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Extra View

The End of the BeginningCdk1 Thresholds and Exit from Mitosis

Fra k W fR ar S gS a G *Biocenter; Division of Molecular Pathophysiology; Innsbruck Medical University;Innsbruck, Austria

*Correspondence to: Stephan Geley; Biocenter, Division of MolecularPathophysiology; Innsbruck Medical University; Fritz-Pregl-Str.3; 6020 Innsbruck,Austria; Tel: +43.512.9003.70365; Fax: +43.512.9003.73960; Email: [email protected]

Original manuscript submitted: 04/25/07Manuscript accepted: 04/27/07

Previously published online as aCell Cycle E-publication:http://www.landesbioscience.com/journals/cc/abstract.php?id=4361

Key WoRdS

cyclin, mitosis, proteolysis, ubiquitin, APC/C

AcKnoWledGementS

We aregrateful to Drs. R. Kofler andC. Plonerfor their help with lentiviral transduction andthe members of the lab for their help and stim-ulating discussion. This work was supportedby grants from the Austrian Science Funds(FWF) P16400-B10, SFB021 Cell prolifera-tion and cell death in tumors and EU grantLSHS-CT-2004-503438 (TRANSFOG).

[Cell Cycle 6:12, 1408-1411, 15 June 2007]; 2007 Landes Bioscience

AbStRActExit from mitosis requires the proteolytic degradation of mitotic cyclins, which is

gated by the APC/C ubiquitin ligase. The coincidence of mitotic cyclin B1 degradatiowith the onset of anaphase intuitively suggested a requirement of cyclin degradation fsister chromatid separation. While this hypothesis has originally been refuted, evidenthat cyclin B1 degradation is required for anaphase during meiosis has been obtainedwhile its requirement for anaphase during mitosis is still more controversial. By studhuman cells engineered to express nondegradable cyclin B1, we have recently shown thstable cyclin B1 affects progression through mitosis at various steps in a dosedependmanner. These experiments suggest that controlled exit from mitosis might involve activity thresholds for important late mitotic events, such as the onset of anaphase, formtion of the spindle midzone, the onset of cytokinesis, cellular abscission and chromosodecondensation.

Finishing a project successfully may sometimes be as, or even more, difficult thinitiating one. This day-to-day experience may also hold true for certain biologicprocesses, which involve large scale, yet reversible, cellular changes to achieve a cerbiological outcome. For cellular division, cells have to reorganise their cytoskeleton, thechromatin state, membrane dynamics and signalling interfaces to the extracellular compartment. All these changes occur during the dramatic events that happen when cells entemitosis. To revert to an interphase state, however, cells have to revert all of these changes inorder to regain the ability to perform their specific physiological tasks or for another rounof cellular replication. Exit from mitosis is a complex transition involving many celluchanges to occur in a timely coordinated manner.1 In addition, different organisms and celltypes might have to organise exit from mitosis slightly differently, in order to be successfThus, although the regulation of exit from mitosis involves conserved signalling proteinthese signalling pathways might be used in a cell type specific manner to coordinate thnuclear division cycle and cytokinesis.

In all eukaryotes exit from mitosis is controlled by proteolysis. The conserved multi-subunit ubiquitin ligase APC/C controls sister chromatid separation and exit from mitosis btargeting securin and mitotic cyclins for proteasome-dependent degradation.2 Linkingsecurin and cyclin degradation ensures that sister chromatid separation is not uncoupledfrom exit from mitosis (and vice versa). Anaphase and exit from mitosis are not oncoupled by being dependent on the APC/C, but separase and cyclin B1 activities mutuallinhibit each other. 3 Cyclin B1 degradation and CDK inactivation are required for the onsetof anaphase in some systems (e.g., during mouse oocyte meiosis, ref. 4) but are essen4) but are essentia) but are essentiafor exit from mitosis in all eukaryotes (see ref. 5 and references therein). In budding yea5 and references therein). In budding yeasand references therein). In budding yeasPds1 (securin) and the mitotic Clb cyclins are the only essential targets for the APC/C.6-8

In vertebrate cells (near complete) exit from mitosis can be triggered in cells arrestedmetaphase by the proteasome inhibitor MG-132 or due to the lack of E1 enzyme activityby using chemical inhibition of CDK activity.9,10 This suggests that after anaphase, mitoticcyclins are the only targets that need to be degraded for initiation of cytokinesis. Whethecompletion of cytokinesis requires proteolysis, be it APC/C dependent or not, is, howevea still unresolved question. At the meta- to anaphase transition, the substrate specificiof the APC/C expands due to replacement of the activator and substrate binding factoCdc20 by its homolog Cdh1/Fzr1. 11,12 Fzr1-activated APC/C continues to polyubiquit-ylate D-box containing substrates but also recognises KEN-box substrates as targetspolyubiquitylation and subsequentdegradation13 but, in contrast to Cdc20, 14,15 Cdh1/Fzris not essential for exit from mitosis in any organism studied thus far.16,17,18 These datasuggest that, among the many mitotic APC/C substrates, only securin and cyclinB1 aressential targets for mitotic proteolysis.

