Finishing mitosis, one step at a time

A skyscraper is built in a strictly ordered series of steps: the foundation is laid, the frame goes up, and only after that are the walls added and the furniture moved in. The same is true for cell division: chromosome duplication comes first, after which the duplicated chromosomes are pulled apart and then packaged in individual cells. In both these cases, the ordering of events depends on central regulatory systems that initiate each event at the appropriate time, based in part on predetermined schedules. These schedules can be adjusted if necessary: when problems delay the completion of an event (such as chromosome duplication), subsequent events (such as mitosis) can be postponed.

Work over the past 20 years has unveiled the basic molecular workings of the control system that orders and coordinates the events of the eukaryotic cell-division cycle1. The central components of this system are the cyclin-dependent kinases (Cdks), the activities of which oscillate during the cell cycle in response to changes in their association with regulatory cyclin subunits. Distinct cyclin–Cdk complexes form at specific cell-cycle stages and initiate the events of the S and M phases. Mitotic cyclin–Cdk complexes drive the particularly striking events of early mitosis: chromosome condensation and resolution, nuclear envelope breakdown, and assembly of the mitotic spindle. Cdks have completed their main functions by metaphase, when all sister-chromatid pairs are bi-orientated on the spindle, pulled towards the poles but held together by sister-chromatid cohesion.

Cdk activity drives cell-cycle progression as far as metaphase, but progression into anaphase and beyond depends on another major regulatory component — a ubiquitin-protein ligase called the anaphase-promoting complex (APC) or cyclosome, which ubiquitinates sev-eral regulatory proteins and thereby targets them to the proteasome for destruction2,3. APC activity oscillates in

response to changes in the association of the APC with the activating subunits Cdc20 or Cdh1: association of the APC with Cdc20 in mid-mitosis leads to the initiation of anaphase, whereas association with Cdh1 in late mitosis maintains APC activity throughout the subsequent G1. A key target of the APC is securin, the destruction of which initiates chromosome segregation by unleashing the protease separase, which destroys sister-chromatid cohesion. The APC also ubiquitinates the mitotic cyclins, the destruction of which inactivates Cdks and allows phosphatases to dephosphorylate the many Cdk sub-strates in the cell. Dephosphorylation of Cdk substrates is required for normal chromosome and spindle move-ments in anaphase, as well as for the subsequent events of telophase: spindle disassembly, reformation of nuclei and decondensation of chromatin.

The final stages of mitosis are therefore governed by two main regulatory mechanisms: dephosphorylation of Cdk substrates and ubiquitination of APC substrates (FIG. 1). In this Review, we describe our present knowledge of the highly conserved molecular circuitry that under-lies these mechanisms and discuss the emerging concept that the correct ordering of late mitotic events depends, at least in part, on the order in which Cdk substrates are dephosphorylated and APC targets are destroyed.

Cyclin oscillations drive mitosisCdk activation in mitosis depends on two or three classes of mitotic cyclins, each defined by its function and the timing of its expression. In animal cells, the first major mitotic cyclin to be expressed is cyclin A, which appears at the onset of S phase, when it is thought to contribute to the stimulation of DNA synthesis. Cyclin A levels remain high after S phase, and in early mitosis it drives the initiation of chromosome condensation and, possibly, nuclear envelope breakdown4,5. Cyclin A is destroyed

Departments of Physiology and Biochemistry & Biophysics, University of California, 600 16th Street, San Francisco, California 94158‑2517, USA.Correspondence to D.O.M. e‑mail: [email protected]:10.1038/nrm2276Published online 3 October 2007

Finishing mitosis, one step at a timeMatt Sullivan and David O. Morgan

Abstract | The final stages of mitosis begin in anaphase, when the mitotic spindle segregates the duplicated chromosomes. Mitotic exit is then completed by disassembly of the spindle and packaging of chromosomes into daughter nuclei. The successful completion of mitosis requires that these events occur in a strict order. Two main mechanisms govern progression through late mitosis: dephosphorylation of cyclin-dependent kinase (Cdk) substrates and destruction of the substrates of the anaphase-promoting complex (APC). Here, we discuss the hypothesis that the order of late mitotic events depends, at least in part, on the order in which different Cdk and APC substrates are dephosphorylated or destroyed, respectively.

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894 | NoveMbeR 2007 | voluMe 8 www.nature.com/reviews/molcellbio

© 2007 Nature Publishing Group

http://www.expasy.org/uniprot/P26309



http://www.expasy.org/uniprot/Q03018

mailto:[email protected]

Nature Reviews | Molecular Cell Biology

Arb

itrar

y un

its

Cdk activity

APC activity

APC substrate

Cdk substrate

UbUbUb

P PP

Earlymitosis

Mitoticexit

MetaphaseG2 G1Anaphase

Hydrophobic patchA short stretch of amino acids on the surface of some cyclins near the Cdk active site. It interacts with RXL motifs on cyclin-specific Cdk substrates or inhibitors.

RXL motifA degenerate sequence motif on some Cdk substrates and inhibitors. It interacts with the hydrophobic patch region of specific cyclins.

during prometaphase6. Next in line is cyclin b, the levels of which rise during G2. Cyclin b promotes the comple-tion of chromosome condensation and spindle assembly, thereby driving cell-cycle progression as far as metaphase. Cyclin b (together with securin) is destroyed during meta-phase, significantly later than cyclin A7. Finally, a third mitotic cyclin, cyclin b3, has been identified in some animal species, although its role in mitosis is unclear8,9. on the basis of studies in Drosophila melanogaster embryos, cyclin b3 is thought to be destroyed in anaphase, later than cyclin b. The three mitotic cyclins are therefore destroyed in a clear sequence: cyclin A, cyclin b and then cyclin b3 (FIG. 2a).

