EMBO. BubR1 acetylation at prometaphase is required for modulating APC/C activity and timing of mitosis EMBO. open

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1 The EMO Journal (2009) 28, & 2009 European Molecular iology Organization Some Rights Reserved /09 ubr1 acetylation at prometaphase is required for modulating P/ activity and timing of mitosis THE EMO JOURNL EMO open This is an open-access article distributed under the terms of the reative ommons ttribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission. Eunhee hoi, Hyerim hoe, Jaewon Min, Ji Yoon hoi, Jimi Kim and Hyunsook Lee* epartment of iological Sciences and Institute of Molecular iology and Genetics, ollege of Natural Sciences, Seoul National University, Seoul, Korea Regulation of ubr1 is central to the control of P/ activity. We have found that ubr1 forms a complex with and is acetylated at lysine 250. Using mass spectrometry and acetylated ubr1-specific antibodies, we have confirmed that ubr1 acetylation occurs at prometaphase. Importantly, ubr1 acetylation was required for checkpoint function, through the inhibition of ubiquitindependent ubr1 degradation. ubr1 degradation began before the onset of anaphase. It was noted that the preanaphase degradation was regulated by ubr1 acetylation. egradation of an acetylation-mimetic form, ubr1, was inhibited and chromosome segregation in cells expressing ubr1 was markedly delayed. y contrast, the acetylation-deficient mutant, ubr1, was unstable, and mitosis was accelerated in ubr1-expressing cells. Furthermore, we found that P/dc20 was responsible for ubr1 degradation during mitosis. On the basis of our collective results, we propose that the acetylation status of ubr1 is a molecular switch that converts ubr1 from an inhibitor to a substrate of the P/ complex, thus providing an efficient way to modulate P/ activity and mitotic timing. The EMO Journal (2009) 28, doi: / emboj ; Published online 30 pril 2009 Subject ategories: proteins; cell cycle Keywords: acetylation; P/; ubr1; dc20; spindle assembly checkpoint (S) Introduction Genetic integrity through accurate chromosome segregation requires the spindle-assembly checkpoint (S; Musacchio and Hardwick, 2002; haradwaj and Yu, 2004; Taylor et al, 2004). The activated S inhibits the anaphase-promoting complex (P/), a multicomplex E3 ligase that is responsible for destroying securin and yclin (Yu, 2002). When *orresponding author. epartment of iological Sciences and Institute of Molecular iology and Genetics, ollege of Natural Sciences, Seoul National University, Seoul, Korea. Tel.: þ ; Fax: þ ; HL212@snu.ac.kr Received: 19 ugust 2008; accepted: 7 pril 2009; published online: 30 pril 2009 this checkpoint fails, cells die or become aneuploid a hallmark of cancer (Rieder and Maiato, 2004; Kops et al, 2005). ubr1 is essential for the S and checkpoint signalling (Yu, 2002). Importantly, ubr1 binds directly to dc20, the W domain-containing coactivator of the P/ complex during mitosis (Tang et al, 2001; Yu, 2002; han and Yen, 2003). dh1 is another coactivator that is activated when it is no longer phosphorylated by dk1, and replaces dc20 in binding and activating the P/ complex. Thus, dc20 serves as a coactivator of the P/ E3 ligase until mitotic exit (Passmore and arford, 2005; Peters, 2006; Thornton and Toczyski, 2006; Yu, 2007). The importance of ubr1 in the S is reflected by the observation that ubr1 forms the mitotic checkpoint complex (M) composed of Mad2, ub3, ubr1, and dc20 and inactivates the P/dc20 complex (Sudakin et al, 2001). recent report suggested an alternative model for S activation, where Mad2 only loads dc20 onto ubr1 and then leaves the complex. onsequently, the ub3ubr1dc20 complex is presented to the P/ complex, followed by dc20 ubiquitination and degradation to maintain the S (Nilsson et al, 2008). In any model presented, ubr1 plays a central role in binding to and regulating the P/dc20 complex. It should be noted that ubr1 binds to P as well as to dc20 (han et al, 1999; Sudakin et al, 2001; Morrow et al, 2005). ubr1, similar to its orthologue Mad3 in yeast (urton and Solomon, 2007; Sczaniecka et al, 2008), has a -box-like motif and two KEN boxes, which serve as destruction signals for the P/ complex (Glotzer et al, 1991; King et al, 1996b; Pfleger and Kirschner, 2000). This suggests that ubr1 can be regulated by P/-mediated destruction. In this vein, it is noteworthy that ubr1 levels decrease late in mitosis (han et al, 1999). However, data from yeast Mad3, a homologue of mammalian ubr1, indicated that ubr1 is not degraded through P/-mediated polyubiquitination. Instead, ubr1 functions as a pseudosubstrate of the P/ complex by competing with genuine substrates of the P/ complex for the same dc20-binding sites (-box and KEN box) (urton and Solomon, 2007). Therefore, it remains to be elucidated whether ubr1 is regulated through destruction by the P/ complex. Regarding the regulation of ubr1 protein levels and its consequences in checkpoint activity, it has been shown that the levels of ubr1 during mitosis are crucial for mitotic timing. sirn knockdown of ubr1 resulted in mitosis acceleration and checkpoint abrogation (Meraldi et al, 2004). In addition, ubr1 is required for the assembly of S components (hen, 2002). Therefore, regulation of ubr1 levels might be an essential regulatory step in checkpoint activity. ubr1 is a kinase, whereas its yeast homologue, Mad3, lacks a kinase domain. The kinase activity of ubr1 is & 2009 European Molecular iology Organization The EMO Journal VOL 28 NO