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Cyclin Degradation and Exit from Mitosis

Exit from mitosis in different organisms is not a uniform processbecause it has to meet specific requirements, e.g., in budding yeast where the daughter bud (and thus the site of cytokinesis) is deter-mined very early in the cell cycle and the mitotic spindle has tobe positioned accordingly for proper chromosome segregation. Itis therefore not surprising, that in this organism exit from mitosis,i.e., events after the onset of anaphase, is controlled by an intricatesignalling complex, called the mitotic exit network (MEN), that

controls the post anaphase activity of CDK in order to preventpremature cytokinesis.19 Basically, this pathway consists of a GTPasecontrolled kinase pathway, whose components appear to be conservedthroughout eukaryotes. In budding yeast, this pathway controlsCDK activity during late mitosis by regulating Cdc14 phosphatase, which dephosphorylates CDK substrates and is essential for exit frommitosis in this organism.20 As cells of other organisms face ratherdifferent requirements (compared to finding the way into a bud forchromosome deposition) these conserved signalling proteins are usedfor different purposes during exit from mitosis. Whether vertebratecells are able to delay exit from mitosis, e.g., to allow adjustment ofthe spindle axis, in relation to the polarity of the cell, is currently notknown. It has been shown, however, that during exit from mitosis

APC/C activity can be turned off, if microtubule function is blockedin anaphase cells.21 In addition, although Cdc14 homologs in otherorganisms are not essential for exit from mitosis, they are requiredfor cytokinesis.22

In human cells, very little is known about the function of mitoticphosphatases and the balance of kinase and phosphatase activitiesduring exit from mitosis. Our observations that different levels ofnondegradable cyclin B1 arrest cells in different stages of mitosissuggest a model where exit from mitosis might be controlled bytimed dephosphorylation of key regulatory proteins. Whether thisdephosphorylation is primarily determined by the decay rate of cylinlevels or the activation and/or recruitment of (specific) phosphatasesis currently not known.

Controlling exit from mitosis by regulating the rate of cyclin B1proteolysis would only make sense if late mitotic Cdk1 substratesacted in a sequential manner. During early mitosis, Cdk1 not onlyactivates proteins by phosphorylation, but also inhibits proteins thatfunction after anaphase and require CDK inactivation. Whetherall these Cdk1 substrates are dephosphorylated precipitously bynonspecific phosphatases as soon as Cdk1 activity drops at the onsetof anaphase, or whether there is a temporal order of substrate dephos-phorylation is poorly understood but several lines of investigationsuggest that it might be.

cdKdependent contRol oF StRuctuReS RequiRedFoR cytoKineSiS

First, progression into anaphase and formation of the centralspindle requiresamong many other thingsthe action of chro-mosome passenger proteins, mitotic motor proteins and othermicrotubule binding and regulatory proteins. Chromosome passengerproteins reside at kinetochores until anaphase upon which they trans-locate to, and help to form, the central spindle.23

In budding yeast, release of the INCENP homolog Sli15 ismediated by Cdc14 phosphatase24 and cells arrested by non-degra-dable cyclin B1 do not release INCENP from kinetochores25 (andunpublished observations). Recently two CDK phosphorylation siteshave been mapped on INCENP, one of which generates a Polo-boxbinding domain.26 INCENP is thus not only an important regulator

of Aurora B kinase but also helps to recruit Plk1 to kinetochoresDephosphorylation of INCENP might thus be an important eventfor the switch from a metaphase to an anaphase spindle. The relocalisation of the Aurora B kinase complex to the central spindle alsrequires the dephosphorylation of Mklp2, a kinesin that is phosphorylated (and inhibited) by CDK in early mitosis.27