A series of distinct cyclins also governs mitosis in bud-ding yeast, although in this species there is considerable overlap in the functions of the different cyclins10. The major S-phase cyclin, Clb5, exhibits features that are remi-niscent of vertebrate cyclin A: its expression increases in late G1, it helps to stimulate chromosome duplication and it appears to have some functions during mitosis. unlike cyclin A, Clb5 is destroyed just before anaphase, together with securin. The major mitotic cyclin, Clb2, is crucial for spindle assembly and progression to metaphase. Some Clb2 protein is destroyed at the same time as Clb5 and securin, but most remains stable until after anaphase.

Cyclin destruction can order late mitotic events. What function is served by destroying different cyclins at dif-ferent stages of mitosis? An appealing model is that each class of cyclin is responsible for driving the phosphoryla-tion of a subset of Cdk substrates, and that each subset is dephosphorylated when its respective cyclin is destroyed.

Specific targets of cyclin A–Cdk, for example, would be expected to be dephosphorylated before anaphase, earlier than the targets of cyclin b–Cdk. Given that Cdk-substrate dephosphorylation drives late mitotic events, it follows that the ordered destruction of different cyclins could help to order the events of late mitosis.

A key requirement of this model is that different cyclins must have different intrinsic functional capacities. Abundant evidence supports this notion10. For example, when individual cyclins are mutated to render them resistant to APC-mediated destruction, the resulting cellular phenotype generally varies depending on which cyclin has been mutated. A particularly striking example is found in embryos of D. melanogaster: stabilization of cyclin A causes an arrest in metaphase, stabilization of cyclin b causes an arrest in anaphase and abnormal chromosome movements, and stabilization of cyclin b3 allows normal anaphase but causes defects in later events8,11 (FIG. 2b). The effects of stabilized cyclins vary in different cell types and species, but the clear implica-tion from these types of studies is that different cyclins are destroyed at different times to promote the correct sequence of late mitotic events.

A mechanism that probably underlies the specializa-tion of cyclin function is that cyclins restrict the substrate specificity or the localization of the associated Cdks10. biochemical studies with mammalian cyclins indicate that several Cdk substrates are phosphorylated rapidly in vitro by cyclin A–Cdk1 but not by cyclin b–Cdk1 (ReF. 12), and cyclin specificity in these cases depends on a docking site in cyclin A (called the hydrophobic patch) that interacts with a small motif (called the RXL (or Cy) motif) that is found on its targets13. Similarly, several Cdk1 substrates in budding yeast are much more rapidly phosphorylated by Clb5–Cdk1 than by Clb2–Cdk1, again as a result of an interaction between the hydrophobic patch in Clb5 and a docking motif in specific substrates14. As might be pre-dicted, many of these Clb5-specific substrates are involved in chromosome duplication, although some have func-tions in the mitotic spindle. As we discuss below, dephos-phorylation of spindle proteins helps to govern spindle behaviour during anaphase.

Phosphatases order dephosphorylationThe timing of Cdk-substrate dephosphorylation is not determined simply by the timing of kinase inactivation: phosphatases must remove phosphates from Cdk targets, and substrate specificity could have a considerable impact on the order in which Cdk substrates are dephosphory-lated. Analysis of phosphatases in late mitosis has lagged far behind that of the kinases, but progress is beginning to be made in this area.

Numerous general phosphatases exist in the cell, and the basal activity of some of these might help to drive Cdk- substrate dephosphorylation when specific cyclin– Cdk complexes are inactivated. There is also evidence that robust dephosphorylation is a result of the activation of specific phosphatases during late mitosis. The classic example is the budding yeast phosphatase Cdc14, which is activated transiently during late mitosis and is required for the dephosphorylation of several Cdk substrates15,16.

Figure 1 | Control of late mitotic events. Progression through mitosis is shown by the cells along the bottom of the figure (chromosomes in blue and spindle microtubules in red). The transition from metaphase to anaphase is triggered by an increase in the activity of the anaphase-promoting complex (APC) (green line; top of figure), a ubiquitin-protein ligase that promotes the assembly of chains of ubiquitin (Ub) on its substrates, thereby targeting them for destruction in the proteasome. The main APC targets are securin, the destruction of which leads to sister-chromatid separation, and cyclins, the destruction of which results in a drop in Cdk activity (blue line). Cdk inactivation allows cellular phosphatases to dephosphorylate Cdk substrates during late mitosis. Cdk-substrate dephosphorylation is required for the events of anaphase and telophase (not shown).

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NATuRe RevIeWS | moleCular Cell biology voluMe 8 | NoveMbeR 2007 | 895






G2 G1Prophase Prometaphase Metaphase Anaphase Telophase

Stable cyclin Ab

a

Stable cyclin B Stable cyclin B3

Prot

ein

leve

l

Cyclin ACyclin BCyclin B3


Stepwise activation of Cdc14. before anaphase, Cdc14 is found in the nucleolus, where it is held in an inactive state by its association with the protein Net1 (also known as Cfi1). beginning in early anaphase, Cdc14 is activated by dissociation from Net1 and by release from the nucleolus17,18. Cdc14 then diffuses throughout the nucleus and out into the cytoplasm to dephosphory-late targets in both locations. Activation and release of Cdc14 from the nucleolus depends on two regulatory mechanisms (FIG. 3). First, Cdc14 is activated by separase, the protease that also triggers sister-chromatid sep-aration. Separase initiates Cdc14 activation by a poorly understood process that depends on several other pro-teins, together referred to as the Cdc14 early anaphase release (FeAR) network19,20. Second, complete Cdc14 activation depends on the mitotic exit network (MeN), a signalling system that includes a small GTPase and a pair of protein kinases that are activated during late mitosis21.