2 E hoi et al stimulated by the microtubule motor protein, ENP-E, and is required for checkpoint maintenance and signalling in metazoans (brieu et al, 2000; Mao et al, 2003, 2005; Weaver et al, 2003). Phosphorylation of ubr1 in mitosis by kinases such as dk1, urora, and Plk1 is essential for monitoring stable spindle attachments to kinetochores and tension. ubr1 phosphorylation is also required for checkpoint signalling (itchfield et al, 2003; Lampson and Kapoor, 2005; Elowe et al, 2007; Wong and Fang, 2007; Huang et al, 2008). In this study, we show that ubr1 is acetylated exclusively at the prometaphase stage. We found that ubr1 is degraded during mitosis through P/dc20-mediated ubiquitination. It is noteworthy that the acetylation of ubr1 inhibits its degradation by the P/dc20 complex during mitosis, and this mechanism controls the timing of mitosis. Our results indicate that the acetylation status of ubr1 provides a molecular switch that controls the timely activation of the P/ complex, thereby serving as a regulatory step for the S. Results ubr1 binds to and is acetylated by at prometaphase kinetochores Protein modifications such as phosphorylation, SUMOylation, and acetylation contribute to the control of cell-cycle progression. While studying the regulation of ubr1 activity, we found that ubr1 might be controlled by acetylation. synchronously growing cells and nocodazole-arrested HeLa cells were compared and tested for the possibility of ubr1 acetylation. Immunoprecipitation (IP) followed by W analysis showed that ubr1 might be acetylated in nocodazole-arrested cells (Figure 1). Next, we tested the binding ability of ubr1 to p300, P, and to verify whether ubr1 was capable of binding to acetyltransferase and being acetylated. In IP and W, ubr1 interacted with but not with p300 or P acetyltransferases (Supplementary Figure 1). It is noteworthy that ubr1 complex formation was detected in nocodazole-arrested cells but was barely detectable in asynchronous cells (Figure 1). This suggests the possibility that acetylates ubr1 and this acetylation might play a role in the control of ubr1 activity. We then examined the location of the ubr1 complex formation because ubr1 localizes to kinetochores in mitosis. The localization of ubr1 to kinetochores is essential for S function; it monitors bipolar spindle attachment and chromosome alignment (itchfield et al, 2003), thereby regulating the P/ complex and the metaphaseanaphase transition (Yu, 2002; han and Yen, 2003). Metaphase chromosomal spreads were prepared and subjected to an immunofluorescence assay with anti- and anti-ubr1 antibodies. The assays showed that ubr1 and colocalized at kinetochores (Figure 1 and ). onsistent with in vivo complex formation between ubr1 and at prometaphase kinetochores, we observed that ubr1 could be acetylated by. Recombinant ubr1, as well as immunoprecipitated ubr1, was acetylated by during in vitro acetylation assays (Figure 1E and F). Next, we asked whether sustained ubr1 acetylation affected mitotic cell-cycle progression. s a first step towards answering this question, we treated cells in mitosis with the pan-h inhibitor trichostatin (TS). HeLa cells were synchronized at prometaphase by nocodazole treatment followed by mitotic shake-off. Subsequently, synchronized cells were either released from the mitotic arrest by washing out nocodazole (Figure 2, left panel), or kept in nocodazole to maintain mitotic arrest (Figure 2, right panel). The effects of TS treatment on ubr1 levels were analysed in both cases, with cycloheximide (HX) treatment included to control for the effects of de novo protein synthesis. ells were collected at the indicated time points and were analysed for ubr1 levels (Figure 2) and cell-cycle profiles (Figure 2). Interestingly, ubr1 levels decreased markedly from the initial time point of analysis in cells progressing through mitosis (Figure 2, left panel, none), and were further decreased by HX treatment (Figure 2, left panel, þ HX). When cells were arrested in mitosis by continuous nocodazole treatment, ubr1 levels were maintained (Figure 2, right panel, none). However, when HX was added, ubr1 levels decreased in the nocodazole-arrested condition (Figure 2, right panel, þ HX). Taken together, these results suggest that, similar to dc20 (Nilsson et al, 2008), ubr1 is not only degraded but also continuously synthesized during mitosis. TS treatment resulted in sustained levels of ubr1 relative to the levels after HX treatment in cells progressing through mitosis (Figure 2, left panel, TS and TS þhx) and in cells blocked for exit from mitosis (Figure 2, right panel, TS and TS þ HX). oncomitantly, mitotic exit was delayed for at least 5 h, until the end of the time course, by treatment with TS (Figure 2, left panel, TS and TS þ HX), whereas control or HX-treated cells exited from mitosis 1 h after nocodazole release (Figure 2, left panel, none and HX). In all cases of continuous nocodazole treatment, cells did not exit from mitosis (Figure 2, right panel). The fact that TS treatment resulted in the stabilization of ubr1 levels suggests that ubr1 might be one of the targets of TS during mitosis. Taken together with the results from Figure 1, ubr1 is likely to be acetylated in mitosis, which probably results in the inhibition of its degradation. In this regard, our observation that ubr1 interacted with H 13 in transfected cells (Supplementary Figure 3) suggests the intriguing possibility that the ubr1 acetylation state might regulate ubr1 function and progression through mitosis. ubr1 Lysine 250 (K250) is acetylated To identify the acetylation site, we made a series of ubr1 deletion constructs, transfected them into 293T cells, and analysed the acetylation domain by IP and W. The N- terminal region of ubr1 contained the acetylation domain (Supplementary Figure 4). Further in vitro acetylation assays showed that ubr1 amino acids (R23) contained acetylated lysine residues (Figure 3 and ). In parallel, mass spectrometry analysis was carried out with full-length ubr1 purified from insect cells and K250 was identified as the site of acetylation (Supplementary Figure 5). y means of in vitro mutagenesis and acetylation assays, we confirmed that K250 was the acetylation site (Figure 3 and Supplementary Figure 6). To verify that K250 was acetylated in vivo, interphase and prometaphase HeLa cells prepared by nocodazole arrest and mitotic shake-off were subjected to IP using anti-ubr1 antibodies and to mass spectrometry analysis. Our findings showed that the K250 site was acetylated 2078 The EMO Journal VOL 28 NO & 2009 European Molecular iology Organization

