THE ANAPHASE-PROMOTING COMPLEX

Oncogene (2005) 24, 314–325

& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc

REVIEW

The anaphase-promoting complex: a key factor in the regulation of cell cycle Anna Castro*,1, Cyril Bernis1, Suzanne Vigneron1, Jean-Claude Labbe´1 and Thierry Lorca*,1 1

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

Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation by the 26S proteasome. Two different ubiquitin ligases play an important role in the cell cycle: the SCF (Skp1/Cullin/ F-box) and the anaphase-promoting complex (APC). In this review, we describe the present knowledge about the APC. We pay particular attention to the latest results concerning APC structure, APC regulation and substrate recognition, and we discuss the implication of these findings 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 sequential degradation of different cell cycle proteins. This degradation is mediated by the ubiquitination pathway. In this process, an ubiquitin chain is covalently attached to the target protein thereby promoting its recognition and degradation by a large cytosolic protease complex named 26S proteasome. Ubiquitination involves three major steps (Hershko and Ciechanover, 1998). First, the ubiquitin-activating enzyme (E1) forms a thiol ester bond between the active-site cysteine and the C-terminal glycine residue of ubiquitin. Second, the activated ubiquitin is transferred by the formation of a new thiol ester linkage to an active-site cysteine residue on the ubiquitin-conjugating enzyme (E2). Finally, the ubiquitin molecule is coupled through an amide isopeptide linkage to an e-amino group of the substrate protein’s lysine residues. This coupling is performed directly by the E2 or in conjunction with a third enzyme, the ubiquitin-ligase (E3). An E3 enzyme may participate in the shuttling of ubiquitin to the substrate either by directly forming a thiol ester bond or by simply bringing the E2 enzyme in close proximity to the substrate. *Correspondence: T Lorca; E-mail: A Castro, [email protected]

[email protected];

Whatever the mechanism used, the E3 enzyme is a key component of this pathway, since it confers substrate specificity. Two different ubiquitin-ligases play an important 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 boundaries by inducing the degradation of different factors at these phases of the cell cycle. This ubiquitin-ligase is a macromolecular complex formed of a cullin subunit, Cul1, a RING-H2 protein, Hrt1/Rbx1, an F-box subunit and a linker subunit, Skp1. In this complex, the F-box specifically recognizes phosphorylated SCF targets. Once bound to the F-box, the SCF substrate will be brought closer to the Cul1/Hrt1 catalytic core of this ubiquitin-ligase by Skp1. Finally, Cul1/Hrt1 will recruit the E2 enzyme, Cdc34, and will induce substrate ubiquitination (Pintard et al., 2004). Unlike the SCF, the APC is mainly required to induce progression and exit from mitosis by inducing proteolysis of different cell cycle regulators including Pds1/ securin and cyclin B. This ubiquitin-ligase is a large complex that contains at least 11 subunits. Similar to the SCF, it contains a catalytic core formed of a cullin subunit, Apc2, and a RING-H2 protein, Apc11. However, substrate recognition is mediated by the APC activators, Cdc20 and Cdh1, in an F-boxindependent pathway. This review focuses on the E3 complex, APC.

Identification of the APC Degradation of cyclin B by ubiquitination is a crucial event required to induce exit from mitosis. As described above, the ubiquitin ligases, E3s, play a prominent role in this pathway by inducing substrate specificity and E2 recruitment. Thus, the identification and the regulation of the E3 ligase involved in cyclin B degradation is of great interest for the general understanding of cell cycle progression. The ubiquitin-ligase involved in cyclin B ubiquitination was first identified as a result of a simultaneous genetic screen in budding yeast (Irniger et al., 1995) and two different biochemical studies in clam and Xenopus egg extracts (King et al., 1995; Sudakin et al., 1995). The screen of Saccharomyces cerevisiae mutants that were defective in cyclin B

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degradation allowed the finding of mutations in the CDC23 and CDC16 genes. These two mutants, as well as the Cdc27 mutant, had previously been shown to cause cells to arrest at metaphase (Hartwell et al., 1970). At the same time, a first biochemical study was developed in extracts from clam oocytes. Fractionation of these extracts showed the presence of a particulate fraction containing a complex of approximately 1500 kDa with a destruction box-specific cyclin/ubiquitin-ligase activity. This complex named Cyclosome was inactive in interphase and was activated during mitosis by Cdk1 (Hershko et al., 1994; Sudakin et al., 1995). A second biochemical study developed in Xenopus egg extracts described the presence of a 20S complex that was named APC and that contained the homologues of the budding yeast CDC16 and CDC27 genes (King et al., 1995). When APC was removed from these extracts by immunoprecipitation with anti-Cdc27 antibodies, no cyclin B-ubiquitination activity was observed. Moreover, immunopurified Cdc27 complexes were sufficient to complement interphase extracts to induce cyclin B ubiquitination. Subsequent work demonstrated that Cdc16p, Cdc23p and Cdc27p are three of the 11 (in vertebrates) core subunits of the APC and that, in addition, this complex is regulated during mitosis by two different activators, Cdc20 and Cdh1. All the results obtained in different species about the role of the APC during the mitotic cell cycle converge on a requirement of this ubiquitin-ligase for cell cycle progression; however, this is not the case when the role of this complex on the meiotic progression is analysed. In this regard, the APC seems to be essential to induce metaphase II-exit, whereas this complex seems to be required for metaphase I –to anaphase I transition in some species like Caenorhabditis elegans (Furuta et al., 2000), mouse (Terret et al., 2003) or yeast (Salah and

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

APC substrates Ubiquitin-dependent degradation of cell cycle factors induced by the APC is a key mechanism used by the cell to tightly regulate different transitions throughout cell division. Thus, the APC orchestrates mitosis by controlling anaphase entry, anaphase progression, exit of mitosis and G1 phase. Moreover, it plays an important role in the formation of the prereplicative complexes required to induce DNA replication. Finally, other additional roles of this E3 in different cell functions have also been described (Figure 1). APC substrates controlling anaphase entry Entry into anaphase is marked by the initiation of sister chromatid separation. Sister chromatids are held together by the multiprotein complex called cohesin. This complex is cleaved by separase that, in turn, is inhibited by securin. At the anaphase entry, the APCdependent degradation of securin enables separase activation and as a consequence cleavage of the cohesin complex, thereby allowing sister chromatid separation (Uhlmann et al., 1999, 2000; Yanagida, 2000). Proteolysis of securin is ensured by APCCdc20 before anaphase onset, but its degradation is maintained until the end of G1 by APCCdh1 (Nasmyth, 2001; Zur and Brandeis, 2001). Initial studies suggested that the sole proteolysis of securin was required for anaphase entry. Accordingly, in budding yeast, the failure of APC or Cdc20 mutants to enter anaphase is bypassed by the unique deletion of the securin 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 subsequent degradation at metaphase of cyclin B, Xkid and securin and at anaphase of the kinesins Kip1 and Cin8. From late anaphase until mitotic exit and throughout G1 phase, the degradation of all these proteins is ensured by APCCdh1. Moreover, this complex will first induce proteolysis of Cdc20, Prc1, Tome-1, Plk1 and Aurora A at mitotic exit and subsequent degradation of Orc1, Cdc6 and Geminin in early G1. Substrates whose degradation starts at the same phase of the cell cycle are equally coloured. The exact phase in which every substrate starts its APC-dependent degradation is represented in lower coloured arrows. Plane lanes represent APCCdc20dependent degradation. Dotted lanes represent APCCdh1-dependent degradation Oncogene


