review

Regulation of the APC and the exit from mitosis

David O. Morgan*†*Department of Physiology, University of California, San Francisco, California 94143-0444, USA

†e-mail: dmorgan@cgl.ucsf.edu

The events of late mitosis, from sister-chromatid separation to cytokinesis, are governed by the anaphase-promoting complex (APC), a multisubunit assembly that triggers the ubiquitin-dependent proteloysis of key regulatory proteins. An intricate regulatory network governs APC activity and helps to ensure that late mitotic events are properly timed and coordinated.

e’ve all seen the movie. The sister chromatids hover at themetaphase plate, and then suddenly break apart and racetoward the spindle poles. The suddenness of the event is

striking: there can be little doubt that a biochemical switch is atwork, ensuring that sister-chromatid separation is rapid, complete,and irreversible.

Chromosome separation is followed by spindle disassembly,reconstruction of interphase structures and, finally, by the comple-tion of cell division by cytokinesis. Here again, at the exit frommitosis, we can readily imagine the need for a regulatory system thattriggers events completely and irreversibly. We can also deduceanother feature of the system: mitotic events are initiated in a coor-dinated and perfectly timed fashion, ensuring that each eventoccurs only after the previous one is complete.

These and other aspects of the regulation of late mitotic eventshave recently begun to yield their secrets. A fundamental principlehas emerged: the initiation and coordination of late mitotic eventsare governed by ubiquitin-dependent proteolysis of key regulatoryproteins1–4. A major step in this destruction is catalysed by a mul-timeric ubiquitin ligase known as the anaphase-promoting com-plex (APC) or cyclosome. APC activity rises abruptly at metaphase,resulting in the destruction of proteins that inhibit sister-chromatidseparation. APC-dependent destruction of additional regulatorsinitiates spindle disassembly, cytokinesis and the resetting of repli-cation origins for the next cell-division cycle. Thus, to understandhow late mitotic events are controlled, we must understand the reg-ulatory network that controls the timing and substrate specificity ofAPC activation.

The anaphase-promoting complexThe APC is thought to be a ubiquitin ligase (or ‘E3’ enzyme), whichcollaborates with a ubiquitin-activating enzyme (E1) and a ubiqui-tin-conjugating enzyme (E2 or UBC) to catalyse the transfer ofubiquitin molecules to lysine side chains on target proteins5. TheAPC (but not the E1 and E2 enzymes) seems to be the only ubiq-uitination component whose activity oscillates during the cellcycle6–11. Thus, most studies of late mitotic regulation focus on theAPC. It should be borne in mind, however, that in many organ-isms, regulation of other steps in the proteolytic pathway has notbeen rigorously excluded.

The vertebrate APC is a 20S complex originally thought to con-tain eight subunits (Apc1–8)7,12,13; an additional component hasrecently been identified, and more are probably on the way14.Homologues of most of the vertebrate subunits are found in theenzyme from budding yeast (Saccharomyces cerevisiae), which con-tains at least twelve subunits15–19. The primary structures of thesesubunits reveal little about their potential functions, except that oneof the conserved subunits (Apc2) shares sequence homology withthe ‘cullins’, a class of proteins that is found in other types of ubiq-

uitin ligases and which may mediate the interaction with the E2enzyme. Several conserved APC subunits contain tetratricopeptide(TPR) repeats that are thought to mediate protein–protein interac-tions. Apart from these minor clues, the massive machine at theheart of late mitotic regulation remains mysterious. What are thefunctions of its individual subunits? Why is the APC so big andcomplex? Does the APC serve as a giant scaffold upon which multi-ple ubiquitination reactions can be compartmentalized or coordi-nated? Does the APC actually possess enzymatic activity, or does itsimply present the target protein to the E2 enzyme?