3 Ehoiet al Input(4%) IP: α- Neg Input (5%) Pre. IP: α-ck P-ubR1 ubr1 W: α- Noc Noc ubr1 PI ubr1 Merge olocalization Merge PI ubr1 Merge E M (μg) W: α-ck W: α- F ubr1 W: α-ck W: α- Figure 1 ubr1 interacts with and is acetylated at prometaphase kinetochores. () ubr1 acetylation was compared between asynchronously growing cells and prometaphase cells. HeLa cells were treated with nocodazole for 13 h (or left untreated) and subjected to mitotic shake-off. IP was carried out with anti-c-k antibodies and subsequent W with anti-ubr1 antibodies. The slower migrating form of ubr1, phospho-ubr1 (as shown in W of input control), was detected with the anti-c-k antibodies. () ubr1 and interact in nocodazole-treated cells. ells were treated with nocodazole for 13 h (or left untreated), and lysates were analysed by IP with anti-, followed by W using anti-ubr1. The blot was then re-probed with anti- to normalize the IP. () ubr1 interacts with at prometaphase kinetochores. Metaphase chromosome spreads were immunostained with anti- (red) and anti-ubr1 (green) antibodies. localization at prometaphase kinetochores was verified by sirn experiments (Supplementary Figure 2). White scale bar, 5 mm. () Images in () were enlarged to visualize the localization of ubr1 and with the chromosome. (E) ubr1 purified from insect cells (left) was subjected to an in vitro acetylation assay in the presence of purified and acetyl-o. The reaction was analysed by W with anti-c-k antibodies (right). One mg each of ubr1 (8.4 pmol) and (10.8 pmol) was employed for the assay, which corresponds to a molar ratio of 1:1.3. The same blot was re-probed with anti-ubr1 or anti- antibodies. (F) ubr1 IPs from 1 mg of HeLa cell lysates were subjected to acetylation assays with or without recombinant (1 mg), followed by W with anti-c-k antibodies. in prometaphase but not in interphase cells (Figure 3). Interestingly, substrate recognition motifs of P/-dependent ubiquitination (Peters, 2006; Thornton and Toczyski, 2006; Yu, 2007), -box (Glotzer et al, 1991; King et al, 1996a; urton and Solomon, 2007) and KEN box motifs (Pfleger and Kirschner, 2000; Murray and Marks, 2001; urton and Solomon, 2007; King et al, 2007) were found adjacent to K250. K250 and these degrons are conserved in vertebrates (Figure 3E), suggesting that ubr1 acetylation might regulate S activity by controlling either the binding affinity of ubr1 to the dc20 coactivator (urton and Solomon, 2007) or the P/-dependent destruction of ubr1 (King et al, 2007). To further confirm the in vivo acetylation of ubr1, we generated a polyclonal antibody that specifically recognizes K250-acetylated ubr1 (Figure 4). To verify that the anti- c-k250 antibody specifically recognizes acetylated ubr1 in M phase, interphase and M phase HeLa cell extracts were subjected to IP with anti-ubr1 antibodies and to W with the antibodies indicated. Similar to the anti-c-k antibody, the anti-c-k250 antibody recognized ubr1 only during M phase (Figure 4). These results confirmed that the anti-c- K250 antibody recognizes acetylated ubr1 in vivo. To further assess the time point when ubr1 is acetylated during the mitotic cell cycle, HeLa cells were synchronized in prometaphase and then released into the cell cycle in the presence of HX. W analysis with anti-acetyl-k250 serum at different time points after release indicated that acetylation occurred exclusively during prometaphase (time 0), but not during the other phases of the mitotic cell cycle (Figure 4). This acetylation showed excellent correlation with ubr1 complex formation (Figure 1). Furthermore, TS-treated cells showed an attenuated level of ubr1 and acetylation for more than 5 h (Figure 4, þ TS, anti-c-k250), suggesting that one of the targets of TS in mitosis might be acetylated ubr1, supporting our interpretation from Figure 2. ubr1 acetylation participates in S activity Substituting K250 with arginine () produces an acetylation-deficient mutant, whereas substituting lysine with glutamine () generates a mutant that mimics the characteristics of acetylated ubr1 (Supplementary Figure 7 and ). Using these in vitro mutagenized constructs, we determined the effect of acetylation on various aspects of S activity. & 2009 European Molecular iology Organization The EMO Journal VOL 28 NO

4 E hoi et al Noc. release Noc Noc (h) Noc. release (h) None None HX HX TS TS TSHX TSHX W: α-lamin / W: α-lamin / None Noc. release Noc HX TS TSHX None HX TS TSHX 5 h 2 h 1 h 0.5 h 0 h 2 N 4 N 2 N 4 N 2 N 4 N 2 N 4 N 2 N 4 N 2 N 4 N 2 N 4 N 2 N 4 N Figure 2 Treatment with TS during mitosis inhibits the degradation of ubr1 and a delay in anaphase entry. () HeLa cells were synchronized at prometaphase by tyminidinenocodazole arrest followed by mitotic shake-off, and then either released into the cell cycle (left panel) or maintained in nocodazole for 5 h (right panel). To assess the effect of H inhibition on ubr1, cells were treated with 100 nm of TS or left untreated for the indicated time points and subjected to W; a total of 100 mg/ml of cycloheximide (HX) was included as a control to suppress de novo protein synthesis. lots were re-probed with an anti-lamin / antibody to normalize protein levels. ed cells () remaining after mitotic shake-off served as controls. () The cell-cycle stage was assessed by propidium iodide staining and flow cytometry at the same time points as in (); 2 N and 4 N N contents are indicated. To assess the checkpoint response to spindle damage, we compared MPM2 staining after nocodazole treatment in cells expressing three different ubr1 constructs (Figure 5). epletion of ubr1 or Mad2 by sirn resulted in the abrogation of S function, as shown by a marked reduction in MPM2 staining (Figure 5, square and cross, respectively). Wild-type and -expressing cells, depleted of endogenous ubr1, restored the MPM2 staining to a level comparable with control cells (Figure 5, triangle and open circle, respectively). In contrast, -expressing (Figure 5, diamond) or -expressing cells depleted of Mad2 expression could not recover the MPM2 staining (Figure 5, closed circle). The importance of ubr1 acetylation for checkpoint function was confirmed by live-cell analysis. ontrol cells, cells expressing wild-type