The anaphase-promoting complex: a key factor in the regulation of cell cycle

REVIEW

The anaphase-promoting complex: a key factor in the regulation of cell

cycle

Anna Castro*,1, Cyril Bernis1, Suzanne Vigneron1, Jean-Claude Labbe1 and Thierry Lorca*,1

1Centre de Recherche de Biochimie Macromoleculaire, CNRS FRE 2593 1919 Route de Mende, 34293 Montpellier cedex 5, France

Events controlling cell division are governed by thedegradation of different regulatory proteins by theubiquitin-dependent pathway. In this pathway, the attach-ment of a polyubiquitin chain to a substrate by anubiquitin-ligase targets this substrate for degradation bythe 26S proteasome. Two different ubiquitin ligases playan important role in the cell cycle: the SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC). Inthis review, we describe the present knowledge about theAPC. We pay particular attention to the latest resultsconcerning APC structure, APC regulation and substraterecognition, and we discuss the implication of thesefindings in the understanding the APC function.Oncogene (2005) 24, 314–325. doi:10.1038/sj.onc.1207973

Keywords: APC; cdc20; cdh1; proteolysis; mitosis

Introduction

Progress through mitosis is governed by the sequentialdegradation of different cell cycle proteins. Thisdegradation is mediated by the ubiquitination pathway.In this process, an ubiquitin chain is covalently attachedto the target protein thereby promoting its recognitionand degradation by a large cytosolic protease complexnamed 26S proteasome. Ubiquitination involves threemajor steps (Hershko and Ciechanover, 1998). First, theubiquitin-activating enzyme (E1) forms a thiol esterbond between the active-site cysteine and the C-terminalglycine residue of ubiquitin. Second, the activatedubiquitin is transferred by the formation of a new thiolester linkage to an active-site cysteine residue on theubiquitin-conjugating enzyme (E2). Finally, the ubiqui-tin molecule is coupled through an amide isopeptidelinkage to an e-amino group of the substrate protein’slysine residues. This coupling is performed directly bythe E2 or in conjunction with a third enzyme, theubiquitin-ligase (E3). An E3 enzyme may participate inthe shuttling of ubiquitin to the substrate either bydirectly forming a thiol ester bond or by simply bringingthe E2 enzyme in close proximity to the substrate.

Whatever the mechanism used, the E3 enzyme is a keycomponent of this pathway, since it confers substratespecificity. Two different ubiquitin-ligases play animportant role in the cell cycle: the SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC).The SCF is active throughout the cell cycle; however,it controls principally the G1/S and G2/M boundariesby inducing the degradation of different factors atthese phases of the cell cycle. This ubiquitin-ligase isa macromolecular complex formed of a cullin subunit,Cul1, a RING-H2 protein, Hrt1/Rbx1, an F-boxsubunit and a linker subunit, Skp1. In this complex,the F-box specifically recognizes phosphorylated SCFtargets. Once bound to the F-box, the SCF substrate willbe brought closer to the Cul1/Hrt1 catalytic core of thisubiquitin-ligase by Skp1. Finally, Cul1/Hrt1 will recruitthe E2 enzyme, Cdc34, and will induce substrateubiquitination (Pintard et al., 2004).

Unlike the SCF, the APC is mainly required to induceprogression and exit from mitosis by inducing proteo-lysis of different cell cycle regulators including Pds1/securin and cyclin B. This ubiquitin-ligase is a largecomplex that contains at least 11 subunits. Similar to theSCF, it contains a catalytic core formed of a cullinsubunit, Apc2, and a RING-H2 protein, Apc11.However, substrate recognition is mediated by theAPC activators, Cdc20 and Cdh1, in an F-box-independent pathway. This review focuses on the E3complex, APC.

Identification of the APC

Degradation of cyclin B by ubiquitination is a crucialevent required to induce exit from mitosis. As describedabove, the ubiquitin ligases, E3s, play a prominent rolein this pathway by inducing substrate specificity and E2recruitment. Thus, the identification and the regulationof the E3 ligase involved in cyclin B degradation is ofgreat interest for the general understanding of cell cycleprogression. The ubiquitin-ligase involved in cyclin Bubiquitination was first identified as a result of asimultaneous genetic screen in budding yeast (Irnigeret al., 1995) and two different biochemical studies inclam and Xenopus egg extracts (King et al., 1995;Sudakin et al., 1995). The screen of Saccharomycescerevisiae mutants that were defective in cyclin B

*Correspondence: T Lorca; E-mail: [email protected];A Castro, [email protected]

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degradation allowed the finding of mutations in theCDC23 and CDC16 genes. These two mutants, as wellas the Cdc27 mutant, had previously been shown tocause cells to arrest at metaphase (Hartwell et al., 1970).At the same time, a first biochemical study wasdeveloped in extracts from clam oocytes. Fractionationof these extracts showed the presence of a particulatefraction containing a complex of approximately1500 kDa with a destruction box-specific cyclin/ubiqui-tin-ligase activity. This complex named Cyclosome wasinactive in interphase and was activated during mitosisby Cdk1 (Hershko et al., 1994; Sudakin et al., 1995). Asecond biochemical study developed in Xenopus eggextracts described the presence of a 20S complex thatwas named APC and that contained the homologues ofthe budding yeast CDC16 and CDC27 genes (King et al.,1995). When APC was removed from these extracts byimmunoprecipitation with anti-Cdc27 antibodies, nocyclin B-ubiquitination activity was observed. More-over, immunopurified Cdc27 complexes were sufficientto complement interphase extracts to induce cyclin Bubiquitination. Subsequent work demonstrated thatCdc16p, Cdc23p and Cdc27p are three of the 11 (invertebrates) core subunits of the APC and that, inaddition, this complex is regulated during mitosis by twodifferent activators, Cdc20 and Cdh1.