APC activity requires Cdc20 or Hct1Recent studies have led to the surprising revelation that the coreAPC, despite its large size and complexity, does not actually containeverything it needs to promote ubiquitination of target proteins.One more subunit is required to complete the enzyme. Two relatedversions of this activating subunit have been identified in numerousorganisms. I will refer to these proteins by their budding yeast namesCdc20 and Hct1/Cdh120,21, although they go by different names inother species (Fizzy and Fizzy-related in Drosophila22–24 and frogs(Xenopus)25, p55Cdc/hCdc20 and hCdh1 in humans11,26,27, Slp1 andSte9/Srw1 in fission yeast (Schizosaccharomyces pombe)28–30). Cell-cycle-dependent increases in APC activity are correlated withincreased binding of Cdc20 or Hct111,27,31, and mutations in theseproteins abolish APC-dependent proteolysis20,21,32,33. VertebrateCdc20 and Hct1, as well as budding yeast Hct1, bind and activate theAPC in vitro11,27,34. An essential role for these proteins in APC activa-tion thus seems clear. Their mechanism of action, however, is not.Cdc20 and Hct1 are related proteins, containing multiple WD40repeats but no other sequence motif that might betray their bio-chemical function; thus, it is not known if the binding of these pro-teins to the APC provides all or part of the active site, or whethertheir binding induces a conformational change that activates a cata-lytic site on the APC or on the associated E2 enzyme.

Cdc20 or Hct1 are present in purified APC preparations at levelsfar lower than those of the core subunits (explaining why they werenot identified as APC subunits), and it is possible to greatly super-activate an active late mitotic or G1 APC by adding Cdc20 or Hct1in vitro11,34. Cdc20 and Hct1 thus seem to be sub-stoichiometricAPC activators whose binding to the APC is a limiting determinantof APC activity.

Why are there two APC-activating subunits? This is a simple butvery important question in late mitotic regulation. Two possibili-ties, which are not mutually exclusive, will form the basis for muchof this review. First, there is some evidence, primarily in buddingyeast, that Cdc20 and Hct1 confer different substrate specificities onthe APC. Second, Cdc20 and Hct1 are regulated by different mech-anisms and thereby alter the responsiveness of the APC to certainregulatory inputs.

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APC substratesThe first step in mitotic exit is sister-chromatid separation, which istriggered, at least in part, by APC-dependent destruction of ‘ana-phase inhibitors’ (or securins) (Fig. 1). These proteins are believedto exist in all eukaryotes, and include the Pds1 protein of buddingyeast, the Cut2 protein of fission yeast, and a recently identifiedPds1 homologue in vertebrates (M. Kirschner et al., personal com-munication). These proteins are destroyed during mitosis in anAPC-dependent fashion, and sister-chromatid separation isblocked in cells expressing non-destructible mutant forms of Pds1or Cut2 (refs 35, 36 and M. Kirschner et al., personal communica-tion). Pds1 and Cut2 function by binding and inhibiting proteins(separins) that promote anaphase. Thus, destruction of Pds1 (orCut2) liberates Esp1 (or Cut1), which then triggers the inactivationof proteins required for sister-chromatid cohesion37,38.

Cells of budding yeast carrying conditional APC mutationsarrest in metaphase with undegraded Pds1. Deletion of PDS1 fromcells with APC mutations allows sister-chromatid separation tooccur despite the absence of APC activity37,39; Pds1 therefore seemsto be the sole substrate whose APC-dependent destruction isrequired for anaphase. Cells completely lacking Pds1 do notundergo premature sister-chromatid separation37; indeed, at lowtemperatures, chromatid separation occurs with normal timing inthe absence of Pds140. Pds1 destruction is therefore required, butnot sufficient, to trigger the initiation of anaphase, and additionalmechanisms, not involving the APC, must also restrain Esp1 func-tion and control the timing of sister-chromatid separation.

Another intriguing feature of this story is that although cells lack-ing Pds1 do not undergo premature sister-chromatid separation,they do display a significant delay in this process at hightemperature37. Thus, Pds1 actually promotes anaphase as well asinhibiting it. A similar conclusion has been reached from studies infission yeast, where Cut2 is essential for sister-chromatid separation41.How can an anaphase inhibitor promote chromatid separation? Theanswer seems to be that Pds1 and Cut2 are required for the full func-

tion of the anaphase promoters Esp1 and Cut1. In fission yeast, forexample, Cut2 does not simply bind and inactivate Cut1, but mayalso be required for correct Cut1 localization38. In other words, Cut2brings Cut1 to its site of action, and then cuts it loose.