ubr1, and expressing cells were blocked in mitosis by nocodazole treatment. y contrast, ubr1-depleted cells (siubr1), Mad2-depleted cells (simad2), cells expressing (siubr1 þ ), and Mad2-depleted cells expressing (simad2 þ ) exited from mitosis despite the presence of nocodazole (Table I). Taken together, these results confirm that ubr1 acetylation is required for S function. We then determined whether ubr1 acetylation affected its phosphorylation in prometaphase. HeLa cells were transfected with,, or wild-type () ubr1 plasmids, synchronized at prometaphase and analysed by IP and W. To avoid proteolytic effects, MG132 treatments were included. The results showed that ubr1 acetylation did not affect the appearance of the characteristic slow-migrating band of ubr1 in prometaphase/metaphase cells after nocodazole treatment (Figure 5). ccurate chromosome segregation is guaranteed by the sensing of kinetochoremicrotubule attachments and tension across sister kinetochores. This signal is transmitted and amplified by phosphorylation events in S components, resulting in inhibition of the P/ complex even when only one chromosome is attached to spindle microtubules. oncerted ubr1 phosphorylations play a crucial role in spindle checkpoint signalling (Gillett and Sorger, 2001; Taylor et al, 2001; hen, 2002; Mao et al, 2003, 2005; Morrow et al, 2005). It has been shown that Plk1 phosphorylates ubr1 in a tension-sensitive manner and that this event is crucial for stable kinetochoremicrotubule attachments (Elowe et al, 2007; Wong and Fang, 2007). To investigate whether ubr1 phosphorylation by Plk1 was affected 2080 The EMO Journal VOL 28 NO & 2009 European Molecular iology Organization

5 Ehoiet al ub3- Mad3 binding ΔR ΔR ΔR ΔR2-4 ΔR W: α-ck W: α-gst ΔR2-2 E 20 KEN1 H. sapiens M. musculus G. gallus X. laevis 221estruction ox H. sapiens M. musculus G. gallus X. laevis 291 KEN2 H. sapiens M. musculus G. gallus X. laevis cetylated ΔR2-2 / K250 ΔR2-1 Interphase M phase ΔR2-2 ΔR2-4 ΔR2-5 W: α-ck ΔR2-1 ΔR2-5 ~ 1,190 1,200 1,210 1,220 1,230 1,240 1,250 1,260 1,270 c (42 a) ,180 1,200 1,220 1,240 1,260 1,280 Figure 3 Identification of the ubr1 acetylation site. () The ubr1 N-terminal region (R2; amino acids 1514) was subdivided into five regions to precisely map the acetylation site. () GST fusions of the recombinant proteins (R2-15) were purified from Escherichia coli and subjected to in vitro acetylation with or without in the reaction. () K250 in R23 was subjected to in vitro mutagenesis with rg (R23 ), purified from E. coli, and subjected to an in vitro acetylation assay. Samples were analysed for acetylation by W with anti- c-k antibodies. () Mass spectrometry analysis to detect in vivo acetylation of ubr1. HeLa cells were treated with nocodazole and prometaphase cells were collected by mitotic shake-off. ed cells (interphase) and prometaphase cells (M phase) were immunoprecipitated with anti-ubr1 antibodies and SSPGE. Excised ubr1 bands were analysed for the acetylated site by mass spectrometry. n additional peak at , which was shifted by acetylation (42-a) from at M phase, confirms that K250 of ubr1 is acetylated at prometaphase in vivo. (E) omparison of ubr1 sequences surrounding K250 among vertebrates. onserved estruction ox (-box)-like sequences and two KEN boxes are marked W: α-gst by acetylation, we utilized a ubr1 antibody that specifically recognizes Plk1-mediated ubr1 phosphorylation (Elowe et al, 2007). In a W of the same blot that was used to assess ubr1 phosphorylation at prometaphase, we observed that ubr1 acetylation did not affect Plk1-mediated phosphorylation of ubr1 in prometaphase (Figure 5, upper panel). inding of ubr1 to dc20 is crucial in the inhibition of P/ activity (Tang et al, 2001; Yu, 2007; Nilsson et al, 2008). Therefore, we tested whether ubr1 acetylation affected its binding to dc20. Our results showed that acetylation of ubr1 did not alter its direct binding to dc20 (Figure 5E). We then questioned whether ubr1 acetylation affected its binding to the dc20 complex in nocodazolearrested cells. The 293T cells transfected with various ubr1 constructs were treated with nocodazole; some were subsequently treated with MG132 to inhibit protein degradation. Immunoprecipitation with 9E10 and subsequent W showed that the formation of a complex between ubr1 and dc20 in prometaphase was not affected by ubr1 acetylation (Figure 5F). ubr1 acetylation at K250 inhibits its ubiquitinationdependent proteolysis cetylation sometimes interferes with ubiquitinationmediated proteolysis by either competing for the same residue or altering protein structure (Minucci and Pelicci, 2006). This possibility is also supported by our observations that ubr1 levels decreased after nocodazole arrest in the presence of HX (Figures 2 and 4), TS treatment produced higher protein levels (Figures 2 and 4), and sirn of resulted in a marked reduction of ubr1 staining at prometaphase kinetochores (Supplementary Figure 2). Therefore, we investigated whether ubr1 acetylation interfered with ubiquitination-mediated proteolysis. HeLa cells were transfected with Myc-, -, or wild-type ubr1 expression constructs and their protein levels assessed in the presence or absence of the MG132 proteasome inhibitor. The results indicated that was present at significantly lower levels than wild-type ubr1 or when de novo synthesis of proteins was inhibited (Figure 6, HX). When the same set of transfected cells was & 2009 European Molecular iology Organization The EMO Journal VOL 28 NO