All the results obtained in different species about therole of the APC during the mitotic cell cycle converge ona requirement of this ubiquitin-ligase for cell cycleprogression; however, this is not the case when the roleof this complex on the meiotic progression is analysed.In this regard, the APC seems to be essential to inducemetaphase II-exit, whereas this complex seems to berequired for metaphase I –to anaphase I transition insome species like Caenorhabditis elegans (Furuta et al.,2000), mouse (Terret et al., 2003) or yeast (Salah and

Nasmyth, 2000) and dispensable in others like inXenopus (Peter et al., 2001; Taieb et al., 2001).

APC substrates

Ubiquitin-dependent degradation of cell cycle factorsinduced by the APC is a key mechanism used by the cellto tightly regulate different transitions throughout celldivision. Thus, the APC orchestrates mitosis bycontrolling anaphase entry, anaphase progression, exitof mitosis and G1 phase. Moreover, it plays animportant role in the formation of the prereplicativecomplexes required to induce DNA replication. Finally,other additional roles of this E3 in different cellfunctions have also been described (Figure 1).

APC substrates controlling anaphase entry

Entry into anaphase is marked by the initiation of sisterchromatid separation. Sister chromatids are heldtogether by the multiprotein complex called cohesin.This complex is cleaved by separase that, in turn, isinhibited by securin. At the anaphase entry, the APC-dependent degradation of securin enables separaseactivation and as a consequence cleavage of the cohesincomplex, thereby allowing sister chromatid separation(Uhlmann et al., 1999, 2000; Yanagida, 2000). Proteolysisof securin is ensured by APCCdc20 before anaphase onset,but its degradation is maintained until the end of G1 byAPCCdh1 (Nasmyth, 2001; Zur and Brandeis, 2001).

Initial studies suggested that the sole proteolysis ofsecurin was required for anaphase entry. Accordingly, inbudding yeast, the failure of APC or Cdc20 mutants toenter anaphase is bypassed by the unique deletion of thesecurin gene, implying that securin is the sole essential

Figure 1 APCCdc20 and APCCdh1-dependent degradation of different cell cycle proteins. APCCdc20 is first activated at the prometaphase–metaphase transition where it will induce cyclin A and Nek2 degradation. This complex is also responsible for the subsequentdegradation at metaphase of cyclin B, Xkid and securin and at anaphase of the kinesins Kip1 and Cin8. From late anaphase untilmitotic exit and throughout G1phase, the degradation of all these proteins is ensured by APCCdh1. Moreover, this complex will firstinduce proteolysis of Cdc20, Prc1, Tome-1, Plk1 and Aurora A at mitotic exit and subsequent degradation of Orc1, Cdc6 and Gemininin early G1. Substrates whose degradation starts at the same phase of the cell cycle are equally coloured. The exact phase in whichevery substrate starts its APC-dependent degradation is represented in lower coloured arrows. Plane lanes represent APCCdc20-dependent degradation. Dotted lanes represent APCCdh1-dependent degradation

The anaphase-promoting complexA Castro et al

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substrate degraded by the APC required to enter thisphase of the cell cycle (Yamamoto et al., 1996; Ciosket al., 1998; Shirayama et al., 1999). Moreover, theaddition of nondegradable cyclin B to Xenopus eggextracts does not prevent sister chromatid separation,whereas the addition of an N-terminal fragment ofthis protein which blocks APC prevents both sisterchromatid separation and exit from mitosis (Hollowayet al., 1993). These results indicate that cyclin Bproteolysis is not essential to promote entry intoanaphase. However, more recent results suggest thatdegradation of both cyclin A and cyclin B is alsorequired to induce sister chromatid disjunction. InDrosophila, the expression of a nondegradable cyclin Ablocks separation of sister chromatids (Parry andO’Farrell, 2001), whereas, in Xenopus egg extracts, thisblock is also observed when high concentrations ofnondegradable cyclin B are added. In the latter case, themechanism by which cyclin B blocks anaphase entryinvolves direct inhibition of separase through itsphosphorylation (Stemmann et al., 2001).

APC substrates controlling anaphase progression

APC also induces degradation of several factors thatare essential for spindle-pole separation and spindledisassembly. One of these factors is the kinesin-relatedprotein Xkid. Xkid plays an important role in bothmeiotic and mitotic cell cycles. At meiosis, thischromokinesin is required to reactivate cyclin B/cdk1complex after meiosis I and to inhibit DNA replicationbetween meiosis I and meiosis II (Perez et al., 2002). Atmitosis, Xkid is required during prometaphase tomaintain the polar ejection force, a force that pusheschromosomes away from the pole and that mediateschromosome congression (Antonio et al., 2000; Funa-biki and Murray, 2000). However, subsequent proteo-lysis of this protein is also essential to allowchromosome movements to the spindle poles through-out anaphase. Accordingly, an excess of nondegradableXkid does not prevent sister chromatid separation butinhibits chromosome movements towards the spindlepoles (Funabiki and Murray, 2000). Xkid degradation ismediated by APCCdc20 and APCCdh1. It starts at anaphaseand is maintained until the end of G1 phase (Levesqueand Compton, 2001; Castro et al., 2003).

Two other motor proteins, the kinesins Kip1 andCin8, are proteolysed by the APC. These proteins arefirst degraded at the initial stages of anaphase and theirprotein levels are subsequently maintained throughoutthe G1 phase. Both kinesins are required to separatethe spindle poles during spindle assembly and meta-phase, but their subsequent degradation at anaphaseis required to allow progression through anaphase(Gordon and Roof, 2001; Hildebrandt and Hoyt, 2001).

Finally, the spindle-associated protein Ase1 is pro-teolysed by the APC. Ase1 is present at greatest levelsduring mitosis and is not detected in G1 phase,suggesting that its destruction could be mediated byCdh1/Hct1-dependent activation of the APC (Juanget al., 1997). In this regard, proteolysis of the Xenopus

ortholog of Ase1, Prc1, is indeed mediated by theAPCCdh1 complex (D Fesquet, personal communication).In budding yeast, Ase1 is associated with the spindlemidzone, and is required for anaphase B, appropriateelongation of the spindle and separation of the spindlepoles. A nondegradable Ase1 mutant delays spindledisassembly (Juang et al., 1997), indicating that removalof this protein from the spindle midzone is required forprogression through anaphase and exit from mitosis.

