PML regulates p53 stability by sequestering Mdm2 to the nucleolus

Transcription

1 regultes stility y sequestering to the nuleolus Ros Bernrdi 1,Pier Polo Sglioni 1,3,Stephn Bergmnn 1,3,Henning F. Horn 2,Kren H. Vousden 2 nd Pier Polo Pndolfi 1,4 The promyeloyti leukemi () tumour-suppressor protein potentites funtion y regulting post-trnsltionl modifitions, suh s CBP-dependent etyltion 1,2 nd Chk2-dependent phosphoryltion, in the -Nuler Body (NB) 3. ws reently shown to intert with the uiquitin-ligse (refs 4 6); however, the mehnism y whih regultes remins unler. Here, we show tht enhnes stility y sequestering to the nuleolus. We found tht fter DNA dmge, nd umulte in the nuleolus in n Arf-independent mnner. In ddition, we found tht the nuleolr loliztion of is dependent on ATR tivtion nd phosphoryltion of y ATR. Notly, in Pml / ells, sequestrtion of to the nuleolus ws impired, s well s stiliztion nd the indution of poptosis. Furthermore, we demonstrte tht physilly ssoites with the nuleolr protein, nd tht knokdown impirs the ility of to lolize to nuleoli fter DNA dmge. These findings demonstrte n unexpeted role of in the nuleolr network for tumour suppression. The mehnisms promoting the up-regultion nd trnsriptionl tivtion of re ritil for modulting its role in tumour suppression. The produt of the mdm2 proto-onogene physilly interts with nd is negtive regultor of in ells tht re not sujeted to stress. It does this y funtioning s uiquitin ligse 7.Disruption of the omplex is hieved in severl wys, suh s phosphoryltion of or (or oth) nd sequestrtion of either or to seprte su-nuler omprtments 7.These mehnisms n e differentilly utilized fter distint ellulr stresses; for exmple, nuleolr sequestrtion of n our fter replitive senesene 8 or fter inhiition of RNA polymerse I y tinomyin D (nuleolr stress) 9. The gene enodes tumour suppressor involved in the pthogenesis of ute promyeloyti leukemi (APL) 1.The protein lolizes to multi-protein su-nuler strutures termed -NBs 11. Numerous proteins ssoite dynmilly with in -NBs fter speifi stimuli. In prtiulr, fter onogeni stress nd DNA dmge indued y γ-irrdition, funtions s trnsriptionl o-tivtor y reruiting it to the -NBs 1,2,12.Here, it filittes etyltion of (on Lys-382), medited y the etyltrnsferse CBP 1,2.However, we found tht -IV (the isoform tht inds ; refs 2, 13) ould funtion s trnsriptionl o-tivtor, even fter sustitution of ritil lysine residues with rginines ( K382R, K3R nd K5R mutnts; see Supplementry Informtion, Fig. S1). These results suggest tht my potentite tivity through dditionl mehnisms. We resoned tht might diretly influene protein stility. Indeed, inresing mounts of indued the umultion of nd trget genes, suh s p21 (Fig. 1), y inresing the hlflife of (Fig. 1). Importntly, overexpression of in / doule-null ( / / / ) mouse emryoni firolsts (MEFs) did not result in the umultion of trnsfeted (Fig. 1), ut prevented the redution in levels used y otrnsfetion of humn (Hdm2; Fig. 1d). These results demonstrte tht n stilize y ntgonizing funtion. The stiliztion of in wild-type nd Pml / primry ells ws exmined fter the introdution of -dependent poptoti stimuli. In Pml / MEFs, the umultion of fter tretment with the topoisomerse inhiitor doxoruiin nd the ross-linking gent mitomyin C ws impired (Fig. 1e). The phosphoryltion of on Ser 18 (Ser 15 in humn ) nd etyltion on Lys 3 nd Lys 382 prlleled the levels of totl in MEFs treted with doxoruiin nd mitomyin C (see Supplementry Informtion, Fig. S2). This suggested tht these post-trnsltionl modifitions were not defetive; thus, they ould not ount for the defet in umultion tht ws oserved in Pml / MEFs. As promoted stiliztion y ounterting the funtion of, we resoned tht the sene of Pml might result in n inrese in