The centralspindlin complex, which consists of Mklp1MgcRacGAP, Ect2 and other proteins then determines the site o

cytokinesis by controlling the activity of the cytokinesis regulatRhoA.28 Like the chromosome passenger proteins, several compnents for the centralspindlin complex are regulated by CDK activitsuch as Mklp1, whose phosphorylation by Cdk1 reduces its kinesinmotor activity.29 Finally, the spindle midzone and the formation othe midbody require the function of the microtubule crosslinkinprotein PRC1, which is phosphorylated during early mitosis an whose dephosphorylation is required for proper localisation anfunction.30,31 In cells expressing nondegradable cyclin B1, a spindmidzone is not formed,32 and chromosome passenger proteinremain at kinetochores, suggesting that cyclin B1 proteolysis trigers the formation of an anaphase spindle.25 Thus, in the presenceof CDK activity formation of cytoskeletal structures important focytokinesis is inhibited.

cdKdependent contRol oF cytoKineSiS ReGulAtoRSSecond, in mitotically arrested cells, e.g., by expression of stab

B-type cyclins, cytokinesis can rapidly be triggered by chemiinhibition of CDK activity.5,33,34 Thus, cytokinesis can btriggered in the absence of instructive or permissive signals originatinfrom the central spindle.33 Rather, the onset of cytokinesis seems todepend only on a decline of CDK activity, suggesting that a centralcytokinesis regulator might be a direct target of CDK activity.

cdK Activity beyond AnAphASe

Third, in fly embryos, exit from mitosis is controlled by thsequential degradation of the mitotic cyclins A, B and B3, anexpression of stable variants of these cyclins arrest early embryocell divisions at metaphase, early anaphase and telophase, respetively.35,36,37 This sequential cyclin degradation strongly suggests thaCDK activity is involved in the regulation of exit from mitosis. CycliB3 is conserved in mice and humans but its expression appears to brestricted to testes and ovaries.38 In chicken B cells, however, cycliB3 is expressed and may be involved in regulating events during efrom mitosis (e.g., chromosome decondensation).39

phoSphAtASeS contRollinG exit FRom mitoSiS

Fourth, Cdc14 phosphatase homologs play important functions invarious species, including human cells.20 Dephosphorylation is a keevent in the completion of cytokinesis, but whether human Cdc14is specific for CDK substrates remains to be determined. In order tocoordinate anaphase with the formation of the central spindle, thonset and completion of cytokinesis, sequential dephosphorylation okey regulators can be envisaged to play an important role.40

evidence FoR cdK contRol oF exit FRom mitoSiSIf progression beyond metaphase is controlled by the pho

phorylation state of CDK substrates, then this model predictthat maintaining Cdk1 activity at different levels during mitosi

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1410 Cell Cycle 2007; Vol. 6 Issue 12

should arrest cells at different phases during late mitosis. To test

this hypothesis, we have generated human cell lines with induciblenondegradable cyclin B1 to maintain Cdk1 activity in mitosis.5 Byusing GFP fused to nondegradable cyclin B1, we were able to corre-late expression levels with three distinct phenotypes. At high levels(~4 times higher than endogenous cyclinB1) cells did not undergovisible anaphase as evaluated by live cell imaging of histone H2B-RFPtagged cells. At physiological levels, nondegradable cyclin B1 arrestedcells in anaphase. In these cells, cytokinesis was blocked, chromo-some passenger proteins remained at kinetochores and a bipolarspindle with merotelically attached chromosomes was present. Cellsexpressing low levels of nondegradable cyclinB1 arrested in telophase,because completion of cytokinesis, i.e., abscission, was blocked,and the chromatin remained condensed in these arrested cells.

When cells arrested by nondegradable cyclinB1 wetreated with a chemical CDK inhibitor, they rapidlyinitiated cytokinesis, even if chromosomes did nosegregate properly. In summary, these data suggethat different levels of Cdk1 activity are required tphosphorylate and inhibit key regulatory substrateduring mitosis. With the exception of separase, thespotential targets are, however, largely unknown.