APC substrates controlling exit from mitosis

Cyclin B is the first known substrate of the APC.Destruction of this protein begins at metaphase andcontinues throughout mitosis and G1phase (Clute andPines, 1999). Both complexes APCCdc20 and APCCdh1

mediate its degradation. Cyclin B proteolysis is requiredto inhibit Cdk1 activity and as consequence to inducedifferent cell processes such as sister chromatid separa-tion, disassembly of the mitotic spindle, chromosomedecondensation, cytokinesis and reformation of thenuclear envelope (Murray and Kirschner, 1989; Lucaet al., 1991; Gallant and Nigg, 1992; Holloway et al.,1993; Surana et al., 1993).

Another APC substrate whose degradation is requiredfor mitotic exit is the Cdc5/Plk1/Polo kinase. Degrada-tion of Cdc5 in budding yeast is mediated by APCCdh1

and expression of a nondegradable Cdc5 arrests cells ina G2-like state, lacking mitotic cyclins and a mitoticspindle, suggesting that destruction of this protein isrequired to inactivate APC-dependent degradation ofmitotic cyclins as cells enter S phase (Charles et al., 1998;Shirayama et al., 1998). Accordingly, immunoprecipita-tion of Plx1 from interphasic Xenopus egg extracts inwhich APC has been activated by the addition ofpurified cyclin B/cdk1 induces premature inactivation ofcyclin B degradation (Brassac et al., 2000).

APC substrates controlling G1 phase

The main APC substrate during G1phase is the APCactivator Cdc20. Cdc20 proteolysis by APCCdh1 inducesAPCCdc20 inactivation and allows the switch fromAPCCdc20 to APCCdh1. APCCdc20 is active in the presenceof high cyclin B/cdk1 activity, whereas Cdh1 phosphor-ylation by cyclin B/cdk1 prevents APC/Cdh1 associa-tion and as a consequence, APCCdh1 activation. Oncecyclin B degradation has started at metaphase, cyclin B/cdk1 activity decreases, Cdh1 is dephosphorylated,APCCdh1 is activated and Cdc20 is degraded (Figure 2).At this stage of the cell cycle, APCCdh1 takes over thedegradation of mitotic cyclins, preventing the prematureaccumulation of these proteins and a premature entryinto S phase (reviewed in Zachariae and Nasmyth,1999).

Besides Cdc20, Aurora A kinase is also degraded bythe APC during G1phase. This proteolysis is exclusivelymediated by APCCdh1 (Littlepage and Ruderman, 2002;Castro et al., 2002a, b). Aurora A is localized to thespindles and its overexpression induces centrosomeduplication. Actually, high levels of the Aurora A


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kinase do not directly alter centrosome duplication butinduces an aberrant mitosis without cytokinesis thatresults in tetraploid cells (Meraldi et al., 2002). ElevatedAurora A levels have been frequently displayed incancer cells indicating that Aurora A proteolysis may beimportant to prevent polyploidy.

A recent report has identified a new G1 substrate ofthe APC, Tome-1, required for degradation of theprotein kinase wee1 and for mitotic entry in Xenopusegg extracts. Tome-1 contains an F-box motif andassociates with Skp1 to induce ubiquitination anddegradation of the wee1 kinase. wee1 is responsible ofthe inhibitory phosphorylation of Cdk1 and its Tome-1-dependent degradation at G2 is probably requiredto induce mitotic entry. Tome-1 degradation during G1allows wee1 accumulation during interphase, thereby

preventing premature activation of cyclin B/cdk1(Ayad et al., 2003).

APC substrates controlling DNA replication

Three different APC substrates controlling DNAreplication have been described: Orc1, Cdc6 andgeminin. These three proteins control prereplicationcomplex formation at the replication origins duringS phase.

The origin recognition complex (ORC) binds theorigins of replication and serves as a platform forsubsequent loading of additional factors such as Cdc6,Cdt1 and MCM proteins rendering DNA competent forreplication. In Drosophila, the levels of the ORC subunitOrc1 are controlled during the cell cycle. Orc1 persists

Figure 2 Temporal pattern of APCCdc20 and APCCdh1 regulation throughout the cell cycle. During G1phase of the cell cycle, APCCdh1 isan active complex. Once G1-cyclins accumulate, Cdh1 becomes phosphorylated and dissociates from the APC. This phosphorylationwill be maintained until anaphase. From G2 to prophase, free APC is kept inactive by its inhibitor Emi1, which associates with Cdc20and prevents APC-Cdc20 binding. At late prophase, Emi1 is degraded and RASSFA1 takes over the role of this inhibitor until lateprometaphase when the latter is also proteolysed. Free APC is then phosphorylated by cyclin B/cdk1 and Plk1 kinases. At metaphase,APCCdc20 is still maintained inactive through direct binding of the checkpoint complex Mad2-Bub3-BubR1 (except for cyclin A andNek2 proteolysis). Once the spindle checkpoint is satisfied, the Mad2-Bub3-BubR1 complex is dissociated from APCCdc20 and thisubiquitin-ligase achieves its full activity, and induces degradation of securin and initiation of cyclin B proteolysis. Continuous cyclin Bdegradation present during anaphase will ensure a decrease in cyclin B/cdk1 activity and a dephosphorylation of Cdh1, which in turn,will induce activation of APCCdh1 and degradation of Cdc20


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from late G1 phase until late mitosis and it is degradedby APCCdh1 at the exit from mitosis (Araki et al., 2003).Cdc6 binds to the ORC complex and mediatessubsequent recruitment of the MCM complex. Similarly,human Cdc6 is degraded in early G1 by APCCdh1

(Petersen et al., 2000). According to these results it hasbeen suggested that replication origins are licensed atmitosis, and that the subsequent synthesis of Cdc6 andOrc1 could be exclusively required for cells that havebeen out of the cell cycle for a long period (Petersenet al., 2000).