In addition to contributing to the initiation of anaphase, theAPC is required for spindle disassembly and cytokinesis. The keyAPC substrates controlling these processes are the mitotic cyclins(cyclins A and B of higher eukaryotes or the Clb proteins of buddingyeast) (Fig. 1). These proteins are well known as activators of thecyclin-dependent kinase Cdk1 (also known as Cdc2 in fission yeastand higher eukaryotes, and Cdc28 in budding yeast), whose activityis required for spindle assembly and the other processes leading upto anaphase42–45. Progress beyond anaphase requires the inactivationof Cdk1 (refs 23,46–50), and this is accomplished primarily byAPC-dependent cyclin destruction.

Control of APC targeting to substrateHow might the APC regulatory system ensure that anaphase occursbefore spindle disassembly and cytokinesis? One obvious possibilityis that the order of events is determined by sequential changes in APCsubstrate targeting; in this scenario, the APC targets anaphase inhib-itors first, after which inhibitors of late mitotic events (cyclins) aredestroyed. Another possibility is that activation of the APC at met-aphase triggers the simultaneous destruction of all of its substrates.This would release the brakes on all late mitotic events, in which casethe order of events would be generated by other mechanisms.

In support of a role for the regulation of APC substrate targeting,genetic evidence in budding yeast suggests that Cdc20 and Hct1 aresubstrate-specific APC activators, such that Cdc20 stimulates Pds1destruction whereas Hct1 targets the major mitotic cyclin Clb2 fordestruction20,21,32,33 (Fig. 1). Mutations in CDC20 cause a metaphasearrest with stable Pds1; overexpression of CDC20 induces thedestruction of Pds1 but not Clb2. On the other hand, deletion ofHCT1 has little effect on Pds1 destruction but causes stabilization

Figure 1 A speculative model of the regulatory network controlling the APC in late mitosis. In embryonic cells, the Cdc20–APC is stimulated by mitotic CDK activity and is responsible for the destruction of anaphase inhibitors and cyclins; the dashed arrows indicate that the ability of Cdk1 and Polo-like kinases to stimulate the

Cdc20–APC is poorly understood and perhaps indirect. In somatic cells, the CDK-inhibited Hct1 subsystem (enclosed in a dashed box) is bolted on to allow stable APC activation in G1. In budding yeast, Cdc20–APC may target some cyclins for destruction, but at least one cyclin type (Clb2) is targeted primarily by the Hct1–APC.

AnaphaseCytokinesisDNA re-replication

S/G2 Metaphase Spindle disassembly

APC

APC

P

APC P

P

P

APC

APC

PPP

APC PP

P Cdc20 Hct1

Hct1 Hct1

Cdc14

Cdc14

Cdc20

Cdc20

Anaphase inhibitors

Sic1Sic1

Mitotic cyclins Cdk1-cyclin

Cdc5/Polo

Lag

Mad2

Incompletespindle

? ? Net1

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of Clb2, and HCT1 overexpression induces destruction of Clb2 butnot Pds1. These results are consistent with the idea that the timingof late mitotic events results from the sequential activation of theAPC by Cdc20 and then Hct1.

It should be mentioned, however, that most studies of cyclindestruction in yeast have focused on Clb2 and ignored the other Clbproteins, many of which are probably targeted for destruction byCdc20. Clb5, for example, disappears earlier than Clb2 and is nottargeted by the Hct1–APC21; there is also evidence that Clb3 is tar-geted by the Cdc20–APC40. Clb2 may therefore represent an unusualmitotic cyclin whose destruction requires Hct1 instead of Cdc20.

In budding yeast, Cdc20 is destroyed in late mitosis and is notpresent at significant levels in G1 cells33,51; in contrast, Hct1 ispresent at normal levels in G1 and associates with active APC31,34,51.Thus, despite the genetic evidence that Cdc20, and not Hct1, targetsPds1 for destruction, it seems likely that Hct1 specificity is broad-ened in G1 to allow activation of the APC towards all substrates,including Pds1. Studies of yeast APC substrate specificity in vitrowould provide critical insight into this issue.

The roles of Cdc20 and Hct1 in substrate targeting are muchmurkier in animal cells. The early embryos of frogs do not appearto contain the Hct1 protein25, indicating that Hct1 is not requiredfor the well known cyclin oscillations that occur in early frogembryonic cell cycles. Similarly, HCT1 is not expressed in early flyembryos24, where Cdc20 is required for cyclin destruction22,23. Hct1appears only in later somatic cell cycles, when the regulated gap(G1) between mitosis and S phase appears. Furthermore, vertebrateCdc20–APC and Hct1–APC both ubiquitinate mitotic cyclins invitro, although they appear to recognize slightly different sequenceson the cyclin target11. Thus, in early embryos (and probably insomatic cells) Cdc20 alone seems capable of promoting the destruc-tion of all APC substrates, including cyclins.