As evidence derived from overexpression expiments has to be considered with caution, w wondered whether a hypomorphic APC/C mutanmight cause the same mitotic arrest phenotypes. Wehave therefore used a tetracycline-inducible shRNexpression system to generate human cell lines wistable, inducible RNAi to target the APC/C subunitCdc27. As can be seen in Figure 1, treatment with1mg/ml doxycycline for 48 hours causes a signicant knockdown of Cdc27 levels, which interfere with cyclin B1 degradation in cells released froa nocodazole-induced mitotic block (Fig 1 A) anarrested cells in mitosis. Histone H2B-GFP cell

expressing TetR and infected with a lentivirus for inducible Cdc2RNAi were treated with doxycyline and monitored by live cefluorescence time lapse microscopy over a period of three day Among cells that became arrested in mitosis, 85% arrested ametaphase, while 15% arrested like stable cyclinB1 expressing cein anaphase (Fig. 1B), suggesting the presence of residual APC/activity sufficient to eliminate securin but not cyclin B1 from theseCdc27 RNAi hypomorphic cells. Thus, when cyclin B1 levels amaintained by low APC/C activity in RNAi hypomorphs, cells arreat different time points in mitosis. These experiments confirm ouprevious findings that complete cyclinB1 degradation is not requirefor the onset of anaphase but for exit from mitosis. Analysis of cellexpressing low amounts of nondegradable cyclin B1 (or endogenocyclin B1 in Cdc27 RNAi hypomorphs) progress well into anapha

but fail to complete cellular abscission, upon which cells often fuand are able to reestablish a bipolar spindle, i.e., they reverse mitosto arrest in a pseudometaphase state.

The mitotic arrest phenotypes observed in cells expressing nondgradable cyclin B1 or Cdc27 knockdown are consistent with the idethat late mitotic events exhibit different sensitivities to remaininCDK activity. While very high levels appear to block the onset oanaphase, lower levels suppress the onset of cytokinesis and asufficient to maintain a bipolar spindle with chromosomes arrangedin a pseudometaphase plate, while the lowest levels are sufficientblock completion of cytokinesis and chromosome decondensation.stepwise removal of CDK activity as a regulatory mechanism for coodinated exit from mitosis has been suggested previously by OFarrand coworkers who have studied the consequences of expressinondegradable versions of the three mitoticDrosophila cyclins (A, Band B3) in fly embryos.36 These cyclins become degraded at speciftimes in mitosis and expression of nondegradable mutants blockeprogression through mitosis at or before metaphase (cyclin A), iearly anaphase (B) and in telophase (B3).

In contrast to fly embryos, which express a mitotic cyclin untilate in mitosis, human mitotic B-type cyclins in somatic cells becomunstable at the onset of anaphase. By quantitative monitoring ocyclin B1 degradation in human cells using a cyclinB1-GFP reportprotein, Clute et al showed that cyclin B1-GFP levels start to declinalready during metaphase, and gradually decline during exit fromitosis to become undetectable only at the onset of cytokinesis.21

Figure 2. Control of exit from mitosis by timed dephosphorylation of CDKsubstrates. Exit from mitosis might be controlled by the specific dephosphor

ylation of key regulators of late mitotic events, such as formation of the central spindle, cytokinesis and cellular abscission. Since key regulators of theseevents have been identified (see text for details) to be CDK substrates that areinhibited during early mitosis, a gradual decline of CDK activity or specificdephosphorylation of these key substrates might control the timed executionof these events during exit from mitosis. P, prophase; PM, prometaphase; M,metaphase; A, anaphase; T/C, telophase/cytokinesis; G 1, G1 phase.

Figure 1. Cdc27 hypomorphic cells arrest in pseudometaphase. U2OS cells expressing thetetracyclinedependent transcriptional silencer tetR,42 were infected with a lentiviral constructexpressing a Cdc27 (APC2) specific shRNA and single clones isolated using puromycinselection. (A) Cells were left untreated or induced using 1mg/ml doxycyline for 48 hoursbefore addition of nocodazole (500nM) for 16 hours. Mitotically arrested and released cellswere harvested and total cell extracts probed for expression of Cdc27 (BD TransductionLaboratories), Cyclin B1 (V152) and GAPDH (Abcam). (B) 48 hours after induction with1mg/ml. U2OS cells expressing H2BGFP were monitored for progression through mitosis.Time is shown in hours:minutes. The white arrow indicates onset of anaphase after a prolonged metaphase.

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In summary, it is likely that specific dephosphorylation of keyregulatory proteins is essential for coordinated exit from mitosis(Fig. 2). A gradual decline in Cdk1 activity might be an importantcontributor to this regulatory pathway which most likely also involvesthe recruitment of phosphatases in a timely coordinated manner ashas been shown recently for CdcA2, a mitotic phosphoprotein which, upon dephosphorylation, is able to recruit PP1-g to anaphasechromosomes.41 As little is known about mitotic phosphatases, it will

be interesting to learn more about how these are controlled duringexit from mitosis.References

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