Geminin prohibits initiation of DNA replication atinappropriate times of the cell cycle by preventing MCMrecruitment at the replication origins. In HeLa cells,geminin is absent during G1 and accumulates during S,G2 and Mphases. A model to explain the role ofgeminin in cell cycle control proposes that duringG1phase the APC is active and that as a consequence,geminin concentration is low. At the G1–S transition,APC is inactivated and geminin begins to accumulate;however, its concentration is not sufficient to inhibit afirst wave of prereplication complex formation andDNA replication begins. As S phase progresses, gemininaccumulates and inhibits subsequent recruitment ofMCMs to the replication origins (McGarry andKirschner, 1998).

Thus, the APC-dependent degradation of these threeproteins acts as a control mechanism required to ensurethe appropriate DNA replication.

Role of the APC in other cellular functions

Finally, the APC also plays an important role in othercell functions such as TGFb signalling by degradingSnoN repressor (Stroschein et al., 2001; Wan et al.,2001), in the re-establishment of the intercentriolarlinkage, by degrading Nek2A kinase (Hames et al.,2001) in the salvage pathway of dTTP synthesis forDNA replication, by degrading thymidine kinase (Ke

and Chang, 2004) in the control of cell proliferation byinducing the degradation of the HOXC10 transcriptionfactor (Gabellini et al., 2003) or in the control of latentreplication of the Bovine Papillomavirus genome bydegrading the viral initiation factor E1 (Mechali et al.,2004).

APC composition and structure

The APC is a large protein complex containing at least11 core subunits (Zachariae et al., 1998b; Gmachl et al.,2000; Yoon et al., 2002) that can further associate withat least three known different activators (Fang et al.,1998a, b; Kallio et al., 1998; Zachariae et al., 1998a;Cooper et al., 2000). The majority of these subunits arestably associated throughout the cell cycle (Peters et al.,1996; Grossberger et al., 1999) except for the differentactivators whose binding to APC is cell cycle regulated(Fang et al., 1998b; Zachariae et al., 1998a; Cooper et al.,2000). Little is known about how APC core subunitswork together to form a functional E3 ligase. Similarly,the exact mechanism used by the different APCactivators to modulate E3 activity is not clear.

Core components of the APC

Compelling data on the composition of the APC showthat the majority of the APC subunits described in yeastare also present in vertebrates, suggesting that the APChas a similar composition in all eukaryotes (Table 1).A total of 11 APC core subunits have been describedin vertebrates versus 13 in yeast. From these, only twosubunits, Apc7 and Apc9, seem to be specific tovertebrate (Yu et al., 1998) and yeast (Zachariae et al.,1998b) respectively, whereas, to date, vertebrate ortho-logs of the recently described yeast Apc13, Apc14 andApc15 components have not been identified (Yoon et al.,2002). Some of these proteins present known structural

Table 1 Subunits of the APC in yeast and vertebrate cells

Core subunits S. Cerevisiae S. Pombe Vertebrates Motif

Apc1 Cut4 Apc1/Tsg24 Rpn1/2 homologyApc2 — Apc2 Cullin homologyCdc27 Nuc2 Apc3/Cdc27 Tetratricopeptide repeatsApc4 Cut20/Lid1 Apc4 —Apc5 — Apc5 —Cdc16 Cut9 Apc6/Cdc16 Tetratricopeptide repeats— — Apc7 Tetratricopeptide repeatsCdc23 Cut23 Apc8/Cdc23 Tetratricopeptide repeatsApc9 — — —Doc1 Apc10 Apc10 Doc domainApc11 Apc11 Apc11 Ring-H2 finger domainCdc26 Hcn1 Cdc26 —Swm1 Apc13 — —— Apc14 — —Mnd2 Apc15 — —

ActivatorsCdc20 Slp1 Cdc20/p55cdc WD40 repeatsCdh1/Hct1 Srw1/Ste9 Cdh1 WD40 repeatsAma1 — — WD40 repeats


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motifs in their amino-acid sequence already describedfor different subunits of the SCF ubiquitin-ligase. Thesestructural similarities between SCF and APC contri-buted significantly to the understanding of the role ofeach component of the APC (Figure 3).

Apc1 Apc1 is the largest subunit of the APC. It wasfirst identified in Xenopus egg extracts by biochemicalpurification (Peters et al., 1996), and in budding yeast ina screen of mutants in which the yeast mitotic cyclin B,Clb2, was stabilized (Zachariae et al., 1996). Sequenceanalysis revealed sequence homology with the BimE(Engle et al., 1990) protein from Aspergillus nidulansand the Tgs24 murine protein (Starborg et al., 1994).Moreover, a homolog of Apc1 in fission yeast, namedCut4, has also been described as a constituent of the 20Scyclosome (Yamashita et al., 1996). The levels of Apc1are constant throughout the cell cycle; however, regula-tion of this protein during mitosis seems to be exerted byphosphorylation (Peters et al., 1996; Jorgensen et al.,2001). Apc1 shares a structural motif with Rpn1 andRpn2, two proteins of the 19S cap from the 26Sproteasome. Rpn1 and Rpn2 present several sequencerepeats formed each of a b-strand followed by ana-helix. A secondary structure prediction of thissequence repeat indicates the presence of a horseshoe-

shaped structure with an inner b-strand surface and anouter a-helix face. In this structure, the inner hydro-phobic b-strand surface would be ideally suited to bindunfolded proteins (Lupas et al., 1997). The function ofthis repetitive sequence in Apc1 protein has not beenelucidated; however, a possible role as a scaffold for theassembly of APC complex or in the interaction withpolyubiquitinated proteins has been suggested (Lupaset al., 1997).

The tetratricopeptide repeat containing proteins FourAPC subunits Apc3/Cdc27, Apc6/Cdc16, Apc7 andApc8/Cdc23 (Sikorski et al., 1990; Lamb et al., 1994)present in their amino-acid sequences randomly re-peated copies of the 34-residue tetratricopeptide motif(TPR motif). The TPR is a structural motif present in awide range of proteins mediating a variety of biologicalprocesses such as cell cycle regulation, transcriptionalcontrol, mitochondrial and peroxisomal protein trans-port and protein folding (reviewed in D’Andrea andRegan, 2003). This motif is thought to mediate protein–protein interactions of multiprotein complexes (D’An-drea and Regan, 2003). In this regard, it has beendemonstrated that Apc3/Cdc27 and Apc7 bind to a C-terminal motif in the amino-acid sequences of APCactivators Cdc20 and Cdh1. Moreover, it has also beenshown that these C-terminal motifs ending in anisoleucine–arginine (IR) dipeptide are essential forCdc20 and Cdh1 to bind the APC (Passmore et al.,2003; Vodermaier et al., 2003). These results indicatethat the TPR-containing APC subunits regulate APCactivity by inducing Cdc20 and Cdh1 association.