The two Cdc14 activation mechanisms seem to act in sequence to trigger the dephosphorylation of distinct populations of Cdk substrates and, thus, dis-tinct mitotic events (FIG. 3). The FeAR network initiates Cdc14 activation and triggers various processes that are important for successful anaphase, including the

segregation of ribosomal DNA22,23, positioning of the anaphase nucleus24 and stabilization of the anaphase spindle25. These events are likely to depend on dephos-phorylation during early anaphase of a limited subset of Cdk targets, including the yeast INCeNP homologue Sli15 (ReF. 26) and the spindle regulators Ase1, Ask1 and Fin1 (ReFs 25,27,28). The FeAR network alone, however, is not sufficient for the dephosphorylation of all Cdc14 targets. In the absence of MeN activity, FeAR-dependent Cdc14 activity rises in early anaphase and an apparently normal anaphase occurs, but Cdc14 activity declines prematurely and the mutant cells arrest at the end of anaphase19. MeN activation is required for complete Cdc14 activation and for the dephosphoryla-tion of numerous Cdk substrates, including the APC activator Cdh1, the transcription factor Swi5 and the Cdk inhibitor Sic1 (all of which help to drive the com-pletion of Cdk inactivation in late mitosis, as described below). So, the two steps in Cdc14 activation might pro-vide a mechanism for the ordered dephosphorylation of different Cdk substrates and, therefore, the ordered execution of distinct events.

Cdc14 homologues have been identified in higher eukaryotes, but they do not seem to have the same central function in late mitosis as in budding yeast29.

Figure 2 | The order of mitotic cyclin destruction. a | The three major mitotic cyclins of animal cells are destroyed at different times during mitosis. Cyclin A is degraded soon after nuclear envelope breakdown, whereas cyclin B degradation (and that of securin) begins immediately after the last sister-chromatid pair is bi-orientated on the spindle at the beginning of metaphase. An additional mitotic cyclin, cyclin B3, has been studied primarily in Drosophila melanogaster embryos, in which it is destroyed after cyclin B. Key mitotic transitions are indicated by bold dashed lines. b | Distinct phenotypes result when different mitotic cyclins are stabilized during mitosis. Stabilized forms of each cyclin were constructed by mutating the sequences that are required for recognition by the anaphase-promoting complex. Expression of the indicated cyclin was induced during G2 of cycle 14 in D. melanogaster embryos, resulting in mitotic arrests (DNA is shown in green and microtubules are shown in red). Stable cyclin A caused an arrest during metaphase, stable cyclin B caused an arrest during anaphase and abnormal chromosome movements, and stable cyclin B3 caused an arrest during late anaphase. Panel b is reproduced with permission from ReF. 11 (2001) Elsevier.

R E V I E W S






Cdc14

Clb2

Cdc20

Cdc55


APC

Separase

SecurinSister-chromatid separation

PP2A

Net1Net1

P P P

Cdk1

+

Partly activeInactive

Dephosphorylation ofearly anaphase targets

Chromosome movements,spindle stability,rDNA segregation

Cdc14

Fully active

Dephosphorylation oflate anaphase targets

Clb2–Cdk1 inactivation,completion of mitosis

Dbf2

Cdc15

Mob1

Tem1

Tem1

GDP

GTP Lte1Bub2

Bfa1

Cdc5

MEN

FEAR

Cdc14

Separase

In fission yeast and Caenorhabditis elegans, for example, the Cdc14 homologue is not required for mitotic exit, although it is crucial for cytokinesis. Furthermore, in these and other species Cdc14 might also help to govern early mitosis, in part through the dephosphorylation of Cdk regulators. Therefore, much remains to be learned about the phosphatases that govern the phosphorylation states of Cdk substrates during late mitosis in animal cells.

Order of Cdk-substrate dephosphorylationDespite the copious hints that late mitotic events depend on the ordered dephosphorylation of Cdk targets, we still know remarkably little about the identities of these targets. Several potential Cdk substrates have been identified, but few have been linked to a specific mitotic process and even fewer have been studied in sufficient detail to assess the precise timing of their dephosphorylation in relation to other substrates. We discuss a few of the best-known examples in this section.

Separase is dephosphorylated before anaphase. In ver-tebrate cells, separase is a well-established Cdk substrate that is dephosphorylated and thereby activated before anaphase (FIG. 4). Cdks phosphorylate separase in early mitosis and thereby inhibit its protease activity, indicating that separase dephosphorylation (in addition to securin destruction) must occur before anaphase can begin30. Inhibition of separase by cyclin A–Cdk may explain the observation that the expression of stabilized cyclin A mutants in flies (FIG. 2b) and in some vertebrate cells blocks the onset of anaphase6,8,11,31. However, separase is not strictly a cyclin A target that is dephosphorylated only when cyclin A is degraded. In vertebrates, cyclin b–Cdk1 also phosphorylates separase (and inhibits it by bind-ing to it)32, and a stabilized cyclin b mutant prevents anaphase in some mammalian cell types7,33,34. In these cases at least, separase is probably dephosphorylated at the end of metaphase, after cyclin b–Cdk1 is inactivated. The phosphatase that acts on separase is not known.

