Chk1 is required for spindle checkpoint function

The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Here, we show that Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. We propose that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling.     

Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing

BubR1 is an important component of the spindle assembly checkpoint, and deregulated BubR1 functions frequently result in chromosomal instability and malignant transformation. We recently demonstrated that BubR1 was modified by sumoylation, and that lysine 250 (K250) functions as the crucial site for this modification. BubR1 sumoylation was neither required for its activation nor for binding to kinetochores. However, ectopically expressed sumoylation-deficient BubR1 mutants were retained on the kintochores even after apparent chromosome congression. The kinetochore retention of the sumoylation-deficient mutant of BubR1 caused an anaphase delay coupled with premature sister chromatid separation. Moreover, BubR1 interacted with unphosphorylated Sgo1, and its sumoylation facilitated the interaction. BubR1 sumoylation was inversely associated with its acetylation during mitotic progression. Trichostatin A, a protein deacetylase inhibitor, significantly compromised BubR1 sumoylation. Combined, these results reveal that BubR1 sumoylation plays an important role in its timely removal from the kinetochores and the checkpoint inactivation, thus allowing normal anaphase entry and chromosome segregation.Key words: BubR1, sumoylation, kinetochores, centromeric cohesion, spindle checkpoint, Sgo1     

Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing

BubR1 is an important component of the spindle assembly checkpoint, and deregulated BubR1 functions frequently result in chromosomal instability and malignant transformation. We recently demonstrated that BubR1 was modified by sumoylation, and that lysine 250 (K250) functions as the crucial site for this modification. BubR1 sumoylation was neither required for its activation nor for binding to kinetochores. However, ectopically expressed sumoylation-deficient BubR1 mutants were retained on the kintochores even after apparent chromosome congression. The kinetochore retention of the sumoylation-deficient mutant of BubR1 caused an anaphase delay coupled with premature sister chromatid separation. Moreover, BubR1 interacted with unphosphorylated Sgo1, and its sumoylation facilitated the interaction. BubR1 sumoylation was inversely associated with its acetylation during mitotic progression. Trichostatin A, a protein deacetylase inhibitor, significantly compromised BubR1 sumoylation. Combined, these results reveal that BubR1 sumoylation plays an important role in its timely removal from the kinetochores and the checkpoint inactivation, thus allowing normal anaphase entry and chromosome segregation.     

BubR1 deficiency results in enhanced activation of MEK and ERKs upon microtubule stresses

Disruption of microtubules activates the spindle checkpoint, of which BubR1 is a major component. Our early studies show that BubR1 haplo-insufficiency results in enhanced mitotic slippage in vitro and tumorigenesis in vivo. OBJECTIVE: Given that both MAPKs/ERKs and MEK play an important role during mitosis, we investigated whether there existed regulatory relationship between the MAPK signalling pathway and BubR1. METHOD AND RESULTS: Here, we have demonstrated that BubR1 deficiency is correlated with enhanced activation of MEK and ERKs after disruption of microtubule dynamics. Specifically, treatment with nocodazole and paclitaxel resulted in hyper-activation of ERKs and MEK in BubR1(+/-) murine embryonic fibroblasts (MEF) compared to that of wild-type MEFs. This enhanced activation of ERKs and MEK was at least partly responsible for more successful proliferation completion when cells were treated with nocodazole. BubR1 knockdown via RNAi resulted in enhanced activation of ERKs and MEK in HeLa cells, correlating with inhibition of PP1, a negative regulator of MEK. Moreover, when BubR1 was partially inactivated due to premature missegregation of chromosomes after Sgo1 depletion, phosphorylation of ERKs and MEK was enhanced in mitotic cells; in contrast, little, if any activated ERKs and MEK were detected in mitotic cells induced by nocodazole. Furthermore, BubR1, activated ERKs and activated MEK all localized to spindle poles during mitosis, and also, the proteins physically interacted with each other. CONCLUSION: Our studies suggest that there exists a cross-talk between spindle checkpoint components and ERKs and MEK and that BubR1 may play an important role in mediating the cross-talk.     

CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.     

Microtubule capture by CENP-E silences BubR1-dependent mitotic checkpoint signaling

The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other components to generate a diffusible "stop anaphase" inhibitor. Not only does direct binding of BubR1 to the centromere-associated kinesin family member CENP-E activate its essential kinase, binding of a motorless fragment of CENP-E is shown here to constitutively activate BubR1 bound at kinetochores, producing checkpoint signaling that is not silenced either by spindle microtubule capture or the tension developed at those kinetochores by other components. Using purified BubR1, microtubules, and CENP-E, microtubule capture by the CENP-E motor domain is shown to silence BubR1 kinase activity in a ternary complex of BubR1-CENP-E-microtubule. Together, this reveals that CENP-E is the signal transducing linker responsible for silencing BubR1-dependent mitotic checkpoint signaling through its capture at kinetochores of spindle microtubules.     

