The 7th International Fission Yeast Meeting: Pombe2013
The spindle assembly checkpoint: progress and persistent puzzles Silke Hauf*1 *Friedrich Miescher Laboratory of the Max Planck Society, Spemannstrasse 39, 72076 Tubingen, ¨ Germany
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract The spindle assembly checkpoint is a conserved mitotic signalling pathway that ensures the equal segregation of chromosomes to daughter cells. Despite intensive work in many model organisms, key features of this safety mechanism remain unexplained. In the present review, I briefly summarize advances made in the last few years, and then focus on unexplored corners of this signalling pathway.
A succinct overview of spindle assembly checkpoint signalling The principles of spindle assembly checkpoint signalling are simple: checkpoint proteins recognize mitotic chromosomes that are so poorly attached to spindle microtubules that their segregation to opposite cell poles is at risk. Attachment between chromosomes and spindle microtubules is mediated by kinetochores, huge protein assemblies on the centromeric region of chromosomes. Checkpoint proteins enrich at unattached kinetochores and initiate a signalling cascade that ultimately blocks the APC/C (anaphase-promoting complex/cyclosome), a ubiquitin ligase that is essential for anaphase onset [1]. Thus the checkpoint delays anaphase until all chromosomes have achieved proper attachment. Although this general scheme has been clear for many years, elucidating the mechanistic basis remains a challenge. Considerable progress has been made in understanding how checkpoint proteins bind to kinetochores and how checkpoint proteins block the action of the APC/C. In the present review, I summarize these advances and then focus on aspects of checkpoint signalling that are equally important, but less understood. The present short review only highlights some facets, but comprehensive and nuanced overviews are available [1–5].
Spindle assembly checkpoint proteins and their functions Mad1, Mad2, Mad3 (or BubR1, depending on the organism), Bub1, Bub3 and Mps1 are the core spindle assembly checkpoint proteins and form a signalling cascade of protein– protein interactions and protein phosphorylation. Mad2 and Mad3/BubR1 bind directly to and thereby disable the activator of the APC/C, Cdc20. This effector complex is called MCC (mitotic checkpoint complex) and in many organisms additionally contains Bub3, which is co-recruited Key words: anaphase-promoting complex (APC), mitosis, mitotic checkpoint, signalling pathway, spindle assembly checkpoint. Abbreviations used: APC/C, anaphase-promoting complex/cyclosome; C-, closed; MCC, mitotic checkpoint complex; O-, open. 1 email [email protected]
Biochem. Soc. Trans. (2013) 41, 1755–1760; doi:10.1042/BST20130240
with Mad3/BubR1. Mad2 undergoes a major conformational change when binding to Cdc20: from open (O) to closed (C) [6–8]. This transformation is facilitated by dimerization with another molecule of (C-)Mad2 that is stably bound to Mad1. Mad1 and Mad2 form a 2:2 complex that, like most other checkpoint proteins, becomes recruited to unattached kinetochores in mitosis. Bub1, Bub3 and Mps1 presumably act upstream of Mad1 in checkpoint signalling. Their functions are only partly understood (see below). Bub1 and Mad3/BubR1 are related proteins [2], have a similar domain structure, and can both bind to Bub3 (in a mutually exclusive way). Bub1 and Mps1 are protein kinases, but only the kinase activity of Mps1 is essential for the checkpoint.
Hierarchical recruitment of checkpoint proteins to unattached kinetochores How checkpoint proteins connect to the unattached kinetochores whose presence they signal has recently been revealed in some detail (reviewed in [3–5]) (Figure 1). The checkpoint kinase Mps1 acts upstream and brings other checkpoint proteins to the kinetochore. Not all relevant substrates of Mps1 are known, but phosphorylation of the outer kinetochore protein KNL1 (also called CASC5, Blinkin, Spc7 and Spc105) is important [9–12]. Mps1 recruitment, presumably to the outer kinetochore protein Ndc80/Hec1 [13,14], is aided by the Aurora B kinase, and Mps1 reinforces its own recruitment by (indirectly) promoting Aurora B localization [15–18]. Phosphorylation of KNL1 by Mps1 provides a binding platform for the Bub1–Bub3 complex, representing the next layer of recruitment [10–12]. Bub1 in turn is required for the kinetochore localization of the Mad1–Mad2 complex, in metazoan cells in conjunction with the RZZ (Rod–ZW10–Zwilch) complex, whose localization also depends on Mps1. Bub1 and Mad1 may interact [19– 21], but artificial recruitment of Bub1 does not co-recruit Mad1 in interphase [11,22], and mitotic kinetochores can be bound to Bub1 without Mad1 being present [20,23]. This indicates that other requirements must be met for Mad1 to localize. Enrichment of Mad3/BubR1 at unattached kinetochores is not universal across organisms, may not C The
C 2013 Biochemical Society Authors Journal compilation
1755
1756
Biochemical Society Transactions (2013) Volume 41, part 6
Figure 1 Assembly of checkpoint proteins at unattached kinetochore Recruitment of checkpoint proteins (blue) to the kinetochore (red). Black arrows show dependencies; yellow circles mark phosphorylation (P) events. Potential conformational changes at the kinetochore that could aid recruitment are shown as irregular shapes with question marks. See the text for details.
of substrates [28,29]. Two KEN-box (Lys-Glu-Asn) motifs within Mad3/BubR1, which normally are recognition motifs for APC/C-mediated degradation, are essential for the inhibitory function. The more N-terminal KEN-box binds directly (and thereby blocks) the KEN-box receptor of Cdc20 [27,28,33]. The second KEN-box seems to inhibit substrate recruitment to the APC/C through an unknown mechanism [27]. It is striking that the second KEN-box is so important, although the MCC forms in its absence and at least some of the mechanisms mentioned above should be functional. Cdc20 as part of the MCC is an APC/C substrate, and much has been discussed about the function of this ubiquitylation, which may suppress Cdc20 levels through degradation [32,34–36], thus aiding the checkpoint, or may lead to disassembly of the MCC and termination of the checkpoint signal [32,37–40]. These functions have sometimes been presented as mutually exclusive, which they may not be [32].
A succinct overview of our nescience Despite all of this progress, we are still ignorant about key aspects of checkpoint signalling (Figure 2): we lack a mechanistic understanding of some steps in the signalling cascade, and we know hardly anything about the molecular basis of the dynamic features of checkpoint signalling [41]. In the following sections, I highlight some of the open questions.
be obligatory for checkpoint activity, and its mechanistic basis remains unclear. Both Bub1 and Mad3/BubR1 contain N-terminal TPR (tetratricopeptide repeat) domains that bind to KNL1, but they seem to play a minor role in kinetochore recruitment (see [24,25] and references therein). Human BubR1 needs interaction with Bub3 for efficient kinetochore binding [24,26,27], indicating that Mad3/BubR1–Bub3 could be recruited in a similar manner to Bub1–Bub3. However, at least in human cells and fission yeast, recruitment of Mad3/BubR1 requires Bub1, but not vice versa, indicating that additional influences are needed.
Molecular basis of APC/C inhibition As for kinetochore recruitment, recent years have seen major advances in understanding how the checkpoint blocks APC/C activity. The structure of the central part of the MCC (Mad2 and part of Mad3 bound to Cdc20) has been solved [28]; and cryo-electron microscopy and biochemical assays have shown where MCC binds the APC/C [29,30]. MCC formation seems to block APC/C–Cdc20 activity in multiple ways: (i) binding of Mad2 to Cdc20 impairs binding of Cdc20 to the APC/C [31,32]; and (ii) additional binding of Mad3/BubR1 allows binding of the so-formed MCC to the APC/C, but Cdc20 as part of this complex cannot act as activator of the APC/C, (a) because substrate recruitment by Cdc20 and the APC/C may be blocked [27,29,33], and (b) because Cdc20 within the MCC may not have the right position on the APC/C to promote ubiquitylation C The
C 2013 Biochemical Society Authors Journal compilation
What is the trigger for checkpoint protein accumulation at kinetochores? Aurora B can phosphorylate kinetochore proteins in a chromosome attachment-dependent manner [42], which, in principle, provides a mechanism to recruit Mps1 and other downstream checkpoint proteins depending on the chromosome-attachment state. However, at least in human cells, Aurora B speeds up, but may not be essential for Mps1 recruitment [16]. Hence Mps1 must have other ways to distinguish between unattached and attached kinetochores. Not all checkpoint proteins need to be influenced directly by the attachment state (Bub1 and Bub3 may simply depend on the presence of Mps1), but a second point of interference could be Mad1, for whose localization Bub1 is required, but not sufficient. One could imagine that microtubules compete with checkpoint proteins for the same binding site or that microtubule attachment creates a conformational change or leads to post-translational modifications that regulate checkpoint protein binding [43,44].