The APC subunit Apc10/Doc1 (see below) alsopossesses an ‘IR tail’, which mediates Apc10–Apc3/Cdc27 and Apc10–Apc7 binding (Wendt et al., 2001;Vodermaier and Peters, 2004), suggesting that theassociation of Apc10/Doc1 to the APC could also beregulated by the TPR-containing proteins.

All these TPR-containing proteins are phosphory-lated during mitosis (Peters et al., 1996) and thisphosphorylation seems to be essential to induce APCactivation at this phase of the cell cycle (Lahav-Baratzet al., 1995). Thus, Apc3/Cdc27 and Apc7 phosphoryla-tion likely modify Apc3/Cdc27-Cdc20 and Apc7-Cdc20binding and thereby increase Cdc20 binding and APCactivation (Vodermaier and Peters, 2004).

APC2 and APC11 Both Apc2 and Apc11 subunitspresent close homology with two other components ofthe ubiquitin-ligase SCF.

The SCF subunit Cdc53 is a member of the cullinfamily (Patton et al., 1998; Zachariae et al., 1998b).These proteins contain a 180-residue domain referred toas the cullin homology domain (Zachariae et al., 1998b).As described above, the C-terminal cullin domain ofCdc53 binds to the RING-H2 finger protein Hrt1/Rbx1that in turn binds to the E2 enzyme tethering thisenzyme to the SCF (Kamura et al., 1999; Seol et al.,1999; Skowyra et al., 1999). The N-terminal domain ofCdc53 binds to Skp1, mediating the association with the

Figure 3 Comparison of the APCCdc20 and the SCF ubiquitin-ligases. APC and SCF both contain a Cullin, a Ring-H2 finger anda WD40 subunit. The cullin and the Ring-H2 finger proteins formthe minimal ubiquitin-ligase module of both E3, and are requiredfor E2 tethering. The APC contains additional subunits such as thetetratricopeptide containing proteins and the Doc protein. Proteinsbelonging to the same family are identically coloured. Interactionof the different APC subunits have been represented taking intoaccount the existent results on the association between differentAPC components. This model was built by integrating the datadescribed in this review obtained in different models


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different F-box proteins, which will directly associatewith phosphorylated substrates (Bai et al., 1996; Pattonet al., 1998).

Like the SCF components Cdc53 and Hrt1/Rbx1,the APC subunits Apc2 and Apc11 contain a cullinand a RING-H2 finger domain, respectively. Thissequence homology suggests that Apc2 and Apc11might fulfill in the APC complex a similar role to thatdeveloped by Cdc53 and Hrt1/Rbx1 in the SCF enzyme(Figure 3).

Recent results demonstrate that this is the case. Tanget al. (2001b) have shown that the Apc11 RING-H2protein directly interacts with the Ubc4 E2 enzyme andinduces E2-dependent ubiquitination of protein sub-strates, whereas binding of Apc11 to the Ubch10 E2enzyme requires the C-terminal cullin domain of Apc2.Thus, at least in the UbcH10-mediated ubiquitination,Apc2 and Apc11 are required to form the minimalubiquitin-ligase module of the APC. Accordingly, aheterodimeric Apc2/Apc11 complex purified frombaculoviral-coinfected insect cells is sufficient to catalysesecurin and cyclin B1 ubiquitination in presence of theUbcH10 enzyme, although this ubiquitination does notpossess substrate specificity (Leverson et al., 2000).

Apc10/Doc1 Apc10/Doc1 was first identified as anAPC subunit in budding yeast in a genetic screen formutants defective in the degradation of mitotic cyclins(Hwang and Murray, 1997). Orthologs of this proteinhave also been found in fission yeast and human(Kominami et al., 1998; Grossberger et al., 1999). ThisAPC subunit is characterized by the presence of a ‘Doc’domain in its amino-acid sequence. This sequence is alsofound in several proteins containing other motifs linkedto ubiquitination such as cullin and HECT motifs(Kominami et al., 1998; Grossberger et al., 1999).No differences in the subunit composition of the APCpurified from temperature-sensitive alleles of the DOC1gene have been observed in budding yeast, indicatingthat Apc10/Doc1 binding to the APC is not required tostabilize this complex (Kominami et al., 1998; Gross-berger et al., 1999). Nevertheless, this APC subunitdirectly associates to Apc11 (Tang et al., 2001b), Apc3/Cdc27 (Wendt et al., 2001) and Apc7 (see above)(Vodermaier and Peters, 2004). The physiological role ofthese direct associations is not clear. However, a recentreport shows that Apc10/Doc1 could act as a processiv-ity factor for the APC (Carroll and Morgan, 2002).Moreover, a role of Apc10/Doc1 in the recognition ofAPC substrates has also been proposed. In this regard,binding of the APC activator Cdh1 with APC substratesis prevented in APC complexes purified from Doc1mutants (Passmore et al., 2003). These results imply thatApc10/Doc1 mediates substrate binding to Cdh1 and tothe APC either direct or indirectly. On the basis of theseresults, it would be interesting to determine if thisbinding is mediated by a conformational change of APCor through direct binding of Apc10/Doc1 to thesubstrate. If Doc1/Apc10 bound the APC substratesdirectly, it would suggest that a Doc1 recognition motifis present in these proteins. In this case, two different

degradation motifs could be present in the substrates tobe recognized by the APC: a first Cdh1-recognitionsequence and a second Apc10/Doc1-recognition motif.

