Bimodal Activation of Bubr1 by Bub3 Sustains Mitotic Checkpoint Signaling

Bimodal activation of BubR1 by Bub3 sustains mitotic PNAS PLUS checkpoint signaling

Joo Seok Hana,b,1, Benjamin Vitrea,b,2, Daniele Fachinettia,b,2, and Don W. Clevelanda,b,c,1 aLudwig Institute for Cancer Research and Departments of bCellular and Molecular Medicine and cMedicine, University of California, San Diego, La Jolla, CA 92093

Contributed by Don W. Cleveland, August 25, 2014 (sent for review May 6, 2014) The mitotic checkpoint (also known as the spindle assembly sites) (18), along with its associated Bub3, but not Mad2, accounts checkpoint) prevents premature anaphase onset through gen- for the inhibition of APC/CCdc20 both in vitro using purified com- eration of an inhibitor of the E3 ubiquitin ligase APC/C, whose ponents and in vivo after induced degradation of either BubR1 or ubiquitination of cyclin B and securin targets them for degrada- Mad2 (13). These latter in vivo efforts were interpreted to dem- tion. Combining in vitro reconstitution and cell-based assays, we onstrate that Mad2 can dissociate from an initial complex with now identify dual mechanisms through which Bub3 promotes mi- Cdc20 (or APC/CCdc20) in an activated conformation capable of totic checkpoint signaling. Bub3 enhances signaling at unattached catalyzing additional Bub3–BubR1–Cdc20 complexes. Thus, we kinetochores not only by facilitating binding of BubR1 but also by proposed that BubR1–Bub3–Cdc20 is the mitotic checkpoint in- enhancing Cdc20 recruitment to kinetochores mediated by BubR1’s hibitor that blocks APC/C-dependent ubiquitination of cyclin B internal Cdc20 binding site. Downstream of kinetochore-produced and securin, through dual catalytic steps, an initial one at kinet- complexes, Bub3 promotes binding of BubR1’s conserved, amino ter- ochores and another acting within the cytoplasm mediated by minal Cdc20 binding domain to a site in Cdc20 that becomes exposed kinetochore-derived, activated Mad2 (13). by initial Mad2 binding. This latter Bub3-stimulated event generates Bub3 was identified as a mitotic checkpoint protein through the final mitotic checkpoint complex of Bub3–BubR1–Cdc20 that se- genetic screening in budding yeast (19), and subsequent work in lectively inhibits ubiquitination of securin and cyclin B by APC/CCdc20. higher eukaryotes also demonstrated a profound defect in this Thus, Bub3 promotes two distinct BubR1-Cdc20 interactions, involving checkpoint in the absence of Bub3 (20–23). Recently, Bub3 has each of the two Cdc20 binding sites of BubR1 and acting at unattached been shown to bind the phospho MELT motif on KNL-1 for kinetochores or cytoplasmically, respectively, to facilitate production kinetochore localization of Bub1 (budding uninhibited by of the mitotic checkpoint inhibitor. benzimidazole 1), disruption of which caused a defective checkpoint (24–27) with Bub1 binding to kinetochores appar- – pon entry into mitosis, each duplicated chromosome aligns ently required for binding of other checkpoint proteins (28 32). Uat metaphase through capture of spindle microtubules by A role for Bub3 in mitotic checkpoint silencing has also been the kinetochore assembled onto its centromere. Premature chro- proposed in fission yeast (33). Bub3 binds to the Gle2-binding- mosome segregation often leads to abnormal chromosome num- sequence (GLEBS) motif of Bub1 and Mad3 (the yeast homolog ber, or aneuploidy, a hallmark of cancer. The mitotic checkpoint of BubR1) in a mutually exclusive manner, with binding mediated β (also known as the spindle assembly checkpoint) is the major cell- through the top face of its -propeller (34). Another GLEBS cycle control mechanism in mitosis. It functions to ensure accurate motif-containing protein, BugZ, was also shown to interact with Bub3, stimulating its mitotic function by promoting its stability chromosome segregation through production of an inhibitory sig- – nal generated by unattached kinetochores (1), thereby delaying and kinetochore loading (35 37). Bub3 not only mediates BubR1 anaphase onset until all of the chromosomes attach to spindle microtubules (2–4). This signaling pathway is initiated by a com- Significance plex of Mad1 (mitotic arrest deficient 1) and Mad2 (mitotic arrest deficient 2) immobilized at unattached kinetochores (5). This The mitotic checkpoint (or the spindle assembly checkpoint) complex then recruits a second Mad2 molecule (5–7) and catalyzes ensures genome integrity by preventing premature chromosome (8–10) its conformational change from open or N1 (inactive) to segregation. The pathway is triggered locally by kinetochores, closed or N2 (active) (11, 12) state. Closed Mad2 can bind Cdc20 multiprotein complexes assembled onto centromeres. Unattached (cell division cycle 20), the mitotic activator of the E3 ubiquitin kinetochores produce Mad2 bound to Cdc20, the mitotic activator ligase APC/C (anaphase promoting complex or cyclosome) that is of the E3 ubiquitin ligase APC/C. The initial Mad2–Cdc20 complex is responsible for advance to anaphase by its ubiquitination and then converted into the final mitotic checkpoint inhibitor Bub3– subsequent proteasome-dependent degradation of cyclin B and BubR1–Cdc20 that blocks APC/C (anaphase promoting complex or CELL BIOLOGY securin. Diffusible Mad2–Cdc20 produced by unattached kinet- cyclosome)-dependent ubiquitination of cyclin B and securin, ochores recruits a complex of Bub3 (budding uninhibited by thereby stabilizing them and preventing an advance to anaphase. benzimidazole 3) and BubR1 (Bub1 related protein 1). It does this In this study, we identify dual mechanisms by which Bub3 pro- either by exposing a previously inaccessible site in Cdc20 for binding motes mitotic checkpoint signaling. Bub3 binding to BubR1 pro- – to BubR1’s N-terminal Cdc20 binding domain (13) and/or by a di- motes two distinct BubR1 Cdc20 interactions, one acting at rect interaction between Cdc20-bound Mad2 and BubR1 (14). unattached kinetochores and the other cytoplasmically to facili- A four-protein complex of Mad2, BubR1, Bub3, and Cdc20, tate production of the mitotic checkpoint inhibitor. named the mitotic checkpoint complex (or MCC), has long been Cdc20 Author contributions: J.S.H., B.V., D.F., and D.W.C. designed research; J.S.H., B.V., and D.F. implicated in the inhibition of APC/C ubiquitination of performed research; J.S.H., B.V., D.F., and D.W.C. analyzed data; and J.S.H. and D.W.C. securin and cyclin B1 (14, 15). However, the identity of the ul- wrote the paper. timate mitotic checkpoint inhibitor remains controversial, with The authors declare no conflict of interest. some investigators arguing that Mad2 plays the predominant role 1To whom correspondence may be addressed. Email: [email protected] or j3han@ Cdc20 (16) and others arguing that the inhibitory activity of APC/C ucsd.edu. is provided by BubR1 (13, 17), or both BubR1 and Mad2 (14, 2B.V. and D.F. contributed equally to this work. 15). We recently demonstrated that the N-terminal Mad3 ho- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mology domain of BubR1 (including one of its two Cdc20 binding 1073/pnas.1416277111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1416277111 PNAS | Published online September 22, 2014 | E4185–E4193 N localization to the kinetochore (38) but is also incorporated into mediated by BubR11–477 (hereafter referred as BubR1 ), which con- the MCC with Cdc20, BubR1, and Mad2 (15). Failure of Bub3 tains BubR1’s N-terminal Cdc20 binding site (Fig. 2A). In contrast, binding to BubR1 has been shown to weaken the mitotic check- Bub3 did not affect Mad2-independent inhibition by BubR1 of free point (39–41). However, it has remained unclear how