Do kinetochores have a role in signalling beyond recruitment? Are kinetochores merely a landing pad for checkpoint proteins, or do they actively contribute to checkpoint signalling? So far, artificial recruitment of single checkpoint proteins to sites other than kinetochores failed to initiate checkpoint activity [45,46]. However, in the absence of kinetochore proteins, some crucial checkpoint proteins were probably not co-recruited. To answer the question above,
The 7th International Fission Yeast Meeting: Pombe2013
Figure 2 Open questions in spindle assembly checkpoint signalling Outline of checkpoint signalling from the kinetochore to the APC/C. Small circles labelled ‘O-’ and ‘C-’ represent O-Mad2 and C-Mad2 respectively. Black arrows highlight unresolved questions. See the text for details.
all checkpoint proteins would need to be experimentally enriched at a locus other than the kinetochore. An argument against enrichment being the only crucial activity of kinetochores can be made since checkpoint signalling works surprisingly well when reduction of an outer kinetochore protein in human cells [47] or deletion of bub3 in fission yeast [48,49] abolishes the visible enrichment of many checkpoint proteins. This could either indicate that only some checkpoint proteins need to be enriched to propagate the signal or that kinetochores provide a specific activity other than enrichment (e.g. initiation of a conformational change), for which a transient interaction may be sufficient.
How is the Mad1–C-Mad2 complex regulated? The Mad1–C-Mad2 complex is a key element in checkpoint signalling. The complex exists throughout the cell cycle, but only promotes checkpoint signalling when unattached kinetochores are present. How is this regulated? The protein p31comet , which structurally resembles Mad2, has been proposed to ‘cap’ Mad1–C-Mad2 to prevent it from signalling [50,51]. Taylor and colleagues, however, did not find evidence for this mechanism [52], but found that the Mps1 kinase promotes the recruitment of O-Mad2 to kinetochore-localized Mad1–Mad2 [53]. They have therefore suggested the alternative hypothesis that Mps1 itself ‘caps’ Mad1:Mad2. This is inspired by findings that Mad1 and Mps1 interact [54,55] and that active Mps1 enhances its own turnover at kinetochores [53,56]. Hence Mps1 may release itself from blocking access to Mad1–Mad2 [3]. Further work is needed to accept or refute this hypothesis.
What is ‘active’ Mad2 and how long-lived is it? When binding to Cdc20, Mad2 rearranges from the Oto the C-conformation, and entraps a stretch of Cdc20, just like a seat belt ties a passenger to the seat [6–8]. In the absence of a binding partner, Mad2 can transition into ‘unliganded’ C-Mad2. The Mad2 conversion reaction is interesting both from the structural point of view and because it is relevant to propagation of the signal from kinetochores to APC/C–Cdc20. At present, it is unclear whether Mad2 binds Cdc20 concomitantly with dimerization or whether ‘active’ Mad2 can dissociate after dimerization and bind Cdc20 later. The hypothetical, dissociating, active species could be C-Mad2 or an O/C-intermediate (I-Mad2 or Mad2*). In favour of the former, constitutive C-Mad2 is more efficient than wild-type Mad2 in inhibiting APC/C– Cdc20 in vitro [57] and when overexpressed in vivo [58,59]. However, at endogenous levels, constitutive C-Mad2 does not inhibit Cdc20 efficiently in fission yeast (A. Malasane, U. Schellhaas and S. Hauf, unpublished work), suggesting that active Mad2 looks different from C-Mad2. There is currently no evidence that diffusion of active Mad2 is needed for efficient signalling. However, amplification of the signal away from kinetochores (see below) may require this. If diffusible active Mad2 exists, it should only have a limited life-time in order to ensure that the checkpoint can be efficiently shut off, raising the question of how its activity is limited.
How does C-Mad2 revert to O-Mad2? Regardless of the activation mechanism, checkpoint signalling creates C-Mad2. Too much C-Mad2 could be problematic in several ways: if it is Cdc20-binding-competent (see above), it C The
C 2013 Biochemical Society Authors Journal compilation
1757
1758
Biochemical Society Transactions (2013) Volume 41, part 6
must be inactivated at the latest when the checkpoint shuts off; if it is Cdc20-binding-incompetent, it poses a risk for the signalling reaction in two opposing ways: (i) because the pool of O-Mad2 (that can be activated) will be depleted, and (ii) since it may provide a template for further Mad2 activation. Hence there are reasons to believe that C-Mad2 is converted back into O-Mad2 when no longer bound to Cdc20. This is corroborated by findings that free Mad2 in the cell is in the Oconformation, even while the checkpoint is active [51,60]. The Mad2 mimic p31comet seems to be involved in releasing Mad2 from the MCC [52]. Owing to its capacity to dimerize with Mad2, it may revert Mad2 from the C-conformation to the Oconformation, mirroring the C-Mad2-dimerization triggered conversion of the O-conformation into the C-conformation. Even if true, this leaves open the question how conversion is accomplished in yeast, where p31comet seems to be lacking [2].
Does signal propagation involve amplification? Because signalling from even a single unattached kinetochore reliably delays anaphase, it has frequently been suggested that the checkpoint signal is amplified away from kinetochores. Initially, Cdc20–C-Mad2 was thought to activate Mad2 away from the kinetochore (similar to Mad1–C-Mad2mediated activation at the kinetochore) [61]. This is possible in vitro [62,63], but no evidence was found in vivo [58]. Furthermore, the dimerization site on Mad2 is occupied by Mad3/BubR1 when C-Mad2–Cdc20 is part of the MCC [28,64]. Hence this amplification is unlikely. More recently, Han, Cleveland and colleagues proposed that Mad2 catalyses BubR1/Mad3 binding to Cdc20 [65]. According to this model, the continued presence of Mad2 in the MCC is not required [35,52,66], and Mad2 can be freed and catalyse formation of new Cdc20–BubR1/Mad3 complexes. Like any other potential amplification, this requires a mechanism to terminate the signal. Mad2 will either need to lose activity over time or will need to be actively converted into inactive Mad2 (see above). Whether amplification is indeed necessary in vivo has not yet been shown conclusively.
being required for kinetochore enrichment of Bub1, Mad3, and Mad1–Mad2 [48,49]. Instead, Bub3 may promote checkpoint inactivation [48]. Furthermore, deletion of fission yeast bub3 rescues the checkpoint defect in cells with KNL1/Spc7 mutations that are unable to recruit Bub1 to kinetochores [10,11]. This hints at a function of Bub3 as inhibitor of Bub1, whose suppressive activity may be relieved at kinetochores. If and how these differences between organisms can be reconciled with a common molecular function will be interesting to see.
What is the molecular basis of fast activation and inactivation? One of the most characteristic and least understood features of the checkpoint is its ability to rapidly switch between active and inactive signalling [69]. Activation occurs within minutes. Yet, Mad2-activation kinetics measured in vitro suggest that it needs hours to generate a signal [62,63]. Clearly, the reaction must be enhanced in vivo in an unknown way. In addition to the kinetics of individual reactions, the design of the system is important [41]. Positive-feedback loops (as between Aurora B and Mps1) can contribute to fast switching. The characteristic turnover of both Cdc20 and the MCC is probably another way to promote switching. If Cdc20 were stable, its activation (after the checkpoint has been active) would entirely rely on disassembly of checkpoint complexes. In contrast, the continuous synthesis of Cdc20 ensures that formation of free Cdc20 is promoted both by disassembly of inhibitory complexes and by resynthesis. Similarly for the MCC, when the signal ceases, inactivation may occur without any time delay, because disassembly mechanisms are continuously in place. These effects could be enhanced by regulating signalling both on the activation and inactivation side. As is very often the case, inactivation [70] has been less studied than activation. If and how the efficiency of inactivating mechanisms (such as p31comet or Cdc20 ubiquitylation) is enhanced when the checkpoint shuts off is largely unknown.