uiquitintion fter DNA dmge. Wild-type nd Pml / ells were trnsfeted with hemgglutinin fused to uiquitin (HA U) nd treted with doxoruiin, s well s the protesome inhiitor MG132 (to prevent degrdtion). Immunopreipittion of endogenous identified mrked inrese in HA U omplexes in Pml /,ells ompred with wild-type ells (Fig. 1f). Therefore, fter DNA dmge in Pml / ells, stility is impired nd uiquitintion inreses. Interestingly, wild-type MEFs treted with doxoruiin nd mitomyin C showed signifint up-regultion of Pml, whih 1 Cner Biology nd Genetis Progrm, Deprtment of Pthology nd Mediine, Slon-Kettering Institute, Memoril Slon-Kettering Cner Center, New York, NY 121, USA. 2 Betson Institute for Cner Reserh, Glsgow G61 1BD, UK. 3 These uthors ontriuted eqully to this work. 4 Correspondene should e ddressed to P.P.P. (e-mil: p-pndolfi@ski.msk.org) Pulished online: 13 June 4, DOI: 1.138/n1147 NATURE CELL BIOLOGY VOLUME 6 NUMBER 7 JULY Nture Pulishing Group

2 Cyloheximide e 1 p expression (fold) Pml / h d Hdm2 Hdm expression (fold) Pml / h Perentge of ontrol Atin expression (fold) Mitomyin C Atin expression (fold) f Uiquitin Pml / Uiquitin Figure 1 medites stiliztion of in the presene of. () Western lot nlysis of H1299 ells trnsfeted with nd inresing mounts of -IV. pe ws otrnsfeted to monitor trnsfetion effiieny. Intensity of the nds quntified y densitometry is expressed s expression (fold) over the levels of lone. (), pe nd - IV were trnsfeted in H1299 ells s indited. At 24 h fter trnsfetion, ells were inuted with yloheximide for the indited times nd nlysed y western lotting. protein levels were ssessed y densitometry. () / / / MEFs were trnsfeted s in nd were nlysed y western lotting. (d) Western lot nlysis of / / / MEFs fter trnsfetion with the indited plsmids. (e) Wild-type nd Pml / MEFs were HA g Cell deth (%) Pml / Cell deth (%) Pml / Mitomyin treted with doxoruiin nd mitomyin C for the indited times. Cell lystes were nlysed y western lotting. protein levels, reltive to untreted ells nd to tin levels, re indited t the ottom of the pnels. (f) Wild-type nd Pml / immortlized MEFs were trnsfeted with ontrol vetor or HA U nd pe. At 24 h fter trnsfetion, ells were treted with doxoruiin for 12 h nd with MG132 for the lst 6 h. ws immunopreipitted nd the western lot ws proed with nti-ha nd nti ntiodies. (g) Wild-type (white rs) nd Pml / (grey rs) MEFs were treted for 12 nd 24 h with doxoruiin (left) or mitomyin C (right). Cell viility ws mesured y trypn lue exlusion. All pnels ( g) re representtive of t lest three independent experiments. oinided with the umultion of (see Supplementry Informtion, Fig. S2). Notly, doxoruiin- nd mitomyin-cindued poptosis ws mrkedly impired in the sene of Pml (Fig. 1g). Therefore, is importnt for the umultion of nd the indution of poptosis fter DNA dmge. To understnd the mehnisms underlying stiliztion y, we nlysed the loliztion of, nd fter DNA dmge. Surprisingly, in WI38 (norml humn emryoni firolsts) ells nd wild-type MEFs treted with doxoruiin, onentrted in the nuleoli, s demonstrted y o-loliztion with nuleolr mrkers (Fig. 2, ). In the mjority of ells, umulted t the periphery of the nuleolus. Aumultion of in the nuleolus ws not ompnied y dispperne of the -NBs, nd this umultion ws lso oserved fter mitomyin C tretment (dt not 666 NATURE CELL BIOLOGY VOLUME 6 NUMBER 7 JULY 4 4 Nture Pulishing Group