Figure 3 | Control of Cdc14 activation by separase and the meN. During metaphase, Cdc14 is held inactive in the nucleolus by an interaction with its inhibitor Net1 (also known as Cfi1). The initial release and activation of Cdc14 during early anaphase is the result of an abrupt increase in Net1 phosphorylation, which is carried out by a complex of the mitotic cyclin Clb2 and the cyclin-dependent kinase, Cdk1 (Clb2–Cdk1)80. Recent studies20 suggest that Net1 phosphorylation is removed before anaphase by the phosphatase PP2ACdc55. By unknown mechanisms, separase and the Cdc14 early anaphase release (FEAR) network reduce PP2ACdc55 activity on Net1, which tilts the balance of activities towards Net1 phosphorylation, thereby initiating Cdc14 activation. Complete Cdc14 activation during late mitosis requires the mitotic exit network (MEN), the central component of which is the GTPase Tem1 (ReF. 21). Tem1 activation might be promoted by the guanine-nucleotide exchange factor Lte1 and is opposed by the GTPase-activating protein complex Bfa1–Bub2. Tem1 activates a protein kinase, Cdc15, which in turn activates a kinase complex, Dbf2–Mob1, which stimulates Cdc14 activity. Activation of the MEN depends on the Polo-like kinase Cdc5, which is activated during mitosis and promotes Tem1 activation by phosphorylating and thereby inhibiting Bfa1–Bub2 (ReF. 81). In addition, Cdc14 dephosphorylates and thereby activates the kinase Cdc15 (ReF. 82). It is not known how MEN activation promotes Cdc14 function. It is unlikely that the MEN simply increases the level of Cdc14 activity. Instead, it might somehow give Cdc14 access to a broader range of targets. For example, separase and the FEAR network might promote the release of Cdc14 within the nucleus, whereas the MEN might allow Cdc14 access to targets in the cytoplasm.

R E V I E W S




Prometaphase Metaphase Anaphase Telophase

Phos

phor

ylat

ion

leve

l

Pre-anaphase substrates(vertebrate separase)Early-anaphase substrates(INCENP, Fin1, Ase1/PRC1)Late-anaphase substrates(Cdh1, Sic1, spindle proteins)

Completion ofsister-chromatidattachment

Sister-chromatidseparation

Initiation ofspindle disassembly,cytokinesis

KinetochoreA large protein structure that assembles on the chromosome and mediates the attachment of the chromosome to microtubules of the mitotic spindle.

Spindle midzoneThe region at the equator of the mitotic spindle where interpolar microtubules overlap. During anaphase, this region helps to organize proteins that govern anaphase spindle behaviour and that control the initiation of cytokinesis.

Dephosphorylation of Cdk substrates in early anaphase. Dephosphorylation of Cdk substrates has been implicated in anaphase chromosome movement and spindle stability (FIG. 4). expression of stabilized cyclin b in some animal cell types, including the fly embryo, results in abnormal chromo-some movements and spindle structure35,36. In budding yeast, anaphase chromosome and spindle behaviours are defective when sister-chromatid segregation is artificially induced under conditions of high Cdk activity and low Cdc14 activity25. one important Cdk substrate in ana-phase is INCeNP (Sli15 in yeast), the regulatory subunit of the kinase Aurora b (Ipl1 in yeast). Dephosphorylation of INCeNP in early anaphase triggers its translocation from kinetochores to the spindle midzone, where it promotes spindle stability26. The dephosphorylation of several addi-tional yeast Cdk substrates has also been implicated in the control of anaphase spindle behaviour. These substrates include: Ask1, a kinetochore protein that contributes to normal anaphase chromosome movements and spindle function25; Fin1, which binds and stabilizes the anaphase spindle27; and Ase1, which helps to organize the spindle midzone during anaphase28. PRC1, the human homologue of Ase1, is also a spindle midzone protein that is activated by dephosphorylation during anaphase37.

The yeast proteins Fin1 and Ase1 are specific targets of Clb5–Cdk1 (ReF. 14), and their dephosphorylation during early anaphase is driven by the combined effects of Clb5

destruction and separase-dependent Cdc14 activation27,28. Thus, in the case of these proteins at least, it is clear that the timing of cyclin destruction helps to determine the timing of substrate dephosphorylation and function.

Dephosphorylation of Cdk substrates in late anaphase. Three key components of the budding yeast cell-cycle control system — Cdh1, Sic1 and Swi5 — are Cdk1 sub-strates that must be dephosphorylated during anaphase for late mitotic progression and for entry into G1. Cdh1 is an APC activator, and its phosphorylation by Cdks restrains its activity from late G1 until anaphase, when it is dephosphorylated and thereby activated38,39. APCCdh1 is required for the complete destruction of Clb2, and so the inactivation of Clb2–Cdk1 is delayed until late anaphase or thereafter. Sic1 has a similar regulatory function to that of Cdh1: it is a tight-binding inhibitor of all Clb–Cdk complexes and its phosphorylation between late G1 and mitosis triggers its destruction40,41. Its dephosphorylation during late mitosis allows Sic1 to accumulate and contrib-ute to Clb2–Cdk1 inactivation42. Swi5 is a transcription factor that drives the expression of SIC1 and other genes. It is inhibited by Cdk1-dependent phosphorylation and is activated by dephosphorylation during late mitosis42,43.

Cdh1, Sic1 and Swi5 are all thought to be dephos-phorylated during late anaphase as a result of Clb2 des-truction and MeN-dependent Cdc14 activation (FIGs 3,4). Phosphorylated Cdh1 and Swi5 are located in the cyto-plasm and are then imported into the nucleus upon dephosphorylation43,44; therefore, their activation might depend on the export of Cdc14 from the nucleus. The full activation and nuclear export of Cdc14 could depend on the MeN, which is activated later than the FeAR-dependent Cdc14 activation of early anaphase. Thus, the local-ization and stepwise activation of Cdc14 might provide a mechanism to introduce a delay between the dephos-phorylation of nuclear targets (such as those involved in spindle stabilization, as mentioned above) and that of cytoplasmic targets (such as Cdh1, Swi5 and proteins involved in later events, including cytokinesis45).