What is the function of Bub1 and Bub3?
Significance
One of the big unknowns in our understanding of checkpoint signalling is the function of Bub1 and Bub3. Bub1 brings Mad1 to kinetochores and could thereby contribute to activating Mad2. Whether artificial recruitment of Mad1 to kinetochores [45] bypasses Bub1 function has not yet been tested. Bub1 is also required to bring Mad3/BubR1 to kinetochores, and may therefore help to incorporate Mad3/BubR1 into the MCC. Surprisingly, however, at least during a mitotic arrest in fission yeast, the MCC can form without Bub1 [67]. Hence the role of Bub1 remains obscure. The situation is even less clear for Bub3. In mammalian cells, Bub3 is part of the MCC. Yet its presence does not seem necessary for the inhibitory activity of the MCC, at least in vitro [66,68]. The importance of Bub3 could be explained by its role in bringing Bub1 and BubR1 to the kinetochore, which may enhance MCC formation. Surprisingly, in fission yeast, Bub3 is not essential for checkpoint activity, despite
Why is it important to understand spindle assembly checkpoint signalling? First and foremost, the spindle assembly checkpoint provides a fascinating study case of cellular signalling. It unites disparate features, i.e. robust signalling, high sensitivity and fast reversibility, in a way that we do not (yet) understand. Efforts to design synthetic signalling pathways often fail, demonstrating how little we still know about the principles of signalling. Much can be gained by exploring a system so complex, sophisticated and, at the same time, conserved and important as the spindle assembly checkpoint. Secondly, as a protector of genome integrity, the spindle assembly checkpoint has a crucial role in cellular physiology. Its malfunction promotes chromosome misdistribution and aneuploidy, which are hallmarks of cancer. Some cancer therapies use the spindle assembly checkpoint as a point of intervention. Cancer development seems to be promoted by checkpoint dysfunction, and
C The
C 2013 Biochemical Society Authors Journal compilation
The 7th International Fission Yeast Meeting: Pombe2013
changes in checkpoint protein levels have often been observed. If we had better ways to predict how these changes affect signalling, the choice of appropriate therapies may become easier.
Acknowledgements I am grateful to Jakob Nilsson and Stephanie Heinrich for critical comments on the text before submission. I sincerely apologize to colleagues whose primary research articles I did not cite owing to space constraints.
Funding The work of my group is supported by the Max Planck Society.
References 1 Primorac, I. and Musacchio, A. (2013) Panta rhei: the APC/C at steady state. J. Cell Biol. 201, 177–189 2 Vleugel, M., Hoogendoorn, E., Snel, B. and Kops, G.J. (2012) Evolution and function of the mitotic checkpoint. Dev. Cell 23, 239–250 3 Lara-Gonzalez, P., Westhorpe, F.G. and Taylor, S.S. (2012) The spindle assembly checkpoint. Curr. Biol. 22, R966–R980 4 Foley, E.A. and Kapoor, T.M. (2013) Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. Nat. Rev. Mol. Cell Biol. 14, 25–37 5 Jia, L., Kim, S. and Yu, H. (2013) Tracking spindle checkpoint signals from kinetochores to APC/C. Trends Biochem. Sci. 38, 302–311 6 Yu, H. (2006) Structural activation of Mad2 in the mitotic spindle checkpoint: the two-state Mad2 model versus the Mad2 template model. J. Cell Biol. 173, 153–157 7 Mapelli, M. and Musacchio, A. (2007) MAD contortions: conformational dimerization boosts spindle checkpoint signaling. Curr. Opin. Struct. Biol. 17, 716–725 8 Luo, X. and Yu, H. (2008) Protein metamorphosis: the two-state behavior of Mad2. Structure 16, 1616–1625 9 London, N., Ceto, S., Ranish, J.A. and Biggins, S. (2012) Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores. Curr. Biol. 22, 900–906 10 Shepperd, L.A., Meadows, J.C., Sochaj, A.M., Lancaster, T.C., Zou, J., Buttrick, G.J., Rappsilber, J., Hardwick, K.G. and Millar, J.B. (2012) Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint. Curr. Biol. 22, 891–899 11 Yamagishi, Y., Yang, C.H., Tanno, Y. and Watanabe, Y. (2012) MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components. Nat. Cell Biol. 14, 746–752 12 Primorac, I., Weir, J.R., Chiroli, E., Gross, F., Hoffmann, I., van Gerwen, S., Ciliberto, A. and Musacchio, A. (2013) Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling. eLife 2, e01030 13 Kemmler, S., Stach, M., Knapp, M., Ortiz, J., Pfannstiel, J., Ruppert, T. and Lechner, J. (2009) Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling. EMBO J. 28, 1099–1110 14 Nijenhuis, W., von Castelmur, E., Littler, D., De Marco, V., Tromer, E., Vleugel, M., van Osch, M.H., Snel, B., Perrakis, A. and Kops, G.J. (2013) A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B. J. Cell Biol. 201, 217–231 15 Santaguida, S., Tighe, A., D’Alise, A.M., Taylor, S.S. and Musacchio, A. (2010) Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J. Cell Biol. 190, 73–87 16 Saurin, A.T., van der Waal, M.S., Medema, R.H., Lens, S.M. and Kops, G.J. (2011) Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis. Nat. Commun. 2, 316
17 Heinrich, S., Windecker, H., Hustedt, N. and Hauf, S. (2012) Mph1 kinetochore localization is crucial and upstream in the hierarchy of spindle assembly checkpoint protein recruitment to kinetochores. J. Cell Sci. 125, 4720–4727 18 van der Waal, M.S., Saurin, A.T., Vromans, M.J., Vleugel, M., Wurzenberger, C., Gerlich, D.W., Medema, R.H., Kops, G.J. and Lens, S.M. (2012) Mps1 promotes rapid centromere accumulation of Aurora B. EMBO Rep. 13, 847–854 19 Brady, D.M. and Hardwick, K.G. (2000) Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function. Curr. Biol. 10, 675–678 20 Sharp-Baker, H. and Chen, R.H. (2001) Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity. J. Cell Biol. 153, 1239–1250 21 Seeley, T.W., Wang, L. and Zhen, J.Y. (1999) Phosphorylation of human MAD1 by the BUB1 kinase in vitro. Biochem. Biophys. Res. Commun. 257, 589–595 22 Ito, D., Saito, Y. and Matsumoto, T. (2012) Centromere-tethered Mps1 pombe homolog (Mph1) kinase is a sufficient marker for recruitment of the spindle checkpoint protein Bub1, but not Mad1. Proc. Natl. Acad. Sci. U.S.A. 109, 209–214 23 Skoufias, D.A., Andreassen, P.R., Lacroix, F.B., Wilson, L. and Margolis, R.L. (2001) Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints. Proc. Natl. Acad. Sci. U.S.A. 98, 4492–4497 24 Malureanu, L.A., Jeganathan, K.B., Hamada, M., Wasilewski, L., Davenport, J. and van Deursen, J.M. (2009) BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/CCdc20 in interphase. Dev. Cell 16, 118–131 25 Krenn, V., Wehenkel, A., Li, X., Santaguida, S. and Musacchio, A. (2012) Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction. J. Cell Biol. 196, 451–467 26 Elowe, S., Dulla, K., Uldschmid, A., Li, X., Dou, Z. and Nigg, E.A. (2010) Uncoupling of the spindle-checkpoint and chromosome-congression functions of BubR1. J. Cell Sci. 123, 84–94 27 Lara-Gonzalez, P., Scott, M.I., Diez, M., Sen, O. and Taylor, S.S. (2011) BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner. J. Cell Sci. 124, 4332–4345 28 Chao, W.C., Kulkarni, K., Zhang, Z., Kong, E.H. and Barford, D. (2012) Structure of the mitotic checkpoint complex. Nature 484, 208–213 29 Herzog, F., Primorac, I., Dube, P., Lenart, P., Sander, B., Mechtler, K., Stark, H. and Peters, J.M. (2009) Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex. Science 323, 1477–1481 30 Izawa, D. and Pines, J. (2011) How APC/C-Cdc20 changes its substrate specificity in mitosis. Nat. Cell Biol. 13, 223–233 31 Izawa, D. and Pines, J. (2012) Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation. J. Cell Biol. 199, 27–37 32 Foster, S.A. and