Bub3 Cdc20 activation of APC/C (inhibition mediated by a BubR1 variant stimulates mitotic checkpoint signaling through binding to BubR1. [BubR1357-1050] containing only its internal Cdc20 binding site— Using cell-based assays and our established in vitro reconstituted hereafter referred to as BubR1C)(Fig. S2). This stimulated in- APC/C activity assay (8, 13), we now have investigated the mech- hibition required a direct interaction between BubR1N with Bub3, anism by which Bub3 contributes to the mitotic checkpoint. We as Bub3 failed to activate Bub3 binding-defective BubR1N-E409K find that Bub3 promotes two distinct BubR1–Cdc20 interactions, (Fig. 2 D–F). The E409K mutation itself did not affect function of involving each of the two Cdc20 binding sites of BubR1 and acting BubR1, as there was no difference between wild-type BubR1N at unattached kinetochores or cytoplasmically, respectively, to fa- and BubR1N-E409K in their ability to inhibit APC/CCdc20 in the ab- cilitate production of the mitotic checkpoint inhibitor. sence of Bub3 (Fig. S3). Consistently, a Bub3 variant (Bub3R183E) that contains a mutation of arginine 183 (corresponding to arginine Results 197 in yeast, a residue required for its interaction with Bub1 or Bub3 Directly Stimulates Production of the APC/CCdc20 Inhibitor of Mad3) (34) bound less to BubR1N and also produced a corre- Cyclin B1 Ubiquitination. To determine the specific contribution spondingly reduced ability to stimulate BubR1N inhibition of APC/ of Bub3 binding to BubR1 in the mitotic checkpoint, endogenous CCdc20 (Fig. S4). Collectively, these data indicate that Bub3 binding BubR1 was depleted in cells using an siRNA and replaced by to BubR1 specifically promotes the Mad3–homology domain expression of inducible, siRNA-resistant, Myc and GFP amino- (BubR1N)-mediated production of an APC/CCdc20 inhibitor. terminally tagged BubR1 variants (Fig. S1C and Fig. 1 A and B). To disrupt binding to Bub3, we converted the conserved gluta- Bub3 Promotes Assembly of the BubR1–Cdc20 Mitotic Checkpoint mate at residue 409 of the GLEBS motif of BubR1 (34) to lysine. Inhibitor. Our previous study found that BubR1 binding to Depletion of Bub3 produced a severe defect in nocodazole-induced, Cdc20 (through the N-terminal Cdc20 binding domain), but not Cdc20 chronic mitotic checkpoint signaling (Fig. S1 A and B), as expected Mad2, is critical for inhibition of APC/C recognition of cyclin B Cdc20 E409K (13). Because Bub3 stimulated the inhibition of APC/C by from prior work (23). The BubR1 variant with a defect in Bub3 N binding gave rise to extensive chromosome misalignment yet BubR1 (Fig. 2), we therefore tested in vitro if Bub3 promoted BubR1N association with Cdc20. Bead-bound APC/CCdc20 was failed to delay anaphase onset in an unperturbed mitosis (Fig. S1 N D and E). Additionally, BubR1E409K allowed much faster mitotic purified after incubating with various combinations of BubR1 , exit than wild-type BubR1 in cells with unattached kinetochores Mad2, and Bub3, and APC/C-associated proteins were then ana- A N produced either by nocodazole-induced microtubule disassembly lyzed (Fig. 3 ; see schematic at top). BubR1 , with or without Bub3, failed to produce a significant amount of BubR1N–Cdc20 (Fig. 1 C and D) or inhibition of the kinetochore-bound, kinesin complex in the absence of Mad2 (Fig. 3A, lanes 3–5), indicating family member CENP-E (Fig. S1 F–H). Taken together, these a Mad2 dependency for establishing an initial BubR1N–Cdc20 results indicate that the Bub3–BubR1 interaction is required for interaction. This Mad2-dependent BubR1N–Cdc20 interaction sustained mitotic checkpoint signaling. was enhanced by twofold by coincubation with Bub3 (Fig. 3 A, Bub3 has been copurified with BubR1 and Mad2 in Cdc20 lanes 6–8, and B), quantitatively accounting for the twofold complex(es) from cells with an active mitotic checkpoint (15, 17, 42), Cdc20 stimulation of APC/C inhibition by Bub3 (Fig. 2C). We suggesting a contribution by Bub3 to the inhibition of Cdc20. To further confirmed that Bub3-stimulated inhibition required its determine the direct contribution of Bub3 to the inhibition of APC/ N Cdc20 Cdc20 binding to BubR1 , as Bub3 was unable to promote APC/C C in vitro, Bub3 and various combinations of full-length BubR1 association with a shorter BubR1N variant lacking the Bub3 bind- (hereafter referred to as BubR1FL) and Mad2 (Fig. 1E) were coin- ing domain (BubR11–363)(Fig.3C). Consistent with in vitro cubated at concentrations approximating physiological (150, 150, results, immunopurified BubR1E409K (Bub3 binding-defective) and 300 nM, respectively) (8, 42). Mad2 and/or BubR1 were then Cdc20 from mitotically arrested cells was less associated with Cdc20 added to bead-bound APC/C (at 2:1 or 1:1 stoichiometries, and APC/C than wild-type BubR1 (Fig. 3 D and E). respectively, relative to Cdc20). APC/C and any associated proteins were recovered, and ubiquitination activity toward cyclin B Bub3-Mediated Kinetochore Recruitment of BubR1 Enhances Mitotic substrate was assayed after addition of E1, the E2 UbcH10, and Checkpoint Signaling. Unattached kinetochores stably bind a ubiquitin (see schematic in Fig. 1F). Incubation of Mad2 and complex of Mad1 and Mad2 (5), which catalytically acts (8, 10) to FL BubR1 at levels approximating their known physiological con- convert additional Mad2 molecules to an active form that binds Cdc20 centrations partially suppressed APC/C -mediated cyclin B Cdc20, thereby initiating mitotic checkpoint signaling. The other ubiquitination, as determined by quantifying unubiquitinated cyclin components of the MCC—BubR1, Bub3 (38), and Cdc20 (43)— B(Fig.1G,lane6,H,andI). Although Bub3 alone or in combination are also recruited to unattached kinetochores (reviewed in ref. with Mad2 produced no APC/CCdc20 inhibition, when added with 44). Because BubR1 relies on Bub3 for its binding to kinetochore BubR1FL and Mad2, it stimulated them to produce complete (38), we tested whether such Bub3-dependent targeting of BubR1 loss of cyclin B ubiquitination (Fig. 1 G,lane7,H,andI). Thus, to kinetochores also promotes mitotic checkpoint signaling beyond Bub3 directly promotes the production of an APC/CCdc20 inhibitor, kinetochore binding-independent Bub3 stimulation of BubR1– independent of any function for it at kinetochores. Cdc20 complex formation (Fig. 3). To produce kinetochore localization of the cytosolic BubR1E409K, GFP-tagged Mis12 or Bub3 Binding Enhances Inhibition of APC/CCdc20 Mediated by the Mad3 Bub3, respectively, was fused in frame to the N or C terminus of the Homology Region of BubR1. BubR1 contains two Cdc20 binding protein (Fig. 4A). After siRNA-mediated reduction in endogenous sites, a conserved site in the N-terminal Mad3–homology domain BubR1, these Bub3 and Mis12 fusion proteins accumulated to levels and an internal site that is present only in higher eukaryotes. The similar to that of the corresponding GFP-tagged BubR1 variants two Cdc20 binding sites of BubR1 have been shown to be able, (Fig. 4B). As expected, BubR1E409K was not kinetochore-associated respectively, to mediate inhibition of Cdc20 activation of APC/C in unless fused to Bub3 or Mis12 (Fig. 4C). Mad2-dependent (13, 41) and Mad2-independent manners (42). Compared with BubR1E409K alone, BubR1 significantly extended We tested the contributions of the individual Cdc20 binding sites of nocodazole-induced mitotic arrest in cells when kinetochore targeted BubR1 in Bub3-stimulated inhibition of APC/CCdc20 ubiquitination through Bub3 but not Mis12 (Fig. 4 D and E). BubR1 localization of cyclin B in vitro. Bub3 enhanced inhibition of APC/CCdc20 to the kinetochore per se was necessary for Bub3 enhancement of