Apc4 and Apc5 Little is known about the role of thesetwo subunits in APC structure and activity. NeitherApc4 nor Apc5 present any known motif in theirsequence that could hint to their function. However,these two subunits, in addition to Apc1, are tightlyassociated to the Apc2/Apc11 ubiquitin-ligase moduleand could therefore connect Apc2/Apc11 with the TPRsubunits (Vodermaier et al., 2003).

Apc9 and Cdc26 Apc9 was identified by biochemicalpurification of yeast APC and no orthologs in otherspecies have been identified so far (Zachariae et al.,1998b). Unlike Apc9, Cdc26 has been described in bothyeast (Yamada et al., 1997; Zachariae et al., 1998b) andvertebrates (Gmachl et al., 2000). The analysis of theamino-acid sequences of these two proteins has notrevealed any known motif. Both subunits seem to berequired to maintain APC structure. Thus, associationof Apc3/Cdc27 with the APC is drastically reduced inApc9 mutants, whereas APC purified from Cdc26mutants presents a reduced levels of Apc3/Cdc27,Apc6/Cdc16 and Apc9 (Zachariae et al., 1998b).

Apc13/Swm1, Apc14 and Apc15/Mnd2 Apc13/Swm1,Apc14 and Apc15/Mnd2 are three new subunits of theAPC that have been recently described in Saccharo-myces pombe (Yoon et al., 2002). Two orthologs of thesesubunits Apc13/Swm1 and Apc15/Mnd2 have also beendescribed in S. cerevisiae (Yoon et al., 2002; Hall et al.,2003). Apc13/Swm1 binds Apc8/Cdc23 and Apc5.Moreover, association of Apc15/Mnd2 with Apc8/Cdc23, and of Apc5 with Apc1 have been demonstrated.These interactions suggest that these subunits could beimportant in the stabilization of the APC structure (Hallet al., 2003). The role of these subunits is unknown sofar. However, it has been suggested that Apc13/Swm1and Apc15/Mnd2 could provide an essential functionfor the APC during meiosis that is not required duringmitosis (Hall et al., 2003).

APC activators: Cdc20, Cdh1 and Ama1

A Cdc20 mutant was originally described in buddingyeast as causing cells to arrest after DNA synthesis atthe G2 stage of the cell cycle (Hartwell and Smith, 1985).However, the first direct connection between this proteinand APC-dependent degradation of the mitotic cyclinswas described in Drosophila in which a mutation in thegene encoding the Cdc20 ortholog, Fizzy, blockedmitotic degradation of cyclin A, B and B3 (Sigristet al., 1995). A subsequent study in this species describedthe presence of a new protein with significant homologyto Fizzy that was named Fizzy-Related, and thatcorresponds to the Drosophila Cdh1/Hct1 ortholog.Fizzy-Related is required for cyclin removal duringG1 in epidermal embryo cells of Drosophila (Sigristand Lehner, 1997). Two other studies simultaneously


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appeared in budding yeast in which similar roles wereascribed to the Cdc20 and Cdh1/Hct1 (Schwab et al.,1997; Visintin et al., 1997). Cdc20 and Cdh1/Hct1belong to a multigene family that includes four Cdh1/Hct1 homologs (Wan and Kirschner, 2001) and a yeastmeiotic-specific APC activator named Ama1 (Cooperet al., 2000). The four Cdh1/Hct1 homologs describedin chicken are differently localized in proliferating,differentiated and postmitotic tissues and they mediateubiquitination of different APC substrates (Wan andKirschner, 2001).

Studies with purified APC from budding yeast andhuman cells demonstrate that both Cdc20 and Cdh1/Hct1 activate the ‘in vitro’ ubiquitination of cyclin B(Kramer et al., 1998; Jaspersen et al., 1999). Moreover,the APC purified from Xenopus egg extracts in whichCdc20 function is blocked presents a clearly inhibitedubiquitination activity (Lorca et al., 1998). The capacityof these activators to stimulate ‘in vitro’ ubiquitinationof mitotic cyclins is dependent on their own associationto the APC. This interaction seems to be mediatedthrough direct binding of the TPR subunits Apc3/Cdc27and Apc7 with the IR C-terminal tails of Cdc20 andCdh1/Hct1 (see above) (Vodermaier et al., 2003). Inaddition to the IR motif, another sequence in theN-terminus of these activators, the C-Box, has indeedbeen found necessary for their association with APC inyeast (Schwab et al., 2001). Thus, further studies arerequired to establish the real mechanism through whichthese proteins bind the APC.

All these results suggest that Cdc20, Cdh1/Hct1 andAma1 act as APC activators, however, the exactmechanism through which these proteins increasesAPC activity is not known. They are all members of aconserved subfamily of WD40 proteins. This motif hasalso been described for a subset of F-box subunits ofthe SCF ubiquitin-ligase. The different F-box proteinscontain two domains: one required for substraterecognition, and another one, the F-box, essential forSkp1 association. Interestingly, in this subset of F-boxproteins, substrate binding to the F-box protein ismediated by the WD40 domain (Patton et al., 1998).Comparison between structures of the SCF and APCsuggests a putative role of these activators in substraterecognition. In this regard, recent results have shownthat different APC substrates can directly and specifi-cally bind to Cdc20 and Cdh1/Hct1 (Fang et al., 1998b;Burton and Solomon, 2001; Sorensen et al., 2001;Pfleger et al., 2001a).

Substrate recognition

The first results indicating that Cdh1/Hct1 couldmediate substrate recognition by the APC throughdirect binding come from the study of Sorensen et al.These authors demonstrated direct binding of cyclin Awith the last Cdh1’s WD40 repeat. The mutation of thisbinding site in Cdh1/Hct1 inhibited cyclin A associationto Cdh1/Hct1 and cyclin A degradation (Sorensen et al.,2001). A second study demonstrated that the APC

substrate, Pds1, whose degradation is exclusivelyinduced by APCCdc20, binds directly Cdc20, whereas itis incapable of associating with Cdh1/Hct1, indicatingthat Cdc20 and Cdh1/Hct1 recognize and bind theirsubstrates differently and selectively (Hilioti et al.,2001). Further specific and direct interactions of Cdc20with Pds1 and of Cdh1/Hct1 with Clb2, Clb3 and Cdc5were also demonstrated in budding yeast (Burton andSolomon, 2001; Schwab et al., 2001). Finally, Pflegeret al. (2001a) showed that in the absence of APC, Cdc20and Cdh1/Hct1 can directly and specifically bind a largenumber of APC substrates including Xkid, securin,geminin, cyclin A and cyclin B.