Clute and Pines52 recently measured the precise timing of cyclinB1 destruction in somatic vertebrate cells injected with a cyclin B1–green fluorescent protein (GFP) fusion protein. They reached thesomewhat startling conclusion that cyclin B1 destruction is initi-ated at the beginning of metaphase and is nearly complete whenanaphase begins. These results seem most consistent with thenotion that general activation of the Cdc20–APC occurs at thebeginning of metaphase, resulting in the simultaneous destructionof Pds1 and cyclin B1. It therefore seems unlikely that the order oflate mitotic events in these cells is controlled simply by sequentialchanges in the activating subunit or substrate specificity of the APC.

There is an added twist to this already confusing scenario. Inanimal embryos and somatic cells, cyclin A destruction occurs earlyin mitosis, whereas cyclin B is destroyed in metaphase23,52–54. Thatthis difference exists in embryos containing Cdc20 but not Hct1further suggests that the timing and specificity of substrate destruc-tion can be determined by factors other than the identity of the acti-vating subunit. The early mitotic destruction of cyclin A also pointsto another fascinating and unresolved mystery: how can cyclin A bedestroyed in early mitosis if Cdc20–APC activation cannot occuruntil spindle assembly is complete at the beginning of metaphase?Is cyclin A targeted by a separate APC population that does not havethis requirement for completion of spindle assembly?

Subcellular localization could well provide another dimensionto APC–substrate targeting. In vertebrate cells, APC subunits areconcentrated at the kinetochores, spindle poles and along the spin-dle itself55,56, whereas Cdc20 is found at the kinetochores through-out mitosis57. There is intriguing evidence that cyclin B destructionand CDK inactivation are initiated at the spindle poles52,58,59. Whatmight be the role of this localization in the control of APC–sub-strate targeting or other aspects of late mitotic regulation?

Switching on anaphaseHow is anaphase initiated? We know that anaphase initiationrequires activation of the Cdc20–APC, but the mechanisms by

which this occurs remain obscure. In yeast and vertebrate somaticcells, Cdc20 accumulates in late S phase through early mitosis, afterwhich APC-dependent destruction of Cdc20 leads to a drop in itsconcentration in G111,27,33,51. Changes in Cdc20 levels are generallyparalleled by changes in the amount of Cdc20 associated with theAPC. Thus, one factor contributing to the activation of Cdc20–APCactivity in mitosis is simply the increased level of the Cdc20 protein.

However, it seems likely that rapid post-translational mecha-nisms generate the abrupt Cdc20–APC activation that is requiredfor the initiation of anaphase. These mechanisms seem to includephosphorylation of Cdc20 and multiple subunits of the APC core(Fig. 1). It has been known for many years that cyclin destruction inembryonic frog extracts is promoted by the activity of Cdk1–cyclinB (ref. 60), and more recent work has suggested that Cdc20 and sev-eral APC subunits undergo CDK-dependent mitotic phosphoryla-tion in a variety of cell types7,12,25,26,61,62. Phosphatase treatmentinactivates partially purified APC, and addition of Cdk1–cyclin Brestores APC activity8. At least one APC subunit (Apc3/Cdc27) isdirectly phosphorylated by Cdk1–cyclin B in vitro63. These lines ofevidence all support the possibility that CDK activity stimulates theAPC, perhaps by direct phosphorylation. However, it should beemphasized that this hypothesis remains shaky: phosphorylationsites on Cdc20 or the APC have not been identified or mutated, andthere is no clear biochemical evidence that direct phosphorylationof Cdc20 or the APC by CDKs influences activity.

In animal cells (and probably in yeast), Cdc20–APC targets cyc-lins as well as anaphase inhibitors for proteolysis. Thus, by promot-ing Cdc20–APC activity, CDKs are sowing the seeds of their owndestruction. How then do they maintain high levels of kinase activ-ity through the early stages of mitosis, and what prevents prematuredestruction of anaphase inhibitors? Studies in embryonic cells sug-gest that additional mechanisms introduce a significant lag phasebetween CDK activation and APC activation6,8 (Fig. 1). Nothing isknown about the molecular basis of this important timing system.