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substrate degraded by the APC required to enter this phase of the cell cycle (Yamamoto et al., 1996; Ciosk et al., 1998; Shirayama et al., 1999). Moreover, the addition of nondegradable cyclin B to Xenopus egg extracts does not prevent sister chromatid separation, whereas the addition of an N-terminal fragment of this protein which blocks APC prevents both sister chromatid separation and exit from mitosis (Holloway et al., 1993). These results indicate that cyclin B proteolysis is not essential to promote entry into anaphase. However, more recent results suggest that degradation of both cyclin A and cyclin B is also required to induce sister chromatid disjunction. In Drosophila, the expression of a nondegradable cyclin A blocks separation of sister chromatids (Parry and O’Farrell, 2001), whereas, in Xenopus egg extracts, this block is also observed when high concentrations of nondegradable cyclin B are added. In the latter case, the mechanism by which cyclin B blocks anaphase entry involves direct inhibition of separase through its phosphorylation (Stemmann et al., 2001). APC substrates controlling anaphase progression APC also induces degradation of several factors that are essential for spindle-pole separation and spindle disassembly. One of these factors is the kinesin-related protein Xkid. Xkid plays an important role in both meiotic and mitotic cell cycles. At meiosis, this chromokinesin is required to reactivate cyclin B/cdk1 complex after meiosis I and to inhibit DNA replication between meiosis I and meiosis II (Perez et al., 2002). At mitosis, Xkid is required during prometaphase to maintain the polar ejection force, a force that pushes chromosomes away from the pole and that mediates chromosome congression (Antonio et al., 2000; Funabiki and Murray, 2000). However, subsequent proteolysis of this protein is also essential to allow chromosome movements to the spindle poles throughout anaphase. Accordingly, an excess of nondegradable Xkid does not prevent sister chromatid separation but inhibits chromosome movements towards the spindle poles (Funabiki and Murray, 2000). Xkid degradation is mediated by APCCdc20 and APCCdh1. It starts at anaphase and is maintained until the end of G1 phase (Levesque and Compton, 2001; Castro et al., 2003). Two other motor proteins, the kinesins Kip1 and Cin8, are proteolysed by the APC. These proteins are first degraded at the initial stages of anaphase and their protein levels are subsequently maintained throughout the G1 phase. Both kinesins are required to separate the spindle poles during spindle assembly and metaphase, but their subsequent degradation at anaphase is required to allow progression through anaphase (Gordon and Roof, 2001; Hildebrandt and Hoyt, 2001). Finally, the spindle-associated protein Ase1 is proteolysed by the APC. Ase1 is present at greatest levels during mitosis and is not detected in G1 phase, suggesting that its destruction could be mediated by Cdh1/Hct1-dependent activation of the APC (Juang et al., 1997). In this regard, proteolysis of the Xenopus Oncogene

ortholog of Ase1, Prc1, is indeed mediated by the APCCdh1 complex (D Fesquet, personal communication). In budding yeast, Ase1 is associated with the spindle midzone, and is required for anaphase B, appropriate elongation of the spindle and separation of the spindle poles. A nondegradable Ase1 mutant delays spindle disassembly (Juang et al., 1997), indicating that removal of this protein from the spindle midzone is required for progression 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 and continues throughout mitosis and G1 phase (Clute and Pines, 1999). Both complexes APCCdc20 and APCCdh1 mediate its degradation. Cyclin B proteolysis is required to inhibit Cdk1 activity and as consequence to induce different cell processes such as sister chromatid separation, disassembly of the mitotic spindle, chromosome decondensation, cytokinesis and reformation of the nuclear envelope (Murray and Kirschner, 1989; Luca et al., 1991; Gallant and Nigg, 1992; Holloway et al., 1993; Surana et al., 1993). Another APC substrate whose degradation is required for mitotic exit is the Cdc5/Plk1/Polo kinase. Degradation of Cdc5 in budding yeast is mediated by APCCdh1 and expression of a nondegradable Cdc5 arrests cells in a G2-like state, lacking mitotic cyclins and a mitotic spindle, suggesting that destruction of this protein is required to inactivate APC-dependent degradation of mitotic cyclins as cells enter S phase (Charles et al., 1998; Shirayama et al., 1998). Accordingly, immunoprecipitation of Plx1 from interphasic Xenopus egg extracts in which APC has been activated by the addition of purified cyclin B/cdk1 induces premature inactivation of cyclin B degradation (Brassac et al., 2000). APC substrates controlling G1 phase The main APC substrate during G1 phase is the APC activator Cdc20. Cdc20 proteolysis by APCCdh1 induces APCCdc20 inactivation and allows the switch from APCCdc20 to APCCdh1. APCCdc20 is active in the presence of high cyclin B/cdk1 activity, whereas Cdh1 phosphorylation by cyclin B/cdk1 prevents APC/Cdh1 association and as a consequence, APCCdh1 activation. Once cyclin 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 the degradation of mitotic cyclins, preventing the premature accumulation of these proteins and a premature entry into S phase (reviewed in Zachariae and Nasmyth, 1999). Besides Cdc20, Aurora A kinase is also degraded by the APC during G1 phase. This proteolysis is exclusively mediated by APCCdh1 (Littlepage and Ruderman, 2002; Castro et al., 2002a, b). Aurora A is localized to the spindles and its overexpression induces centrosome duplication. Actually, high levels of the Aurora A


