BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.     

Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint

The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase in response to misaligned sister chromatids during mitosis. Upon checkpoint activation, Mad2 binds directly to Cdc20 and inhibits the anaphase-promoting complex or cyclosome (APC/C). Cdc20 binding triggers a dramatic conformational change of Mad2. Consistent with an earlier report, we show herein that depletion of p31(comet) (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31(comet) protein antagonizes the ability of Mad2 to inhibit APC/C(Cdc20) in vitro and in Xenopus egg extracts. Interestingly, p31(comet) binds selectively to the Cdc20-bound conformation of Mad2. Binding of p31(comet) to Mad2 does not prevent the interaction between Mad2 and Cdc20 in vitro. During checkpoint inactivation in HeLa cells, p31(comet) forms a transient complex with APC/C(Cdc20)-bound Mad2. Purified p31(comet) enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2-Cdc20 interaction. Therefore, our results suggest that p31(comet) counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint.     

Spindle checkpoint protein Bub1 is required for kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase activity

The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1-Mad2. However, Bub1 differs from Mad1-Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.     

Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31(comet)

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase-anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31(comet) delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31(comet) and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.     

Identification of an Overlapping Binding Domain on Cdc20 for Mad2 and Anaphase-Promoting Complex: Model for Spindle Checkpoint Regulation

Activation of the anaphase-promoting complex (APC) is required for anaphase initiation and for exit from mitosis in mammalian cells. Cdc20, which specifically recognizes APC substrates involved in the metaphase-to-anaphase transition, plays a pivotal role in APC activation through direct interaction with the APC. The activation of the APC by Cdc20 is prevented by the interaction of Cdc20 with Mad2 when the spindle checkpoint is activated. Using deletion mutagenesis and peptide mapping, we have identified the sequences in Cdc20 that target it to Mad2 and the APC, respectively. These sequences are distinct but overlapping, providing a possible structural explanation for the internal modulation of the APC-Cdc20 complex by Mad2. In the course of these studies, a truncation mutant of Cdc20 (1-153) that constitutively binds Mad2 but fails to bind the APC was identified. Overexpression of this mutant induces the formation of multinucleated cells and increases their susceptibility to undergoing apoptosis when treated with microtubule-inhibiting drugs. Our experiments demonstrate that disruption of the Mad2-Cdc20 interaction perturbs the mitotic checkpoint, leading to premature activation of the APC, sensitizing the cells to the cytotoxic effects of microtubule-inhibiting drugs.     

C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast <Emphasis Type="Italic">S</Emphasis>c<Emphasis Type="Italic">hizosaccharomyces pombe</Emphasis>

The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-?CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.     

Kinase activity of fission yeast Mph1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6-8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.     

Mad2 phosphorylation regulates its association with Mad1 and the APC/C

Improper attachment of the mitotic spindle to the kinetochores of paired sister chromatids in mitosis is monitored by a checkpoint that leads to an arrest in early metaphase. This arrest requires the inhibitory association of Mad2 with the anaphase promoting complex/cyclosome (APC/C). It is not known how the association of Mad2 with the kinetochore and the APC/C is regulated in mitosis. Here, we demonstrate that human Mad2 is modified through phosphorylation on multiple serine residues in vivo in a cell cycle dependent manner and that only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo. A Mad2 mutant containing serine to aspartic acid mutations mimicking the C-terminal phosphorylation events fails to interact with Mad1 or the APC/C and acts as a dominant-negative antagonist of wild-type Mad2. These data suggest that the phosphorylation state of Mad2 regulates its checkpoint activity by modulating its association with Mad1 and the APC/C.     

The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction

The spindle assembly checkpoint (SAC) is required to block sister chromatid separation until all chromosomes are properly attached to the mitotic apparatus. The SAC prevents cells from entering anaphase by inhibiting the ubiquitylation of cyclin B1 and securin by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The target of the SAC is the essential APC/C activator Cdc20. It is unclear how the SAC inactivates Cdc20 but most current models suggest that Cdc20 forms a stable complex with the Mad2 checkpoint protein. Here we show that most Cdc20 is not in a complex with Mad2; instead Mad2 is required for Cdc20 to form a complex with another checkpoint protein, BubR1. We further show that during the SAC, the APC/C ubiquitylates Cdc20 to target it for degradation. Thus, ubiquitylation of human Cdc20 is not required to release it from the checkpoint complex, but to degrade it to maintain mitotic arrest.     

