiovision Software, version 4.5 (Carl Zeiss). Immunofluoresene. an A549 ells were grown on glass overslips. After treatment with CPT (1 µm) for 2 or 4 h, ells were fixe with 4 % paraformalehye for 1 min an permeailize with Triton X-1 (.5%). Slie ulture hamers were washe with phosphate-uffere saline (PBS) an loke with PBS-ontaining 2% BSA, inuate with an antioy speifi to, ps1981,, p-ps, p-ps2, p-ps46 or -pt68 (1:5 1:2 ilution), followe y Alexa 594- or 647 (re)-onjugate anti-rait, Alexa 594- (1:2 ilution) or 647 (re)-onjugate anti-goat (1:2 ilution), an Alexa 488 (green)-onjugate anti-mouse (1:2 ilution) or rait (1:2 ilution) seonary antioies (Moleular Proes). Cells were ounterstaine with 4,6-iamiino-2-phenylinole (DAPI; Sigma) to show the nulei. Speifi staining was visualize an images were apture with a Leia SP2 AOBS onfoal laser sanning mirosope. To analyse quantitative o-loalization, we use ~1 ell images ranomly apture y onfoal mirosopy. The volume an perentage of p-ps2 o-loalize with were measure using the Veloity software (ver. 6.1, Improvision, Perkin-Elmer). To measure foi-positive ells, we use ~2 ells ranomly apture y onfoal mirosopy. The perentages of onsiering foi-positive ells were alulate from ells ontaining at least five foi. Eah error ar presente is the mean of stanar eviation. Immunopreipitation an immunolotting. All experiments were performe as esrie previously 23. Briefly, ells were washe twie with PBS an lyse with lysis uffer ontaining protease inhiitors at 4 C for 2 min. The lysates were entrifuge at 16, g for 1 min to remove ell eris. Total protein onentration was etermine as esrie aove. Protein samples were first preleare with a nonspeifi antioy. Preleare lysates were then inuate with an antioy y rotating at 4 C overnight followe y the aition of µl of 5% protein A- or protein G-sepharose slurry an rotating for 1 h. Protein A/G eas were ollete an washe with lysis uffer four times. Immunopreipitates were resolve y 6 or 8% or 1 or 12% SDS polyarylamie gel eletrophoresis (PAGE) an analyse y analysis. For analysis, the protein samples were sujete to SDS PAGE an transferre onto nitroellulose memranes (Bio-Ra). Memranes were loke for 1 h in 3% ovine serum alumin (BSA) in Tris-uffere saline ontaining.1% Tween-2 (TBST) an inuate for 1 h with primary antioy ilute in TBST ontaining 1% BSA. After three washes with TBST, memranes were inuate for 1 h with horseraish peroxiase-onjugate seonary antioies (1:3, or 1

11 nature ommuniations DOI: 1.138/nomms28 ARTICLE 1:5, ilution) in TBST ontaining 3% BSA. The immunolots were visualize y an enhane hemiluminesene kit otaine from Santa Cruz Biotehnology or West-Q ECL Platinum Solution otaine from GenDEPOT. TUNEL assay. Cells were grown on glass overslips. After treatment with CPT (1 µm) for 48 or 6 h, ells were fixe with 4% paraformalehye solution an permeailize with Triton X-1 (.2%). For TUNEL assay, ellular apoptosis assay was etermine y enzymati laelling of DNA stran reaks with a TUNEL assay kit (the DeaEn Fluorometri TUNEL System, Promega) aoring to the manufaturer s instrutions. Nulei were staine with DAPI (olour was inverte to re). Merge images (yellow) were onsiere as apoptoti ells an ounte uner a onfoal laser sanning mirosope (Leia SP2 AOBS). Chromatin retention assay. These experiments were performe as esrie previously 59 with some moifiations. Cells (1 1 7 ) were suspene for 5 min on ie in µl of frationation uffer (5 mm HEPES, ph 7.5, mm NaCl, 1 mm EDTA) ontaining.2% Noniet P-4 (Noniet P-4), supplemente with protease inhiitors (5 µg ml 1 eah of pepstatin, leupeptin an aprotonin) an phosphatase inhiitors. Following entrifugation at 1, g for 5 min, the supernatant was ollete (fration I) an pellets were washe with the same uffer. After the washing step, the washe sample was ollete as efore (fration II), an the nulear pellets were further extrate for 4 min on ie with µl of frationation uffer ontaining.5% Noniet P-4. The extrats were larifie y entrifugation at 16, g for min (fration III). The pellets were finally lyse in frationation uffer II an oile for 5 min (fration IV). After etermining the onentration of all frations, samples were separate on 8% or 1% SDS PAGE gels an lotte onto nitroellulose memranes (Boi-Ra). Memranes were loke for 1 h in 