Re-evaluating the role of Tao1 in the spindle checkpoint

The spindle checkpoint restrains anaphase onset and mitotic exit until all chromosomes are stably attached to the mitotic spindle via their kinetochores. The Tao1 protein kinase was recently reported as a novel spindle checkpoint component. When an siRNA was used to repress Tao1, the essential spindle checkpoint component Mad2 failed to localise to kinetochores, and cells rapidly exited mitosis. Tao1 was also shown to interact with BubR1, another essential checkpoint component, and be rapidly degraded after mitosis, a feature typical of many mitotic regulators. Here, we identify four different siRNAs that repress Tao1 protein levels as efficiently as the previously reported siRNA. However, these siRNAs do not override the spindle checkpoint. We also present data indicating that Tao1 does not interact with BubR1 and that it is not rapidly degraded after mitosis. We show that the previously reported siRNA not only represses Tao1 but also dramatically reduces Mad2 protein levels. Crucially, expression of exogenous Mad2, but not Tao1, rescued the spindle checkpoint phenotype induced by this siRNA. Thus, the key functional data implicating Tao1 in the spindle checkpoint can be explained by an off-target siRNA phenomenon that results in Mad2 inhibition. Taken together, our data do not support the notion that Tao1 is a component of the spindle checkpoint.     

Cdk1 phosphorylation of BubR1 controls spindle checkpoint arrest and Plk1-mediated formation of the 3F3/2 epitope

Accurate chromosome segregation is controlled by the spindle checkpoint, which senses kinetochore– microtubule attachments and tension across sister kinetochores. An important step in the tension-signaling pathway involves the phosphorylation of an unknown protein by polo-like kinase 1/Xenopus laevis polo-like kinase 1 (Plx1) on kinetochores lacking tension to generate the 3F3/2 phosphoepitope. We report here that the checkpoint protein BubR1 interacts with Plx1 and that phosphorylation of BubR1 by Plx1 generates the 3F3/2 epitope. Formation of the BubR1 3F3/2 epitope by Plx1 requires a prior phosphorylation of BubR1 on Thr 605 by cyclin-dependant kinase 1 (Cdk1). This priming phosphorylation of BubR1 by Cdk1 is required for checkpoint-mediated mitotic arrest and for recruitment of Plx1 and the checkpoint protein Mad2 to unattached kinetochores. Biochemically, formation of the 3F3/2 phosphoepitope by Cdk1 and Plx1 greatly enhances the kinase activity of BubR1. Thus, Cdk1-mediated phosphorylation of BubR1 controls checkpoint arrest and promotes the formation of the kinetochore 3F3/2 epitope.     

Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Delta579Delta610) was resistant to paclitaxel-induced degradation. Expression of BubR1Delta579Delta610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.     

CENP-E--dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint

How the state of spindle microtubule capture at the kinetochore is translated into mitotic checkpoint signaling remains largely unknown. In this paper, we demonstrate that the kinetochore-associated mitotic kinase BubR1 phosphorylates itself in human cells and that this autophosphorylation is dependent on its binding partner, the kinetochore motor CENP-E. This CENP-E-dependent BubR1 autophosphorylation at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single chromosome loss. Replacing endogenous BubR1 with a nonphosphorylatable BubR1 mutant, as well as depletion of CENP-E, the BubR1 kinase activator, results in metaphase chromosome misalignment and a decrease of Aurora B-mediated Ndc80 phosphorylation at kinetochores. Furthermore, expressing a phosphomimetic BubR1 mutant substantially reduces the incidence of polar chromosomes in CENP-E-depleted cells. Thus, the state of CENP-E-dependent BubR1 autophosphorylation in response to spindle microtubule capture by CENP-E is important for kinetochore function in achieving accurate chromosome segregation.     

Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension

Genetic evidence is mounting that survivin plays a crucial role in mitosis, but its exact role in human cell division remains elusive. We show that mammalian cells lacking survivin are unable to align their chromosomes, fail to recruit Aurora B to kinetochores and become polyploid at a very high frequency. Survivin-depleted cells enter mitosis with normal kinetics, but are delayed in prometaphase in a BubR1/Mad2-dependent fashion. Nonetheless, these cells exit mitosis prior to completion of chromosome congression and without sister chromatid segregation, indicating that the spindle assembly checkpoint is not fully functional. Indeed, in survivin-depleted cells, BubR1 and Mad2 are prematurely displaced from kinetochores, yet no tension is generated at kinetochores. Importantly, these cells fail to respond to drugs that prevent tension, but do arrest in mitosis after depolymerization of the mitotic spindle. This demonstrates that survivin is not required for initial checkpoint activation, or for sustained checkpoint activation by loss of microtubules. However, stable association of BubR1 to kinetochores and sustained checkpoint signalling in response to lack of tension crucially depend on survivin.     