6 E hoi et al ΔR2-4 W: α-gst W: α-c-k250 W: α-ck cetylated cetylated ΔR2-4 / I M I M I M IP: α-ubr1 α-ck α-c-k250 Noc. release HX ubr1 (h) W: α-c-k250 W: α-ctin Noc. release Noc. Release (h) W: α-c-k250 None TS W: α-lamin / Figure 4 ntibodies raised against acetylated ubr1 detect ubr1 exclusively at prometaphase. The anti-c-k250 antibodies specifically recognize ubr1 acetylated at K250. () Recombinant proteins R23 and R23 were subjected to in vitro acetylation and W. Similar to the anti-c-k antibody, anti-c-k250 antibodies detect only acetylated ubr1 and not. ntibodies raised against acetyl-k250 do not recognize autoacetylated, whereas the anti-c-k antibody does recognize it. The same blot was re-probed with anti-gstantibodies as loading controls. () HeLa cells in interphase (I) and M phase (M) were subjected to IP with anti-ubr1, anti-c-k, or anti-c-k250 antibodies and then immunoblotted with anti-ubr1 antibodies. ubr1 acetylation at K250 was detected in the M phase. () Levels of ubr1 and ubr1 acetylation after nocodazole release in the presence of HX. HeLa cells were synchronized (time point 0), then washed and released into the cell cycle in the presence of 100 mg/ml of HX. Lysates were prepared at the indicated time points after release and analysed by W with anti-ubr1 and anti-c-k250 antibodies. The same blot was re-probed with anti-ctin antibodies. () ubr1 acetylation in mitosis is sustained by treating the cells with the pan-h inhibitor TS. W with anti-c-k250 detects acetylated ubr1 exclusively at prometaphasearrested cells (left panel). The right panel shows cells released from nocodazole arrest in the presence of 100 nm TS, which resulted in the attenuation of ubr1 acetylation (c-k250). The same blot was re-probed with anti-ubr1 and anti-lamin /. treated with MG132, protein levels were restored to wild-type levels (Figure 6, MG132). Furthermore, ubiquitination of the acetylation-defective mutant was consistently higher than that of the wild type, whereas ubiquitination of was markedly lower (Figure 6). Immunofluorescence assays substantiated the observation that ubr1 acetylation correlates with ubr1 levels. The intensity of staining was markedly higher than that of at kinetochores when normalized to REST staining (Figure 6 and ; Supplementary Figure 8). When the proteasome inhibitor, MG132, was included 3 h before fixation, signals at kinetochores were restored (Figure 6 and ), suggesting that the checkpoint defects in expressing cells are because of insufficient amounts of ubr1 at kinetochores, resulting from acetylation-deficient ubr1 being susceptible to ubiquitin-mediated proteolysis. The localization of ubr1 at kinetochores was not altered by acetylation, which was corroborated by the finding that ub3 binding was not affected by ubr1 acetylation (Supplementary Figure 9). ubr1 acetylation/deacetylation regulates its degradation and timing in anaphase entry Next, we questioned whether ubr1 acetylation and degradation affected the timing of mitosis. For this, we adopted live-cell analysis coupled with fluorescence measurements, which have earlier been used efficiently to measure the proteolysis of proteins in mitosis (Lindon and Pines, 2004; Pines and Lindon, 2005; Nilsson et al, 2008). Wild-type-, -, or -expression plasmids tagged with sred at their N-termini were transfected into HeLaH2 cells depleted of endogenous ubr1 by sirn. The transfected cells were synchronized and subjected to time-lapse imaging (Figure 7). The level of wild-type ubr1 declined during mitosis; it began to decline before the onset of anaphase and declined further throughout mitosis (Figure 7, blue line and 7, upper panel). Mitosis took an average of 57 min from nuclear envelope breakdown (NE) to anaphase onset in wild-type ubr1-rescued cells (Figure 7, top panel; Supplementary movie 1 and Supplementary Figure 11) The EMO Journal VOL 28 NO & 2009 European Molecular iology Organization

7 Ehoiet al Infection sirn Nocodazole siubr1 simad d-gfp d-gfp d-gfp d- d- d- d- W: α-mad2 W: α-ctin IP: 9E10 MPM2 staining (%) h 13 h 25 h 36 h 48 h Hours after nocodazole treatment ontrol siubr1 simad2 siubr1 siubr1 siubr1 simad2 W: α-ps676 W: 9E10 W: α-c-k Noc MG132 E 35S-FL 35S-ΔR1 Input (5%) ΔR1 IP: α-dc20 ΔR1 pcn Lysate F IP: 9E10 pcn3myc pcn3myc W: 9E10 W: α-c-k250 W: α-dc20 W: α-dc20 Input (5%) Noc MG132 W: 9E10 W: α-dc20 W: α-ctin Figure 5 ubr1 acetylation is required for checkpoint activity. () ubr1 acetylation is required for checkpoint activity. () Schematic illustration of the experiment. HeLa cells were infected with 100 MOI of GFP-fused ubr1- (d-), (d-), or (d-)-expressing adenovirus. denovirus expressing GFP only was employed for control. Five hours later, cells were depleted of endogenous ubr1 by synthetic siubr1 targeting the 3 0 UTR (siubr1) or simad2 targeting Mad2, as marked. Nineteen hours post sirn transfection, cells were treated with nocodazole (200 ng/ml) and aliquots of cells were collected at the indicated time points for W () and measuring mitotic index in (). Western analysis was used to determine the levels of ubr1 and Mad2 after sirn transfection and adenovirus infection as indicated. () omparison of the mitotic index of cells indicated after nocodazole treatment, measured by MPM2FS analysis (avis et al, 1983). () cetylation of ubr1 does not directly affect its phosphorylation. HeLa cells were transfected with the indicated plasmids and treated with nocodazole for 13 h. fter mitotic shake-off, cells were washed with PS five times and incubated in the presence or absence of 15 mm of MG132 for 1.5 h. ed cells () were included for control. Lysates were prepared and subjected to IP (9E10), followed by W with anti-p-s676 ubr1, which detects Plk-1-mediated phosphorylated ubr1. The same blot was re-probed with 9E10 (middle panel) and anti-c-k250 antibodies. (E) ubr1 acetylation at K250 does not alter dc20 binding. Wild-type-, -, and encoding constructs were translated in vitro in the presence of [ 35 S]-methionine and incubated with in vitro-translated dc20. dc20 complexes were immunoprecipitated using an anti-dc20 antibody and analysed by SSPGE. R1 (amino acids 1322), which lacks the dc20- interacting domain, a pcn3myc empty vector, and reticulocyte lysate alone were included as negative controls. 5% of the in vitro-translated products were included as input controls. (F) 293Tcells transfected with various ubr1 constructs were arrested with nocodazole, treated with MG132 for 3 h, and analysed for complex formation with dc20 by IP with 9E10 and by W with anti-dc20 antibodies. In the lysis buffer, 10 mm of TS was added. The same blot was re-probed with an anti-c-k250 antibody. ells transfected with an empty vector (pcn3myc) were included as a negative control. Untransfected control cells took 53 min (Figure 7; Supplementary Figure 11) and ubr1-depleted cells took 25 min (Figure 7; Supplementary Figures 10 and 11) from NE to anaphase onset. Meanwhile, intensities declined faster than wild-type ubr1-rescued cells (Figure 7, red line). Furthermore, chromosomes segregated prematurely without proper alignment at the metaphase plane (Figure 7, middle panel), and mitotic timing was & 2009 European Molecular iology Organization The EMO Journal VOL 28 NO