Compelling data indicate that the selective bindingof the APC activators with each substrate is mediatedby the presence in the substrate amino-acid sequence ofdifferent ‘degradation motifs’ specifically recognized byCdc20 and/or Cdh1/Hct1. The first ‘degradation signal’was described in the cyclin B protein. This signal, named‘Dbox’, was necessary to induce cyclin B degradationand when fused to a foreign protein, was also sufficientto generate a similar proteolytic pattern to that observedfor cyclin B (Glotzer et al., 1991). Subsequent studiesdemonstrated a Dbox-dependent degradation of otherAPC substrates such as cyclin A (Lorca et al., 1992;Sorensen et al., 2001), Nek2 (Hames et al., 2001) andAurora A (Castro et al., 2002a, b).

A second degradation motif, named the ‘KENbox’was subsequently described by Pfleger and Kirschner(2000). This sequence is present in the APC activatorCdc20 which is itself an APCCdh1 substrate. Cdc20 lacksa Dbox and its APCCdh1-dependent degradation ismediated by the KENbox. Like the Dbox, the KENboxsequence is a transposable motif that induces proteo-lysis of the hybrid protein and which acts as a specificrecognition signal for APCCdh1. The authors alsodemonstrated that Cdh1/Hct1 specifically interacts withsubstrates containing a KENbox and/or a Dbox,whereas Cdc20 exclusively binds Dbox-containing sub-strates (Pfleger and Kirschner, 2000).

A third degradation signal, the ‘GxENbox’, hasrecently been described (Castro et al., 2003). This motifwas shown in the amino-acid sequence of the chromo-kinesin Xkid and mediates degradation of this proteinby both APCCdc20 and APCCdh1. Deletion of this motif inXkid prevents its APCCdc20 and APCCdh1-dependentdegradation. Moreover, like other proteolytic sequences,when fused, it is capable to induce degradation of aforeign protein with a similar timing to that observed forXkid.

A particularly interesting case is degradation ofAurora A. Aurora A proteolysis is exclusively mediatedby APCCdh1 and requires the presence of a doubledegradation motif: the Dbox and a new degradationsignal named ‘DAD’ (for Dbox Activating Domain) or‘Abox’. Mutation of either of these two domainsprevents destruction of Aurora A (Castro et al.,2002a, b; Littlepage and Ruderman, 2002). A doublerecognition motif has also been described for otherproteins such as cyclin A (Geley et al., 2001) and Nek2(Hames et al., 2001). These results suggest that


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degradation of these proteins is mediated by either adouble motif formed by a bipartite sequence placed intwo distal parts of the substrate or by the presence oftwo different degradation signals that could be recog-nized by two different APC subunits. In this regard,a recent study demonstrated a cell cycle-dependentDbox-binding activity of the APC in the absence ofCdc20 or Cdh1, indicating that another APC subunitbesides Cdc20 and Cdh1 possesses Dbox receptoractivity (Yamano et al., 2004). One possible candidatefor the Dbox receptor of the APC is the core subunitApc10/Doc1. As described above, binding of substrateswith APC is prevented in APC complexes purified fromApc10/Doc1 mutants (Passmore et al., 2003). Theseresults imply that Apc10/Doc1 directly or indirectlymediates substrate binding to Cdh1 and the APC. Thus,it is possible that two different APC receptors, Cdc20 orCdh1/Hct1 and Apc10/Doc1, recognize two differentdegradation signals in the substrate and thereby indu-cing APC binding and ubiquitination.

APC regulation

Phosphorylation of APC is one of the mechanisms usedby the cell to modulate APC activity. As describedabove, the core subunits of the APC, Apc1, Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23, are phos-phorylated during mitosis (Peters et al., 1996). Thisphosphorylation modulates Cdc20 binding to the APCand APC activity. In this regard, three different kinaseshave been described to phosphorylate APC: cyclinB/cdk1, Plk1 and PKA (Kotani et al., 1998; Rudnerand Murray, 2000; Golan et al., 2002; Kraft et al., 2003).

Cyclin B/cdk1-mediated phosphorylation of APC inXenopus egg extracts requires Suc1. This conclusion isbased on the observation that the immunodepletion ininterphase Xenopus egg extracts of the Suc1 ortholog,Xe-p9, at the beginning of mitosis, prevents phosphor-ylation of Apc3/Cdc27 by this kinase (Patra andDunphy, 1998). Both, ‘in vitro’ and ‘in vivo’ phosphor-ylation of Apc3/Cdc27, Apc6/Cdc16 and Apc8/Cdc23by cyclin B/cdk1 increases Cdc20 binding (Kotani et al.,1998; Kraft et al., 2003) and APC activation (Kraft et al.,2003). Unlike cyclin B/cdk1, the role of APC phosphor-ylation by Plk1 does not stimulate either Cdc20 bindingor APC activity; however, when combined with cyclinB/cdk1, both kinases act synergistically to increase APCubiquitination activity (Golan et al., 2002; Kraft et al.,2003). In vitro phosphorylation of APC by PKA inhibitsubiquitination of cyclin B even in the presence of theregulatory factors Cdc20 and Cdh1/Hct1 (Kotani et al.,1998). In addition, yeast APC mutants are suppressedby mutations in the PKA pathway that lower its kinaseactivity (Yamashita et al., 1996). Taken together, theseevidence suggests that cyclin B/cdk1- and Plk1-mediatedphosphorylations induce APC activation, whereas PKAphosphorylation inhibits its activity.