3 Nuleolin DAPI Merge Nuleolr (perentge of ells ) Wl p19-arf DAPI Merge Nuleolr (perentge of ells ) 6 4 Mefs Nuleolr (perentge of ells ) MEFs Nuleolr (perentge of ells ) ATR-DN firolsts ffeine ffeine DOXY DOXY d e -ATR f Doxyylin Doxyylin Flg Flg DN-ATR -ATR Cffeine Doxyylin 32P PATR IP: nti-flg 32P His His Figure 2 umultes in the nuleolus nd is phosphorylted y ATR fter DNA dmge. WI38 humn firolsts () nd MEFs () were ultured for 36 nd 24 h, respetively, in the presene of doxoruiin. Both nd nuleolin (), nd nd p19-arf (), were deteted y indiret immunofluoresene mirosopy. Cell nulei were visulized with DAPI (lue). Fields were merged to ssess protein ololiztion. Upper pnels show ontrol ells, ottom pnels show ells treted with doxoruiin. Grphs to the right show the perentge of ells displying nuleolr stining fter tretment with doxoruiin. () Left, MEFs treted with doxoruiin in the presene of sene of ffeine for 12 h. Right, ATR-DN firolsts treted with doxoruiin in the presene of sene of doxyyline for 12 h. In oth pnels, n verge of 3 ells were sored for nuleolr. The pnels represent four independent experiments. (d) ATR-DN firolsts were trnsfeted with -IV. At 24 h fter trnsfetion, ells were treted with doxoruiin nd doxyyline, s indited, for 12 h. ws immunopreipitted with n nti-flg ntiody. Western lot nlysis ws performed with n nti-p-atr ntiody nd susequently with n nti-flg ntiody. (e) ATR kinse ssy with purified His. U2OS ells expressing induile Flg - ATR or Flg ATR-DN were treted with doxyyline for 48 h. Anti-Flg immunopreipittes were inuted with His in the presene of 32 P-ATP. Kinse retions were nlysed y utordiogrphy nd y western lotting with nti-flg nd nti-his ntiodies to mesure protein levels. (f) Wild-type ATR ws immunopreipitted s in e. Kinse ssys were performed with or without ffeine. Pnels f re representtive of three independent experiments. shown). Six hours fter doxoruiin tretment, nuleoli ould not e deteted y immunofluoresene mirosopy with nuleolr mrkers (dt not shown), s noted fter other ellulr stresses 14.However, nuleoli reformed t lter times (onwrds of 12 h), when ws found to ololize with nuleolr proteins (Fig. 2, ). SUMO-1 lolized to the nuleolus with fter doxoruiin tretment (dt not shown), suggesting tht nuleolr my e SUMO-modified. ATM nd ATR kinses re the mjor regultors of sde of ellulr responses to DNA dmge tht result in either ell-yle rrest nd DNA repir or poptosis 15.Therefore, we tested whether umultion in the nuleolus ws prt of n ATM- or ATR-dependent hekpoint response. A dose of ffeine tht is known to inhiit oth ATM nd ATR 16 mrkedly loked the loliztion of to the nuleolus fter doxoruiin tretment (Fig. 2, left pnel). To determine whether loliztion ws dependent on the funtion of ATM, ATR, or oth, Atm / primry ells were used 17. loliztion in the nuleolus ws unffeted y Atm intivtion (dt not shown). A humn ell line stly trnsfeted with NATURE CELL BIOLOGY VOLUME 6 NUMBER 7 JULY Nture Pulishing Group

4 NPM Hdm2 Merge d Blot: nti (h) IP: nti- Nuleolin DAPI Merge Nuleolr (perentge of ells) e Nuleolin DAPI Merge f Nuleoplsm Nuleoli NPM Pml / Pml / Figure 3 nd ololize in the nuleolus fter DNA dmge. () Triple stining of, NPM nd Hdm2 in WI38 ells treted with doxoruiin for 36 h. Cells were stined with nti- (lue), nti-npm (red) nd nti-hdm2 (green) ntiodies nd nlysed y onfol mirosopy. (, ) nd lolize to the nuleolus in Arf / MEFs treted with doxoruiin. 18 h fter tretment, ells were stined with nti- ntiody (green), nti-nuleolin ntiody (red) (), nti- ntiody (green) nd nti-nuleolin ntiody (red) () efore nlysis y onfol immunofluoresene mirosopy. Nulei were stined with DAPI (lue). (d) Extrts of WI38 ells treted for 24 or 36 h with doxoruiin were immunopreipitted with nti- ntiody. The input ws 5% of tht used in the immunopreipittion (top). Western lot nlysis ws rried out with n nti- ntiody. The pnel is representtive of two independent experiments. (e, f) nuleolr umultion is impired in Pml / ells. (e) Grphi representtion of the perentge of wild-type (white olumns) nd Pml / MEFs (lk olumns) displying nuleolr stining fter inution with doxoruiin for 12 nd 24 h. More thn 4 