Other dephosphorylation events in late mitosis. The identities of Cdk substrates involved in the major events of late anaphase and telophase are not yet clear. Spindle assembly probably depends on the phosphorylation of many microtubule regulators and motor proteins, and it is reasonable to expect — although it has not been proven — that dephosphorylation of these same proteins is required for spindle disassembly during telophase. Similar mechanisms probably apply to the proteins that are phosphorylated to drive chromosome condensation and to the proteins of the nuclear pore and lamina that trigger nuclear envelope breakdown — the dephospho-rylation of these same proteins is likely to be required to reverse these events.

How the dephosphorylation of such proteins is delayed until after anaphase also remains unclear. In budding yeast, in which a subpopulation of the mitotic cyclin Clb2 is stable until the end of anaphase, a likely answer is that the proteins involved in telophase events are Clb2 targets that remain phosphorylated until Clb2 has been destroyed.

Figure 4 | The order of Cdk substrate dephosphorylation during mitosis. The curves represent the phosphorylation levels of various cyclin-dependent kinase (Cdk) substrates, which are dephosphorylated at different times as a result of differences in the timing of cyclin destruction (FIG. 2) and the activation of phosphatases such as Cdc14 in budding yeast (FIG. 3). Some substrates, of which vertebrate separase is presently the sole known example, must be dephosphorylated during late metaphase, before anaphase can begin. Several other substrates (such as INCENP, Fin1 and Ase1 (known as PRC1 in mammals)) are dephosphorylated at about this time or during early anaphase; these proteins help to govern chromosome and spindle behaviour during anaphase. A large number of substrates are dephosphorylated later during anaphase: these include regulatory proteins such as Cdh1 and Sic1, the dephosphorylation of which completes Cdk inactivation, and spindle proteins, the dephosphorylation of which leads to spindle disassembly. Other major telophase events, such as chromosome decondensation, nuclear envelope formation and the initiation of cytokinesis, also depend on Cdk-substrate dephosphorylation during late anaphase or thereafter. The metaphase- to-anaphase transition is indicated with a bold dashed line.

R E V I E W S






Apc1

Apc5

Cdc23

Cdc16

Cdc27

Apc4Apc2

Doc1 Apc11

E2

Ub

Cdc20orCdh1

Target

NH2

26S proteasomeA large protease complex that binds polyubiquitinated proteins and degrades them.

Destruction box(D-box). A degenerate sequence motif (RXXLXXXXN) that is in most APC targets. The D-box mediates an interaction with APC activator subunits and is required for target destruction.

KEN boxA degenerate sequence motif (KeNXXXN) that is in some APC targets. The KeN box mediates an interaction with the APC activator Cdh1.

Polo-like kinase-1(Plk1). A protein kinase that is activated during early mitosis and that helps to promote certain mitotic events, such as spindle assembly.

Aurora kinases A and BRelated protein kinases that are activated during early mitosis and govern spindle assembly, chromosome attachment to kinetochores and other mitotic processes.

In animal cells, however, the major mitotic cyclin, cyclin b, is destroyed in parallel with securin during metaphase. If dephosphorylation of cyclin b targets promotes telo-phase events, then why are these targets not dephosphoryl-ated until long after cyclin b is gone? The answer might lie in the activity and specificity of mitotic phosphatases, which remain largely unexplored in animal cells.

Order of APC-substrate destruction The sequence of Cdk-substrate dephosphorylation depends, in part, on the order in which cyclins are destroyed. What, then, determines the order in which cyclins and other APC targets are destroyed? Multiple mechanisms are involved, but a central issue is the intrin-sic ability of different isoforms of the APC to recognize and ubiquitinate different targets. Here, we discuss some of the mechanisms by which APC-substrate specificity can change at different stages of mitosis.

like other ubiquitin-protein ligases, the APC catalyses the transfer of ubiquitin from an e2–ubiquitin conjugate to a lys side chain on a protein target (BOX 1). ubiquitin itself can also be modified, resulting in polyubiquitin chains that direct the target to the 26s proteasome for destruction. The APC reaction is processive, such that multiple ubiquitins are attached before the substrate dissociates.

The efficiency and processivity of the APC reaction are determined, at least in part, by the affinity of protein-target binding to the APC. For example, a decrease in the affin-ity of a substrate for the active site of the APC probably reduces processivity — and thereby reduces the length of polyubiquitin chains on the protein and, hence, its rate of destruction. Currently, there is only a rudimentary understanding of how the APC interacts with its sub-strates. APC targets contain short amino-acid sequence

motifs, called the destruction (D-) box and KeN box, that are required for target destruction during mitosis46,47. These motifs interact directly with the activator subunits Cdc20 or Cdh1 (ReFs 48,49), which are then thought to recruit the bound substrate to the APC for ubiquitination. There is also evidence that the APC itself contains binding sites for the D-box, although these sites are not understood in any detail50,51.

APC activators order substrate degradation. The two APC activators, Cdc20 and Cdh1, bind directly to APC sub-strates and recruit them to the APC core for ubiquitina-tion. The APC is activated first by Cdc20 before anaphase and then by Cdh1 during late anaphase. Interestingly, the two activators have different substrate specificities: Cdc20 binds a limited range of targets (primarily securin and cyclins), whereas Cdh1 has a broader specificity that includes Cdc20 targets and various additional proteins that are not recognized by Cdc20 (including Cdc20 itself, Polo-like kinase-1 (Plk1), Aurora kinases A and B and, in yeast, the spindle proteins Ase1 and Fin1)2,3. Thus, the ubiquitination of APCCdc20 targets occurs earlier than that of APCCdh1 targets, thereby providing a clear mechanism of APC-substrate ordering (FIG. 5).