Morgan, D.O. (2012) The APC/C subunit Mnd2/Apc15 promotes Cdc20 autoubiquitination and spindle assembly checkpoint inactivation. Mol. Cell 47, 921–932 33 Burton, J.L. and Solomon, M.J. (2007) Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint. Genes Dev. 21, 655–667 34 Pan, J. and Chen, R.H. (2004) Spindle checkpoint regulates Cdc20p stability in Saccharomyces cerevisiae. Genes Dev. 18, 1439–1451 35 Nilsson, J., Yekezare, M., Minshull, J. and Pines, J. (2008) The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nat. Cell Biol. 10, 1411–1420 36 Ge, S., Skaar, J.R. and Pagano, M. (2009) APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation. Cell Cycle 8, 167–171 37 Reddy, S.K., Rape, M., Margansky, W.A. and Kirschner, M.W. (2007) Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 446, 921–925 38 Mansfeld, J., Collin, P., Collins, M.O., Choudhary, J.S. and Pines, J. (2011) APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nat. Cell Biol. 13, 1234–1243 39 Uzunova, K., Dye, B.T., Schutz, H., Ladurner, R., Petzold, G., Toyoda, Y., Jarvis, M.A., Brown, N.G., Poser, I., Novatchkova, M. et al. (2012) APC15 mediates CDC20 autoubiquitylation by APC/CMCC and disassembly of the mitotic checkpoint complex. Nat. Struct. Mol. Biol. 19, 1116–1123 40 Ma, H.T. and Poon, R.Y. (2011) Orderly inactivation of the key checkpoint protein mitotic arrest deficient 2 (MAD2) during mitotic progression. J. Biol. Chem. 286, 13052–13059 C The
C 2013 Biochemical Society Authors Journal compilation
1759
1760
Biochemical Society Transactions (2013) Volume 41, part 6
41 Ciliberto, A. and Shah, J.V. (2009) A quantitative systems view of the spindle assembly checkpoint. EMBO J. 28, 2162–2173 42 Lampson, M.A. and Cheeseman, I.M. (2011) Sensing centromere tension: Aurora B and the regulation of kinetochore function. Trends Cell Biol. 21, 133–140 43 Burke, D.J. and Stukenberg, P.T. (2008) Linking kinetochore-microtubule binding to the spindle checkpoint. Dev. Cell 14, 474–479 44 Espeut, J., Cheerambathur, D.K., Krenning, L., Oegema, K. and Desai, A. (2012) Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore. J. Cell Biol. 196, 469–482 45 Maldonado, M. and Kapoor, T.M. (2011) Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation. Nat. Cell Biol. 13, 475–482 46 Rischitor, P.E., May, K.M. and Hardwick, K.G. (2007) Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes. PLoS ONE 2, e1342 47 Martin-Lluesma, S., Stucke, V.M. and Nigg, E.A. (2002) Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2. Science 297, 2267–2270 48 Vanoosthuyse, V., Meadows, J.C., van der Sar, S.J., Millar, J.B. and Hardwick, K.G. (2009) Bub3p facilitates spindle checkpoint silencing in fission yeast. Mol. Biol. Cell 20, 5096–5105 49 Windecker, H., Langegger, M., Heinrich, S. and Hauf, S. (2009) Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin. EMBO Rep. 10, 1022–1028 50 Vink, M., Simonetta, M., Transidico, P., Ferrari, K., Mapelli, M., De Antoni, A., Massimiliano, L., Ciliberto, A., Faretta, M., Salmon, E.D. and Musacchio, A. (2006) In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics. Curr. Biol. 16, 755–766 51 Fava, L.L., Kaulich, M., Nigg, E.A. and Santamaria, A. (2011) Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint. EMBO J. 30, 3322–3336 52 Westhorpe, F.G., Tighe, A., Lara-Gonzalez, P. and Taylor, S.S. (2011) p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit. J. Cell Sci. 124, 3905–3916 53 Hewitt, L., Tighe, A., Santaguida, S., White, A.M., Jones, C.D., Musacchio, A., Green, S. and Taylor, S.S. (2010) Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex. J. Cell Biol. 190, 25–34 54 Althoff, F., Karess, R.E. and Lehner, C.F. (2012) Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1. Mol. Biol. Cell 23, 2275–2291 55 Lince-Faria, M., Maffini, S., Orr, B., Ding, Y., Claudia, F., Sunkel, C.E., Tavares, A., Johansen, J., Johansen, K.M. and Maiato, H. (2009) Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator. J. Cell Biol. 184, 647–657 56 Jelluma, N., Dansen, T.B., Sliedrecht, T., Kwiatkowski, N.P. and Kops, G.J. (2010) Release of Mps1 from kinetochores is crucial for timely anaphase onset. J. Cell Biol. 191, 281–290 57 Yang, M., Li, B., Liu, C.J., Tomchick, D.R., Machius, M., Rizo, J., Yu, H. and Luo, X. (2008) Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer. PLoS Biol. 6, e50
C The
C 2013 Biochemical Society Authors Journal compilation
58 Mariani, L., Chiroli, E., Nezi, L., Muller, H., Piatti, S., Musacchio, A. and Ciliberto, A. (2012) Role of the Mad2 dimerization interface in the spindle assembly checkpoint independent of kinetochores. Curr. Biol. 22, 1900–1908 59 Oliveira, R.A., Hamilton, R.S., Pauli, A., Davis, I. and Nasmyth, K. (2010) Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei. Nat. Cell Biol. 12, 185–192 60 Luo, X., Tang, Z., Xia, G., Wassmann, K., Matsumoto, T., Rizo, J. and Yu, H. (2004) The Mad2 spindle checkpoint protein has two distinct natively folded states. Nat. Struct. Mol. Biol. 11, 338–345 61 De Antoni, A., Pearson, C.G., Cimini, D., Canman, J.C., Sala, V., Nezi, L., Mapelli, M., Sironi, L., Faretta, M., Salmon, E.D. and Musacchio, A. (2005) The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint. Curr. Biol. 15, 214–225 62 Lad, L., Lichtsteiner, S., Hartman, J.J., Wood, K.W. and Sakowicz, R. (2009) Kinetic analysis of Mad2–Cdc20 formation: conformational changes in Mad2 are catalyzed by a C-Mad2–ligand complex. Biochemistry 48, 9503–9515 63 Simonetta, M., Manzoni, R., Mosca, R., Mapelli, M., Massimiliano, L., Vink, M., Novak, B., Musacchio, A. and Ciliberto, A. (2009) The influence of catalysis on mad2 activation dynamics. PLoS Biol. 7, e10 64 Tipton, A.R., Wang, K., Link, L., Bellizzi, J.J., Huang, H., Yen, T. and Liu, S.T. (2011) BUBR1 and closed MAD2 (C-MAD2) interact directly to assemble a functional mitotic checkpoint complex. J. Biol. Chem. 286, 21173–21179 65 Han, J.S., Holland, A.J., Fachinetti, D., Kulukian, A., Cetin, B. and Cleveland, D.W. (2013) Catalytic assembly of the mitotic checkpoint inhibitor BubR1–Cdc20 by a Mad2-induced functional switch in Cdc20. Mol. Cell 51, 92–104 66 Kulukian, A., Han, J.S. and Cleveland, D.W. (2009) Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding. Dev. Cell 16, 105–117 67 Sczaniecka, M., Feoktistova, A., May, K.M., Chen, J.S., Blyth, J., Gould, K.L. and Hardwick, K.G. (2008) The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C). J. Biol. Chem. 283, 23039–23047 68 Fang, G. (2002) Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex. Mol. Biol. Cell 13, 755–766 69 Hagting, A., Den Elzen, N., Vodermaier, H.C., Waizenegger, I.C., Peters, J.M. and Pines, J. (2002) Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1. J. Cell Biol. 157, 1125–1137 70 Kops, G.J. and Shah, J.V. (2012) Connecting up and clearing out: how kinetochore attachment silences the spindle assembly checkpoint. Chromosoma 121, 509–525
Received 8 October 2013 doi:10.1042/BST20130240
Copyright of Biochemical Society Transactions is the property of Portland Press Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
The spindle assembly checkpoint: progress and persistent puzzles Silke Hauf*1 *Friedrich Miescher Laboratory of the Max Planck Society, Spemannstrasse 39, 72076 Tubingen, ¨ Germany
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract The spindle assembly checkpoint is a conserved mitotic signalling pathway that ensures the equal segregation of chromosomes to daughter cells. Despite intensive work in many model organisms, key features of this safety mechanism remain unexplained. In the present review, I briefly summarize advances made in the last few years, and then focus on unexplored corners of this signalling pathway.