ells were nlysed y onfol immunofluoresene mirosopy in three independent experiments. (f) Western lot nlysis of, NPM nd from nuleoplsmi nd nuleolr extrts of wild-type nd Pml / MEFs treted with doxoruiin for 12 h. tetryline-induile dominnt-negtive form of ATR (ATR-DN; see Methods) ws then used 18.Indution of ATR-DN y doxyyline signifintly redued the umultion of in the nuleolus fter doxoruiin tretment (Fig. 2, right). Endogenous immunopreipitted from doxoruiin-treted wild-type MEFs, ws reognized with n ntiody ginst proteins phosphorylted y ATM or ATR (dt not shown). In ddition, ws overexpressed in ATR- DN firolsts treted with doxoruiin in the presene or sene of doxyyline. Immunopreipitted ws reognized y the ntiphospho-atm/atr ntiody, ut only fter tretment with doxoruiin nd in the sene of doxyyline (Fig. 2d). Finlly, ATR kinse ssys were performed with purified His-tgged (His ) nd ell extrts from U2OS ells expressing induile wild-type or dominnt-negtive ATR 19. ws phosphorylted fter indution of wild-type, ut not dominnt-negtive, ATR (Fig. 2e). Furthermore, this phosphoryltion ws inhiited y ffeine (Fig. 2f), demonstrting tht is diret trget of ATR. In onlusion, is phosphorylted nd umultes in the nuleolus fter DNA dmge s prt of hekpoint response tht depends on ATR tivtion. The loliztion of nd fter DNA dmge ws exmined. umulted in the nuleolus fter doxoruiin tretment nd ololized with nuleolr, s shown y triple-stining of Hdm2, nd nuleophosmin (NPM; Fig. 3). Importntly, nuleolr loliztion of oth nd fter doxoruiin tretment lso ourred in Arf / MEFs (Fig. 3, ). Furthermore, endogenous nd were oimmunopreipitted from WI38 ells fter DNA dmge t times when they were found to ololize in the nuleolus (Fig. 3d; lso see Fig. 2). In ontrst, did not umulte in the nuleolus fter doxoruiin tretment (dt not shown). Together, these dt demonstrte tht fter DNA dmge, is sequestered in the nuleolus in n ARF-independent mnner, where it interts with. Therefore, we tested whether sequestrtion of to the nuleolus fter DNA dmge ws -dependent. The perentge of Pml / MEFs displying nuleolr fter doxoruiin tretment ws mrkedly redued when ompred with wild-type MEFs (Fig. 3e). To further quntify these differenes, nuleoli were isolted from wild-type nd Pml / MEFs efore nd fter doxoruiin tretment. ws found in the nuleoplsm of oth wild-type nd Pml / ells (Fig. 3f). 668 NATURE CELL BIOLOGY VOLUME 6 NUMBER 7 JULY 4 4 Nture Pulishing Group

5 Flg Flg DAPI Merge Flg Flg Untrnsfeted IP: nti- Flg mutnts inding FL- MT- DAPI Merge FL- M r (K) GST-inding ssy GST MT d FL- MT- FL- MT Hdm2 Figure 4 interts diretly with nd stilizes through sequestrtion of. () H1299 ells were trnsfeted with -IV nd s indited. 48 h fter trnsfetion, ell extrts were immunopreipitted with n nti-flg ntiody (right). Cell lystes were loded s ontrol (left). Western lot nlysis ws performed with nti-flg nd nti- ntiodies s indited. The sterisk indites nonspeifi nd. The pnel is representtive of three independent experiments. () GST-inding ssy with GST nd in vitro-trnslted. 35 S-lelled full-length -IV (indited s 1) nd deletion mutnts of -IV (indited s 2 5) were inuted with GST or GST. The input represents 5% of wht ws used in the ssy. A shemti representtion of - nd -inding regions of is displyed t the ottom. () Upper pnels, H1299 ells were left untrnsfeted or trnsfeted with full-length -IV (FL-) nd mutnt 5 (MT-) nd nlysed y immunofluoresene mirosopy with nti- (red) nd nti- (green) ntiodies. Nulei re stined with DAPI (lue). Arrows indite ololiztion of nd MT- in the ytoplsm. Lower pnels, H1299 ells trnsfeted with FL- or MT-, s well s, were nlysed y immunofluoresene mirosopy with nti- (green) nd nti- (red) ntiodies. Nulei re stined with DAPI (lue). (d) Western lot nlysis of H1299 