Differential substrate targeting by APCCdc20 and APCCdh1 is presumably achieved by differential recogni-tion of APC substrates by each activator. Cdh1-specific targets often contain a KeN-box motif that is not recog-nized by Cdc20 (ReF. 47). The main recognition motif, the D-box, is found in most APC substrates, although many of these substrates are recognized only by APCCdh1 and not by APCCdc20. It seems likely that subtle differences in D-box sequences, or sequence context, can influ-ence substrate recognition by different APC isoforms.

Box 1 | APC enzymology

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin-protein ligase of the RING-domain family72,73. Like other members of this family, it binds two substrates: the protein target (such as securin or cyclin) and an E2 ubiquitin-conjugating enzyme that is covalently linked to the C terminus of a 76-residue protein called ubiquitin (Ub; see figure). The APC stimulates transfer of the C terminus of ubiquitin from the E2–ubiquitin conjugate to a Lys side chain on the target substrate. In subsequent reactions, it can catalyse the transfer of ubiquitin to a Lys in another molecule of ubiquitin, resulting in the formation of polyubiquitin chains74,75. Long chains that are linked at Lys48 of ubiquitin are efficiently recognized by the 26S proteasome, resulting in target destruction76. Clusters of short chains might also drive proteasome-dependent destruction74.

Multiubiquitination by the APC is processive, so multiple Lys residues can be modified during a single substrate-binding event66,77. Processivity probably depends on differences in the interaction affinities of the enzyme with its two substrates. Thus, the APC might bind a protein target relatively tightly (that is, with a low dissociation rate) while binding an E2 with relatively low affinity. Therefore, the protein target would remain bound to the APC as a series of E2–ubiquitin conjugates bind, unload their ubiquitin on the target and dissociate to make way for the next E2–ubiquitin77,78.

The APC contains 12 or 13 subunits2,3. In yeast, nine subunits are essential for normal activity and are thought to interact as shown in the figure79. The Apc11 subunit contains the RING domain and probably binds the E2–ubiquitin conjugate. Protein targets are recruited by the activator subunits Cdc20 or Cdh1, which interact with specific APC core subunits. Substrates may also interact directly with core subunits such as Doc1 (ReFs 50,51).

R E V I E W S



G1Prophase Prometaphase Metaphase Anaphase Telophase

Nuclearenvelopebreakdown


Cdc20 Plk1 Aurora A Aurora BCyclin BSecurin

Prot

ein

leve

l

Cdc20

APC

Cdc20

APC

Cdh1

APC

SAC

Completion ofsister-chromatidattachment

Sister-chromatidseparation

Initiation ofspindle disassembly,cytokinesis

Cyclin A Nek2AHOXC10

Spindle-assembly checkpointA regulatory system that monitors chromosome attachment to the mitotic spindle and delays APC activation until all chromosomes are correctly bi-orientated.

Adding to this complexity is the fact that several APC targets contain destruction sequences that bear little resemblance to either the KeN box or the D-box52–56.

budding yeast display an interesting case of APC- substrate ordering that is not easily explained. In this species, APCCdc20 triggers complete destruction of the cyclin Clb5 before anaphase, but the levels of the major mitotic cyclin Clb2 are only partly reduced57,58. APCCdh1 activation later during anaphase is required to drive the destruction of the remaining Clb2. A subpopulation of Clb2 might be protected from APCCdc20 by extrinsic factors such as localization59.

Spindle-assembly checkpoint orders substrate degradation. In budding yeast, the major Cdc20-specific targets (securin and Clb5) are degraded together before anaphase. In ani-mal cells, however, some Cdc20-specific targets (cyclin A, the kinase Nek2A and the transcription factor HoXC10) are degraded during prometaphase6,31,60,61, whereas others (securin and cyclin b) are not degraded until meta-phase7. This ordering of APCCdc20 targets in animal cells depends on a regulatory system called the spindle-assembly checkpoint (SAC), which prevents the destruction of

securin and cyclin b until all sister-chromatid pairs are correctly attached to the spindle62.

APCCdc20 is first activated during prometaphase, which leads to the ubiquitination of prometaphase targets such as cyclin A. However, the unattached sister chromatids that exist in prometaphase cells generate signals that allow inhibitory SAC components, such as Mad2, to bind Cdc20 and block its ability to interact with substrates. by some mysterious mechanism, Mad2 and other SAC pro-teins prevent the ubiquitination of some APCCdc20 targets (securin and cyclin b) while allowing the ubiquitination of others (cyclin A and Nek2A) (FIG. 5).