A succinct overview of spindle assembly checkpoint signalling The principles of spindle assembly checkpoint signalling are simple: checkpoint proteins recognize mitotic chromosomes that are so poorly attached to spindle microtubules that their segregation to opposite cell poles is at risk. Attachment between chromosomes and spindle microtubules is mediated by kinetochores, huge protein assemblies on the centromeric region of chromosomes. Checkpoint proteins enrich at unattached kinetochores and initiate a signalling cascade that ultimately blocks the APC/C (anaphase-promoting complex/cyclosome), a ubiquitin ligase that is essential for anaphase onset [1]. Thus the checkpoint delays anaphase until all chromosomes have achieved proper attachment. Although this general scheme has been clear for many years, elucidating the mechanistic basis remains a challenge. Considerable progress has been made in understanding how checkpoint proteins bind to kinetochores and how checkpoint proteins block the action of the APC/C. In the present review, I summarize these advances and then focus on aspects of checkpoint signalling that are equally important, but less understood. The present short review only highlights some facets, but comprehensive and nuanced overviews are available [1–5].
Spindle assembly checkpoint proteins and their functions Mad1, Mad2, Mad3 (or BubR1, depending on the organism), Bub1, Bub3 and Mps1 are the core spindle assembly checkpoint proteins and form a signalling cascade of protein– protein interactions and protein phosphorylation. Mad2 and Mad3/BubR1 bind directly to and thereby disable the activator of the APC/C, Cdc20. This effector complex is called MCC (mitotic checkpoint complex) and in many organisms additionally contains Bub3, which is co-recruited Key words: anaphase-promoting complex (APC), mitosis, mitotic checkpoint, signalling pathway, spindle assembly checkpoint. Abbreviations used: APC/C, anaphase-promoting complex/cyclosome; C-, closed; MCC, mitotic checkpoint complex; O-, open. 1 email [email protected]
Biochem. Soc. Trans. (2013) 41, 1755–1760; doi:10.1042/BST20130240
with Mad3/BubR1. Mad2 undergoes a major conformational change when binding to Cdc20: from open (O) to closed (C) [6–8]. This transformation is facilitated by dimerization with another molecule of (C-)Mad2 that is stably bound to Mad1. Mad1 and Mad2 form a 2:2 complex that, like most other checkpoint proteins, becomes recruited to unattached kinetochores in mitosis. Bub1, Bub3 and Mps1 presumably act upstream of Mad1 in checkpoint signalling. Their functions are only partly understood (see below). Bub1 and Mad3/BubR1 are related proteins [2], have a similar domain structure, and can both bind to Bub3 (in a mutually exclusive way). Bub1 and Mps1 are protein kinases, but only the kinase activity of Mps1 is essential for the checkpoint.
Hierarchical recruitment of checkpoint proteins to unattached kinetochores How checkpoint proteins connect to the unattached kinetochores whose presence they signal has recently been revealed in some detail (reviewed in [3–5]) (Figure 1). The checkpoint kinase Mps1 acts upstream and brings other checkpoint proteins to the kinetochore. Not all relevant substrates of Mps1 are known, but phosphorylation of the outer kinetochore protein KNL1 (also called CASC5, Blinkin, Spc7 and Spc105) is important [9–12]. Mps1 recruitment, presumably to the outer kinetochore protein Ndc80/Hec1 [13,14], is aided by the Aurora B kinase, and Mps1 reinforces its own recruitment by (indirectly) promoting Aurora B localization [15–18]. Phosphorylation of KNL1 by Mps1 provides a binding platform for the Bub1–Bub3 complex, representing the next layer of recruitment [10–12]. Bub1 in turn is required for the kinetochore localization of the Mad1–Mad2 complex, in metazoan cells in conjunction with the RZZ (Rod–ZW10–Zwilch) complex, whose localization also depends on Mps1. Bub1 and Mad1 may interact [19– 21], but artificial recruitment of Bub1 does not co-recruit Mad1 in interphase [11,22], and mitotic kinetochores can be bound to Bub1 without Mad1 being present [20,23]. This indicates that other requirements must be met for Mad1 to localize. Enrichment of Mad3/BubR1 at unattached kinetochores is not universal across organisms, may not C The
C 2013 Biochemical Society Authors Journal compilation
1755
1756
Biochemical Society Transactions (2013) Volume 41, part 6
Figure 1 Assembly of checkpoint proteins at unattached kinetochore Recruitment of checkpoint proteins (blue) to the kinetochore (red). Black arrows show dependencies; yellow circles mark phosphorylation (P) events. Potential conformational changes at the kinetochore that could aid recruitment are shown as irregular shapes with question marks. See the text for details.
of substrates [28,29]. Two KEN-box (Lys-Glu-Asn) motifs within Mad3/BubR1, which normally are recognition motifs for APC/C-mediated degradation, are essential for the inhibitory function. The more N-terminal KEN-box binds directly (and thereby blocks) the KEN-box receptor of Cdc20 [27,28,33]. The second KEN-box seems to inhibit substrate recruitment to the APC/C through an unknown mechanism [27]. It is striking that the second KEN-box is so important, although the MCC forms in its absence and at least some of the mechanisms mentioned above should be functional. Cdc20 as part of the MCC is an APC/C substrate, and much has been discussed about the function of this ubiquitylation, which may suppress Cdc20 levels through degradation [32,34–36], thus aiding the checkpoint, or may lead to disassembly of the MCC and termination of the checkpoint signal [32,37–40]. These functions have sometimes been presented as mutually exclusive, which they may not be [32].
A succinct overview of our nescience Despite all of this progress, we are still ignorant about key aspects of checkpoint signalling (Figure 2): we lack a mechanistic understanding of some steps in the signalling cascade, and we know hardly anything about the molecular basis of the dynamic features of checkpoint signalling [41]. In the following sections, I highlight some of the open questions.
be obligatory for checkpoint activity, and its mechanistic basis remains unclear. Both Bub1 and Mad3/BubR1 contain N-terminal TPR (tetratricopeptide repeat) domains that bind to KNL1, but they seem to play a minor role in kinetochore recruitment (see [24,25] and references therein). Human BubR1 needs interaction with Bub3 for efficient kinetochore binding [24,26,27], indicating that Mad3/BubR1–Bub3 could be recruited in a similar manner to Bub1–Bub3. However, at least in human cells and fission yeast, recruitment of Mad3/BubR1 requires Bub1, but not vice versa, indicating that additional influences are needed.
Molecular basis of APC/C inhibition As for kinetochore recruitment, recent years have seen major advances in understanding how the checkpoint blocks APC/C activity. The structure of the central part of the MCC (Mad2 and part of Mad3 bound to Cdc20) has been solved [28]; and cryo-electron microscopy and biochemical assays have shown where MCC binds the APC/C [29,30]. MCC formation seems to block APC/C–Cdc20 activity in multiple ways: (i) binding of Mad2 to Cdc20 impairs binding of Cdc20 to the APC/C [31,32]; and (ii) additional binding of Mad3/BubR1 allows binding of the so-formed MCC to the APC/C, but Cdc20 as part of this complex cannot act as activator of the APC/C, (a) because substrate recruitment by Cdc20 and the APC/C may be blocked [27,29,33], and (b) because Cdc20 within the MCC may not have the right position on the APC/C to promote ubiquitylation C The
C 2013 Biochemical Society Authors Journal compilation
What is the trigger for checkpoint protein accumulation at kinetochores? Aurora B can phosphorylate kinetochore proteins in a chromosome attachment-dependent manner [42], which, in principle, provides a mechanism to recruit Mps1 and other downstream checkpoint proteins depending on the chromosome-attachment state. However, at least in human cells, Aurora B speeds up, but may not be essential for Mps1 recruitment [16]. Hence Mps1 must have other ways to distinguish between unattached and attached kinetochores. Not all checkpoint proteins need to be influenced directly by the attachment state (Bub1 and Bub3 may simply depend on the presence of Mps1), but a second point of interference could be Mad1, for whose localization Bub1 is required, but not sufficient. One could imagine that microtubules compete with checkpoint proteins for the same binding site or that microtubule attachment creates a conformational change or leads to post-translational modifications that regulate checkpoint protein binding [43,44].