ell lystes 24 h fter trnsfetion with (left pnel) or Hdm2 (right pnel), s well s inresing mounts of FL- nd MT-. pe ws otrnsfeted to monitor trnsfetion effiieny. The pnels re representtive of three independent experiments. However, fter doxoruiin tretment, perentge of ould e deteted in the nuleolr frtion of wild-type ells, ut not Pml / ells (Fig. 3f). These findings demonstrte tht is ritil for nuleolr umultion of fter DNA dmge. Our dt imply tht my promote stiliztion y sequestering. To test this hypothesis, we ssessed whether nd intert diretly nd, if so, whether mutnt of tht inds, ut not, stilizes. nd interted in - null H1299 ells, even in the sene of (Fig. 4). Glutthione S- trnsferse (GST) pull-down ssys with GST or GST, s well s deletion mutnts of, demonstrted tht the two proteins intert diretly. The inding site for resided round the oiled-oil motif of, whih is shred y ll isoforms (Fig. 4)., however, inds to the extreme roxyl terminus of -IV 13 (Fig. 4). Next, mutnt ( mutnt 5 in Fig. 4; MT-) tht inds, ut not, ws exmined. This mutnt lks the nuler loliztion signl (NLS) nd forms ytoplsmi ggregtes when overexpressed (Fig. 4). MT- ws used in these experiments (rther thn other isoforms), euse ll isoforms hve een reported to reruit to the -NB.MT- reruited endogenous to the ytoplsm (Fig. 4, top), ut filed to reruit otrnsfeted (Fig. 4, ottom). Similr results were otined with Pml / MEFs (dt not shown). Notly, MT- stilized to the sme extent s full-length (FL-; Fig. 4d, left). Interestingly, oth FL- nd MT- promoted the umultion of Hdm2 (Fig. 4d, right), suggesting tht n inhiit Hdm2 self-uiquitintion/degrdtion, s previously shown for ARF 21 nd the riosoml protein (ref. 9). Together, these results suggest tht n promote umultion of y sequestering in the sene of diret / inding. During replitive senesene, the tumour-suppressor protein ARF inds to nd sequesters it in the nuleolus 8.Nuleolr NATURE CELL BIOLOGY VOLUME 6 NUMBER 7 JULY Nture Pulishing Group

6 Flg -lv -lv Flg IP: nti-flg inding C C IP: nti- Flg e d IP: nti- Pml / Pml / Nuleolin HSP9 sirna-1 IP: nti- sirna-2 Whole-ell extrts % in o-ip Nuleolr extrts sirna WCE NE sirna-2 HPRT IP: nti-his His 35 S Cells with /nuleolin ololiztion (%) f * -1 ** -2 Nuleolin Merge -1 Figure 5 filittes nuleolr loliztion fter DNA dmge. () NIH 3T3 ells trnsfeted with ontrol vetor or -IV were immunopreipitted with n nti-flg ntiody. Western lot nlysis ws performed with nti-flg nd nti- ntiodies. () H1299 ells were trnsfeted with full-length nd mutnt proteins, whih were then immunopreipitted with nti-flg ntiody. Western lot nlysis ws performed with nti-flg nd nti- ntiodies. () In-vitro-trnslted ws inuted with nti-his ntiody with or without ffinity-purified His. The omplex ws resolved y SDS PAGE, stined with Coomssie lue nd nlysed y utordiogrphy. (d) Top, whole-ell extrts from immortlized wild-type MEFs treted for 18 h with doxoruiin were immunopreipitted with n nti- ntiody. Western lot nlysis ws performed with nti- nd nti- ntiodies. Pnels re representtive of four independent experiments. Bottom, nuleolr extrts from wild-type nd Pml / MEFs were immunopreipitted with n nti- ntiody. Pnels re representtive of two independent experiments. s re 5 1% of tht used in the immunopreipittions. The grph indites the mount of o-immunopreipitted with s perentge of the totl mount of in inputs. WCE, whole-ell extrts; NE, nuleolr extrts. (e) WI38 ells were trnsfeted with ontrol sirna or inresing mounts of -speifi sirna-1 nd sirnai- 2. At 48 h fter trnsfetion, western lot nlysis for nuleolin, HSP9 nd (left) nd RT PCR for nd HPRT for normliztion (right) were performed. (f) WI38 ells were trnsfeted with ontrol or sirna- 1 nd sirnai-2 for 48 h nd treted with doxoruiin for the lst 36 h. Cells