Anaphase control by spindle assembly and DNA damageInsight into the control of anaphase initiation has come from recentstudies of the spindle-assembly checkpoint, the signalling systemthat blocks the metaphase-to-anaphase transition in the presence ofspindle damage or unattached kinetochores64–66. The spindle check-point arrests the cell cycle in metaphase by blocking activation ofthe Cdc20–APC. The protein Mad2, an essential component of thespindle-checkpoint system, associates with Cdc20 in yeast and ver-tebrates, and this association seems essential for the ability of spin-dle damage to inhibit APC activation28,57,61,67,68. In frog embryoextracts, added Mad2 binds the Cdc20–APC and inhibits APCactivity68. Interestingly, purified recombinant Mad2 exists in eithera monomeric or a tetrameric state, and only the tetramer inhibitsCdc20–APC activation, suggesting that the spindle checkpoint mayactivate Mad2 by altering its structure.

Mad2 and another checkpoint component, Bub1, are concen-trated at unattached kinetochores in vertebrate cells69–71. This leadsto a fascinating question: does localization of Mad2 to a single unat-tached kinetochore inhibit the entire complement of Cdc20–APCcomplexes in the cell? Does the unattached kinetochore catalyse theactivation of Mad2, which is then released to find and bind Cdc20–APC complexes at other sites? How is Mad2 throughout the cellinactivated when the last kinetochore is attached to the spindle?

The spindle-assembly checkpoint is clearly positioned to be akey regulator of anaphase onset, not just in cells encounteringabnormal spindle defects but also in normal cells progressingthrough mitosis. Might this system be responsible for the normaltriggering of anaphase that follows successful assembly of the spin-dle? Consistent with this possibility, destruction of cyclin B1 in ver-tebrate cells begins immediately after the last chromatid pairreaches the metaphase plate, and cyclin destruction can be arrestedin mid-metaphase by disruption of spindle structure with taxol52.

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Inhibition of the checkpoint signalling pathway in mammaliansomatic cells leads to a slight advancement in the onset of sister-chromatid separation70,72. However, frog embryo cells divide nor-mally despite lacking the spindle-checkpoint system and, in bud-ding yeast, the checkpoint is not essential for cell growth (althoughmutations in the pathway may cause a slight advancement in thetiming of anaphase). Thus, the successful completion of spindleassembly seems to limit the onset of anaphase in some cell types, butit is clear that additional mechanisms are also involved. Perhaps theinitiation of anaphase is also controlled by the timer that generatesthe lag phase between CDK activation and activation of the APC.

In budding yeast, initiation of anaphase is restrained not only byspindle damage, but also by damage to the DNA. The DNA damagecheckpoint may act primarily by inhibiting the destruction of Pds1.Most cells lacking Pds1 proceed unimpeded through mitosis afterDNA damage39. Interestingly, Pds1 is phosphorylated in response toDNA damage73, and an intriguing possibility is that Pds1 phospho-rylation blocks its ubiquitination by the Cdc20–APC.

Regulation of Hct1 by CDKs: switching on G1The most striking difference between Cdc20 and Hct1 lies not intheir substrate specificity but in their regulation. Whereas theCdc20–APC appears to be stimulated by mitotic CDK activity (asdescribed above), the Hct1–APC is inhibited by this activity (Fig. 1).In budding yeast, phosphorylation of Hct1 by Cdk1 blocks the abil-ity of Hct1 to activate the APC, and overexpression of a mutantHct1 lacking these phosphorylation sites causes premature cyclindestruction in vivo31,34,74. Cdk1 thus blocks its own inactivation bythe Hct1–APC, explaining the inverse correlation between theactivities of Cdk1 and Hct1–APC during the cell cycle.