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Figure 2 Temporal pattern of APCCdc20 and APCCdh1 regulation throughout the cell cycle. During G1 phase of the cell cycle, APCCdh1 is an active complex. Once G1-cyclins accumulate, Cdh1 becomes phosphorylated and dissociates from the APC. This phosphorylation will be maintained until anaphase. From G2 to prophase, free APC is kept inactive by its inhibitor Emi1, which associates with Cdc20 and prevents APC-Cdc20 binding. At late prophase, Emi1 is degraded and RASSFA1 takes over the role of this inhibitor until late prometaphase 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 and Nek2 proteolysis). Once the spindle checkpoint is satisfied, the Mad2-Bub3-BubR1 complex is dissociated from APCCdc20 and this ubiquitin-ligase achieves its full activity, and induces degradation of securin and initiation of cyclin B proteolysis. Continuous cyclin B degradation 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

kinase do not directly alter centrosome duplication but induces an aberrant mitosis without cytokinesis that results in tetraploid cells (Meraldi et al., 2002). Elevated Aurora A levels have been frequently displayed in cancer cells indicating that Aurora A proteolysis may be important to prevent polyploidy. A recent report has identified a new G1 substrate of the APC, Tome-1, required for degradation of the protein kinase wee1 and for mitotic entry in Xenopus egg extracts. Tome-1 contains an F-box motif and associates with Skp1 to induce ubiquitination and degradation of the wee1 kinase. wee1 is responsible of the inhibitory phosphorylation of Cdk1 and its Tome1-dependent degradation at G2 is probably required to induce mitotic entry. Tome-1 degradation during G1 allows 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 DNA replication have been described: Orc1, Cdc6 and geminin. These three proteins control prereplication complex formation at the replication origins during S phase. The origin recognition complex (ORC) binds the origins of replication and serves as a platform for subsequent loading of additional factors such as Cdc6, Cdt1 and MCM proteins rendering DNA competent for replication. In Drosophila, the levels of the ORC subunit Orc1 are controlled during the cell cycle. Orc1 persists Oncogene