were stined with nti-nuleolin nd nti- ntiodies. The grph expresses the perentge of ells with nuleolin- ololiztion in sirna-trnsfeted smples versus ontrol sirna-trnsfeted ells (ontrol oligonuleotide-treted ells = 1%; P =.2; P =.3). Dt is representtive of three independent experiments. loliztion of fter tinomyin D tretment, however, is ARF-independent nd is medited y the nuleolr protein (ref. 9). Atinomyin D tretment lso indued the loliztion of to the nuleolus (dt not shown). Therefore, we hypothesized tht oth nd might intert with fter DNA dmge, nd tht might medite nuleolr loliztion of. Endogenous o-immunopreipitted with overexpressed in NIH 3T3 firolsts (Fig. 5) nd H1299 ells (dt not shown). deletion mutnts tht lked the C-terminl region ould no longer intert with (Fig. 5), suggesting tht inding does not overlp with the -inding region (Fig. 4). In vitro inding ssys demonstrted tht nd n intert diretly (Fig. 5). Thus, we resoned tht endogenous nd might intert in the nuleolus fter doxoruiin tretment. Low levels of were o-immunopreipitted with n nti- ntiody in wild-type MEFs fter doxoruiin tretment (Fig. 5d, top). Importntly, the mount of from isolted nuleoli tht o-immunopreipitted with ws onsiderly higher thn tht from whole-ell extrts (Fig. 5d, ompre ottom nd top pnels, nd see grph). Thus nd intert speifilly in the nuleoli. Next, we ssessed whether is required for the nuleolr umultion of. Smll-interfering RNAs (sirnas) knoked-down expression in WI38 ells. Two sirnas were tested, nd oth produed similr redutions in expression (up to 5%), while leving other 67 NATURE CELL BIOLOGY VOLUME 6 NUMBER 7 JULY 4 4 Nture Pulishing Group

7 nuleolr proteins unffeted (Fig. 5e). nuleolr loliztion ws nlysed in ells treted with ontrol sirnas or sirnas fter doxoruiin tretment. The numer of ells displying ololiztion of with nuleolin (or NPM; dt not shown) ws redued mrkedly in sirna-treted ells (Fig. 5f). As interts with fter tretment with tinomyin D 9,22, we resoned tht nd might lso intert fter doxoruiin tretment; furthermore, might e required for this intertion. Overexpressed nd o-immunopreipitted from oth wild-type nd Pml / ells; however, doxoruiin tretment only inresed this intertion in the presene of (see Supplementry Informtion, Fig. S3). Together, these results demonstrte tht interts with in the nuleolus nd tht this intertion is importnt for the loliztion of to the nuleolus. Our nlysis leds to three mjor onlusions. First, (until now onsidered to e NB protein) n e found in distint suellulr omprtment (the nuleolus) fter ellulr stress. It is importnt to note tht umultes in the nuleolus fter tretment with speifi DNA-dmging gents (for exmple, doxoruiin, ut not γ-irrdition 23 ; see Supplementry Informtion, Fig. S4). The nuleolr loliztion of is triggered y tivtion of the hekpoint kinse ATR, onsistent with ATR tivtion eing mjor event in the indution of S/G2 hekpoints fter exposure to topoisomerse inhiitors 24. This oservtion, long with the finding tht is diret trget of Chk2 phosphoryltion fter γ-irrdition 25,suggests tht the loliztion nd funtion of re regulted y different hekpoints in response to distint poptoti stimuli. Seond, the umultion of in the nuleolus is ARF-independent nd, t lest in prt, dependent on the nuleolr protein. As nuleolr umultion of is redued ut not rogted fter knokdown, this suggests tht other proteins my lso funtion to dok t the nuleolus. Finlly, is required for nuleolr loliztion nd, in turn, stiliztion fter DNA dmge. As n intert diretly with nd, it is possile tht oth proteins prtiipte in lrger nuleolr omplex to indue sequestrtion. Our dt dd omplexity to the originl working model y whih regultes funtion in the NB, nd the -NB represents site for modifition nd tivtion of (refs 26, 27). METHODS