Evidence that the spindle assembly checkpoint does not regulate APC(Fzy) activity in Drosophila female meiosis

The spindle assembly checkpoint (SAC) plays an important role in mitotic cells to sense improper chromosome attachment to spindle microtubules and to inhibit APC(Fzy)-dependent destruction of cyclin B and Securin; consequent initiation of anaphase until correct attachments are made. In Drosophila , SAC genes have been found to play a role in ensuring proper chromosome segregation in meiosis, possibly reflecting a similar role for the SAC in APC(Fzy) inhibition during meiosis. We found that loss of function mutations in SAC genes, Mad2, zwilch, and mps1, do not lead to the predicted rise in APC(Fzy)-dependent degradation of cyclin B either globally throughout the egg or locally on the meiotic spindle. Further, the SAC is not responsible for the inability of APC(Fzy) to target cyclin B and promote anaphase in metaphase II arrested eggs from cort mutant females. Our findings support the argument that SAC proteins play checkpoint independent roles in Drosophila female meiosis and that other mechanisms must function to control APC activity.     

Structural activation of Mad2 in the mitotic spindle checkpoint: the two-state Mad2 model versus the Mad2 template model

The inheritance of a normal assortment of chromosomes during each cell division relies on a cell-cycle surveillance system called the mitotic spindle checkpoint. The existence of sister chromatids that do not achieve proper bipolar attachment to the mitotic spindle in a cell activates this checkpoint, which inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C) and delays the onset of anaphase. The mitotic arrest deficiency 2 (Mad2) spindle checkpoint protein inhibits APC/C through binding to its mitotic-specific activator, Cdc20. Binding of Mad2 to Cdc20 involves a large conformational change of Mad2 and requires the Mad1-Mad2 interaction in vivo. Two related but distinct models of Mad1-assisted activation of Mad2, the "two-state Mad2" and the "Mad2 template" models, have been proposed. I review the recent structural, biochemical, and cell biological data on Mad2, discuss the differences between the two models, and propose experiments that test their key principles.     

The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.     

Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests

The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1^Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1^Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20^Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1^Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.     

Budding yeast Bub2 is localized at spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2.

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.     

Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint

The mitotic checkpoint monitors kinetochore–microtubule attachment and prevents anaphase until all kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to kinetochores. Unattached, checkpoint-active kinetochores accumulate multiple checkpoint proteins, which are depleted from kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached kinetochores, it is not known what changes at kinetochores are essential for anaphase promoting complex/cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, we show that recruiting the checkpoint protein Mad1 to metaphase kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 or BubR1. Furthermore, Mad2 binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of our checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at kinetochores.     

Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble 'degron' signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.     

Kinetochore Localization of Spindle Checkpoint Proteins: Who Controls Whom?

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.     

The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C)

Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.     

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.     

Regulation of Chk1

The execution of the mitotic program with high fidelity is dependent upon precise spatiotemporal regulation of posttranslational protein modifications. For example, the timely polyubiquitination of critical mitotic regulators by Anaphase Promoting Complex/Cyclosome (APC/C) is essential for the metaphase to anaphase transition and mitotic exit. The spindle assembly checkpoint prevents unscheduled activity of APC/C-Cdc20 in early mitosis, allowing bipolar attachment of kinetochores to mitotic spindle and facilitating equal segregation of sister chromatids. The critical effector of the spindle checkpoint, Mitotic arrest deficient 2 (Mad2), is recruited to unattached kinetochores forming a complex with other regulatory proteins to efficiently and cooperatively inhibit APC/C-Cdc20. A weakened and/or dysfunctional spindle checkpoint has been linked to the development of genomic instability in both cell culture and animal models, and evidence suggests that aberrant regulation of the spindle checkpoint plays a critical role in human carcinogenesis. Recent studies have illuminated a network of both degradative and non-degradative ubiquitination events that regulate the metaphase to anaphase transition and mitotic exit. Within this context, our recent work showed that the HECT (Homologous to E6-AP C-terminus)-family E3 ligase Smurf2 (Smad specific ubiquitin regulatory factor 2), known as a negative regulator of transforming growth factor-beta (TGF-β) signaling, is required for a functional spindle checkpoint by promoting the functional localization and stability of Mad2. Here we discuss putative models explaining the role of Smurf2 as a new regulator in the spindle checkpoint. The dynamic mitotic localization of Smurf2 to the centrosome and other critical mitotic structures provides implications about mitotic checkpoint control dependent on various ubiquitination events. Finally, deregulated Smurf2 activity may contribute to carcinogenesis by perturbed mitotic control.