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from late G1 phase until late mitosis and it is degraded by APCCdh1 at the exit from mitosis (Araki et al., 2003). Cdc6 binds to the ORC complex and mediates subsequent recruitment of the MCM complex. Similarly, human Cdc6 is degraded in early G1 by APCCdh1 (Petersen et al., 2000). According to these results it has been suggested that replication origins are licensed at mitosis, and that the subsequent synthesis of Cdc6 and Orc1 could be exclusively required for cells that have been out of the cell cycle for a long period (Petersen et al., 2000). Geminin prohibits initiation of DNA replication at inappropriate times of the cell cycle by preventing MCM recruitment at the replication origins. In HeLa cells, geminin is absent during G1 and accumulates during S, G2 and M phases. A model to explain the role of geminin in cell cycle control proposes that during G1 phase 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 a first wave of prereplication complex formation and DNA replication begins. As S phase progresses, geminin accumulates and inhibits subsequent recruitment of MCMs to the replication origins (McGarry and Kirschner, 1998). Thus, the APC-dependent degradation of these three proteins acts as a control mechanism required to ensure the appropriate DNA replication. Role of the APC in other cellular functions Finally, the APC also plays an important role in other cell functions such as TGFb signalling by degrading SnoN repressor (Stroschein et al., 2001; Wan et al., 2001), in the re-establishment of the intercentriolar linkage, by degrading Nek2A kinase (Hames et al., 2001) in the salvage pathway of dTTP synthesis for DNA replication, by degrading thymidine kinase (Ke

and Chang, 2004) in the control of cell proliferation by inducing the degradation of the HOXC10 transcription factor (Gabellini et al., 2003) or in the control of latent replication of the Bovine Papillomavirus genome by degrading the viral initiation factor E1 (Mechali et al., 2004).

APC composition and structure The APC is a large protein complex containing at least 11 core subunits (Zachariae et al., 1998b; Gmachl et al., 2000; Yoon et al., 2002) that can further associate with at 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 are stably associated throughout the cell cycle (Peters et al., 1996; Grossberger et al., 1999) except for the different activators 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 subunits work together to form a functional E3 ligase. Similarly, the exact mechanism used by the different APC activators to modulate E3 activity is not clear. Core components of the APC Compelling data on the composition of the APC show that the majority of the APC subunits described in yeast are also present in vertebrates, suggesting that the APC has a similar composition in all eukaryotes (Table 1). A total of 11 APC core subunits have been described in vertebrates versus 13 in yeast. From these, only two subunits, Apc7 and Apc9, seem to be specific to vertebrate (Yu et al., 1998) and yeast (Zachariae et al., 1998b) respectively, whereas, to date, vertebrate orthologs of the recently described yeast Apc13, Apc14 and Apc15 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 Apc2 Cdc27 Apc4 Apc5 Cdc16 — Cdc23 Apc9 Doc1 Apc11 Cdc26 Swm1 — Mnd2

Cut4 — Nuc2 Cut20/Lid1 — Cut9 — Cut23 — Apc10 Apc11 Hcn1 Apc13 Apc14 Apc15

Apc1/Tsg24 Apc2 Apc3/Cdc27 Apc4 Apc5 Apc6/Cdc16 Apc7 Apc8/Cdc23 — Apc10 Apc11 Cdc26 — — —

Rpn1/2 homology Cullin homology Tetratricopeptide repeats — — Tetratricopeptide repeats Tetratricopeptide repeats Tetratricopeptide repeats — Doc domain Ring-H2 finger domain — — — —

Cdc20 Cdh1/Hct1 Ama1

Slp1 Srw1/Ste9 —

Cdc20/p55cdc Cdh1 —

WD40 repeats WD40 repeats WD40 repeats

Activators

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motifs in their amino-acid sequence already described for different subunits of the SCF ubiquitin-ligase. These structural similarities between SCF and APC contributed significantly to the understanding of the role of each component of the APC (Figure 3). Apc1 Apc1 is the largest subunit of the APC. It was first identified in Xenopus egg extracts by biochemical purification (Peters et al., 1996), and in budding yeast in a screen of mutants in which the yeast mitotic cyclin B, Clb2, was stabilized (Zachariae et al., 1996). Sequence analysis revealed sequence homology with the BimE (Engle et al., 1990) protein from Aspergillus nidulans and the Tgs24 murine protein (Starborg et al., 1994). Moreover, a homolog of Apc1 in fission yeast, named Cut4, has also been described as a constituent of the 20S cyclosome (Yamashita et al., 1996). The levels of Apc1 are constant throughout the cell cycle; however, regulation of this protein during mitosis seems to be exerted by phosphorylation (Peters et al., 1996; Jorgensen et al., 2001). Apc1 shares a structural motif with Rpn1 and Rpn2, two proteins of the 19S cap from the 26S proteasome. Rpn1 and Rpn2 present several sequence repeats formed each of a b-strand followed by an a-helix. A secondary structure prediction of this sequence repeat indicates the presence of a horseshoe-