E4186 | www.pnas.org/cgi/doi/10.1073/pnas.1416277111 Han et al. mitotic checkpoint signaling, as the Bub3-dependent extended mi- the E409K mutation in BubR1’s GLEBS motif disrupts the au- PNAS PLUS totic arrest largely disappeared (Fig. 4F)whenBubR1E409K was fused thentic BubR1–Bub3 interaction between the fused Bub3 and to a Bub3 variant (Bub3R183E) defective in kinetochore localization BubR1E409K. As a consequence, it is unlikely that Bub3 fusion to (Fig. 4G). Although the Bub3 fusion enables kinetochore binding of the C terminus of BubR1E409K directly facilitates BubR1 binding BubR1E409K through Bub3 binding to Bub1 at the kinetochore, to APC/CCdc20.

A C Bub3 Binding Domain Add Doxycycline to Begin filming Myc-GFP- (GLEBS motif) Transfect HeLa cells induce expression FL with BubR1 siRNA of BubR1 variant Add Nocodazole BubR1 KEN1 KEN2 Myc-GFP N-C20 BD I-C20 BD Kinase (h): 0 24 47 48 1 360 500 700 1050 aa E409K

B D BubR1 RNAi + E FL Myc-GFP- Expression of GFP-BubR1FL FL WT E409K Parental BubR1 None Wild type 100 E409K kDa GST-BubR1His-Bub3His-Cdc20His-Mad2 80 n>57 Myc 170 200 GST- FL 60 97 BubR1 Bub3 32 66 40 His-Cdc20 Tubulin 47 45 His-Bub3 20 1 2 3 4 5 6

Cells in mitosis (%) 29 0 His-Mad2 0 250 500 750 1000 1250 20 Time in mitotic arrest (min) F 20 Cdc20 Active 1hr 20 G APC APC iAPC/C ++++ + ++ Cdc20 - ++++ ++ FL BubR1 - -- 1x 1x 1x 1x BubR1 Bub3 CyB U Mad2 - - 2x - - 2x 2x N C 3 E1 UbcUbc Bub3 - - 1x - 1x - 1x Mad2 1hr 1hr 1hr CyB U U U U U U Wash out Assay for APC/C Activity unbound components

E1 E1 N C BubR1FL Open Mad2 Closed Mad2 UbcUbc UbcH10

3 Bub3 B-(Ub)n cyclin APC/C bound U Ubiquitin APC to Cdc27 Ab 20 Cdc20 CyB Myc-cyclin B beads 1-102 cyclin B 1 2 3 4 5 6 7

H APC/C inhibition (%) I iAPC/C + + + Cdc20 + + + 100 BubR1FL - + + 80 Mad2 - + + +

Bub3 CELL BIOLOGY 60 - - Ubiquitination 0 15 30 45 60 0 15 30 45 60 0 15 30 45 60 40 time (min) 20 0 iAPC/C + + + + + + Cdc20 + + + + + + BubR1FL - - + + + + B-(Ub)n cyclin Mad2 - + - - + + Bub3 - + - + - + cyclin B

Fig. 1. Bub3 directly promotes Mad2-dependent BubR1 inhibition of APC/C. (A) Schematic of functional domains of BubR1. (B) E409K mutation in BubR1 disrupts the BubR1–Bub3 interaction, analyzed by GFP immunoprecipitation. (C) Schematic for the protocol used to replace endogenous BubR1 with a GFP- tagged version and to determine duration of nocodazole-induced mitotic arrest in HeLa cells. (D) Time-lapse microscopy was used to determine nocodazole (100 ng/mL)-induced mitotic duration after replacing endogenous BubR1 with GFP–BubR1 variants using siRNA. (E) Coomassie staining of purified checkpoint components. (F) In vitro APC/C activity assay for checkpoint protein-mediated inhibition of APC/C-mediated cyclin B ubiquitination. (G) Effect on APC/CCdc20- mediated ubiquitination of cyclin B1–102 by various combinations of BubR1, Mad2, and Bub3. (H) Quantification from G. Data represent mean ± SEM, n = 3. (I) Time titration for the inhibition of APC/C ubiquitination of cyclin B by Mad2 and BubR1 in the presence or absence of Bub3.

Han et al. PNAS | Published online September 22, 2014 | E4187 BubR1N N Mad2 A BubR1 (Mad3 homology domain) B N 3 1 26 304 380 420 477 aa Affinity recover 20 1 hr 1 hr KEN1 KEN2 APC APC/C and E409K assay activity GLEBS iAPC/C ++++ + ++++ ++++ BubR1N +Mad2 Cdc20 - ++++ ++++ ++++ C 100 n=3 N BubR1N +Mad2+Bub3 BubR1 - - .2x.5x1x - .2x.5x1x - --- Mad2 - - .2x.5x1x - .2x.5x1x - .2x.5x1x 75 Mad2+Bub3 Bub3 ------.2x.5x1x - .2x.5x1x 50

APC/C Inhibition (%) Inhibition APC/C 0 cyclin B-(Ub)n 0x 0.2x 0.4x 0.6x 0.8x 1x cyclin B Input (Ratio to Cdc20) 1 2 3 4 5 6 7 8 9 10 11 12 13

D GST- E iAPC/C ++++ + + F BubR1N - Bub3 Cdc20 - ++++ + *** kDa WT E409K BubR1N -- WT E409K 100 + Bub3 Mad2 116 -- ++++ 75 ns 97 Bub3 - - - ++- 50 66 25

APC/C Inhibition Inhibition (%) APC/C 0 WT E409K

cyclin B-(Ub)n BubR1N cyclin B

1 2 3 4 5 6

Fig. 2. Bub3 binding promotes the conserved N-terminal Mad3 homology domain of BubR1-mediated inhibition of APC/CCdc20.(A) Schematic for the Mad3 homology domain of BubR1 (BubR1N,1–477 aa). (B) Inhibition of APC/CCdc20 activity by various combinations of checkpoint proteins. (C) Quantification from B.(D) Coomassie staining of purified wild-type or E409K mutant BubR1N.(E) Test for requirement of Bub3 binding to BubR1 for Bub3 stimulation of BubR1N. (F) Quantification from E. Data represent mean ± SEM. ***P < 0.001 from Student t test.

Despite kinetochore binding-derived stimulation of sustained The Internal Cdc20 Binding Site of BubR1 Accelerates Cdc20 Recruitment checkpoint signaling, cells expressing Bub3-tagged BubR1E409K to Kinetochores. Full-length BubR1 has been shown (13) to sustain exited mitosis in nocodazole significantly faster than cells express- more robust mitotic checkpoint signaling than the kinetochore ing wild-type BubR1 (Fig. 4E, compare blue and purple lines). localization-competent variant BubR1N, which contains the Bub3 Thus, we reasoned that the kinetochore binding-independent Bub3 and N-terminal Cdc20 binding sites but is missing the internal stimulation of the BubR1–Cdc20 interaction documented earlier Cdc20 binding site and the kinase domain. Although the role of the (Fig. 3) was necessary for sustained mitotic checkpoint signaling. In BubR1 kinase domain remains controversial [with evidence that support of this, wild-type BubR1 fused to Bub3 or Mis12 instead of kinase activity is stimulated by binding to CENP-E (45) and con- BubR1E409K mediated an even further extension of mitotic arrest flicting with evidence that it is an inactive pseudokinase (46)], regardless of the way it was bound to kinetochores (Fig. S5). BubR1 has been recently proposed to be required for Cdc20 re- Next, we asked whether kinetochore localization of the cruitment to the Drosophila melanogaster kinetochore (47, 48). N-terminal Cdc20 binding site of BubR1 was sufficient for its kinet- These findings led us to test whether BubR1 localization to the ochore localization-dependent enhancement of mitotic checkpoint kinetochore enhances recruitment of Cdc20 to kinetochores in signaling. A GFP-tagged N-terminal BubR1 variant (BubR11–363 human cells. Using siRNA we replaced endogenous BubR1 with that lacks the Bub3 binding site) was directly fused to Bub3 or Mis12 an siRNA-resistant MycGFP–BubR1variantandmeasuredCdc20 (Fig. 5A). After suppressing endogenous BubR1 with siRNA, each intensity at the kinetochore during mitotic arrest produced by ad- Bub3 or Mis12 fusion protein accumulated to a level similar to un- dition of monastrol, an inhibitor for the mitotic kinesin Eg5. Cdc20 tagged BubR11–363 (Fig. 5B). Bub3- and Mis12-tagged BubR11–363 intensity at the kinetochore was reduced at least 75% upon de- variants bound to kinetochores, whereas BubR11–363 did not (Fig. pletion of endogenous BubR1 (Fig. 6 A and B). Expression of re- 5C). Both full-length BubR1 and the Bub3–BubR11–363 fusion combinant wild-type BubR1 or the kinetochore binding-competent bound dynamically, with similar turnover kinetics measured BubR1 variant (BubR1E409K–Bub3) fully restored the Cdc20 level by fluorescence recovery after photo bleaching (FRAP) during at kinetochores, whereas expression of the kinetochore binding- nocodazole-induced mitotic arrest (Fig. 5 D, E,andG). In contrast, deficient BubR1 variant (BubR1E409K) did not. FRAP measurements with Mis12-tagged BubR11–363 revealed it to be Next, we assessed the contribution to kinetochore binding of bound stably to kinetochores, with little exchange with time (Fig. 5 F Cdc20 and to sustained mitotic checkpoint arrest provided by and G). However, unlike full-length cytosolic BubR1 (BubR1E409K) each of the two Cdc20 binding sites of BubR1. Expression of (Fig. 4E), neither enabling dynamic nor stable association of a BubR1 variant lacking the N-terminal Cdc20 binding site BubR11–363 with kinetochores extended the time of nocodazole- [BubR1Δ(1–356)] fully restored Cdc20 levels at kinetochores in induced mitotic arrest (Fig. 5 H and I). This indicates a requirement BubR1-depleted cells. On the other hand, expression of a BubR1 for additional domains of BubR1 for kinetochore binding- variant lacking the internal Cdc20 binding site [BubR1Δ(525–700)] dependent enhancement of sustained mitotic checkpoint signaling. did not (Fig. 6 C and D). Importantly, wild-type BubR1 mediated

E4188 | www.pnas.org/cgi/doi/10.1073/pnas.1416277111 Han et al. ( PNAS PLUS

A N B 2.5 ** Mad2 BubR1 3 APC/C peptide N 2.0 20 1hr 15 min 20 Immunoblot APC APC 3 N analysis 1.5 Affinity recover APC/C 1.0 iAPC/C ++++++++ Cdc20 bound to APC/C BubR1 bound to APC/C 0.5

Cdc20 - +++++++ ( N + +++++ BubR1 -- 0.0 Mad2 ----- +++ Fold 0x 1x 2x Bub3 -- - 1x 2x - 1x 2x Input ratio (Bub3 to BubR1N ) Cdc27 Cdc20 D +Nocodazole

APC/C- bound BubR1N Pull-down 12345678 Myc-GFP- Input GFP Mock BubR1FL WT E409K WT E409K WT E409K C GLEBS Myc 150 KEN1 KEN2 40 BubR11-477 Bub3 1 26 304 380 420 477 aa Cdc20 * BubR11-363 KEN1 KEN2 50 1 26 304 363 aa 150 ( * Cdc27 2.5 GAPDH 30 2.0 ns

1.5 E 1 1.0 0.8 0.6 Cdc20 bound to APC/C BubR1 bound to APC/C 0.5 ( 0.4 0.0 Fold Bub3

- + - + to GFP-BubR1) 0.2

++ ++Mad2 Fold (Cdc20 bound ++ ++APC/CCdc20 0 1-477aa 1-363aa BubR1N WT E409K Myc-GFP-BubR1

Fig. 3. Bub3 binding promotes the Mad2-dependent N-terminal Mad3 homology domain of BubR1 binding to Cdc20. (A) In vitro assay of Bub3-dependent effects on binding of APC/C-bound Cdc20 to the N-terminal Cdc20 binding site of BubR1. (B) Quantification from A.(C) In vitro test if Bub3 stimulation of the BubR1–Cdc20 interaction in A required the Bub3 binding domain of BubR1. (D) Assay (using GFP immunoprecipitation of extracts from nocodazole-induced mitotic HeLa cells) to measure E409K GFP–BubR1 mutant association with Bub3, Cdc20, and the APC/C subunit Cdc27. (E) Quantification from D. Data represent mean ± SEM. *P < 0.05 and **P < 0.01 from Student t test, n = 3. longer mitotic arrest of nocodazole-treated cells than did with Cdc20 independently of kinetochore localization. Second, BubR1Δ(525–700) (Fig. 6E), suggesting a role of BubR1–Bub3- we have further demonstrated that Bub3 binding also mediates mediated kinetochore binding of Cdc20 in sustaining mitotic Cdc20 localization to the kinetochore via BubR1’s internal Cdc20 checkpoint signaling. Kinetochore levels of Mad1 and Mad2 binding site as a means to reinforce the kinetochore-dependent were indistinguishable in cells supported by the various BubR1 first step in mitotic checkpoint inhibitor generation. variant regardless of the levels of kinetochore binding of BubR1 Recently, we demonstrated that BubR1, but not Mad2, bind- CELL BIOLOGY or Cdc20 (Fig. S6). This outcome was inconsistent with the ing (through the N-terminal Cdc20 binding site) is critical for possibility that a less efficient Mad1–Mad2-mediated initiation inhibiting APC/CCdc20 recognition of cyclin B (13). Our evidence of checkpoint signaling was the result of reduction in Mad1– in the current study has established that Bub3 promotes this Mad2 at unattached kinetochores. BubR1–Cdc20 interaction, thereby uncovering a previously un- known role for Bub3 in production of the mitotic checkpoint Discussion inhibitor through action at the most downstream step of the A prevailing hypothesis for Bub3’s function in generation of the signaling pathway. Two previous studies proposed that the first mitotic checkpoint inhibitor is that it simply delivers BubR1 to KEN box in the N-terminal Cdc20 binding site of BubR1 is re- the kinetochore and thus elevates the local concentration of sponsible for BubR1 binding to Cdc20 (14, 41). Based on this, it is BubR1 to facilitate rapid initial production of an anaphase in- plausible that Bub3 binding may alter the conformation of the hibitor (e.g., refs. 2, 40, 49). Here we have established that Bub3’s initially rod-shaped BubR1 N terminus (34, 50) in a way to either role is much more than this. Our evidence identifies a dual mode promote the initial assembly or stabilization of a BubR1–Cdc20 of stimulation by Bub3 of BubR1’s mitotic checkpoint function to complex for generating the characteristic, selective inhibition produce the mitotic checkpoint inhibitor (modeled in Fig. 6F). of APC/CCdc20. First, Bub3’s binding to BubR1 directly stimulates assembly of the In addition to its stimulatory function in cytosolic assembly of BubR1–Cdc20 mitotic checkpoint inhibitor through facilitating the mitotic checkpoint inhibitor (Figs. 1 and 2), our evidence es- association of the conserved Mad3–homology domain of BubR1 tablishes that Bub3 also mediates BubR1-dependent kinetochore

Han et al. PNAS | Published online September 22, 2014 | E4189 GFP-tagged BubR1FL None BubR1FL A B E E409K FL FL BubR1E409K -Bub3 Mis12-BubR1E409K FL FL FL FL FL 100 i) BubR1E409K E409K E409K E409K n>60 Kinetochore BubR1 BubR1 BubR1 Mis12- - 80 230 Bub3 BubR1 FL 150 GFP 60 ii) KMN Mis12 BubR1E409K 100 Kinetochore 50 Tubulin 40

FL 20 iii) Bub1 BubR1E409K Bub3 Cells in mitosis (%)

Kinetochore 0 0 100 200 300 400 500 600 Time in mitotic arrest (min) KT signal of C GFP-BubR1 ACA DNA MERGE GFP-BubR1 F

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G KT signal of E409K FL + GFP-BubR1 ACA DNA MERGE GFP-BubR1 -

Mis12- BubR1 FL E409K ++ Bub3 BubR1 D Add Doxycycline to Begin filming Transfect HeLa cells induce expression

Add Nocodazole -

of BubR1 variant FL with BubR1 siRNA E409K +/ R183E -

(h): 0 24 47 48 Bub3 BubR1

Fig. 4. Enabling a cytosolic BubR1 to localize dynamically to the kinetochore enhances mitotic checkpoint signaling.(A) Schematic showing three tests for assessing BubR1’s role at kinetochores, including (i) blocking BubR1 binding at kinetochores with the E409K mutation, (ii) tethering BubR1 to kinetochores by fusion to Mis12, and (iii) directly tethering BubR1 to Bub3 to mediate kinetochore association. (B) Levels of accumulation of various GFP–BubR1 variants after expression in HeLa cells. (C) Localization of BubR1FL or Bub3 or Mis12-tagged cytosolic BubR1E409K.(D) Schematic of the protocol used in E to determine duration of nocodazole-induced mitotic arrest in cells expressing recombinant BubR1 variants. (E) Test of mitotic checkpoint function of various BubR1 variants. Time-lapse microscopy was used to determine nocodazole-induced mitotic duration after replacing endogenous BubR1 with GFP–BubR1 variants using siRNA. (F) Quantitation of mitotic timing in individual cells assayed as in E.(G) Indirect immunofluorescence to assess kinetochore binding of BubR1E409K fused to Bub3 with or without an additional R183E mutation in Bub3. recruitment of Cdc20 as a means to further strengthen initiation BubR1 variant to the kinetochore (by fusing it to Bub3) signifi- of mitotic checkpoint inhibitor production (Fig. 6F). How BubR1 cantly strengthened sustained mitotic checkpoint signaling (Fig. and Cdc20 recruitment to kinetochores serves to power check- 4E). This evidence adds further support for our model for point signaling is not settled, with two (not mutually exclusive) production of a diffusible mitotic checkpoint inhibitor in which mechanisms likely. First, recruitment at kinetochores of Cdc20 by the majority of Mad2-associated Cdc20 complexes diffuse through- BubR1’s internal Cdc20 binding site may facilitate initial forma- out the mitotic cytoplasm, followed by capture by cytosolic BubR1 to tion of Mad2–Cdc20 complexes whose assembly is enhanced by produce the bona fide inhibitor of APC/CCdc20 recognition of cyclin the elevated local concentration at those kinetochores of Cdc20. B and securin (13). Alternatively, the internal Cdc20 binding site may serve as a Finally, we note that BubR1 at the unattached kinetochores docking site of preassembled Mad2–Cdc20 for its handover to the has been reported to recruit PP2A-B56α, which in turn affects N-terminal Cdc20 binding site of BubR1 for the assembly of the kinetochore–microtubule attachment (51–53) through a region BubR1–Cdc20 mitotic checkpoint inhibitor. Evaluating between similar to the one required for its recruitment of Cdc20 for mi- these hypotheses will now require testing (i) whether the two totic checkpoint signaling. This raises an interesting, now test- Cdc20 binding sites of BubR1 associate with Cdc20 in- able, possibility that there is crosstalk between these two distinct dependently of each other, including whether a transient complex protein–protein interactions at kinetochores, one to initiate mi- forms in which both Cdc20 binding sites are associated with one totic checkpoint signaling and the other to coordinate microtubule– molecule of Cdc20 as an intermediate form, and (ii) whether the kinetochore attachment to silence that signaling. internal Cdc20 binding site of BubR1 binds to Mad2–Cdc20. Our study has further revealed that BubR1’s diffusion into the Materials and Methods cytosol, as well as its kinetochore localization, is crucial for mi- Constructs. Full-length and fragments of the human BubR1 ORF were cloned into either a pcDNA5/FRT (FLP recombination target)/TO (tetracycline resistance totic checkpoint signaling, as indicated by the fact that tethering – E409K operon)-based vector (Invitrogen) modified to contain an amino-terminal Myc cytosolic BubR1 (BubR1 ) stably to the kinetochore (via LAP epitope tag for mammalian cell expression or a pFastBac1-based vector Mis12) was even more detrimental to mitotic checkpoint sig- (Invitrogen) modified to contain an amino-terminal GST–human rhinovirus naling than was keeping BubR1 only in the cytosol (Fig. 4E). By (HRV) 3C site for insect cell expression. The LAP tag consists of GFP–HRV 3C contrast, enabling dynamic attachment of an otherwise cytosolic (LEVLFQGP)–6xHis. All other DNA constructs were previously described (8, 42).