Cell cycle-regulated phosphorylation of Cdc20 hasalso been observed (Weinstein, 1997; Lorca et al., 1998;Chung and Chen, 2003), however this phosphorylation

seems not to be required to induce activation of the APCbut probably to allow APC inhibition by the spindlecheckpoint. Accordingly, recent results have demon-strated that this APC activator is phosphorylated inThr 64 and Thr 68 by MAPK during mitosis and that aThr-to-Ala mutation in these residues decreases spindlecheckpoint-dependent inhibition of the APC by redu-cing its affinity for the spindle checkpoint proteins(Chung and Chen, 2003). Two proteins of the spindlecheckpoint, Mad2 and BubR1, are capable of inhibitingAPCCdc20. Different studies have demonstrated that thisinhibition is mediated by direct binding of Mad2 andBubR1 to Cdc20. Thus, Fang et al. (1998a) have shownthat the microinjection of recombinant Mad2 proteininto Xenopus embryos arrests cells at mitosis withinactive APC. Moreover, despite the fact that the twodifferent forms of Mad2, monomer and tetramer, bindCdc20, only the tetramer is capable of inhibitingactivation of APC (Fang et al., 1998a). Purified BubR1also binds directly to recombinant Cdc20 and inhibitsAPC and this inhibition is 12-fold more potent thanMad2. However, both Mad2 and BubR1 mutuallypromote each other’s binding and act synergistically todecrease APC activity (Fang, 2002). The presence ofBubR1-Bub3-Cdc20, Mad2-Cdc20 and BubR1-Bub3-Mad2-Cdc20 APC-inhibitory complexes has also beendemonstrated in vivo in spindle checkpoint-activatedcells (Sudakin et al., 2001; Tang et al., 2001a). BubR1-Bub3-Mad2-Cdc20 inhibitory activity has been deter-mined 3000-fold greater than recombinant Mad2oligomers (Sudakin et al., 2001). This complex is presentin both interphase and mitotic cells, however ininterphasic cells it is not associated to APC, thus itsAPC-inhibitory capacity is limited to mitosis (Sudakinet al., 2001). A third inhibitor, Mad2L2, specificallyinhibits the APCCdh1 complex. Like Mad2, Mad2L2forms monomers and oligomers; however, both arecapable of inhibiting APCCdh1. ‘In vitro’ translatedMad2L2 selectively binds APCCdh1 but not APCCdc20.This protein does not affect either Cdh1 or substratebinding to the APC, but inhibits substrate ubiquitina-tion. Finally, injection of this protein in Xenopusembryos causes a post-Mid Bastula Transition (MBT)-gastrulation arrest. This is consistent with Mad2L2inhibiting endogenous APCCdh1, since this ubiquitin-ligase is only present after MBT in Xenopus embryos(Pfleger et al., 2001b).

Besides the spindle checkpoint-dependent inhibitorsof the APC and Mad2L2, two other proteins, Emi1 andRASSF1A, have been recently described as negativeregulators of this ubiquitin-ligase (Reimann et al., 2001;Song et al., 2004).

Emi1 inhibits both APCCdh1 and APCCdc20 by directlybinding to Cdc20 and Cdh1 and thereby preventingsubstrate-Cdc20/Cdh1-APC associations. In humancells, Emi1 accumulates in late G1 and is destroyedearly in mitosis by the SCF (Hsu et al., 2002; Margottin-Goguet et al., 2003). Its overexpression in thesecells induces cyclin A accumulation and acceleratesS phase entry. In addition, Emi1 immunodepletion fromcycling Xenopus egg extracts strongly delays cyclin B


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accumulation and mitotic entry, whereas its additionstabilizes APC substrates and causes a mitotic block(Reimann et al., 2001). Thus, Emi1 may participate inS phase entry by promoting accumulation of cyclin Athrough APCCdh1 inhibition and in the subsequententrance into mitosis by inducing cyclin B accumulationthrough APCCdc20 inhibition.

RASSF1 is a tumour suppressor gene that isfrequently silenced in different tumour types as a resultof a hypermethylation of CpG islands in its promoter.Two major isoforms of RASSF1, A and C, are producedfrom the RASSF1 gene. RASSF1A contains a cysteine-rich diacylglycerol-binding domain (C1 domain) and aRas-association (RA) domain. RASSF1C contains theRA domain but the C1 domain is replaced by a distinctamino-acid sequence. RASSF1C is thought to play arole in RAS-mediated function, whereas RASSF1A isimplicated in the regulation of cell cycle. A recent studyby Song et al. has reported that RASSF1A acts at earlyprometaphase to prevent degradation of mitotic cyclins,and to delay progression beyond metaphase by inhibit-ing APCCdc20 complex. Accordingly, overexpression ofthis protein in HeLa cells induces stabilization of cyclinA and cyclin B and mitotic arrest at prometaphase,whereas, its depletion by RNA interference acceleratesmitotic cyclin degradation and mitotic progression.

Moreover, RASSF1A interacts with Cdc20 and inhibitsAPC activity. Finally, they demonstrate that thisinhibition is independent of the spindle checkpointAPC inhibitors and Emi1 (Song et al., 2004).

In conclusion, APC activity is subjected to tightregulation to ensure the correct timing of proteindegradation during the mitotic cell cycle (Figure 2).Thus, APC activity is negatively controlled by differentinhibitors. First, Emi1 ensures S and Mphase entries byallowing accumulation of cyclin A and cyclin B from Suntil prophase. Subsequently, from prophase untilprometaphase, this role is developed by the RASSF1AAPC inhibitor and finally inhibition of the APC atmetaphase is taken over by the spindle checkpointpathway. Besides this negative control, APC is alsopositively regulated first by phosphorylation of thedifferent core subunits during early mitosis and sub-sequently by its association to the APC activatorsCdc20 from prophase to anaphase, and Cdh1/Hct1 fromanaphase through mitotic exit and into G1.

Acknowledgements

We are indebted to Dr Catherine Bonne-Andrea, Dr CarstenJanke and Dr May Morris for the critical reading of thismanuscript. This work was supported by the Ligue NationaleContre le Cancer (Equipe Labellisee).

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The anaphase-promoting complex: a key factor in the regulation of cell cycle

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