KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) delays anaphase onset until kinetochores accomplish bioriented microtubule attachments [1]. Although several centromeric and kinetochore kinases, including Aurora B, regulate kinetochore-microtubule attachment and/or SAC activation [2-4], the molecular mechanism that translates bioriented attachment into SAC silencing remains unclear [5]. Employing a method to rapidly induce exact gene replacement in budding yeast [6], we show here that the binding of protein phosphatase 1?(PP1/Glc7) to the evolutionarily conserved RVSF motif of the kinetochore protein Spc105 (KNL1/Blinkin/CASC5) is essential for viability by silencing the SAC, while it plays an auxiliary nonessential role for physical chromosome segregation. Although Aurora B may inhibit this binding, persistent PP1-Spc105 interaction does not affect chromosome segregation and is insufficient to silence the SAC in the absence of microtubules, indicating that dynamic regulation of this?interaction is dispensable. However, the amount of PP1 targeted to kinetochores must be finely tuned, because recruitment of either no or one extra copy of PP1 to Spc105 is detrimental, illustrating the vital impact of targeting an exiguous fraction of PP1 to the kinetochore. We propose that the PP1-Spc105 interaction enables local regulation of dynamic phosphorylation and dephosphorylation at the kinetochore to couple microtubule attachment and SAC silencing.     

MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components

The genomic stability of all organisms depends on the precise partition of chromosomes to daughter cells. The spindle assembly checkpoint (SAC) senses unattached kinetochores and prevents premature entry to anaphase, thus ensuring that all chromosomes attach to opposite spindle poles (bi-orientation) during mitosis. MPS1 is an evolutionarily conserved protein kinase required for the SAC and chromosome bi-orientation. Yet, its primary cellular substrate has remained elusive. We show that fission yeast Mph1 (MPS1 homologue) phosphorylates the kinetochore protein Spc7 (KNL1/Blinkin homologue) at the MELT repeat sequences. This phosphorylation promotes the in vitro binding to the Bub1-Bub3 complex, which is required for kinetochore-based SAC activation (Mad1-Mad2-Mad3 localization) and chromosome alignment. Accordingly, a non-phosphorylatable spc7-12A mutation abolishes kinetochore targeting of Bub1-Bub3, whereas a phospho-mimetic spc7-12E mutation forces them to localize at kinetochores throughout the entire cell cycle, even in the absence of Mph1. Thus, MPS1/Mph1 kinase locating at the unattached kinetochores initially creates a mark, which is crucial for SAC activation and chromosome bi-orientation. This mechanism seems to be conserved in human cells.     

Structure and substrate recruitment of the human spindle checkpoint kinase Bub1

In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.     

Bub1 maintains centromeric cohesion by activation of the spindle checkpoint

Bub1 is a component of the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures genome stability by delaying anaphase until all the chromosomes are stably attached to spindle microtubules via their kinetochores. To define Bub1's role in chromosome segregation, embryogenesis, and tissue homeostasis, we generated a mouse strain in which BUB1 can be inactivated by administration of tamoxifen, thereby bypassing the preimplantation lethality associated with the Bub1 null phenotype. We show that Bub1 is essential for postimplantation embryogenesis and proliferation of primary embryonic fibroblasts. Bub1 inactivation in adult males inhibits proliferation in seminiferous tubules, reducing sperm production and causing infertility. In culture, Bub1-deficient fibroblasts fail to align their chromosomes or sustain SAC function, yielding a highly aberrant mitosis that prevents further cell divisions. Centromeres in Bub1-deficient cells also separate prematurely; however, we show that this is a consequence of SAC dysfunction rather than a direct role for Bub1 in protecting centromeric cohesion.     

KEN-box-dependent degradation of the Bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome

The spindle checkpoint is a cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a protein serine-threonine kinase that plays multiple roles in chromosome segregation and the spindle checkpoint. In response to misaligned chromosomes, Bub1 directly inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator Cdc20. The protein level and the kinase activity of Bub1 are regulated during the cell cycle; they peak in mitosis and are low in G1/S phase. Here we show that Bub1 is degraded during mitotic exit and that degradation of Bub1 is mediated by APC/C in complex with its activator Cdh1 (APC/C(Cdh1)). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1, whereas depletion of Cdh1 by RNA interference increases the level of the endogenous Bub1 protein. Bub1 is ubiquitinated by immunopurified APC/C(Cdh1) in vitro. We further identify two KEN-box motifs on Bub1 that are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells but fails to elicit a cell cycle phenotype, indicating that degradation of Bub1 by APC/C(Cdh1) is not required for mitotic exit. Nevertheless, our study clearly demonstrates that Bub1, an APC/C inhibitor, is also an APC/C substrate. The antagonistic relationship between Bub1 and APC/C may help to prevent the premature accumulation of Bub1 during G1.     