E4190 | www.pnas.org/cgi/doi/10.1073/pnas.1416277111 Han et al. KT signal of PNAS PLUS A B C GFP-BubR1 ACA DNA MERGE GFP-BubR1 1-363 BubR1 1-363 i) 1-363 - Kinetochore

1-363 1-363 BubR1

KMN 1-363 GFP-tagged ii) Mis12 BubR1 BubR1 BubR1 Mis12-BubR1 -Bub3 1-363 ++

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80 GFP BubR1 - Bub3 60 iii) Bub1 BubR11-363 Bub3 1-363 50 Tubulin + Kinetochore Mis12- BubR1 D E F 0” 0” 24” 0” 0” 24” 0” 0” 24” 1-363 FL 6” 48” 6” 48” 6” 48” 1-363

12” 66” 12” 66” 12” 66” GFP-BubR1 5 μm GFP-BubR1 -Bub3 Mis12-GFP-BubR1

G H Add Doxycycline to BubR1FL (t1/2=10.0 s, n=16) Begin filming Transfect HeLa cells induce expression 1-363 100 BubR1 -Bub3 (t1/2=12.5 s, n=13) with BubR1 siRNA of BubR1 variant Add Nocodazole Mis12-BubR11-363 (t1/2 =n/a, n=15) 80 (h): 0 24 47 48

60 I 40 100 n>50 None BubR11-477 20 1-363 (100 % prebleach) 80 BubR1 Fluorescence recovery Fluorescence 0 BubR11-363 -Bub3 020406080 60 Mis12-BubR11-363 Time (sec) 40

20 Cells in mitosis (%)

0 0 200 400 600 800 Time in mitotic arrest (min)

Fig. 5. Targeting a cytosolic BubR1 variant with only its N-terminal Cdc20 binding site to kinetochores does not enhance mitotic checkpoint signaling. (A) N Schematics for (i) blocking BubR1 (BubR11–363) binding to kinetochores by deleting the Bub3 binding site or by driving its kinetochore localization through fusion to (ii)Mis12or(iii) Bub3. (B) Levels of accumulation of GFP–BubR1 variants after expression in HeLa cells. (C) Intracellular localization of Bub3 or Mis12- tagged cytosolic BubR11–363.(D–F)FRAPanalysisofGFP–BubR1 variants in a nocodazole-induced mitotic cell. (D) Wild-type GFP–BubR1, (E)GFP–BubR11–363–Bub3, and (F)GFP–Mis12–BubR11–363.(G) Quantification from D–F. n/a, not available; t1/2, time (seconds) for 50% recovery of fluorescence. (H) Schematic for the protocol used to determine mitotic timing. (I) Mitotic checkpoint function of BubR1 N terminus variants, analyzed by time-lapse microscopy.

Antibodies. The antibodies used in this study are as follows: BubR1 (SBR1.1, projection created using softWoRx. Movies were assembled and analyzed using a gift from S. Taylor (University of Manchester, Manchester, England); QuickTime (Apple) or FIJI (Image J, National Institutes of Health) software. A300-386, Bethyl Laboratories), Bub3 (SB3.2, a gift from S. Taylor), Mad2 (A300-300A, Bethyl Laboratories), Cdc20 [A301-180A, Bethyl Laboratories; FRAP. The FRAP experiment was performed on HeLa Cells seeded 24 h before SC-13162, Santa Cruz Biotechnology (for immunofluorescence)], Mad1 the experiment in 35-mm glass-bottom culture dishes (Mat Tek Corporation). (BB3-8, a gift from A. Musacchio, Max Planck Institute, Dortmund, Ger- Growth medium was changed to CO2-independent medium before imaging. many), ACA (Antibodies Inc.), Myc (16-213, Millipore), Cdc27 (54), α-Tubulin Images were collected, at 37 °C, with an Olympus 100X/1.35, UPlan Apo (DM1α, Sigma-Aldrich), GST (SC-33613, Santa Cruz Biotechnology), and His objective using a DeltaVision Core system (Applied Precision) equipped with (A00186, GenScript). a Coolsnap camera (Roper). Photobleaching was performed using a Quanti-

fiable Laser Module (Applied Precision) with the 488-nm laser line. Images CELL BIOLOGY Generation of Stable Cell Lines and RNAi. Parental Flp–In TRex–HeLa or –DLD-1 were taken on a single plan, on the GFP channel, every second before the parental cells that stably express mRFP-tagged histone H2B (H2B–mRFP) laser event. A 1-s laser event was performed, and images were acquired with were as previously described (55, 56). Stable, isogenic cell lines expressing increasing time interval following photo bleaching starting with 500-ms MycGFP–BubR1 were generated using FRT/Flp-mediated recombination (57). intervals. Fluorescent intensity was measured, using FIJI (ImageJ, National Expression of MycGFP–BubR1 was induced with 1 μg/mL tetracycline. siRNAs Institutes of Health), in a circle surrounding the GFP kinetochore signal, and directed against the 3′ untranslated region of BubR1 (5′-CUGUAUGUGCU- a background was measured from an equivalent area adjacent to the ki- GUAAUUUA-3′) or Bub3 (58) were purchased from Thermo Fisher Scientific netochore signal. Background subtraction and normalization of the mea- (Dharmacon). Cells were transfected with 50 nM of oligonucleotides using sured signal was done using Excel software (Microsoft). The fitting of the Lipofectamine RNAiMAX (Invitrogen). We added tetracycline 24 h after data was done using Prism software (GraphPad), using a least square poly- transfection to express MycGFP–BubR1 for 24 h before collecting cells for nomial equation, and the recovery half-time was measured from the curve. immunoblotting or analyzing by time-lapse microscopy. Indirect Immunofluorescence. Cells were fixed in 4% (vol/vol) formaldehyde at Live-Cell Microscopy. To determine mitotic timing, cells were seeded onto μ-Slide room temperature for 10 min with or without preextraction by 0.1% Triton

(ibidi) and 48 h posttransfection transferred to supplemented CO2-independent X-100 for 60 s. Incubations with primary antibodies were conducted in blocking media (Invitrogen). Cells were maintained at 37 °C in an environmental control buffer for 1 h at room temperature. Immunofluorescence images were col- station and images collected using a Deltavision RT system (Applied Precision) lected using a Deltavision Core system (Applied Precision). For quantification of with a 40 × 1.35 NA oil lens at 3–5-min time intervals. For each time point, 6 × kinetochore signal intensity, undeconvolved 2D maximum intensity projections 3 μMor6× 4 μM z sections were acquired for RFP and maximum intensity were saved as unscaled 16-bit tagged image file format (TIFF) images and signal