Prometaphase targets may be recognized by the APC through a unique mechanism that does not require bind-ing to Cdc20 (ReF. 60). The prometaphase target Nek2A has a dipeptide sequence (Met-Arg, MR) at its C terminus that resembles a sequence (Ile-Arg, IR) that is found at the C termini of the APC activators Cdc20 and Cdh1. The IR sequence motif is required for association of the activ-ators with specific subunits of the APC63. The MR motif of Nek2A appears to interact directly with the APC core as well, apparently bypassing the need for an activator to recruit the substrate. However, Cdc20 is still required for

Figure 5 | Three windows of aPC-dependent destruction in human cells. Three different anaphase-promoting complex (APC) isoforms predominate during different stages of mitosis and promote the ordered destruction of three different groups of proteins. The APC is first activated during early prometaphase by its activator subunit Cdc20. This form of the APC is initially inhibited by components of the spindle-assembly checkpoint (SAC), which interact with Cdc20 and block its ability to recruit substrates to the APC. By unknown mechanisms, SAC-inhibited APC retains the ability to promote the ubiquitination of several prometaphase targets, including cyclin A, the kinase Nek2A and the transcription factor HOXC10. When the last sister-chromatid pair is attached to the spindle and the SAC is inactivated, fully activated APCCdc20 then targets an additional group of proteins — including cyclin B and securin — for destruction during metaphase. Cyclin destruction leads to cyclin-dependent kinase (Cdk) inactivation, which leads to the dephosphorylation and activation of the second APC activator, Cdh1. APCCdh1 has broader substrate specificity than APCCdc20 and triggers the destruction of various additional targets. These targets are destroyed at different times during late mitosis, presumably providing mechanisms that help to order late mitotic events. APCCdh1 targets include Cdc20, Plk1 and the Aurora kinases, which are destroyed in the order shown in the figure64. There may also be substrate ordering for the substrates of APCCdc20. In Drosophila embryos, for example, cyclin B and cyclin B3 are APCCdc20 targets that are destroyed at different times, as shown in FIG. 2. Key mitotic transitions are indicated with bold dashed lines.

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Nek2A ubiquitination, implying that Cdc20 can activate the APC independently of its substrate-recruiting func-tion, perhaps by inducing some conformational change in the APC core. Such a mechanism could help to explain why the SAC does not prevent the ubiquitination of prometaphase targets such as Nek2A. That is, the SAC-inhibited form of Cdc20 may not be able to bind substrates but may still be able to drive an activating conformational change in the APC, which allows the ubiquitination of proteins that are recruited by activator-independent mechanisms. Cyclin A does not have an IR-like motif at its C terminus, but may contain some other sequence motif that allows it to interact with the APC independ-ently of Cdc20. There is evidence that the APC can bind D-box sequences independently of activators50,51, and so it is possible that the D-box sequences of prometaphase targets have a particularly high affinity for binding sites on the APC core.

APCCdh1 targets are degraded at different times. The activ-ation of APCCdh1 in anaphase does not lead to synchro-nous destruction of all of its targets. In human cells, for example, different APCCdh1 substrates disappear at differ-ent times: Cdc20 is degraded first, followed by Plk1, then Aurora A and finally Aurora b64,65 (FIG. 5). The ordering of APCCdh1 targets probably depends on two general classes of mechanism.

First, APCCdh1 might have different intrinsic activities towards different substrates, such that some substrates are more rapidly and processively modified than others, leading to their earlier degradation. There are consider-able differences in the intrinsic activity of APCCdh1 towards different substrates. Recent studies, for example, reveal that Cdc20, Plk1 and Aurora A are multiubiquitinated in vitro at decreasing rates and processivities that correlate with the order of their disappearance in the cell66. The order-ing of substrate destruction is therefore likely to depend, in part, on differences in the affinity of substrates for the APC. In a sea of multiple different substrates, competition for binding to the APC would initially result in more interactions with substrates that have the highest affini-ties. In addition, given that the affinity of APC-substrate binding helps to determine the processivity of multiubiq-uitination (BOX 1), high-affinity substrates will be modi-fied with longer polyubiquitin chains. upon release from the APC, substrates that are more extensively modified have a greater chance of retaining a sufficiently long chain for recognition by the proteasome, even in the face of abundant de-ubiquitinating activities in the cell.

What factors underlie these differences between APCCdh1 substrates? one of the key factors is likely to be the relative affinity of destruction sequences on the substrate for binding sites on Cdh1 or the APC core. In addition, the position and density of available lys residues on a substrate could affect the pattern of ubiq-uitination and, therefore, the likelihood of destruction by the proteasome.

A second mechanism for ordering APCCdh1 substrates depends on extrinsic factors that modulate the recogni-tion of some substrates, thereby changing the timing of their degradation. Changes in the phosphorylation state

of substrates, for example, could be crucial in some cases. The phosphorylation of the replication protein Cdc6 by cyclin e–Cdk2 protects it against APC-dependent ubiquitination, providing a mechanism that allows Cdc6 accumulation and thus enables origin licensing when cells enter the cycle from quiescence67. Another potentially important extrinsic factor is the subcellular localization of the APC and its substrates. The APC is found on the spindle, kinetochores and centrosomes in mitosis, and APC substrates at these sites may be more rapidly ubiqui-tinated and destroyed in the cell. In Drosophila embryos, for example, the destruction of cyclin b during late mitosis begins around the centrosomes and spindle68.

We do not yet understand the importance of ordered APCCdh1-substrate destruction during late mitosis. In budding yeast, only the destruction of securin and cyc-lins is essential for mitotic progression69, and Cdh1 is not required for viability. Destruction of other substrates, including most Cdh1-specific targets, is not absolutely essential but might enhance the robustness of chromo-some segregation, telophase events and cytokinesis. The spindle-stabilizing proteins Ase1 and Fin1, for example, are destroyed at the end of anaphase, presumably contrib-uting to spindle disassembly; however, non-degradable mutant forms of these proteins have only minor effects on spindle behaviour27,70. Similarly, the destruction of Cdh1-specific targets such as the Polo-like kinase Cdc5 of yeast or Plk1 of vertebrates might not be essential but might make important contributions to the control of the substrates of these kinases.