8 E hoi et al shortened to 2540 min (Figure 7 and ; Supplementary movie 2 and Supplementary Figure 11). In contrast, intensities for did not decline for more than 3 h (n ¼ 40), the chromosomes never segregated in those cells during the recording, and the cells subsequently died (Figure 7, purple line and 7, third row; Supplementary movie 3 and Supplementary Figure 11). Taken together, these results indicate that ubr1 destruction begins before the onset of anaphase and that ubr1 acetylation regulates its proteolysis. From these observations, we speculated that Table I Frequency of exit from mitosis in 200 ng/ml nocodazole Percentage of cells exiting from mitosis (total cell number analysed) ontrol 0% (n ¼ 198) siubr1 100% (n ¼ 128) simad2 100% (n ¼ 97) siubr1 6% (n ¼ 18) siubr1 93% (n ¼ 27) siubr1 0% (n ¼ 16) simad2 94% (n ¼ 17) Experimental schemes are similar to those described in Figure 5. Nocodazole was continuously present and live-cell images were taken for 18 h after the start of nocodazole treatment. The number of cells exiting from mitosis during the time course was counted. Total cell numbers analysed (n) are indicated. MG132 None MG132 HX 9E10 REST PI Merge W: 9E10 W: α-lamin/ ubr1 acetylation and the timed degradation of ubr1 control the correct timing for anaphase entry. cetylated ubr1 is an inhibitor of the P/ complex, whereas unacetylated ubr1 becomes a substrate of P/-dependent proteolysis We have shown that ubr1 acetylation occurs at prometaphase kinetochores and that ubr1 degradation began before the onset of anaphase. These results indicate that ubr1 might become a substrate of the P/ complex when the S is satisfied. To test this hypothesis, we investigated whether ubr1 was ubiquitinated by P/dc20 complex. HeLa cells were cotransfected with Myc-ubR1- and H- dc20-encoding plasmids and subjected to IP with 9E10 and W with anti-ubiquitin antibodies. The results showed that ubr1 was polyubiquitinated by ectopic expression of dc20 in a dose-dependent manner (Figure 8). When the P complex was inhibited by sirn of P3, the level of ubr1 remained constant for 60 min after nocodazole release in the presence of HX, whereas it declined in control cells (Figure 8). This result was corroborated by the finding from live-cell assay for proteolysis that depleting P3 expression abrogated the degradation of ubr1, and concomitantly cells did not enter anaphase for more than 5 h (Supplementary Figure 12 and Supplementary movie 7). When we compared the timing of ubr1 degradation with ubr1/rest signal ratio trl vector HUbi (Ub)nubR1 IP: 9E10 W: 125 IP: 9E10 W: 9E10 MG132 Figure 6 ubr1 acetylation inhibits its polyubiquitination-dependent proteolysis. () Two mg of wild-type Myc-ubR1-, --, or -encoding expression plasmids was transfected into HeLa cells. The cells were then synchronized and arrested in mitosis by continuous nocodazole treatment. The cells were then treated with MG132 or HX for 3 h in the presence of nocodazole before lysis. Total cell lysates were analysed by W with 9E10 to detect the ectopically expressed ubr1. The same blot was re-probed with an anti-lamin / antibody. () ubr1 acetylation results in the inhibition of polyubiquitination-dependent proteolysis. 293T cells were cotransfected with the plasmids encoding wild type,, or with HUbiquitin, and proteins were immunoprecipitated with 9E10 and analysed by W using a 125 anti-h antibody to detect polyubiquitination of ubr1 [(Ub)nubR1]. The blot was re-probed with 9E10 to control for the amount of immunoprecipitation. () HeLa cells were transfected as in (). t 16 h post transfection, the cells were treated with 100 mm of monastrol to arrest the cells in prometaphase without disturbing microtubule structures. fter 5 h, cells were treated with MG132 ( þ MG132) or left untreated. ells were then fixed and co-stained with 9E10 (red) and REST (green). Other phases of mitosis are illustrated in Supplementary Fig 8. ars, 5 mm. () Quantification of results in (). Histograms summarize three different ubr1 intensities at kinetochores in the absence or presence of MG132, measured by staining with 9E10 and normalized for REST staining. Results are from two independent experiments (mean±s.e.m.; nx8 prometaphase cells per experiment, Po0.01) The EMO Journal VOL 28 NO & 2009 European Molecular iology Organization

9 Ehoiet al Transfection Thymidine block Release Start End Fluorescence (sred) 1.20E E E E E E E00 5 T=0 T=13 T=26 T=34 T= NE to anaphase onset (min) NE naphase onset * eath siubr1 siubr1 siubr Time relative to NE (mins) sred ubr1 sred ubr1 sred ubr1 NE to anaphase onset (min) NE Prometa Meta naphase 00:00 00:10 00:15 00:30 00:50 00:55 01:00 NE 00:00 00:05 00:10 00:20 00:25 00:30 NE Prometa Prometa naphase 00:35 eath 00:00 00:10 00:15 01:15 01:20 01:30 02:35 ontrol siubr1 siubr1 ontrol siubr1 siubr1 Figure 7 ubr1 acetylation inhibits its degradation and regulates the timing of anaphase onset. The HeLa cells stably expressing histone H2 GFP (HeLaH2) were co-transfected with synthetic siubr1 targeting the 3 0 UTR and,, or expression plasmids, respectively, tagged with sred at the N-termini using lipofectamine TM 2000 and subjected to time-lapse microscopy. () Schematic illustration of the experiment. t 13 h after transfection, cells were synchronized at the S phase through the addition of 2 mm thymidine to the culture. fter a further 13 h, cells were washed from the thymidine block and released into the cell cycle. Images were taken starting at 8 h after release and were processed for 18 h. () Fluorescence intensities (red) and mitotic timing (segregation of green) were measured and scored from NE. Mean red fluorescence was measured