The human mitotic checkpoint protein BubR1 regulates chromosome-spindle attachments

Loss or gain of whole chromosomes, the form of chromosomal instability (CIN) most commonly associated with human cancers, is expected to arise from the failure to accurately segregate chromosomes in mitosis. The mitotic checkpoint is one pathway that prevents segregation errors by blocking the onset of anaphase until all chromosomes make proper attachments to the spindle. Another process that prevents errors is stabilization and destabilization of connections between chromosomes and spindle microtubules. An outstanding question is how these two pathways are coordinated to ensure accurate chromosome segregation. Here we show that in human cells depleted of BubR1 - a critical component of the mitotic checkpoint that can directly regulate the onset of anaphase - chromosomes do not form stable attachments to spindle microtubules. Attachments in these cells are restored by inhibition of Aurora kinase, which is known to stabilize kinetochore-microtubule attachments. Loss of BubR1 function thus perturbs regulation of attachments rather than the ability of kinetochores to bind to microtubules. Consistent with this finding, depletion of BubR1 increases phosphorylation of CENP-A, a kinetochore-specific Aurora kinase substrate. We propose that BubR1 links regulation of chromosome-spindle attachment to mitotic checkpoint signalling.     

The mitotic checkpoint gene BubR1 has two distinct functions in mitosis

BubR1 is one of two putative vertebrate homologs of the yeast spindle checkpoint protein Bub1. We have used deletion and point mutants to elucidate the functions of BubR1 in mitosis. The nocodazole-activated spindle checkpoint of HeLa cells was disrupted by expression of a 39 amino acid fragment (residues 382-420) of BubR1 containing the Bub3-binding GLEBS motif. In contrast, we observed normal checkpoint function in a truncation mutant comprising residues 1-477, despite the lack of the C-terminal BubR1 kinase domain. In the absence of nocodazole, expression of the 477 amino acid fragment slowed progress through prometaphase of mitosis, causing accumulation of mitotic cells. This accumulation was also seen in a kinase dead mutant. The prolongation of mitosis required both kinetochore binding and an intact, functional spindle checkpoint. The prolongation of mitosis by kinase deficient BubR1 constructs indicates a crucial role for the BubR1 C-terminal kinase domain in chromosome movement, in addition to the role of the N-terminus in the checkpoint.     

Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.     

Regulation of APC-Cdc20 by the spindle checkpoint

The spindle checkpoint ensures the fidelity of chromosome segregation in mitosis and meiosis. In response to defects in the mitotic apparatus, it blocks the activity of the anaphase-promoting complex, a large ubiquitin ligase required for chromosome segregation. Recent studies indicate that the spindle checkpoint monitors both the attachment of chromosomes to the mitotic spindle and the tension across the sister chromatid generated by microtubules. Upon checkpoint activation, checkpoint protein complexes containing BubR1(Mad3), Bub3, Mad2 and Cdc20 directly bind to the anaphase-promoting complex and inhibit its ligase activity. Therefore, the checkpoint proteins form a complex intracellular signalling network to inhibit the anaphase-promoting complex.     

Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest in Drosophila melanogaster

The spindle checkpoint is a surveillance mechanism that regulates the metaphase-anaphase transition during somatic cell division through inhibition of the APC/C ensuring proper chromosome segregation. We show that the conserved spindle checkpoint protein BubR1 is required during early embryonic development. BubR1 is maternally provided and localises to kinetochores from prophase to metaphase during syncytial divisions similarly to somatic cells. To determine BubR1 function during embryogenesis, we generated a new hypomorphic semi-viable female sterile allele. Mutant females lay eggs containing undetectable levels of BubR1 show early developmental arrest, abnormal syncytial nuclear divisions, defects in chromosome congression, premature sister chromatids separation, irregular chromosome distribution and asynchronous divisions. Nuclei in BubR1 mutant embryos do not arrest in response to spindle damage suggesting that BubR1 performs a checkpoint function during syncytial divisions. Furthermore, we find that in wild-type embryos BubR1 localises to the kinetochores of condensed polar body chromosomes. This localisation is functional because in mutant embryos, polar body chromatin undergoes cycles of condensation-decondensation with additional rounds of DNA replication. Our results suggest that BubR1 is required for normal synchrony and progression of syncytial nuclei through mitosis and to maintain the mitotic arrest of the polar body chromosomes after completion of meiosis.     

Mad2-Independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1.

The mitotic checkpoint blocks the activation of the anaphase-promoting complex (APC) until all sister chromatids have achieved bipolar attachment to the spindle. A checkpoint complex containing BubR1 and Bub3 has been purified from mitotic human cells. Upon checkpoint activation, the BubR1-Bub3 complex interacts with Cdc20. In the absence of Mad2, BubR1 inhibits the activity of APC by blocking the binding of Cdc20 to APC. Surprisingly, the kinase activity of BubR1 is not required for the inhibition of APCCdc20. BubR1 also prevents the activation of APCCdc20 in Xenopus egg extracts, and restores the mitotic arrest in Cdc20-overexpressing cells treated with nocodazole. Because BubR1 also interacts with the mitotic motor CENP-E, the ability of BubR1 to inhibit APC may be regulated by kinetochore tension or occupancy.     

Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex

The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC.     

Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint

The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis.