Protein Kinases Involved in Mitotic Spindle Checkpoint Regulation

Kaldis: Cell Cycle RegulationDOI 10.1007/b138827/Published online: 23 September 2005 © Springer-Verlag Berlin Heidelberg 2005 Protein Kinases Involved in Mitotic Spindle Checkpoint Regul

P Kaldis: Cell Cycle Regulation

DOI 10.1007/b138827/Published online: 23 September 2005

© Springer-Verlag Berlin Heidelberg 2005

Protein Kinases Involved in Mitotic Spindle

Checkpoint Regulation

Ingrid Hoffmann

Cell Cycle Control and Carcinogenesis (F045), German Cancer Research Center (DKFZ),

Im Neuenheimer Feld 242, 69120 Heidelberg, Germany

Ingrid.Hoffmann@dkfz.de

Abstract A number of checkpoint controls function to preserve the genome by restraining cell cycle progression until prerequisite events have been properly completed Chromo-some attachment to the mitotic spindle is monitored by the spindle assembly checkpoint Sister chromatid separation in anaphase is initiated only once all chromosomes have been attached to both poles of the spindle Premature separation of sister chromatids leads to the loss or gain of chromosomes in daughter cells (aneuploidy), a prevalent form of ge-netic instability of human cancer The spindle assembly checkpoint ensures that cells with misaligned chromosomes do not exit mitosis and divide to form aneuploid cells A num-ber of protein kinases and checkpoint phosphoproteins are required for the function of the spindle assembly checkpoint This review discusses the recent progress in under-standing the role of protein kinases of the mitotic checkpoint complex in the surveillance pathway of the checkpoint.

1

Introduction

During cell division, accurate transmission of the genome is essential for survival Entry into mitosis is controlled by checkpoints that monitor DNA damage and replication, whereas exit from mitosis is controlled by check-points that monitor assembly and position of the mitotic spindle The mitotic spindle checkpoint is activated by the lack of microtubule occupancy and ten-sion at the kinetochores and leads to cell cycle arrest in prometaphase It

is a tightly conserved signal transduction pathway that prevents sister chro-matid separation until all chromosomes achieve bipolar attachment to the mitotic spindle The presence of even a single misaligned or unattached chro-mosome is sufficient to activate the checkpoint In response to defects in the mitotic apparatus, it blocks the activity of the anaphase-promoting com-plex or cyclosome (APC/C), a large multisubunit ubiquitin ligase required

for chromosome segregation After all sister chromatids have achieved bi-orientation, the APC/C in association with one of its substrate-binding

cofac-tors, Cdc20, tags the anaphase-inhibiting protein securin with polyubiquitin chains, leading to its degradation by the proteasome (Peters 2002; Harper

et al 2002) Sister chromatids are held together by cohesin and cleavage of

cohesin will result in loss of sister chromatid cohesion and the onset of sis-ter chromatid separation (Nasmyth 2002) Degradation of securin activates separase, a protease which cleaves the Scc1 subunit of cohesin

2

The Spindle Assembly Checkpoint

The molecular components of the spindle assembly checkpoint were

identi-fied initially in Saccharomyces cerevisiae (Gorbsky 2001; Shah and Cleveland

2000) They include Mad1-3 (mitotic arrest deficiency) (Li and Murray 1991), Bub1-3 (Hoyt et al 1991; Roberts et al 1994), and Mps1 (Weiss and Winey 1996) (Table 1) Homologues of these checkpoint proteins were later found

in other organisms, including mammals Checkpoint proteins accumulate at unattached kinetochores in prometaphase, but disappear from kinetochores later in mitosis or meiosis upon microtubule attachment and tension In higher eukaryotes the checkpoint control proteins comprise Mad1, Mad2, Bub3 and the protein kinases Bub1, BubR1 (Mad3 in budding yeast), and Mps1 In add-ition to these basic checkpoint components, other proteins such as CENP-E (a member of the kinesin superfamily) (Abrieu et al 2000; Yao et al 2000), Rod, ZW10 (Chan et al 2000), Aurora B (Biggins and Murray 2001; Kallio et al 2002; Ditchfield et al 2003) and mitogen-activated protein kinase (MAPK) (Shapiro

et al 1998; Zecevic et al 1998) play a role in the spindle checkpoint (Table 1)

Table 1 Proteins involved in mitotic spindle checkpoint regulation

Protein Proposed function in the spindle assembly checkpoint

Mad1 Coiled-coil protein, binds to Mad2 and recruits Mad2 to kinetochores

phosphorylated by Mps1 and Bub1 upon checkpoint activation Mad2 Binds to Mad1, binds and inhibits APC/CCdc20

BubR1 (Mad3) Protein kinase, binds to Bub3 and APC/CCdc20 , binds to the mitotic

motor protein CENP-E Bub1 Protein kinase, binds to and recruits Bub3, Mad1 and Mad2

Bub3 Contains WD-40 repeats, binds to Bub1 and BubR1

Mps1 Protein kinase, essential for establishment and maintenance of the

spindle checkpoint Aurora B Protein kinase, binds to INCENP, other substrates: CENP-A, Rec8,

vimentin, desmin, the kinesin MCAK, histone H3 Aurora C Protein kinase, binds to INCENP and Aurora B

MAPK Protein kinase

Rod Identified in Drosophila, binds to Zw10

Zw10 Identified in Drosophila, binds to Rod

Subcellular localization studies have placed all these checkpoint proteins at the kinetochores Ablation or suppression of function of any of these proteins substantially compromises mitotic checkpoint control (Lew and Burke 2003) These checkpoint control proteins form a complex intracellular network, the mitotic checkpoint complex (MCC), to block the action of APC/CCdc20 Mad2 interacts with other components of the spindle checkpoint and plays

a key role in the signaling pathway of the checkpoint In interphase, it binds to Mad1 and is preferentially found on the nuclear periphery (Chen et al 1999) This localization is strictly dependent on Mad1 since in a fission yeast strain lacking Mad1, Mad2 is no longer found on the nuclear periphery (Ikui et al 2002) Upon the onset of mitosis, Mad2 translocates into the nucleus and is guided to unattached kinetochores by Mad1 (Chen et al 1999) Recruiting Mad2 to kinetochores is the only known function of Mad1 to date From early mitosis on, Mad2 is found in a complex with its target, Cdc20 Human Mad2

is modified through phosphorylation on multiple serine residues in vivo in

a cell cycle-dependent manner Only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo (Wassmann et al 2003) Injection of anti-Mad2

antibodies drives prophase cells into a premature anaphase and overrides the arrest induced by microtubule depolymerization, indicating that the check-point activation in situations with unattached kinetochores requires Mad2 (Gorbsky et al 1998) A Mad2 mutant containing serine to aspartic acid muta-tions 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 (Wassmann et al 2003) Although yeast strains lacking Mad2 are vi-able, deletion of Mad2 in mouse causes cell lethality Mad2-/- mouse cells do

not arrest in response to spindle damage, show widespread chromosome mis-segregation, and undergo apoptosis during initiation of gastrulation (Dobles

et al 2000)

Upon checkpoint activation, both BubR1-Bub3 and Mad2 are capable of blocking the activity of APC/C through their direct binding to Cdc20 (Yu

2002; Bharadwaj and Yu 2004) Binding of spindle microtubules to kine-tochores, disrupts the interaction between Mad1 and Mad2 and ultimately disables the arrest (Fig 1a)

3

Regulation of the Spindle Checkpoint by Protein Kinases

3.1

Bub1

Bub1 is a protein kinase and an essential checkpoint component that re-sides at kinetochores during mitosis Bub1 was first described in a genetic screen searching for budding yeast mutants that were sensitive to the

spin-Fig 1 Functions of protein kinases in the spindle checkpoint Attachment of chromo-somes to the mitotic spindle is monitored by the spindle checkpoint Sensing mechanisms may involve Aurora B/Ipl1 and CENP-E Upon checkpoint activation both BubR1-Bub3

and Mad2 interact with Cdc20 and lead to an inhibition of APC Inactivation of the checkpoint occurs upon bipolar attachment to the mitotic spindle APC is activated and ubiquitinates securin Degradation of securin activates a protease called separase which cleaves cohesin, resulting in a loss of sister chromatid cohesion, leading to the the onset

of anaphase

dle poison benomyl (Hoyt et al 1991) Bub1 binds to Bub3 throughout the cell cycle and phosphorylates Bub3 in vitro (Roberts et al 1994) Overexpression

of a dominant allele of Bub1 in yeast causes a mitotic delay without spin-dle damage that is dependent on the functions of Bub2, Bub3, and Mad1-3 (Farr and Hoyt 1998) In yeast, the Bub1–Bub3 complex interacts with Mad1 when the spindle checkpoint is activated (Brady and Hardwick 2000) In ver-tebrates, Bub1 is required for the kinetochore localization of Mad1 and Mad2 (Sharp-Baker and Chen 2001; Johnson et al 2004; Vigneron et al 2004) This activity of Bub1 seems to be independent of its kinase activity since a kinase-inactive mutant of Bub1 is fully capable of recruiting Mad1 and Mad2 to

the kinetochores in Xenopus egg extracts (Sharp-Baker and Chen 2001)

Im-munodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1-3, Bub3, BubR1 and the kineto-chore motor protein CENP-E (Sharp-Baker and Chen 2001; Johnson et al 2004) Recently, it was shown that mammalian Bub1 also has a downstream function in the spindle checkpoint since it directly phosphorylates Cdc20 (Tang et al 2004) HeLa cells depleted for Bub1 by RNA interference (RNAi) are defective in checkpoint signaling Bub1 directly phosphorylates Cdc20

in vitro and in vivo and inhibits the ubiquitin ligase activity of APC/CCdc20 catalytically (Tang et al 2004) Six Ser/Thr residues in Cdc20 were

phospho-rylated by Bub1 in vitro Ectopic expression of a Cdc20 protein where all six residues were mutated to alanine is refractory to Bub1-mediated phosphory-lation and inhibition Overexpression of this Cdc20 mutant protein impairs the function of the spindle checkpoint Bub1 function seems to be regulated

by several upstream kinases Bub1 becomes hyperphosphorylated and its ki-nase activity is induced specifically at unattached chromosomes (Chen 2004) MAPK contributes to this phosphorylation, as inhibiting MAPK or altering MAPK consensus sites in Bub1 abolishes the phosphorylation and activation

on chromosomes The activation of Bub1 seems to be important in maintain-ing the checkpoint towards late prometaphase when the cell contains only

a few kinetochores or a single unattached kinetochore It has been shown that the MAPK downstream target Rsk activates cytosolic Bub1 during frog oocyte maturation (Schwab et al 2001), but Rsk does not seem to be in-volved in Bub1 phosphorylation at kinetochores, because immunodepletion

of Rsk does not have an effect on the phosphorylation Fission yeast Bub1

is phosphorylated during mitosis and the protein is a substrate for Cdc2 (Yamaguchi et al 2003) Mutation at four putative Cdc2 sites abolishes the checkpoint function (Yamaguchi et al 2003) Both Cdc2 and MAPK have similar consensus phosphorylation sites In egg extracts, inhibition of MAPK abolishes Bub1 phosphorylation without an effect on Cdc2 activity, indicat-ing that Cdc2 is not involved in Bub1 phosphorylation in the frog Finally, Bub1 has a noncheckpoint function at the kinetochores and preserves cohe-sion through the MEI-S332/shugoshin family of proteins (Salic et al 2004;

Tang et al 2004)

BubR1

BubR1 was isolated as a Mad3/Bub1-related protein kinase on the basis of

its similarities with the N-terminal domain of the yeast checkpoint protein Mad3 (Taylor et al 1998) Thus, BubR1 is thought to be the homologue of Mad3; in higher eukaryotes BubR1 directly binds to CENP-E (Chan et al 1998) It is a mitosis-specific kinase and is inactive during interphase (Chan

et al 1999) Microinjection of antibodies against BubR1 into HeLa cells ab-rogated mitotic arrest after nocodazole-induced spindle disassembly (Chan

et al 1998; Chan et al 1999) In Xenopus egg extracts, immunodepletion of

BubR1 also prevented mitotic arrest in response to spindle damage (Chen 2002) BubR1 accumulates and becomes hyperphosphorylated at unattached kinetochores Immunodepletion of BubR1 greatly reduces kinetochore bind-ing of Bub1, Bub3, Mad1, Mad2, and CENP-E These defects can be rescued

by wild-type, kinase-dead, or a truncated BubR1 protein that lacks its ki-nase domain, indicating that the kiki-nase activity of BubR1 is not essential for the spindle checkpoint in egg extracts (Chen 2002) Whether phosphorylation

of BubR1 leads to an activation of the kinase is not known BubR1 accu-mulates to a higher level and becomes hyperphosphorylated at unattached kinetochores compared with that at metaphase kinetochores This phospho-rylation requires Mad1 or its downstream effector, but not Mad2 (Chen 2002) Expression of a kinase-inactive mutant of BubR1 abolished mitotic arrest induced by microtubule disassembly (Chan et al 1999) RNAi-mediated de-pletion of BubR1 causes severe chromosome misalignment and results in the loss of kinetochore-microtubule attachment (Chan et al 1999) Attachment in these cells can be restored by inhibition of Aurora kinase, which is known to stabilize kinetochore-microtubule interactions BubR1 similar to Mad2 also associates and can phosphorylate Cdc20 in vitro leading to inactivation of the APC/C (Sudakin et al 2001; Tang et al 2001; Fang 2002) Both Mad2 and

BubR1 can indirectly bind to Cdc20 in vitro and either independently or co-operatively inhibit polyubiquitination of APC/CCdc20substrates Quantitative analysis indicates that BubR1 binds to Cdc20 with a higher affinity and is more potent than Mad2 in inhibiting the activation of APC by Cdc20 (Fang 2002) The two pathways seem to act synergistically since inactivation of ei-ther Mad2 or BubR1 by microinjection of inhibitors shows that the activity

of both proteins is required for the metaphase delay at 23◦C (Shannon et al. 2002) But why does the cell need two different inhibitors of APC in the same checkpoint pathway? It is possible that binding of Cdc20 to BubR1 recruits Cdc20 to kinetochores, where BubR1 promotes the formation of the Mad2– Cdc20 complex, which subsequently diffuses away from kinetochores and inhibits APC throughout the cell Alternatively, BubR1 and Mad2 might

in-hibit APC in response to different checkpoint signals In Xenopus egg extracts,

CENP-E dependent activation of BubR1 kinase activity at kinetochores is

ne-cessary for establishing the mitotic checkpoint (Mao et al 2003) Although BubR1 binds to CENP-E, its kinase activity seems to be required for Mad2, but not CENP-E, recruitment to kinetochores (Mao et al 2003) Graded reduc-tion of BubR1 expression in mouse embryonic fibroblasts causes increased aneuploidy and senescence (Baker et al 2004) Male and female mutant mice have defects in meiotic chromosome segregation and are infertile (Baker et al 2004) BubR1-knockout mice die during early development (beyond day 8.5 in utero) as a result of an increased prevalence of apoptosis (Baker et al 2004) Downregulation of BubR1 by RNAi is associated with the formation of poly-ploidy This seems to be the result of a prolonged mitotic arrest which leads to

a decrease in BubR1 levels and a concomitant increase in polyploid cells (Shin

et al 2003) These data suggest that BubR1 is not only a sensor that monitors the mitotic checkpoint but that it is also involved in apoptotic signaling and chromosome instability

3.3

Aurora B

The Ipl1/Aurora family of protein kinases plays multiple roles in mitosis,

including chromosome segregation and cell division (Meraldi et al 2004b)

In budding yeast, Ipl1 ensures accurate chromosome segregation by resolv-ing syntelic orientations, possible by monitorresolv-ing tension at centromeres and destabilizing inappropriately bound microtubules (Tanaka et al 2002) In

an Ipl1 mutant, the checkpoint remains functional when triggered by dis-ruption of the spindle by nocodazole (Biggins and Murray 2001) Higher eukaryotes express three Aurora kinases, Aurora A, Aurora B, and Aurora C Aurora A and Aurora B have very distinct localizations and functions Au-rora A is involved in regulation of microtubule nucleation at centrosomes Aurora B is found at the inner centromeric regions of chromosomes from prophase through the metaphase–anaphase transition as part of a “chromoso-mal passenger protein complex,” where it appears to promote correct bipolar microtubule-kinetochore attachments Thus, Aurora B seems to be involved in several mitotic processes, including chromosome condensation through phos-phorylation of histone H3, chromosome alignment, kinetochore disjunction, the spindle assembly checkpoint, and cytokinesis After anaphase onset, Au-rora B relocalizes to the central microtubules of the anaphase spindle and then to the midbody during the completion of cytokinesis (Meraldi et al 2004b) Aurora B requires the association to the inner centromere protein and

a founding member of the chromosome passenger proteins, INCENP, for its localization (Adams et al 2000) For over a decade INCENP has been im-plicated in the regulation of cytokinesis as it localizes to the cortex in late anaphase/telophase, before cleavage furrow ingression and before the

recruit-ment of myosin (Cooke et al 1987) Aurora B also affects the localization of

a taxin family member, TACC1, to the midbody, leading to abnormal cell

di-vision and multinucleated cells (Delaval et al 2004) In addition, survivin,

a conserved inhibitor of apoptosis also seems to be required for the localization

of Aurora B (Romano et al 2003) The role of Aurora B kinase activity has been adressed by ectopically expressing a kinase-negative version, Aurora BK109R,

by RNAi, and by small molecule inhibitors One report described that cells ex-pressing Aurora BK109Rcompleted mitosis, but failed to undergo cytokinesis, suggesting that Aurora B activity is not required for chromosome segregation (Tatsuka et al 1998) However, another study revealed that the kinase-negative mutant prevents chromosome alignment owing to the failure of kinetochore-microtubule interactions (Murata-Hori and Wang 2002) Ablation of Aurora B function by RNAi results in a dramatic increase in polyploidy as assayed by the presence of binucleate and multinucleated cells (Ditchfield et al 2003; Scrittori

et al 2005) In addition, treatment with short interfering RNA (siRNA) affected the prometaphase–metaphase transition, yielding a significant increase in the percentage of mitotic cells at prometaphase in transfected cells in comparison with control cells (Scrittori et al 2005) Microinjection of Aurora B anti-bodies blocked chromosome alignment and segregation, leading to abrogation

of the spindle checkpoint (Gorbsky 2001) However, in these approaches it is difficult to distinguish between effects due to the lack of protein itself, where Aurora B-containing complexes and subcomplexes do not form, and those simply due to lack of kinase acitivity, where substrate phosphorylation is the initial defect The use of the small molecule inhibitor ZM447439 (AstraZeneca) which inhibits Aurora B kinase activity allowed to study of the phenotype which is solely due to the lack of kinase activity When ZM447439 was added to mammalian cell cultures, cells entered mitosis and formed a mitotic spindle, but phosphorylation of histone H3 was reduced, the spindle was disorganized, and cytokinesis was blocked (Ditchfield et al 2003) The use of ZM447439

in Xenopus egg cycling extracts made it possible to study the effect of the

inhibitor in a system on the basic cell cycle machinery in the absence of func-tional checkpoints Checkpoint pathways including the spindle checkpoint do

not opperate in Xenopus early embryonic cell cycles Gadea and Ruderman

(2005) found that ZM447439 had striking effects on chromosome morphology since chromosome condensation began to schedule but then failed to progress properly owing to premature decondensation during mid-mitosis ZM447439 strongly interfered with mitotic spindle assembly by inhibiting the formation

of microtubules that are nucleated/stabilized by chromatin Another inhibitor,

hesperadin, causes defects in mitosis and cytokinesis and inhibits Aurora B

in vitro (Hauf et al 2003) The use of each of these inhibitors phenocopies Aurora B RNAi A possible substrate of Aurora B is the Kin1 kinesin MCAK (Andrews et al 2004) Aurora B inhibits the microtubule depolymerizing activ-ity of MCAK in vitro Moreover, disruption of Aurora B function by expression

of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK PP1 is known to antagonize Aurora B activity and inhibit its kinase activity (Francisco and Chan 1994; Sassoon et al 1999) Another known substrate of

Aurora B is the GTPase activating protein, MgcRacGAP, which colocalizes with the kinase at the midbody Aurora B phosphorylates MgcRacGAP at serine residues and that this modification induces latent GAP activity towards RhoA

in vitro (Minoshima et al 2003), thus functionally converting the protein Little is known about the localization and function of the third mammalian Aurora kinase family member, Aurora C Aurora C was reported to express

in testis and some human cancer cell lines with the highest level detected at G2/M (Kimura et al 1999) Recent studies indicate that Aurora C is a

chromo-some passenger protein similar to Aurora B (Li et al 2004; Sasai et al 2004) Aurora C is tightly bound to mitotic chromosomes where it interacts with Au-rora B and INCENP Elevated expression of AuAu-rora C in cancer cells might play a critical role in perturbing the chromosomal passenger complexes In the future, it would seem important to analyze in greater detail the Aurora C pathway in mammalian cells