Do kinetochores have a role in signalling beyond recruitment? Are kinetochores merely a landing pad for checkpoint proteins, or do they actively contribute to checkpoint signalling? So far, artificial recruitment of single checkpoint proteins to sites other than kinetochores failed to initiate checkpoint activity [45,46]. However, in the absence of kinetochore proteins, some crucial checkpoint proteins were probably not co-recruited. To answer the question above,
The 7th International Fission Yeast Meeting: Pombe2013
Figure 2 Open questions in spindle assembly checkpoint signalling Outline of checkpoint signalling from the kinetochore to the APC/C. Small circles labelled ‘O-’ and ‘C-’ represent O-Mad2 and C-Mad2 respectively. Black arrows highlight unresolved questions. See the text for details.
all checkpoint proteins would need to be experimentally enriched at a locus other than the kinetochore. An argument against enrichment being the only crucial activity of kinetochores can be made since checkpoint signalling works surprisingly well when reduction of an outer kinetochore protein in human cells [47] or deletion of bub3 in fission yeast [48,49] abolishes the visible enrichment of many checkpoint proteins. This could either indicate that only some checkpoint proteins need to be enriched to propagate the signal or that kinetochores provide a specific activity other than enrichment (e.g. initiation of a conformational change), for which a transient interaction may be sufficient.
How is the Mad1–C-Mad2 complex regulated? The Mad1–C-Mad2 complex is a key element in checkpoint signalling. The complex exists throughout the cell cycle, but only promotes checkpoint signalling when unattached kinetochores are present. How is this regulated? The protein p31comet , which structurally resembles Mad2, has been proposed to ‘cap’ Mad1–C-Mad2 to prevent it from signalling [50,51]. Taylor and colleagues, however, did not find evidence for this mechanism [52], but found that the Mps1 kinase promotes the recruitment of O-Mad2 to kinetochore-localized Mad1–Mad2 [53]. They have therefore suggested the alternative hypothesis that Mps1 itself ‘caps’ Mad1:Mad2. This is inspired by findings that Mad1 and Mps1 interact [54,55] and that active Mps1 enhances its own turnover at kinetochores [53,56]. Hence Mps1 may release itself from blocking access to Mad1–Mad2 [3]. Further work is needed to accept or refute this hypothesis.
What is ‘active’ Mad2 and how long-lived is it? When binding to Cdc20, Mad2 rearranges from the Oto the C-conformation, and entraps a stretch of Cdc20, just like a seat belt ties a passenger to the seat [6–8]. In the absence of a binding partner, Mad2 can transition into ‘unliganded’ C-Mad2. The Mad2 conversion reaction is interesting both from the structural point of view and because it is relevant to propagation of the signal from kinetochores to APC/C–Cdc20. At present, it is unclear whether Mad2 binds Cdc20 concomitantly with dimerization or whether ‘active’ Mad2 can dissociate after dimerization and bind Cdc20 later. The hypothetical, dissociating, active species could be C-Mad2 or an O/C-intermediate (I-Mad2 or Mad2*). In favour of the former, constitutive C-Mad2 is more efficient than wild-type Mad2 in inhibiting APC/C– Cdc20 in vitro [57] and when overexpressed in vivo [58,59]. However, at endogenous levels, constitutive C-Mad2 does not inhibit Cdc20 efficiently in fission yeast (A. Malasane, U. Schellhaas and S. Hauf, unpublished work), suggesting that active Mad2 looks different from C-Mad2. There is currently no evidence that diffusion of active Mad2 is needed for efficient signalling. However, amplification of the signal away from kinetochores (see below) may require this. If diffusible active Mad2 exists, it should only have a limited life-time in order to ensure that the checkpoint can be efficiently shut off, raising the question of how its activity is limited.
How does C-Mad2 revert to O-Mad2? Regardless of the activation mechanism, checkpoint signalling creates C-Mad2. Too much C-Mad2 could be problematic in several ways: if it is Cdc20-binding-competent (see above), it C The
C 2013 Biochemical Society Authors Journal compilation
1757
1758
Biochemical Society Transactions (2013) Volume 41, part 6
must be inactivated at the latest when the checkpoint shuts off; if it is Cdc20-binding-incompetent, it poses a risk for the signalling reaction in two opposing ways: (i) because the pool of O-Mad2 (that can be activated) will be depleted, and (ii) since it may provide a template for further Mad2 activation. Hence there are reasons to believe that C-Mad2 is converted back into O-Mad2 when no longer bound to Cdc20. This is corroborated by findings that free Mad2 in the cell is in the Oconformation, even while the checkpoint is active [51,60]. The Mad2 mimic p31comet seems to be involved in releasing Mad2 from the MCC [52]. Owing to its capacity to dimerize with Mad2, it may revert Mad2 from the C-conformation to the Oconformation, mirroring the C-Mad2-dimerization triggered conversion of the O-conformation into the C-conformation. Even if true, this leaves open the question how conversion is accomplished in yeast, where p31comet seems to be lacking [2].
Does signal propagation involve amplification? Because signalling from even a single unattached kinetochore reliably delays anaphase, it has frequently been suggested that the checkpoint signal is amplified away from kinetochores. Initially, Cdc20–C-Mad2 was thought to activate Mad2 away from the kinetochore (similar to Mad1–C-Mad2mediated activation at the kinetochore) [61]. This is possible in vitro [62,63], but no evidence was found in vivo [58]. Furthermore, the dimerization site on Mad2 is occupied by Mad3/BubR1 when C-Mad2–Cdc20 is part of the MCC [28,64]. Hence this amplification is unlikely. More recently, Han, Cleveland and colleagues proposed that Mad2 catalyses BubR1/Mad3 binding to Cdc20 [65]. According to this model, the continued presence of Mad2 in the MCC is not required [35,52,66], and Mad2 can be freed and catalyse formation of new Cdc20–BubR1/Mad3 complexes. Like any other potential amplification, this requires a mechanism to terminate the signal. Mad2 will either need to lose activity over time or will need to be actively converted into inactive Mad2 (see above). Whether amplification is indeed necessary in vivo has not yet been shown conclusively.
being required for kinetochore enrichment of Bub1, Mad3, and Mad1–Mad2 [48,49]. Instead, Bub3 may promote checkpoint inactivation [48]. Furthermore, deletion of fission yeast bub3 rescues the checkpoint defect in cells with KNL1/Spc7 mutations that are unable to recruit Bub1 to kinetochores [10,11]. This hints at a function of Bub3 as inhibitor of Bub1, whose suppressive activity may be relieved at kinetochores. If and how these differences between organisms can be reconciled with a common molecular function will be interesting to see.
What is the molecular basis of fast activation and inactivation? One of the most characteristic and least understood features of the checkpoint is its ability to rapidly switch between active and inactive signalling [69]. Activation occurs within minutes. Yet, Mad2-activation kinetics measured in vitro suggest that it needs hours to generate a signal [62,63]. Clearly, the reaction must be enhanced in vivo in an unknown way. In addition to the kinetics of individual reactions, the design of the system is important [41]. Positive-feedback loops (as between Aurora B and Mps1) can contribute to fast switching. The characteristic turnover of both Cdc20 and the MCC is probably another way to promote switching. If Cdc20 were stable, its activation (after the checkpoint has been active) would entirely rely on disassembly of checkpoint complexes. In contrast, the continuous synthesis of Cdc20 ensures that formation of free Cdc20 is promoted both by disassembly of inhibitory complexes and by resynthesis. Similarly for the MCC, when the signal ceases, inactivation may occur without any time delay, because disassembly mechanisms are continuously in place. These effects could be enhanced by regulating signalling both on the activation and inactivation side. As is very often the case, inactivation [70] has been less studied than activation. If and how the efficiency of inactivating mechanisms (such as p31comet or Cdc20 ubiquitylation) is enhanced when the checkpoint shuts off is largely unknown.