The mutual antagonism between Cdk1 and its inhibitor Hct1–APC would be expected to generate a bistable biochemical switch: adrop in CDK activity to some threshold would lead to the suddenand complete activation of the Hct1–APC. The mechanism that setsthe threshold for this switch is not known, although one might pre-dict that the level of phosphatase activity opposing Cdk1 would beimportant. The switch-like features of CDK inactivation in buddingyeast are enhanced by a similar antagonistic relationship betweenCdk1 and Sic1, a protein that binds and inhibits Cdk1–cyclin com-plexes in late mitosis and G1. Phosphorylation of Sic1 by Cdk1destabilizes Sic1, and phosphorylation of the transcription factorSwi5 reduces SIC1 expression75–78. Because of these relationships,Sic1 levels are low from S phase to early mitosis but rise abruptly asCDK activity declines in late mitosis (Fig. 1).

The regulatory differences between Cdc20 and Hct1 may explainsome of the cell-cycle differences between embryos containing onlyCdc20 and somatic cells containing both Cdc20 and Hct1. In theembryonic cell cycle, speed is of the essence and mitosis is followedimmediately by the next S phase. It is therefore important in thesecells that mitotic cyclin accumulation begins immediately aftermitosis. This is precisely the expected result when CDK activity pro-motes Cdc20–APC activity, as the decline in CDK activity in latemitosis would be expected to lead to immediate APC inactivationand cyclin stabilization. The situation is expected to be quite differ-ent in somatic cells containing Hct1 as well as Cdc20: despite thedecline in CDK activity after mitosis, activation of the Hct1–APCleads to continued suppression of cyclin accumulation and theappearance of a G1 phase. Favourable growth conditions then stim-ulate the production of G1 cyclins, which are not APC targets andtherefore induce APC-resistant CDK activity. Perhaps this activityphosphorylates and inactivates the Hct1–APC in late G1, allowingmitotic cyclins to accumulate.

Why does Hct1 follow Cdc20?In budding yeast, the correct order of late mitotic events is achieved(at least in part) by mechanisms that ensure that Hct1 is activated

after Cdc20. The ordering of Cdc20 and Hct1 activation seems to bebased on the ability of Cdc20 to promote the destruction of proteinsthat inhibit Hct1 activation. In fact, the best-known Cdc20 target,the anaphase inhibitor Pds1, may also be an inhibitor of cyclindestruction: overexpression of a non-degradable mutant form ofPds1 blocks not only sister separation but also prevents cyclindestruction and cytokinesis35,40.

Cells carrying mutations in CDC20 arrest in metaphase withundegraded Pds1, whereas pds1∆ cells with mutations in CDC20arrest after anaphase with stable Clb2 (refs 32,39). Thus, Cdc20 isrequired for cyclin destruction even in the absence of Pds1, suggest-ing that Cdc20 must promote the destruction of some other inhib-itor of Hct1–APC activation.

What might this inhibitor be? One likely possibility, as discussedearlier, is that Cdc20 in budding yeast, like its homologues in ani-mal cells, directly promotes cyclin destruction. Although Hct1 isrequired for destruction of Clb2, Cdc20 may target other mitoticcyclins such as Clb3 and Clb5. The destruction of other cyclins byCdc20 would partially lower CDK activity (despite Clb2 stability),causing partial dephosphorylation of Hct1 (and Sic1), and perhapstriggering the switch that activates the Hct1–APC (and causes Sic1accumulation).

Because Cdc20 activation and Pds1 destruction both seem to berequired for activation of the Hct1–APC, any checkpoint mecha-nism that directly inhibits Cdc20 activation (the spindle check-point, for example) or Pds1 destruction (the DNA damagecheckpoint, perhaps) will also indirectly inhibit cyclin destruction.Interestingly, recent evidence suggests that this indirect mechanismis only partly responsible for the inhibition of cyclin destructionafter spindle damage; an additional mechanism, involving the reg-ulatory protein Bub2, directly inhibits Hct1–APC activation40,79,80.

In somatic animal cells, where Cdc20 almost certainly targetscyclins, the order of Cdc20 and Hct1 activation is simply a predict-able consequence of the hypothesis that Cdc20 is activated by CDKsand Hct1 is inhibited. Activation of CDKs in early mitosis leads firstto Cdc20 activation, which then brings on cyclin destruction andCDK inactivation, leading finally to stable Hct1 activation in latemitosis and G1.