Cell ulture. MEFs were prepred from emryos t dy 13.5 of development (E13.5). Erly pssge (3 5) MEFs were used in ll experiments, exept s indited. Wild-type nd Pml / MEFs were immortlized using 3T9 protool nd mintined in ulture up to pssge 3. funtion ws onsidered norml, s the protein ws indued y doxoruiin tretment nd ells displyed sensitivity to doxoruiin. H1299, NIH 3T3, 293T nd WI38 ells were otined from the Amerin Type Culture Colletion. The GM847 ATR-DN (dominnt negtive) ell line n SV4-immortlized humn firolst line expressing the ATR dominnt-negtive kinse under the ontrol of tetryline-induile system ws kindly provided y W. Cliy 17. U2OS wild-type ATR nd ATR- DN were kindly provided y S. Shreier 18.Indution of wild-type ATR nd ATR-DN ws hieved y 48-h-tretment with 1 µm doxyyline (Sigm, Sint Louis, MO). The Atm-null ell line 743 ws kind gift from M. Turker 16. G. Lozno nd M. vn Lohuizen kindly provided mdm2 / / / nd Arf / MEFs, respetively. Primry nd estlished ells were ultured in DMEM supplemented with 1% foetl ovine serum, 4.5 mg ml 1 gluose nd L-glutmine. Cells were treted with.5 2 µm doxoruiin depending on the sensitivity of the ell type (.5 1 µm for MEFs or immortlized MEFs; 2 µm for WI38 ells; 1 µm for NIH3T3 ells; 1 µm for Atm-null ells; nd 1 µm for ATR-DN ells) nd 5 mg ml 1 mitomyin C, (Sigm). Cyloheximide nd ffeine (Sigm) were used t 25 µm nd 4 mm, respetively, for the indited times. MG132 (Cliohem, L Joll, CA) ws used t 1 µm. Plsmids, ell trnsfetion nd trnstivtion ssys. pcmv-tg2b -IV expresses isoform IV under the ontrol of the CMV promoter. pcmv - nd pcmv K5R ( mutnt in whih lysines 37, 372, 373, 381 nd 382 re sustituted y rginines) expression vetors were generous gift from W. Gu. PCMV K3R nd pcmv K382R were reted y site-direted mutgenesis to introdue Lys Arg sustitution t position 3 or 382, respetively. -deletion mutnts were s desried 2,exept for mutnt 5, whih ws reted y site-direted mutgenesis. CMV Hdm2 ws gift from G. Lozno. pbx lu, p21 lu nd pmdm2 lu reporter plsmids were kind gift from C. Di Como nd C. Prives. GST ws from A. Weismnn. His ws reted y loning -IV into pet-33 (Novgen, Mdison, WI). Trnsient trnsfetions were performed with the Effetene trnsfetion regent (Qigen, Vleni, CA) ording to the mnufturer s protool. For trnsfetions in H1299 ells, 5 ng of were otrnsfeted with 5, 1 nd 5 ng of -IV, with or without 5 ng of Hdm2. pe (1 ng) or renill- (5 ng) expressing plsmids were otrnsfeted to normlize for trnsfetion effiieny. For trnstivtion ssys, ng of reporter plsmid ws used nd luiferse tivity ws ssyed 24 h post-trnsfetion. For intertion, wild-type nd Pml / immortlized MEFs were trnsfeted with 2 µg CMV nd 4 µg CMV. 24 h post-trnsfetion, ells were treted with 1 µm doxoruiin for 18 h. Western lotting, immunopreipittion, in vitro protein intertions nd ATR kinse ssy. For western lot nlysis, ells were lysed in ie-old RIPA uffer ontining omplete protese inhiitor oktil (Boehringer Mnnheim, Mnnheim, Germny). Lystes were entrifuged to ler ell deris nd 4 µg of lyste ws used for nlysis. The following ntiodies were used: nti-humn- (DO-1; Snt Cruz, Snt Cruz, CA), nti-mouse- (CM5; Novostr, Newstle, UK), nti-phospo- (Ser 15; New Englnd Biols, Beverly, MA), nti-flg-m2 (Sigm), nti- (SMP14; Snt Cruz), ntihumn- (PG-M3; Snt Cruz), rit nti- (kindly provided y K. S. Chng), nti-mouse (S36 nd S37 monolonl ntiodies; kindly provided y S. Lowe), nti- (Clonteh, Plo Alto, CA), nti phospho-ser/thr- ATM/ATR (Cell Signling, Beverly, MA) nd nti-ha (Covne, Berkeley, CA). For o-immunopreipittion, ells were lysed in immunopreipittion (IP) uffer (15 mm NCl, 5 mm Tris-HCl t ph 7.5 nd 1% NP-4), supplemented with omplete protese-inhiitor oktil. The lyste ws prelered y inution with protein-g or protein-a Sephrose eds (Amershm Biosienes, Pistwy, NY) nd inuted with nti-flg ntiody, ntihumn ntiody (PG-M3) or nti- ntiody 9 overnight t 4 C efore inution with protein-g or protein-a Sephrose for 1 h. Immunopreipittes were wshed five times with ie-old IP uffer nd resolved y SDS PAGE. Western lotting ws performed ording to stndrd proedures. For GST pull-down ssys, in vitro-trnslted produts were generted using the TNT Coupled System (Promeg, Mdison, WI). 