Bub1-mediated adaptation of the spindle checkpoint

During cell division, the spindle checkpoint ensures accurate chromosome segregation by monitoring the kinetochore-microtubule interaction and delaying the onset of anaphase until each pair of sister chromosomes is properly attached to microtubules. The spindle checkpoint is deactivated as chromosomes start moving toward the spindles in anaphase, but the mechanisms by which this deactivation and adaptation to prolonged mitotic arrest occur remain obscure. Our results strongly suggest that Cdc28-mediated phosphorylation of Bub1 at T566 plays an important role for the degradation of Bub1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest.     

Crystal structure of the spindle assembly checkpoint protein Bub3

Bub3 is one of at least six proteins that transmit the spindle assembly checkpoint signal. These proteins delay cell cycle progression from metaphase to anaphase in response to attachment defects between kinetochores and spindle microtubules and to tension defects between sister chromatids. To explore the molecular interactions mediated by Bub3, we have determined the crystal structure of the Saccharomyces cerevisiae protein Bub3p at 2.35 A resolution. Bub3p is a seven-blade beta-propeller, although its sequence diverges from that of other WD40 family members. Several loops are substantially elongated, but extra domains or insertions are not present at the termini. In particular, two extended loops project from the top face of the propeller, forming a cleft. Amino acid residues across the top face and one aspect of the lateral surface (spanning blades 5-6) are highly conserved among Bub3 proteins. We propose that these conserved surfaces are the loci for key interactions with conserved motifs in spindle checkpoint proteins Bub1 and Mad3/BubR1. Comparison of the Bub3 sequence to the WD40 protein, Rae1, shows high sequence conservation along the same surfaces. Rae1 interaction with Bub1 is, therefore, likely to involve a similar mode of binding.     

Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint

Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast. We have found that the spindle checkpoint kinase Bub1p becomes phosphorylated by Cdc2p during spindle damage in mitotic cells. Cdc2p directly phosphorylates Bub1p in vitro at the CDK consensus sites. A Bub1p mutant that cannot be phosphorylated by Cdc2p is checkpoint defective, indicating that Cdc2p-dependent Bub1p phosphorylation is required to activate the checkpoint after spindle damage. The kinase activity of Bub1p is required, but is not sufficient, for complete spindle checkpoint function. The role of Bub1p in maintaining centromeric localization of Rec8p during meiosis I is entirely dependent upon its kinase activity, suggesting that Bub1p kinase activity is essential for establishing proper kinetochore function. Finally, we show that there is a Bub1p-dependent meiotic checkpoint, which is activated in recombination mutants.     

Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR-KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.     

Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function

The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus (see [1] [2] [3] [4] for reviews). The Mad and Bub proteins were identified as key components of the spindle checkpoint through budding yeast genetics [5] [6] and are highly conserved [3]. Most of the spindle checkpoint proteins have been localised to kinetochores, yet almost nothing is known about the molecular events which take place there. Mad1p forms a tight complex with Mad2p [7], and has been shown to recruit Mad2p to kinetochores [8]. Similarly, Bub3p binds to Bub1p [9] and may target it to kinetochores [10]. Here, we show that budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and that this complex is found at significantly higher levels once the spindle checkpoint is activated. We find that formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, we identify a conserved motif within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex formation. Mutation of this motif abolishes checkpoint function, indicating that formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle checkpoint mechanism.     

Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint

Kinetochore attachment to the ends of dynamic microtubules is a conserved feature of mitotic spindle organization that is thought to be critical for proper chromosome segregation. Although kinetochores have been described to transition from lateral to end-on attachments, the phase of lateral attachment has been difficult to study in yeast due to its transient nature. We have previously described a kinetochore mutant, DAM1-765, which exhibits lateral attachments and misregulation of microtubule length. Here we show that the misregulation of microtubule length in DAM1-765 cells occurs despite localization of microtubule associated proteins Bik1, Stu2, Cin8 and Kip3 to microtubules. DAM1-765 kinetochores recruit the spindle checkpoint protein Bub1, however Bub1 localization to DAM1-765 kinetochores is not sufficient to cause a cell cycle arrest. Interestingly, the DAM1-765 mutation rescues the temperature sensitivity of a biorientationdeficient ipl1-321 mutant, and DAM1-765 chromosome loss rates are similar to wild-type cells. the spindle checkpoint in DAM1-765 cells responds properly to unattached kinetochores created by nocodazole treatment and loss of tension caused by a cohesin mutant. progression of DAM1-765 cells through mitosis therefore suggests that satisfaction of the checkpoint depends more highly on biorientation of sister kinetochores than on achievement of a specific interaction between kinetochores and microtubule plus ends.Key words: spindle assembly checkpoint, kinetochore-microtubule attachments, biorientation, DAM1-765     

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.     