3% BSA in TBST an inuate for 1 h with primary antioy (1:5 or 1:1,) ilute in TBST ontaining 1% BSA. After three washes with TBST, memranes were inuate for 1 h with seonary antioies (1:3,) in TBST ontaining 3% BSA. The immunolots were visualize y an enhane hemiluminesene kit otaine from GenDEPOT. Statistial analysis. All ata are expresse as means an stanar eviations from at least three eterminations. The statistial signifiane of ifferene in nulear o-loalization of proteins examine in ells (y immunofluoresene) an the perentage of apoptoti nulei (y TUNEL assay) etween two groups was analyse with two-sie unpaire Stuent s t-tests when the varianes were equal with Grahpa PRISM (Ver.4.2) statistial software (San Diego, CA). All statistial tests were two-sie, an P-values less than.5 were onsiere statistially signifiant. Referenes 1. Shiloh, Y. an relate protein kinases: safeguaring genome integrity. Nat. Rev. Caner 3, (23). 2. Kastan, M. B. & Bartek, J. 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12 nature ommuniations DOI: 1.138/nomms Lee, J. H. & Paull, T. T. Diret ativation of the protein kinase y the Mre11/Ra5/Ns1 omplex. Siene 34, (24).. Lee, J. H. & Paull, T. T. Ativation an regulation of kinase ativity in response to DNA oule-stran reaks. Onogene 26, (27). 54. Uziel, T. et al. Requirement of the MRN omplex for ativation y DNA amage. EMBO J. 22, (23).. Horejsi, Z. et al. Distint funtional omains of Ns1 moulate the timing an magnitue of ativation after low oses of ionizing raiation. Onogene 23, (24). 56. Bakkenist, C. J. & Kastan, M. B. DNA amage ativates through intermoleular autophosphorylation an imer issoiation. Nature 421, (23). 57. Lee, J. H. & Paull, T. T. ativation y DNA oule-stran reaks through the Mre11-Ra5-Ns1 omplex. Siene 38, 1 4 (). 58. Bartkova, J. et al. DNA amage response as a aniate anti-aner arrier in early human tumourigenesis. Nature 434, (). 59. Anegeko, Y. et al. Nulear retention of at sites of DNA oule stran reaks. J. Biol. Chem. 276, (21). Aknowlegements We thank T. Unger, D.W. Meek, M.B. Kastan, R.A. DePinho, B. Vogelstein an L.K. Su for proviing reagents or support, an Z. Xu for tehnial assistane. This work was supporte in part y R1 grant CA (to M.C.T.H.) from the National Caner Institute (NCI), National Institutes of Health (NIH); a grant from the Avon Founation for Women (to M.C.T.H.), a Sientifi Sholar Awar from the Marsha Rivkin Center for Ovarian Caner Researh (to Y.M.C.), the Ann Shreier Researh Awar from the Ovarian Caner Researh Fun (to S.H.P.), NIH grants CA5519 an CA13499 an a grant RP11465 from Caner Researh Institute of Texas (to D.J.C.). The sponsors ha no role in the esign, onut or reporting of the stuy. Author ontriutions The experiments were oneive an esigne y M.C.-T.H., Y.M.C. an S.-H.P; experiments were performe y Y.M.C., S.-H.P., W.-B.T. an M.C.-T.H; laser miro-irraiation an imaging was performe y S.-Y.W. an supervise y D.J.C.; ata were analyse y Y.M.C., S.-H.P. an M.C.-T.H; essential resoures an reagents were provie y J.S.B. an M.-A.I. The paper was written y M.C.-T.H. an Y.M.C. Aitional information Supplementary Information aompanies this paper at natureommuniations Competing finanial interests: The authors elare no ompeting finanial interests. Reprints an permission information is availale online at reprintsanpermissions/ How to ite this artile: Chung, Y. M. et al. signalling links to the p apoptoti pathway following DNA amage. Nat. Commun. 3:1 oi: 1.138/nomms28 (212). 12

13 DOI: 1.138/nomms2465 Erratum: signalling links to the p apoptoti pathway following DNA amage Young Min Chung, See-Hyoung Park, Wen-Bin Tsai, Shih-Ya Wang, Masa-Aki Ikea, Jonathan S. Berek, Davi J. Chen & Mikey C.-T. Hu Nature Communiations 3:1 oi: 1.138/nomms28 (212); Pulishe 14 Aug 212; Upate 26 Feruary 213 The immunolot images in Fig. 1f of this Artile were inavertently upliate from panel g uring the proution proess. The orret version of Fig. 1 appears elow. a p-ps (h) (h) 4 p-ps 2 4 (h) p-ps2 p-ps46 p HK2 13 p-ps p-ps2 p-ps46 (L) p-ps p (L) (L) 4 p-ps2 2 4 (h) e p-ps (h) f (h) HK2 13 p-ps2 p-ps2 p-ps46 p-ps46 p p (H) (H) g (h) p-ps p-ps2 p-ps46 h HK (h) p-ps46 HK2 (H) 13 (L) (L) NATURE COMMUNICATIONS 4:2 DOI: 1.138/nomms & 213 Mamillan Pulishers Limite. All rights reserve.