Regulation of cyclin destruction by Cdc14 and Cdc5In budding yeast, mutations in a large number of genes cause a latemitotic arrest in which anaphase has occurred but spindle disas-sembly and cytokinesis have not. The products of this late mitoticfamily of genes include several protein kinases (Cdc5, Cdc15, Dbf2,Dbf20), a protein phosphatase (Cdc14), a small GTPase (Tem1), aguanine-nucleotide exchange factor (Lte1), and at least one proteinof uncertain biochemical function (Mob1)81–89. In addition to caus-ing similar late mitotic phenotypes when mutated, these genes dis-play a wide range of genetic interactions with each other83,87,88,90,suggesting that they encode members of a regulatory network withoverlapping functions in the control of mitotic exit.

Late mitotic mutants arrest after anaphase with high Clb2 levels,low levels of Pds1, and negligible levels of Hct1–APC activity10,49,90.Overexpression of the CDK inhibitor Sic1 overrides the arrest inmost of these mutants, suggesting that their primary defect is adefect in CDK inactivation10,90. Overexpression of CDC5 or CDC14causes premature activation of the Hct1–APC and Clb2destruction10,34,91. In total, the evidence all points to the hypothesisthat the late mitotic gene products are required for the activation ofmitotic cyclin destruction.

Only one of the late mitotic gene products, the phosphataseCdc14, has been implicated convincingly in the direct control ofcyclin destruction (Fig. 1). Cdc14 catalyses dephosphorylation ofHct1 at the inhibitory CDK sites, contributing to Hct1–APCactivation34. Cdc14 also removes CDK-dependent phosphates fromSic1 and Swi5, leading to Sic1 accumulation91. Cdc14 is thus posi-tioned to be a key regulator of Cdk1 inactivation in late mitosis.

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Recent studies suggest that Cdc14 function is restrained duringmost of the cell cycle by an association with the nucleolar proteinNet1/Cfi1, which sequesters Cdc14 in the nucleolus and may alsoinhibit its phosphatase activity92–94. In late mitosis, dissociation ofthe Net1–Cdc14 complex, and the consequent dispersal of activeCdc14 throughout the cell, helps initiate cyclin destruction. Furtherstudies of Cdc14 regulation, by Net1 and other components, willcertainly provide valuable insights into late mitotic control.

It is intriguing that CDC14 overexpression does not induce cyclindestruction in a cdc20 mutant91. Cdc14 might therefore exert directessential actions on Cdc20 as well as on Hct1. Alternatively, as Cdc20seems to be required for the destruction of some inhibitor of Hct1activation, then perhaps the destruction of this inhibitor is essentialfor Cdc14 to function in the cell (the simplest possibility, that Net1is targeted for destruction by Cdc20, seems not to be the case92,93).

Cdc5 is another late mitotic gene product of special interest, inpart because it is a member of the highly conserved family of Polo-related protein kinases, whose members have been implicated in thecontrol of mitosis in a wide range of eukaryotes95. In Drosophila,mammals, Xenopus, and fission yeast (but not in budding yeast),these kinases are required for the normal maturation of the centro-somes and mitotic spindle at the onset of mitosis96–98. More impor-tant in the present context, Polo-like kinases in many species are alsorequired for mitotic exit. Like Cdc5 in budding yeast, the Polo-likekinases of fission yeast and Drosophila are required for cytokinesisbut not for anaphase98,99, whereas in Xenopus this kinase is requiredfor the activation of cyclin destruction in egg extracts100. Thus, theevidence that Cdc5 promotes cyclin destruction by the APC in bud-ding yeast is consistent with studies in other eukaryotes and supportsa conserved role for Polo-like kinases in the control of mitotic exit.

We know very little about the mechanism by which Cdc5 andthe Polo-like kinases activate cyclin destruction. One speculativepossibility is that these kinases act on the APC core rather than onHct1 or Cdc20 (Fig. 1). The vertebrate Polo homologue Plk phos-phorylates core APC subunits in vitro and causes APC activation62,although effective phosphorylation by Plk may require the presence

of Cdk1 (ref. 63). In budding yeast, the APC core from cdc5 mutantsis less responsive to Hct1 than is the active G1 APC34, also suggestingthat Cdc5 is required for some APC modification that enhances itsactivation by Hct1.