Han et al. PNAS | Published online September 22, 2014 | E4191 ACCdc20 ACA MERGE Cdc20 ACA MERGE Parental Parental None None FL BubR1 Full-length FL E409K Δ (1-356) BubR1 FL E409K BubR1 RNAi + Expression of GFP-BubR1 BubR1 RNAi + Expression of GFP-BubR1 BubR1 - Bub3 Δ (525-700)

n=32 B 125 D 125 n=32 n=30 n=30 n=29 n=28 100 100

75 75

50 n=32 50 n=29 n=34 n=32 25 25 Cdc20 intensity (%) KT at Cdc20 intensity at KT (%) KT intensity at Cdc20 0 0 - ) FL FL E409K FL None None E409K Parental Δ(1-356) Parental (525-700 BubR1 BubR1 BubR1 Full-length Δ Bub3 BubR1 RNAi + BubR1 RNAi + Expression of GFP-BubR1 Expression of GFP-BubR1 E BubR1 RNAi + F Expression of GFP-BubR1 Kinetochore Cytosol Parental None Full-length O-Mad2 Δ(525-700) Mad1 100 Mad2 C-Mad2 80 n>47 Cdc20 Cdc20 Cdc20 60 BubR1 Mad2 Cdc20 40 Bub3 BubR1 Bub3 20 Cdc20 Cells in mitosis (%) 0 BubR1 Bub3 action at Bub3 action in 0 250 500 750 1000 1250 kinetochores the cytosol Bub3 Time in mitotic arrest (min) to enhance Cdc20 to enhance Cdc20 recruitment and capture from tether BubR1 Mad2 by BubR1

Fig. 6. Bub3 mediates kinetochore recruitment of Cdc20 via BubR1’s internal Cdc20 binding site to enhance mitotic checkpoint signaling. (A) Effect of kinetochore localization of BubR1 on kinetochore localization of Cdc20 in HeLa cells arrested in mitosis with monastrol (100 μM). (B) Quantification of Cdc20 intensity on kinetochores from A.(C) Indirect immunofluorescence assay of Cdc20 binding to kinetochores in the presence of BubR1 variants with either of its Cdc20 binding sites. (D) Quantification of Cdc20 intensity on kinetochores from C.(E) Duration of mitosis after reducing endogenous BubR1 (with siRNA) and expression of BubR1FL or BubR1 deleted in its internal Cdc20 binding site. Time-lapse microscopy was used to determine nocodazole-induced duration of mitotic arrest after replacing endogenous BubR1 with a MycGFP–BubR1 in DLD-1 cells. (F) A model for dual modes of BubR1 activation by Bub3 for generating the mitotic checkpoint inhibitor. Mitotic checkpoint signaling is promoted (a) at kinetochores by Bub3-dependent recruitment to those kinetochores of BubR1 and Cdc20 (through binding of the internal Cdc20 binding site of BubR1 to Cdc20) and (b) in the cytosol by Bub3 stimulation of Mad2-dependent BubR1– Cdc20 formation (through binding of the conserved N-terminal Cdc20 binding site of BubR1). intensities determined using MetaMorph (Molecular Devices). A 12 × 12 pixel (Invitrogen) and affinity purified over nickel–nitrilotriacetic acid beads circle was drawn around a centromere [marked by anti-centromere antibodies (Qiagen) or Glutathione Sepharose beads (GE Healthcare Life Sciences). (ACA) staining] and an identical circle drawn adjacent to the structure (back- His–Mad2 and other GST-tagged proteins were expressed from Rosetta ground). The integrated signal intensity of each individual centromere was Escherichia coli after induction with isopropyl beta-D-1-thiogalactopyranoside, calculated by subtracting the fluorescence intensity of the background from and purified. APC/C was immunoprecipitated from interphase Xenopus egg the intensity of the adjacent centromere. About 20 centromeres were averaged extracts as previously described (8). to provide the average fluorescence intensity for each individual cell. APC/C Ubiquitination Activity Assay. The APC/C ubiquitination activity assay Protein Purification. GST or His-tagged human BubR1, Bub3, and Cdc20 were was performed as previously described (59) and activity assessed by expressed in Sf9/High-Five insect cells using the Bac-to-Bac expression system ubiquitination-derived depletion of the cyclin B1–102 substrate. Quantitative

E4192 | www.pnas.org/cgi/doi/10.1073/pnas.1416277111 Han et al. PNAS PLUS analysis of cyclin B1–102 depletion was performed as previously described of TBS buffer. The APC/C complex was eluted from the beads by Cdc27 peptide (13). Briefly, the level of cyclin B1–102 was determined against a series of competition (2 mg/mL) as described and analyzed by immunoblotting. dilution of the proteins. ACKNOWLEDGMENTS. We thank Hongtao Yu, Stephen Taylor, and Andrea APC/C Binding Assay. APC/C was immunoprecipitated from Xenopus inter- Musacchio for providing reagents and Jennifer Santini of the University of phase egg extracts for 2 h at 4 °C using a peptide-derived anti-Cdc27 antibody California, San Diego Neuroscience Microscopy Shared Facility (National In- crosslinked to Affiprep Protein A (Bio-Rad) beads. The APC/C beads were stitute of Neurological Disorders and Stroke P30 NS047101). B.V. and D.F. were supported in part by postdoctoral fellowships from the Human Frontiers washed with Tris-buffered saline (TBS) buffer supplemented with 0.4 M KCl Science Program and the European Molecular Biology Organization, respec- and 0.1% Triton X-100 and incubated with Cdc20 and checkpoint proteins tively. This work was supported by National Institutes of Health Grant GM20513 sequentially or simultaneously for the indicated time at room temperature. (to D.W.C.). Salary support for D.W.C. was provided by the Ludwig Institute for Unbound proteins were removed by washing the beads twice with 20 volumes Cancer Research.