What is the function of Bub1 and Bub3?
Significance
One of the big unknowns in our understanding of checkpoint signalling is the function of Bub1 and Bub3. Bub1 brings Mad1 to kinetochores and could thereby contribute to activating Mad2. Whether artificial recruitment of Mad1 to kinetochores [45] bypasses Bub1 function has not yet been tested. Bub1 is also required to bring Mad3/BubR1 to kinetochores, and may therefore help to incorporate Mad3/BubR1 into the MCC. Surprisingly, however, at least during a mitotic arrest in fission yeast, the MCC can form without Bub1 [67]. Hence the role of Bub1 remains obscure. The situation is even less clear for Bub3. In mammalian cells, Bub3 is part of the MCC. Yet its presence does not seem necessary for the inhibitory activity of the MCC, at least in vitro [66,68]. The importance of Bub3 could be explained by its role in bringing Bub1 and BubR1 to the kinetochore, which may enhance MCC formation. Surprisingly, in fission yeast, Bub3 is not essential for checkpoint activity, despite
Why is it important to understand spindle assembly checkpoint signalling? First and foremost, the spindle assembly checkpoint provides a fascinating study case of cellular signalling. It unites disparate features, i.e. robust signalling, high sensitivity and fast reversibility, in a way that we do not (yet) understand. Efforts to design synthetic signalling pathways often fail, demonstrating how little we still know about the principles of signalling. Much can be gained by exploring a system so complex, sophisticated and, at the same time, conserved and important as the spindle assembly checkpoint. Secondly, as a protector of genome integrity, the spindle assembly checkpoint has a crucial role in cellular physiology. Its malfunction promotes chromosome misdistribution and aneuploidy, which are hallmarks of cancer. Some cancer therapies use the spindle assembly checkpoint as a point of intervention. Cancer development seems to be promoted by checkpoint dysfunction, and
C The
C 2013 Biochemical Society Authors Journal compilation
The 7th International Fission Yeast Meeting: Pombe2013
changes in checkpoint protein levels have often been observed. If we had better ways to predict how these changes affect signalling, the choice of appropriate therapies may become easier.
Acknowledgements I am grateful to Jakob Nilsson and Stephanie Heinrich for critical comments on the text before submission. I sincerely apologize to colleagues whose primary research articles I did not cite owing to space constraints.
Funding The work of my group is supported by the Max Planck Society.
References 1 Primorac, I. and Musacchio, A. (2013) Panta rhei: the APC/C at steady state. J. Cell Biol. 201, 177–189 2 Vleugel, M., Hoogendoorn, E., Snel, B. and Kops, G.J. (2012) Evolution and function of the mitotic checkpoint. Dev. Cell 23, 239–250 3 Lara-Gonzalez, P., Westhorpe, F.G. and Taylor, S.S. (2012) The spindle assembly checkpoint. Curr. Biol. 22, R966–R980 4 Foley, E.A. and Kapoor, T.M. (2013) Microtubule attachment and spindle assembly checkpoint signalling at the kinetochore. Nat. Rev. Mol. Cell Biol. 14, 25–37 5 Jia, L., Kim, S. and Yu, H. (2013) Tracking spindle checkpoint signals from kinetochores to APC/C. Trends Biochem. Sci. 38, 302–311 6 Yu, H. (2006) Structural activation of Mad2 in the mitotic spindle checkpoint: the two-state Mad2 model versus the Mad2 template model. J. Cell Biol. 173, 153–157 7 Mapelli, M. and Musacchio, A. (2007) MAD contortions: conformational dimerization boosts spindle checkpoint signaling. Curr. Opin. Struct. Biol. 17, 716–725 8 Luo, X. and Yu, H. (2008) Protein metamorphosis: the two-state behavior of Mad2. Structure 16, 1616–1625 9 London, N., Ceto, S., Ranish, J.A. and Biggins, S. (2012) Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores. Curr. Biol. 22, 900–906 10 Shepperd, L.A., Meadows, J.C., Sochaj, A.M., Lancaster, T.C., Zou, J., Buttrick, G.J., Rappsilber, J., Hardwick, K.G. and Millar, J.B. (2012) Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint. Curr. Biol. 22, 891–899 11 Yamagishi, Y., Yang, C.H., Tanno, Y. and Watanabe, Y. (2012) MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components. Nat. Cell Biol. 14, 746–752 12 Primorac, I., Weir, J.R., Chiroli, E., Gross, F., Hoffmann, I., van Gerwen, S., Ciliberto, A. and Musacchio, A. (2013) Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling. eLife 2, e01030 13 Kemmler, S., Stach, M., Knapp, M., Ortiz, J., Pfannstiel, J., Ruppert, T. and Lechner, J. (2009) Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling. EMBO J. 28, 1099–1110 14 Nijenhuis, W., von Castelmur, E., Littler, D., De Marco, V., Tromer, E., Vleugel, M., van Osch, M.H., Snel, B., Perrakis, A. and Kops, G.J. (2013) A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B. J. Cell Biol. 201, 217–231 15 Santaguida, S., Tighe, A., D’Alise, A.M., Taylor, S.S. and Musacchio, A. (2010) Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine. J. Cell Biol. 190, 73–87 16 Saurin, A.T., van der Waal, M.S., Medema, R.H., Lens, S.M. and Kops, G.J. (2011) Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis. Nat. Commun. 2, 316
17 Heinrich, S., Windecker, H., Hustedt, N. and Hauf, S. (2012) Mph1 kinetochore localization is crucial and upstream in the hierarchy of spindle assembly checkpoint protein recruitment to kinetochores. J. Cell Sci. 125, 4720–4727 18 van der Waal, M.S., Saurin, A.T., Vromans, M.J., Vleugel, M., Wurzenberger, C., Gerlich, D.W., Medema, R.H., Kops, G.J. and Lens, S.M. (2012) Mps1 promotes rapid centromere accumulation of Aurora B. EMBO Rep. 13, 847–854 19 Brady, D.M. and Hardwick, K.G. (2000) Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function. Curr. Biol. 10, 675–678 20 Sharp-Baker, H. and Chen, R.H. (2001) Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity. J. Cell Biol. 153, 1239–1250 21 Seeley, T.W., Wang, L. and Zhen, J.Y. (1999) Phosphorylation of human MAD1 by the BUB1 kinase in vitro. Biochem. Biophys. Res. Commun. 257, 589–595 22 Ito, D., Saito, Y. and Matsumoto, T. (2012) Centromere-tethered Mps1 pombe homolog (Mph1) kinase is a sufficient marker for recruitment of the spindle checkpoint protein Bub1, but not Mad1. Proc. Natl. Acad. Sci. U.S.A. 109, 209–214 23 Skoufias, D.A., Andreassen, P.R., Lacroix, F.B., Wilson, L. and Margolis, R.L. (2001) Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints. Proc. Natl. Acad. Sci. U.S.A. 98, 4492–4497 24 Malureanu, L.A., Jeganathan, K.B., Hamada, M., Wasilewski, L., Davenport, J. and van Deursen, J.M. (2009) BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/CCdc20 in interphase. Dev. Cell 16, 118–131 25 Krenn, V., Wehenkel, A., Li, X., Santaguida, S. and Musacchio, A. (2012) Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction. J. Cell Biol. 196, 451–467 26 Elowe, S., Dulla, K., Uldschmid, A., Li, X., Dou, Z. and Nigg, E.A. (2010) Uncoupling of the spindle-checkpoint and chromosome-congression functions of BubR1. J. Cell Sci. 123, 84–94 27 Lara-Gonzalez, P., Scott, M.I., Diez, M., Sen, O. and Taylor, S.S. (2011) BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner. J. Cell Sci. 124, 4332–4345 28 Chao, W.C., Kulkarni, K., Zhang, Z., Kong, E.H. and Barford, D. (2012) Structure of the mitotic checkpoint complex. Nature 484, 208–213 29 Herzog, F., Primorac, I., Dube, P., Lenart, P., Sander, B., Mechtler, K., Stark, H. and