The observation that the Polo-like kinase is required for cyclindestruction in Xenopus eggs100, which contain Cdc20 but not Hct1,is consistent with the notion that this kinase has a general stimula-tory effect on the APC core. This leads to another perplexing ques-tion: If Cdc5 acts on the APC core to enhance the function of bothCdc20 and Hct1, then why does it promote only cyclin destructionand not that of Pds1? Might modification of the APC core serve notonly to stimulate its activity but also to target that activity to specificsubstrates?

Perhaps the key unanswered question about the late mitotic geneproducts is not how they promote cyclin destruction, but why? Whatis the function of this network of proteins? The transition from ana-phase to G1 involves a sequence of complex events, and the latemitotic regulatory network may provide the signalling system thatmonitors these events and ensures that they are correctly timed andcoordinated. For example, recent studies have revealed that spindledefects act through the late mitotic network to directly inhibit cyclindestruction40,79,80, perhaps allowing the cell to delay cytokinesis if thespindle fails to function correctly after the initiation of anaphase.

Unsolved mysteriesThe outlines of a remarkably complex regulatory system are cominginto view, but many key questions about this system remain unan-swered. In particular, we do not have a clear picture of the mecha-nisms that trigger Cdc20–APC activation and anaphase, and ourunderstanding of the mechanisms that ensure the correct orderingof events remains sketchy, despite evidence that some control maybe exerted at the level of APC substrate targeting.

An understanding of the precise timing and spatial organizationof late mitotic regulatory events will only be possible with the fur-ther development of high-resolution methods for analysis of theseevents in single cells101. The recent analysis of GFP-tagged cyclin Bin living cells52,59 has provided wonderful insights into the exact tim-ing and location of cyclin B destruction, and similar analyses ofother APC targets will undoubtedly be fruitful.

With many of the pieces in place, it is now possible to beginlooking at the late mitotic regulatory system as a whole, rather thanfocusing too intently on its individual components. The complexnetwork of interactions in this system almost certainly leads toemergent properties that greatly enhance the effectiveness of thesystem in controlling late mitotic events. For example, we havealready discussed how the inhibition of Hct1 and Sic1 by Cdk1 pro-vides a simple biochemical switch, and additional switches willundoubtedly be discovered in this system. A key unresolved issue isthe mechanism that causes sudden, synchronous sister-chromatidseparation at anaphase (Fig. 2). Do feedback loops help to promoterapid all-or-none Pds1 destruction, or is the suddenness of ana-phase the result of other mechanisms? What are the mechanismsthat determine the threshold at which these switches are triggered,and how is this threshold regulated?

An essential regulatory system like the one governing exit frommitosis is expected to perform effectively even if mutation or sto-chastic variation leads to changes in the concentration or biochem-ical properties of some components102,103. There are indications thatthe late mitotic system can in fact tolerate such changes. Changes inthe expression level of many components have little effect onmitotic exit; indeed, Hct1 or Sic1 can be deleted with only minoreffects on the timing of exit. Is the robustness of the system due sim-ply to overlapping or redundant functions of some components, oris it also due to complex networking properties of the system as awhole? These properties may only emerge when all the regulatoryinteractions have been mapped and comprehensive models of thesystem have been developed. h

Figure 2 Late anaphase in a newt lung cell. The separation of sister chromatids inanaphase, as well as the complex events of mitotic exit that follow, are dependent onthe destruction of anaphase inhibitors and mitotic cyclins. The sudden onset, perfectcoordination, and sheer beauty of late mitotic events cannot be fully appreciated in asingle snapshot; for the complete video see: http://cellbio.nature.com. (Photograph:courtesy of V. Skeen and E.D. Salmon).

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ACKNOWLEDGEMENTS

This review is a distillation of many enjoyable conversations with members of my laboratory (Julia

Charles, Sue Jaspersen, Jamison Nourse, Catherine Takizawa, and Rachel Tinker-Kulberg) and the

laboratory of Andrew Murray (Hironori Funabiki, Adam Rudner, and Alex Szidon). I also thank Jan-

Michael Peters and Kim Nasmyth for valuable comments on the manuscript, and I thank the many

colleagues who provided their results before publication. Work in my laboratory is supported by the

National Insitute of General Medical Sciences.

A supplementary video is available on Nature Cell Biology’s World-Wide Web site (http://

cellbio.nature.com).

NATURE CELL BIOLOGY | VOL 1 | JUNE 1999 | cellbio.nature.com E53© 1999 Macmillan Magazines Ltd