and plotted on the Y-axis as pixel values using Image J. The X-axis represents the time from NE in minutes. The curves shown are representative examples of several transfected cells from at least three separate experiments. rrows indicate the timing of anaphase onset. () aptured images of () at indicated time points (marked at lower right). NE is marked as 00:00. ubr1 fluorescence (sred) images are shown in the bottom panels in grayscale to facilitate the measurement of fluorescence intensities for ubr1 (Supplementary movie 1), (Supplementary movie 2), and (Supplementary movie 3), respectively. In the case of -rescued cells, cells were arrested in prometaphase for hours, then the cells frequently died. In the -rescued cell, anaphase began without alignment of chromosomes in metaphase plane, and the mitotic timing was shortened to 25 min. Stages in mitosis were determined from chromosome morphology. It should be noted that the fluorescence intensity around kinetochores declined before the onset of anaphase in - and -expressing cells. Fluorescence intensity around kinetochores in -expressing cell did not decline for hours. () ox plot distributions of statistical mitotic timings from NE to anaphase onset for control HeLa cells (n ¼ 60); after depletion of ubr1 (siubr1) (n ¼ 65); ubr1-transfected cells rescued from ubr1 depletion (n ¼ 21); transfection after sirn of ubr1 (n ¼ 43); and transfection after siubr1 (n ¼ 40). The bars in the box are the median values. Outliers (open circle) and suspected outliers (asterisk) as determined by statistical analysis are shown. ox plots were drawn again on a smaller Y-axis scale, without -rescued data, in the right panel to facilitate the comparisons of mitotic timing. ata are from three independent experiments analysing 2065 transfected cells using SPSS software. the degradation of other players in mitosis, such as dc20, yclin, Plk1, and urora, we found that ubr1 degradation began before that of yclin (Supplementary Figure 13). Next, we tested whether dc20 was responsible for ubr1 degradation during mitosis. To prevent the cells from exiting mitosis before the analysis began, HeLa cells were transfected with an expression construct to force moderate expression of & 2009 European Molecular iology Organization The EMO Journal VOL 28 NO

10 E hoi et al H20 MycubR1 sigfp sip3#1 sip3#2 HX Mr(K) (min) W: α-p3 W: α-ctin Input (5%) IP: 9E10 W: α-ubi IP: 9E10 W: 9E10 W: 9E10 W: 125 sigfp sidc20#1 sidc20#2 sidh1 W: α-dh1 W: α-dc20 W: α-lamin/ sigfp sidc20#1 sidc20#2 sidh1 HX (min) W: α-yclin W: α-lamin/ E 1.00E04 NE 1.00E04 NE Fluorescence (sred) 8.00E E E E E siubr1_ken1,2 () siubr1_ken1,2 () siubr1_ken1,2 () siubr F 95 P/ c ubr1 Prometaphase P/ inhibited Time relative to NE (min) Ub Ub Ub Fluorescence (sred) ipolar KTMT achieved 8.00E E E E E Ub Ub Ub ubr1 P/ naphase onset P/ activation siubr1_xx siubr1_xx siubr1_xx siubr Time relative to NE (min) Figure 8 K250 acetylation functions as a switch between P/ inhibition and P/-dependent ubr1 destruction. () 293T cells were cotransfected with Myc-tagged ubr1 (1 mg each) and H-tagged 20 expression constructs in a dose-dependent manner (0, 1, and 2 mg). Lysates were then subjected to IP with 9E10 and to W with anti-ubiquitin antibody. The blot was re-probed with 9E10 to control for the amount of IP. () ubr1 protein levels were compared after knockdown expressions of P3 to assess whether the P/ complex was responsible for ubr1 degradation. HeLa cells were transfected with synthetic sirns. t 60 h later, cells were treated with 200 ng/ml nocodazole for 12 h, and the mitotic cells were collected by mitotic shake-off. Lysates were prepared before and after HX addition for 1 h. Note that after nocodazole release for 60 min in the presence of HX, P3 appears as a faster migrating form, indicating that P3 is highly modified in prometaphase. () W analysis of dc20 and dh1 after transfection of sirns, as indicated. These cells were used to measure ubr1 levels in (). () Experiments were carried out in a manner similar to (), except that, to block the exit from mitosis, a yclin expression construct was transfected simultaneously with sirns. The lysates for W were collected at 0, 15, 30, and 60 min after nocodazole release and HX treatment. (E) Intensities of fluorescence, which reflects the level of sredubr1 in mitotic cells, were recorded against time relative to NE. The -box or the KEN boxes were destroyed by in vitro mutagenesis in sred by substituting for KEN boxes (left panel) and RXXL to XX for the -box (right panel). The curves shown are representative of at least 20 individual cells from at least two separate experiments. fluorescence intensity curve for was included in all experimentsasacontrol.(f) Our working model for ubr1 acetylation and the regulation of P/ activity. ubr1 is in a complex with dc20 and P/ through its KEN1, KEN2, and -box domains. ubr1 acetylation/deacetylation changes the surface structure of degrons in relation to P/ dc20 complex. When kinetochores are not yet attached to spindles (prometaphase), ubr1 is acetylated by. ubr1 acetylation prevents it from being a substrate of the P/ complex, and acetylated ubr1 is a potent inhibitor of the P/dc20 complex. When all kinetochores are stably attached to spindles, ubr1 is no longer acetylated because of a change in the stoichiometry of the ubr1 complex or its deacetylation. The conformational change allows the -box and KEN box degrons of ubr1 to be recognized as a substrate of the P/-dc20 complex. In our model, the mode of dc20 binding to P/ complex and ubr1 changes, depending on the status of ubr1 acetylation The EMO Journal VOL 28 NO & 2009 European Molecular iology Organization