Peters, J.M. (2009) Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex. Science 323, 1477–1481 30 Izawa, D. and Pines, J. (2011) How APC/C-Cdc20 changes its substrate specificity in mitosis. Nat. Cell Biol. 13, 223–233 31 Izawa, D. and Pines, J. (2012) Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation. J. Cell Biol. 199, 27–37 32 Foster, S.A. and Morgan, D.O. (2012) The APC/C subunit Mnd2/Apc15 promotes Cdc20 autoubiquitination and spindle assembly checkpoint inactivation. Mol. Cell 47, 921–932 33 Burton, J.L. and Solomon, M.J. (2007) Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint. Genes Dev. 21, 655–667 34 Pan, J. and Chen, R.H. (2004) Spindle checkpoint regulates Cdc20p stability in Saccharomyces cerevisiae. Genes Dev. 18, 1439–1451 35 Nilsson, J., Yekezare, M., Minshull, J. and Pines, J. (2008) The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nat. Cell Biol. 10, 1411–1420 36 Ge, S., Skaar, J.R. and Pagano, M. (2009) APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation. Cell Cycle 8, 167–171 37 Reddy, S.K., Rape, M., Margansky, W.A. and Kirschner, M.W. (2007) Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 446, 921–925 38 Mansfeld, J., Collin, P., Collins, M.O., Choudhary, J.S. and Pines, J. (2011) APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nat. Cell Biol. 13, 1234–1243 39 Uzunova, K., Dye, B.T., Schutz, H., Ladurner, R., Petzold, G., Toyoda, Y., Jarvis, M.A., Brown, N.G., Poser, I., Novatchkova, M. et al. (2012) APC15 mediates CDC20 autoubiquitylation by APC/CMCC and disassembly of the mitotic checkpoint complex. Nat. Struct. Mol. Biol. 19, 1116–1123 40 Ma, H.T. and Poon, R.Y. (2011) Orderly inactivation of the key checkpoint protein mitotic arrest deficient 2 (MAD2) during mitotic progression. J. Biol. Chem. 286, 13052–13059 C The
C 2013 Biochemical Society Authors Journal compilation
1759
1760
Biochemical Society Transactions (2013) Volume 41, part 6
41 Ciliberto, A. and Shah, J.V. (2009) A quantitative systems view of the spindle assembly checkpoint. EMBO J. 28, 2162–2173 42 Lampson, M.A. and Cheeseman, I.M. (2011) Sensing centromere tension: Aurora B and the regulation of kinetochore function. Trends Cell Biol. 21, 133–140 43 Burke, D.J. and Stukenberg, P.T. (2008) Linking kinetochore-microtubule binding to the spindle checkpoint. Dev. Cell 14, 474–479 44 Espeut, J., Cheerambathur, D.K., Krenning, L., Oegema, K. and Desai, A. (2012) Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore. J. Cell Biol. 196, 469–482 45 Maldonado, M. and Kapoor, T.M. (2011) Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation. Nat. Cell Biol. 13, 475–482 46 Rischitor, P.E., May, K.M. and Hardwick, K.G. (2007) Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes. PLoS ONE 2, e1342 47 Martin-Lluesma, S., Stucke, V.M. and Nigg, E.A. (2002) Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2. Science 297, 2267–2270 48 Vanoosthuyse, V., Meadows, J.C., van der Sar, S.J., Millar, J.B. and Hardwick, K.G. (2009) Bub3p facilitates spindle checkpoint silencing in fission yeast. Mol. Biol. Cell 20, 5096–5105 49 Windecker, H., Langegger, M., Heinrich, S. and Hauf, S. (2009) Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin. EMBO Rep. 10, 1022–1028 50 Vink, M., Simonetta, M., Transidico, P., Ferrari, K., Mapelli, M., De Antoni, A., Massimiliano, L., Ciliberto, A., Faretta, M., Salmon, E.D. and Musacchio, A. (2006) In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics. Curr. Biol. 16, 755–766 51 Fava, L.L., Kaulich, M., Nigg, E.A. and Santamaria, A. (2011) Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint. EMBO J. 30, 3322–3336 52 Westhorpe, F.G., Tighe, A., Lara-Gonzalez, P. and Taylor, S.S. (2011) p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit. J. Cell Sci. 124, 3905–3916 53 Hewitt, L., Tighe, A., Santaguida, S., White, A.M., Jones, C.D., Musacchio, A., Green, S. and Taylor, S.S. (2010) Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex. J. Cell Biol. 190, 25–34 54 Althoff, F., Karess, R.E. and Lehner, C.F. (2012) Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1. Mol. Biol. Cell 23, 2275–2291 55 Lince-Faria, M., Maffini, S., Orr, B., Ding, Y., Claudia, F., Sunkel, C.E., Tavares, A., Johansen, J., Johansen, K.M. and Maiato, H. (2009) Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator. J. Cell Biol. 184, 647–657 56 Jelluma, N., Dansen, T.B., Sliedrecht, T., Kwiatkowski, N.P. and Kops, G.J. (2010) Release of Mps1 from kinetochores is crucial for timely anaphase onset. J. Cell Biol. 191, 281–290 57 Yang, M., Li, B., Liu, C.J., Tomchick, D.R., Machius, M., Rizo, J., Yu, H. and Luo, X. (2008) Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer. PLoS Biol. 6, e50
C The
C 2013 Biochemical Society Authors Journal compilation
58 Mariani, L., Chiroli, E., Nezi, L., Muller, H., Piatti, S., Musacchio, A. and Ciliberto, A. (2012) Role of the Mad2 dimerization interface in the spindle assembly checkpoint independent of kinetochores. Curr. Biol. 22, 1900–1908 59 Oliveira, R.A., Hamilton, R.S., Pauli, A., Davis, I. and Nasmyth, K. (2010) Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei. Nat. Cell Biol. 12, 185–192 60 Luo, X., Tang, Z., Xia, G., Wassmann, K., Matsumoto, T., Rizo, J. and Yu, H. (2004) The Mad2 spindle checkpoint protein has two distinct natively folded states. Nat. Struct. Mol. Biol. 11, 338–345 61 De Antoni, A., Pearson, C.G., Cimini, D., Canman, J.C., Sala, V., Nezi, L., Mapelli, M., Sironi, L., Faretta, M., Salmon, E.D. and Musacchio, A. (2005) The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint. Curr. Biol. 15, 214–225 62 Lad, L., Lichtsteiner, S., Hartman, J.J., Wood, K.W. and Sakowicz, R. (2009) Kinetic analysis of Mad2–Cdc20 formation: conformational changes in Mad2 are catalyzed by a C-Mad2–ligand complex. Biochemistry 48, 9503–9515 63 Simonetta, M., Manzoni, R., Mosca, R., Mapelli, M., Massimiliano, L., Vink, M., Novak, B., Musacchio, A. and Ciliberto, A. (2009) The influence of catalysis on mad2 activation dynamics. PLoS Biol. 7, e10 64 Tipton, A.R., Wang, K., Link, L., Bellizzi, J.J., Huang, H., Yen, T. and Liu, S.T. (2011) BUBR1 and closed MAD2 (C-MAD2) interact directly to assemble a functional mitotic checkpoint complex. J. Biol. Chem. 286, 21173–21179 65 Han, J.S., Holland, A.J., Fachinetti, D., Kulukian, A., Cetin, B. and Cleveland, D.W. (2013) Catalytic assembly of the mitotic checkpoint inhibitor BubR1–Cdc20 by a Mad2-induced functional switch in Cdc20. Mol. Cell 51, 92–104 66 Kulukian, A., Han, J.S. and Cleveland, D.W. (2009) Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding. Dev. Cell 16, 105–117 67 Sczaniecka, M., Feoktistova, A., May, K.M., Chen, J.S., Blyth, J., Gould, K.L. and Hardwick, K.G. (2008) The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C). J. Biol. Chem. 283, 23039–23047 68 Fang, G. (2002) Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex. Mol. Biol. Cell 13, 755–766 69 Hagting, A., Den Elzen, N., Vodermaier, H.C., Waizenegger, I.C., Peters, J.M. and Pines, J. (2002) Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1. J. Cell Biol. 157, 1125–1137 70 Kops, G.J. and Shah, J.V. (2012) Connecting up and clearing out: how kinetochore attachment silences the spindle assembly checkpoint. Chromosoma 121, 509–525
Received 8 October 2013 doi:10.1042/BST20130240
Copyright of Biochemical Society Transactions is the property of Portland Press Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