Phosphorylation and activation of Bub1 on unattached chromosomes facilitate the spindle checkpoint

The spindle checkpoint inhibits anaphase until all kinetochores have attached properly to spindle microtubules. The protein kinase Bub1 is an essential checkpoint component that resides at kinetochores during mitosis. It is shown herein that Xenopus Bub1 becomes hyperphosphorylated and the kinase is activated on unattached chromosomes. MAP kinase (MAPK) contributes to this phosphorylation, as inhibiting MAPK or altering MAPK consensus sites in Bub1 to alanine or valine (Bub1(5AV)) abolishes the phosphorylation and activation on chromosomes. Both Bub1 and Bub1(5AV) support the checkpoint under an optimal condition for spindle checkpoint activation. However, Bub1, but not Bub1(5AV), supports the checkpoint at a relatively low concentration of nuclei or the microtubule inhibitor nocodazole. Similar to Bub1(5AV), Bub1 without the kinase domain (Bub1(deltaKD)) is also partially compromised in its checkpoint function and in its ability to recruit other checkpoint proteins to kinetochores. This study suggests that activation of Bub1 at kinetochores enhances the efficiency of the spindle checkpoint and is probably important in maintaining the checkpoint toward late prometaphase when the cell contains only a few or a single unattached kinetochore.     

A dual role for Bub1 in the spindle checkpoint and chromosome congression

The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore-microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. In this study, we use live-cell imaging coupled with RNA interference to investigate the functions of human Bub1. We find that Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore-microtubule attachments.     

Bub1 and BubR1: at the interface between chromosome attachment and the spindle checkpoint

The spindle checkpoint ensures genome fidelity by temporarily halting chromosome segregation and the ensuing mitotic exit until the last kinetochore is productively attached to the mitotic spindle. At the interface between proper chromosome attachment and the metaphase-to-anaphase transition are the mammalian spindle checkpoint kinases. Compelling evidence indicates that the checkpoint kinases Bub1 and BubR1 have the added task of regulating kinetochore-microtubule attachments. However, the debate on the requirement of kinase activity is in full swing. This minireview summarizes recent advances in our understanding of the core spindle checkpoint kinases Bub1 and BubR1 and considers evidence that supports and opposes the role of kinase activity in regulating their functions during mitosis.     

Mal3, the fission yeast EB1 homologue, cooperates with Bub1 spindle checkpoint to prevent monopolar attachment

Bipolar microtubule attachment is central to genome stability. Here, we investigate the mitotic role of the fission yeast EB1 homologue Mal3. Mal3 shows dynamic inward movement along the spindle, initial emergence at the spindle pole body (SPB) and translocation towards the equatorial plane, followed by sudden disappearance. Deletion of Mal3 results in early mitotic delay, which is dependent on the Bub1, but not the Mad2, spindle checkpoint. Consistently, Bub1, but not Mad2, shows prolonged kinetochore localization. Double mutants between mal3 and a subset of checkpoint mutants, including bub1, bub3, mad3 and mph1, but not mad1 or mad2, show massive chromosome mis-segregation defects. In mal3bub1 mutants, both sister centromeres tend to remain in close proximity to one of the separating SPBs. Further analysis indicates that mis-segregated centromeres are exclusively associated with the mother SPB. Mal3, therefore, has a role in preventing monopolar attachment in cooperation with the Bub1/Bub3/Mad3/Mph1-dependent checkpoint.     

Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores

Dynamic attachment of microtubules to kinetochores during mitosis generates pulling force, or tension, required for the high fidelity of chromosome separation. A lack of tension activates the spindle checkpoint and delays the anaphase onset. A key step in the tension-response pathway involves the phosphorylation of the 3F3/2 epitope by an unknown kinase on untensed kinetochores. Using a rephosphorylation assay in Xenopus laevis extracts, we identified the kinetochore-associated Polo-like kinase Plx1 as the kinase both necessary and sufficient for this phosphorylation. Indeed, Plx1 is the physiological 3F3/2 kinase involved in checkpoint response, as immunodepletion of Plx1 from checkpoint extracts abolished the 3F3/2 signal and blocked association of xMad2, xBubR1, xNdc80, and xNuf2 with kinetochores. Interestingly, the kinetochore localization of Plx1 is under the control of the checkpoint protein xMps1, as immunodepletion of xMps1 prevents binding of Plx1 to kinetochores. Thus, Plx1 couples the tension signal to cellular responses through phosphorylating the 3F3/2 epitope and targeting structural and checkpoint proteins to kinetochores.     

Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores

Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.