11 Ehoiet al yclin. sirn for GFP, dc20, or dh1 was simultaneously transfected. fter 16 h of transfection, cells were synchronized at prometaphase, followed by release in the presence of HX. ell lysates were prepared at indicated time points after release and the levels of ubr1 and ylin were analysed. The results showed that ubr1 degradation was inhibited in cells depleted of dc20, whereas control cells (sigfp) showed significant decreases in ubr1 compared with initial analysis after nocodazole release. yclin levels in the same blot showed that cells were in mitosis for at least 30 min after nocodazole release (Figure 8). These results suggest that ubr1 is degraded through P/dc20-dependent ubiquitination during mitosis. dh1, a homologue of dc20, serves as a coactivator of P/ complex during mitotic exit. Therefore, we asked whether dh1 was also responsible for ubr1 degradation. When cells were depleted of dh1 (sidh1), ubr1 levels decreased during mitosis. However, in dh1-depleted cells, ubr1 levels remained similar between 30 and 60 min after nocodazole release, whereas control cells (sigfp) had a continuous decrease after release (Figure 8). It should be noted that the level of yclin declined between 30 and 60 min after release, indicating that cells are exiting from mitosis within 60 min after release. The result suggests that ubr1 is subjected to a second round of degradation during mitotic exit through P/dh1-mediated ubiquitination. If the destruction of ubr1 by the P/dc20 complex is important for mitotic timing, the proteolysis of ubr1 might depend on its degrons, the -box and KEN boxes. To test this hypothesis, we made ubr1 constructs in which the -box or KEN boxes were destroyed in sred-, -, and - expression vectors. HeLa cells depleted of endogenous ubr1 and transfected with various plasmids were subjected to live-cell analysis for proteolysis, as shown in Figure 7. The result showed that both KEN1 and KEN2- mutated (KEN1, 2 ()) or -box-mutated,, and were stable during the recorded times, whereas with intact degrons began to degrade 20 min after NE (Figure 8E). We also observed that when KEN boxes or the -box was destroyed in ubr1, cells were not able to maintain the S, and the mitosis was accelerated, although ubr1 level remained constant (Supplementary movie 6). To confirm that ubr1 degradation depended on the -box and KEN boxes, we carried out W analysis in HeLa cells transfected with various constructs. s it was impossible to obtain a similar mitotic index and synchronize the cells after ubr1 sirn and,, or transfection, we compared the level of ectopically expressed proteins in the presence of endogenous ubr1 (Supplementary Figure 14). The result corroborated the finding that the KEN box- or - box-deleted mutants were stable (Supplementary Figure 14, middle and right panels). ollectively, these results suggest that the P/ complex is responsible for ubr1 degradation, and ubr1 degradation requires the -box and KEN boxes. iscussion In this study, we provide compelling evidence that ubr1 is acetylated at K250 by specifically at prometaphase. cetylated ubr1 is a potent inhibitor of the P/dc20 complex, whereas unacetylated (or deacetylated) ubr1 readily becomes a substrate of P/-dependent proteolysis. Thus, ubr1 acetylation/deacetylation serves as a molecular switch for the conversion of ubr1 from inhibiting the P/ complex to becoming its substrate. This Janus-like mode of action provides an efficient way of controlling chromosome segregation after checking that everything is ready, which is a crucial requirement for accurate sister chromatid separation. similar mode of switching from an inhibitor to a substrate of the P/ complex has been reported in Emi1, an inhibitor of the P/dh1 complex in interphase (Miller et al, 2006). Interestingly, a recent report suggested that dc20 serves as a substrate of P/ complex when the S is activated and as a coactivator of P/ complex during the metaphaseanaphase transition (Nilsson et al, 2008). Our working hypothesis is as follows: ubr1 acetylation at K250 takes place at prometaphase, which inhibits the P/ dc20 complex until all the kinetochores are stably attached with bipolar spindles. This monitoring of microtubule attachment probably involves the motor protein ENP-E (han et al, 1999; brieu et al, 2000; Yao et al, 2000; Mao et al, 2003, 2005; Morrow et al, 2005; Zhang et al, 2008). When the spindle checkpoint is satisfied, ubr1 is no longer acetylated and the unacetylated form of ubr1 replaces acetylated ubr1. This process might involve the deacetylation of ubr1, because we observed that H 13 could bind to ubr1. However, whether dynamic acetylation and deacetylation of ubr1 take place during mitosis has not yet been investigated. nother possibility is that detaches from ubr1 after all of the kinetochores are stably attached to spindles, because the ubr1 complex involving ENP-E and microtubules is very likely to change its stoichiometry. In support of the first view, a recent report showed that H3 localizes to spindle microtubules and is required for kinetochoremicrotubule attachments (Ishii et al, 2008). s for the latter view, we have shown that the ubr1 interaction takes place exclusively at prometaphase kinetochores (Figure 1). Turning the P/dc20 complex on and off The timed proteolysis of key regulators of the cell cycle is vital for progression through mitosis. Regulated proteolysis in cell division is largely dependent on the P/ E3 ligase activity. For P/ activation, the W40 domain-containing coactivator dc20 or its homologue dh1 is required (Peters, 2006; Thornton and Toczyski, 2006; Yu, 2007). It was suggested that coactivators recognize the destruction signals (-box or KEN box) and transfer them to the P/ complex for polyubiquitination. However, accumulating evidence indicates that both the -box and the KEN box degrons are recognized by the P/ complex as well. Furthermore, it was convincingly shown in Xenopus extracts that destruction signals are recognized and bound by the P but not the dc20 coactivator (Yamano et al, 2004). Therefore, it is possible that ubr1 binds to dc20 and the P simultaneously through its degrons. It is noteworthy that a recent structural analysis using single-particle electron microscopy showed that the P/ interaction domain partially overlaps with the dc20 binding site on ubr1 (Herzog et al, 2009). How, then, can ubr1 avoid degradation while binding to and inhibiting the P/dc20 complex? When two KEN boxes or a -box was destroyed in ubr1,, or & 2009 European Molecular iology Organization The EMO Journal VOL 28 NO