The three windows of APC-substrate degradation. In summary, APC-substrate degradation in animal cells occurs in a series of three mitotic windows (FIG. 5). First, during prometaphase, substrates such as cyclin A are ubiquitinated by APCCdc20 through mechanisms that may not require activator-dependent substrate recruitment. Second, the complete attachment of chromosomes to the spindle turns off the SAC, triggering APCCdc20-dependent ubiquitination of metaphase targets such as securin and cyclin b. Third, activation of APCCdh1 during anaphase allows continued destruction of metaphase targets and also promotes the ubiquitination of Cdh1-specific targets. Different Cdh1 targets are ubiquitinated at different times owing to intrinsic differences in their affinity for the APC or owing to other factors, such as substrate phosphoryla-tion and localization, that influence their interactions with the APC.

Future directionsThe hypothesis that the destruction of each class of cyclin leads to dephosphorylation of the specific targets of that cyclin is appealing but simplistic, and future models must incorporate the quantitative complexities of kinase– substrate interactions in the cell. First, each cyclin–Cdk complex is likely to possess different intrinsic activities towards its different substrates, primarily as a result of different affinities for those substrates. Indeed, studies of large numbers of yeast Cdk substrates in vitro reveal a remarkably broad range of activities14,71. Second, it is important to consider the concentrations of substrates

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in the cell, which are probably sufficiently high for most cyclin–Cdk complexes to be saturated with substrate. under these conditions, destruction of a specific cyclin to 1% of its normal level might have more dramatic effects on some substrates than others: low-affinity substrates might be dephosphorylated rapidly, whereas phospho-rylation of high-affinity substrates could be maintained until the kinase is completely inactivated. Third, there is the issue of the stoichiometry of phosphorylation and its functional impact. What fraction of the population of a specific substrate must be dephosphorylated to trigger a specific late mitotic event? This fraction is likely to vary in different cases: it is conceivable that dephosphorylation of 1% of some substrate populations is sufficient to initi-ate certain processes, whereas other substrate populations may have to be completely dephosphorylated.

Similarly, we cannot hope for a deep understanding of phosphatase function during late mitosis without more quantitative analyses of phosphatase–substrate inter-actions. Cdc14, for example, has a preference in vitro for Cdk phosphosites (that is, a phosphoserine followed by a Pro residue), but studies with various Cdk substrates in vitro reveal a broad range of activities (l. Holt and D.o.M., unpublished observations), and some Cdk targets do not appear to be targets of Cdc14. Thus, some order-ing of Cdk-substrate dephosphorylation may result from intrinsic differences in the activity of Cdc14 towards its various substrates, not to mention the activities of other phosphatases that have not yet been discovered.

Studies of the importance of APC-substrate degrada-tion in vivo have often focused on the phenotype that results if a substrate is not degraded during mitosis. Future work should begin to address the related but distinct ques-tion of what happens when the order of APC-substrate destruction is altered. What is the effect on mitosis, for example, if cyclin b is degraded during prometaphase with Nek2A, if cyclin A is degraded late at anaphase onset, or if Aurora A is degraded before Cdc20? once we have a better understanding of the sequence motifs that determine the timing of substrate destruction, it may become possible to address these questions by swapping destruction motifs between substrates that are degraded at early and late stages.

The importance of the timing of Cdk-substrate dephosphorylation is not as tractable as that of APC-substrate destruction because it is not easy to create

mutant substrates that cannot be dephosphorylated. Substitution of phosphorylation sites with acidic resi-dues sometimes provides clues about the consequences of continued phosphorylation, but additional methods will be required to allow systematic analyses of many substrates. Perhaps a better understanding of the phos-phatases that govern Cdk substrates will lead to experi-mental approaches for constructing substrates that are resistant to dephosphorylation.

ConclusionsThe onset of anaphase and the completion of mitosis are governed by dephosphorylation of Cdk substrates and ubiquitination of APC substrates. A growing body of evidence suggests that the substrates of Cdks and the APC are modified at different times during mitosis, and the clear implication of this evidence is that the ordering of Cdk and APC substrates helps to establish the correct order of late mitotic events. A great deal of further effort is required, however, to prove that substrate ordering is important. We need more detailed information about the exact time at which Cdk substrates are dephos-phorylated or APC targets are destroyed. Similarly, a complete understanding of the regulation of these sub-strates requires quantitative analyses of the activities and affinities of the Cdks and the APC for all of their targets. A complete picture of this regulatory system must also include the identities and activities of the phosphatases and de-ubiquitinating enzymes that oppose Cdk and APC functions in the cell. only with this information in hand will it be possible to thoroughly assess the impor-tance of intrinsic enzyme–substrate interactions in the timing of substrate modifications.

The cell is guided through the steps of mitosis by an immensely complex regulatory system that is assembled from multiple Cdk and APC activities. Studies of this regulatory system have been directed primarily towards the mechanisms that generate oscillations in the activity of these enzymes, on the assumption that these oscillations provide an accurate reflection of changes in the phospho-rylation or ubiquitination states of Cdk or APC substrates, respectively. A complete understanding of substrate modi-fications and functions, however, will come only from a more thorough knowledge of the specific interactions between cell-cycle regulatory enzymes and all of their major substrates.

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DATABASESUniProtKB: http://ca.expasy.org/sprotAse1 | Cdc14 | Cdc20 | Cdh1 | Cdk1 | Clb2 | Clb5 | Fin1 | Net1 | securin | separase | Sic1 | Swi5

FURTHER INFORMATIONDavid O. Morgan’s homepage: http://www.ucsf.edu/morgan

all liNks are aCTive iN The oNliNe Pdf

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http://ca.expasy.org/sprot














http://www.ucsf.edu/morgan

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Finishing mitosis, one step at a time