35 S-lelled wild-type nd mutnts were inuted with GST nd GST in IP uffer for 2 h t 4 C. Beds were wshed five times with IP uffer, nd proteins were eluted with SDS smple uffer nd resolved y SDS PAGE efore utordiogrphy. For ATR kinse ssys, U2OS ells expressing wild-type ATR or ATR-DN were treted with doxyylin for 48 h nd ATR ws immunopreipitted from 2 mg of ell extrt with nti-flg ntiody. Kinse ssys were performed s previously desried 18 with 1 µg purified His- s sustrte. Proteins were seprted y SDS PAGE, trnsferred onto memrnes nd exposed for utordiogrphy, s well s western lot nlysis with n nti-flg ntiody. Isoltion of nuleoli. Nuleoli were prepred from wild-type nd Pml / immortlized MEFs grown on 1 14 m pltes, s desried 28.Isolted nuleoli were resuspended in RIPA uffer nd entrifuged t 13,g to eliminte insolule mteril. For oimmunopreipittions, nuleoli were resuspended in IP uffer nd immunopreipitted with nti- ntiody. Immunofluoresene nd onfol mirosopy. Cells were grown on overslips nd treted s indited. Cells were fixed in 4% prformldehyde for 1 min efore permeiliztion in PBS ontining.1% Triton X-1 for 2 min. The ntiodies used for immunofluoresene mirosopy were: nti-humn (PG-M3; Snt Cruz), nti-mouse (S36 nd S37 monolonl ntiodies), nti- (SMP14; Snt Cruz), nti-nuleolin (4E2; Reserh Dignostis, NATURE CELL BIOLOGY VOLUME 6 NUMBER 7 JULY Nture Pulishing Group

8 Flnders, NJ), nti-p19 Arf (R562; Am, Cmridge, MA), nti- (A-3; Onogene Reserh Produts, Drmstdt, Germny). Doule stining ws performed s indited. For detetion, ells were inuted with FITC-onjugted nti-mouse ntiodies nd Cy5-onjugted nti-rit ntiodies (Jkson ImmunoReserh Lortories, West Grove, PA). For triple stining, the ntiodies used were: rit nti- (kindly provided y K. S. Chng), mouse nti- (SMP14; Snt Cruz) nd got nti-b23 (C-19; Snt Cruz). The seondry ntiodies used for triple stining were: Alex Fluor-45 nti-rit, Alex Fluor-488 nti-mouse nd Cy5-onjugted nti-got (Moleulr Proes, Eugene, OR). Slides were nlysed y onfol mirosopy. sirna. sirna oligonuleotides for were otined from Dhrmon (Lfyette, CO) in 2 -deproteted, nneled nd deslted duplex form. The trget sequenes were nuleotides (sirna-1) nd (sirna-2). WI38 ells were trnsfeted with Lipofetmine (Invitrogen, Crlsd, CA) ording to the mnufturer s protool. Cells were treted with doxoruiin 12 h fter sirna-trnsfetion, fixed nd nlysed 36 h fter tretment. Primers for RT PCR nlysis were: 5 -GCGCAGGATCAAGGTGAA-3 ; 5 -TTATTTCGGAGGAAGGAT-3. Note: Supplementry Informtion is ville on the Nture Cell Biology wesite. ACKNOWLEDGEMENTS We re indeted to I. Guernh, A. Guo, P. Slomoni, F. Bernssol, nd S. Grisendi for suggestions during the ourse of this work nd ritil reding of the mnusript. We re grteful to W. Cliy, C. Di Como, W. Gu, A. Levine, S. Lowe, G. Lozno, C. Prives, S. Shreier, M. Turker, M. vn Lohuizen nd A. Weismnn for regents nd dvie. We thnk Y. Hupt for useful disussion. R.B. nd P.P.S. were supported y T32 trining grnts from the Ntionl Institutes of Helth. P.P.S. is lso reipient of n ASCO Young Investigtor Awrd nd CALGB Onology Fellows Awrd. This work ws supported y the wrd of Ntionl Institutes of Helth grnt RO1 CA to P.P.P. COMPETING FINANCIAL INTERESTS The uthors delre tht they hve no ompeting finnil interests. 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