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Han et al. PNAS | Published online September 22, 2014 | E4193 Supporting Information

Han et al. 10.1073/pnas.1416277111

A B Nocodazole st e RNAi Control 100 n>100 Bub3 GAPDH carr Input (%) 100 100 50 25 13 100 80 Bub3 60 Tubulin control) (% 40 on of mito 20

Dura 0 Control Bub3 RNAi C D E 60

MycGFP- 30 e FL WT E409K

BubR1 ) BubR1

RNAi - + - + (min

t 40 Dox - + - + 20 BubR1 150 100 eonse 50 Tubulin 10 20 40

Bub3 from NEBD to Time aignment defect (%) anaphas Mitosis with chromosom Mad2 25 0 0 WT E409K WT E409K

F H n>60 100 Transfect HeLa cells E409K WT Add Doxycycline Add CENP-E inhibitor with BubR1 siRNA 80

(h): 0 24 46 48 60

Induce expression of Begin Filming 40 wild type (WT) or E409K MycGFP-BubR1 Cells arrested in metaphase (%) 20

0 0 200 400 600 800 G MycGFP-BubR1 0’ 9’ 21’ 111’ 448’ 496’ Time (min) tor WT WT

0’ 9’ 21’ 147’ 165’ 189’ P-E inhibi P-E CEN E409K

Fig. S1. Bub3 (budding uninhibited by benzimidazole 3), binding to BubR1 promotes mitotic checkpoint signaling. (A) Down-regulation of Bub3 by siRNA in HeLa cells. (B) Mitotic checkpoint defect upon Bub3 depletion, analyzed by time-lapse microscopy. (C) Replacement of endogenous BubR1 with recombinant MycGFP–BubR1. siRNA was used to deplete endogenous BubR1, and tetracycline (1 μg/mL) was added to induce expression of recombinant BubR1. Note that siRNA did not cause off-target down-regulation of other checkpoint proteins. (D and E) Endogenous BubR1 was replaced with recombinant BubR1 in HeLa cell as described in C, and time-lapse microscopy was used to determine D. Duration of unperturbed mitotic progression or (E) mitosis with chromosome misalignment. More than 100 cells were counted in each set. (F) Schematic for the protocol used to determine the duration of mitosis in the CENP-E inhibitor (GSK923295, 200 nM)-treated HeLa cells by time-lapse microscopy after replacing endogenous BubR1 with MycGFP–BubR1. (G and H) Representative images (G) and duration of mitosis (H) of HeLa cells treated with the CENP-E inhibitor. More than 60 cells were analyzed for each set over three independent ex- periments.

Han et al. www.pnas.org/cgi/content/short/1416277111 1of4 A Inactive Mad2 APC BubR1 C Affinity recover 1hr 1hr 20 APC/C and assay activity 3

iAPC/C ++++ + + Cdc20 - ++++ + BubR1C - - ++++ Mad2 - - - - ++ Bub3 - - - ++- unubiquitinated cyclin B 1 2 3 4 5 6 B 100 - Bub3 75 +Bub3 tion (%)

25 APC/C Inhibi 0 BubR1C BubR1C + Mad2

Fig. S2. Bub3 binding does not stimulate BubR1’s internal Cdc20 (cell division cycle 20) binding-site–mediated inhibition of APC/C (anaphase promoting C complex or cyclosome) activation by Cdc20. (A) In vitro generation of an inhibitor for Cdc20 activation of APC/C. Various combinations of BubR1 (BubR1357–1,050), Mad2 (mitotic arrest deficient 2), and Bub3 were incubated with free Cdc20, followed by further incubation with APC/C. (B) Inhibitory activity was measured in A by depletion of unubiquitinated cyclin B1–102. Data represent mean ± SEM (n = 3).

A iAPC/C ++++ + ++++ Cdc20 - ++++ ++++ BubR1N - - .2x .5x 1x - .2x .5x 1x WT E409K Mad2 - - .2x .5x 1x - .2x .5x 1x cyclin B-(Ub)n cyclin B 1 2 3 4 5 6 7 8 9

B 120 BubR1N (WT) + Mad2 100 BubR1N (E409K) + Mad2 80 60 40 20 0 0x 0.2x 0.4x 0.6x 0.8x 1x

Fig. S3. E409K mutation does not affect Mad2-dependent BubR1 inhibition of APC/CCdc20.(A) Test to determine if E409K mutation affected inhibitory function of BubR1. Wild-type or E409K BubR1N was incubated with Mad2 and bead-bound APC/CCdc20 followed by removal of components unbound to APC/C before APC/C activity assay. (B) Plot of percent of APC/C inhibition. Error bars represent SEM (n = 3).

Han et al. www.pnas.org/cgi/content/short/1416277111 2of4 A 6His-Bub3 B Input GST pull-down WT R183E 200 GST-BubR1N + +++ 116 His-Bub3 WT + + + 97 R183E + + + 66 116 97 45 GST-BubR1N 66

45 Bub3 (WT 29 or R183E) 20 C iAPC/C ++++ + Cdc20 - ++++ BubR1N - - 1x 1x 1x Mad2 - - 1x 1x 1x WT - - - 1x - Bub3 R183E --- - 1x cyclin B-(Ub)n

cyclin B

Fig. S4. BubR1 binding-defective Bub3 cannot stimulate BubR1-mediated inhibition of APC/CCdc20.(A) Purified recombinant wild-type and R183E Bub3, assessed by Coomassie blue staining. (B)GST–BubR1N was incubated with wild-type or R183E Bub3 for 1 h at room temperature. The GST protein and associated Bub3 were analyzed by immunoblotting. (C) Test of stimulation of BubR1N by wild-type or R183E Bub3. Bead-bound APC/C was incubated with BubR1N and Mad2 with or without wild-type or R183E Bub3 before APC/C activity assay.

BubR1 RNAi + Expression of GFP-tagged protein

FL FL None Mis12 Mis12-BubR1E409K Mis12-BubR1 FL FL FL BubR1E409K -Bub3 BubR1 -Bub3 BubR1

100 n>60 80

20 Cells in mitosis (%) 0 0 100200300400500600 Time in mitotic arrest (min)

Fig. S5. Bub3 binding to the GLEBS motif in BubR1 is required for maximal stimulation of BubR1’s checkpoint function. Test of mitotic checkpoint function of Mis12 or various BubR1 variants. Time-lapse microscopy was used to determine nocodazole-induced mitotic duration after replacing endogenous BubR1 with GFP–Mis12 or GFP–BubR1 variants using siRNA. Data of GFP–Mis12–BubR1E409K and GFP–BubR1E409K–Bub3 in Fig. 4E were used again here for comparison.

Han et al. www.pnas.org/cgi/content/short/1416277111 3of4 A Mad1 ACA MERGE B Mad1 125 n=33 n=23 n=24 n=30 n=16

Parental 100 T (%) t K 75

None 50

FL 25 Mad1 intensity a

BubR1 0 l FL ta FL None E409K FL aren E409K

FL P E409K BubR1 BubR1 BubR1 - Bub3

BubR1 BubR1 RNAi + Expression of GFP-BubR1 FL E409K BubR1 RNAi + Expression of GFP-BubR1 Bub3 BubR1 - CDMad2 ACA MERGE Mad2 125 n=15 n=18 n=31 n=18 Parental n=26 100

75 None 50

FL 25 Mad2 (%) intensity KT at BubR1 0 l FL a FL E409K FL None E409K FL E409K Parent BubR1 BubR1 BubR1 - Bub3 BubR1 BubR1 RNAi + Expression of GFP-BubR1 FL E409K BubR1 RNAi + Expression of GFP-BubR1 BubR1 - Bub3

Fig. S6. Kinetochore localization of Mad1 and Mad2 is independent of BubR1 localization to the kinetochore. (A) Effect of kinetochore localization of BubR1 on kinetochore localization of Mad1 in a mitotic HeLa cell arrested by monastrol (100 μM). (B) Quantification of Mad1 intensity on kinetochore from A.(C) Effect of kinetochore localization of BubR1 on kinetochore localization of Mad1 in a mitotic cell arrested by monastrol (100 μM). (D) Quantification of Mad1 intensity on kinetochore from C.

Han et al. www.pnas.org/cgi/content/short/1416277111 4of4