3.4

Mps1

The Mps1 (monopolar spindle 1) protein kinase was first described in S cere-visiae, where it is implicated in the duplication of the spindle pole body (Winey

et al 1991) Mps1 was identified initially as a centrosomal protein required for the assembly of bipolar spindles, but it was later shown to play a role in the spindle checkpoint as well In yeast, overexpression of Mps1p or a mutant form

of Bub1p, Bub-5p, causes mitotic arrest in the apparent absence of defects in spindle assembly (Hardwick et al 1996; Farr and Hoyt 1998) The mitotic ar-rest of each is dependent on all other checkpoint proteins and both Mps1p and Bub1-5p induced arrests are interdependent These results also suggest that the activation of Mps1 is an early event in checkpoint signaling and may lead

to the recruitment of other checkpoint proteins to kinetochores Using Xeno-pus egg extracts, it was shown that the XenoXeno-pus homologue of yeast Mps1p

is a kinetochore-associated protein kinase, whose activity is necessary to es-tablish and maintain the spindle checkpoint (Abrieu et al 2001) Since high levels of Mad2 overcome checkpoint loss in Mps1-depleted extracts, Mps1 acts upstream of Mad2-mediated inhibition of APC/C Mps1 is essential for the

checkpoint because it is required for recruitment and retention of active

CENP-E at kinetochores, which is in turn required for association of both Mad1 and Mad 2 (Abrieu et al 2001) The human homologue of yeast Mps1p, hMps1, is

a cell cycle-regulated kinase and both its protein levels and its kinase activity peak during progression through the M phase (Stucke et al 2002) Microinjec-tion of anti-hMps1 antibodies and silencing of the kinase by siRNA interferes with the spindle assembly checkpoint (Stucke et al 2002) hMps1 is hyperphos-phorylated in mitosis and is dephoshyperphos-phorylated when cells exit mitosis (Liu et al

2003) Similar to the Xenopus homologue, human Mps1 is required for the

re-cruitment of Mad1 and Mad2 to kinetochores (Liu et al 2003) and it associates

with APC/C in both interphase and mitosis (Liu et al 2003) Thus, it is possible

that Mps1 phosphorylates the APC/C during mitosis and that these

modifica-tions may be part of the mechanism by which the checkpoint inhibits APC/C.

A recent study of Drosophila Mps1 revealed that Mps1 is required for the

spin-dle checkpoint by demonstrating that embryos harbor a transposon insertion mutation In this mutation, called Mps11, the single fly Mps1 orthologue is disrupted; therefore, Mps11embryos do not undergo a mitotic arrest in re-sponse to the microtubule poison colcemid (Fischer et al 2004) In addition the authors showed that the metaphase-to-anaphase transition is accelerated

in Mps11embryos similar to what was observed for other spindle checkpoint proteins in vertebrate cells (Meraldi et al 2004a) Finally, as with any protein kinase, the identification of substrates is critical, particularly those substrates that reveal their specific functions in the various stages of mitosis Thus, it is possible that Mps1 phosphorylates the APC/C during mitosis and that these

modifications may be part of the mechanism by which the checkpoint inhibits the APC/C.

3.5

Mitogen-activated protein kinase

MAPKs are serine/threonine-specific protein kinases that are activated in

response to extracellular signals and play important roles as effectors for di-verse cellular functions, including growth, differentiation, movement, and secretion (L’Allemain 1994) In vertebrate cells, a small fraction of MAPK is activated and enriched at kinetochores during mitosis, and the level of ac-tive MAPK decreases at and after metaphase (Shapiro et al 1998; Zecevic

et al 1998) MAPK activity is important for the spindle checkpoint both in egg extracts and in somatic cells It has also been shown that MAPK interacts with the kinetochore motor protein CENP-E and that MAPK phosphorylates CENP-E in vitro to create a tension-sensitive epitope that is recognized by

a monoclonal antibody (Zecevic et al 1998) In Xenopus, MAPK also

con-tributes to Cdc20 phosphorylation, and this phosphorylation is required for the checkpoint proteins to bind and inhibit Cdc20 (Chung and Chen 2003) Cdc20 mutants that are phosphorylation-deficient are able to activate the APC

in X laevis egg extracts (Chung and Chen 2003) Thus, dephosphorylation of

Cdc20 at multiple MAPK sites may provide a mechanism to disassemble the existing spindle checkpoint complex

4

The Spindle Checkpoint and Cancer

The survival of a cell depends on the accuracy of mitosis and errors in the mechanisms controlling mitosis can lead to genomic instability Cancer cells