Figure 3 Comparison of the APCCdc20 and the SCF ubiquitinligases. APC and SCF both contain a Cullin, a Ring-H2 finger and a WD40 subunit. The cullin and the Ring-H2 finger proteins form the minimal ubiquitin-ligase module of both E3, and are required for E2 tethering. The APC contains additional subunits such as the tetratricopeptide containing proteins and the Doc protein. Proteins belonging to the same family are identically coloured. Interaction of the different APC subunits have been represented taking into account the existent results on the association between different APC components. This model was built by integrating the data described in this review obtained in different models

shaped structure with an inner b-strand surface and an outer a-helix face. In this structure, the inner hydrophobic b-strand surface would be ideally suited to bind unfolded proteins (Lupas et al., 1997). The function of this repetitive sequence in Apc1 protein has not been elucidated; however, a possible role as a scaffold for the assembly of APC complex or in the interaction with polyubiquitinated proteins has been suggested (Lupas et al., 1997). The tetratricopeptide repeat containing proteins Four APC subunits Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23 (Sikorski et al., 1990; Lamb et al., 1994) present in their amino-acid sequences randomly repeated copies of the 34-residue tetratricopeptide motif (TPR motif). The TPR is a structural motif present in a wide range of proteins mediating a variety of biological processes such as cell cycle regulation, transcriptional control, mitochondrial and peroxisomal protein transport and protein folding (reviewed in D’Andrea and Regan, 2003). This motif is thought to mediate protein– protein interactions of multiprotein complexes (D’Andrea and Regan, 2003). In this regard, it has been demonstrated that Apc3/Cdc27 and Apc7 bind to a Cterminal motif in the amino-acid sequences of APC activators Cdc20 and Cdh1. Moreover, it has also been shown that these C-terminal motifs ending in an isoleucine–arginine (IR) dipeptide are essential for Cdc20 and Cdh1 to bind the APC (Passmore et al., 2003; Vodermaier et al., 2003). These results indicate that the TPR-containing APC subunits regulate APC activity by inducing Cdc20 and Cdh1 association. The APC subunit Apc10/Doc1 (see below) also possesses an ‘IR tail’, which mediates Apc10–Apc3/ Cdc27 and Apc10–Apc7 binding (Wendt et al., 2001; Vodermaier and Peters, 2004), suggesting that the association of Apc10/Doc1 to the APC could also be regulated by the TPR-containing proteins. All these TPR-containing proteins are phosphorylated during mitosis (Peters et al., 1996) and this phosphorylation seems to be essential to induce APC activation at this phase of the cell cycle (Lahav-Baratz et al., 1995). Thus, Apc3/Cdc27 and Apc7 phosphorylation likely modify Apc3/Cdc27-Cdc20 and Apc7-Cdc20 binding and thereby increase Cdc20 binding and APC activation (Vodermaier and Peters, 2004). APC2 and APC11 Both Apc2 and Apc11 subunits present close homology with two other components of the ubiquitin-ligase SCF. The SCF subunit Cdc53 is a member of the cullin family (Patton et al., 1998; Zachariae et al., 1998b). These proteins contain a 180-residue domain referred to as the cullin homology domain (Zachariae et al., 1998b). As described above, the C-terminal cullin domain of Cdc53 binds to the RING-H2 finger protein Hrt1/Rbx1 that in turn binds to the E2 enzyme tethering this enzyme to the SCF (Kamura et al., 1999; Seol et al., 1999; Skowyra et al., 1999). The N-terminal domain of Cdc53 binds to Skp1, mediating the association with the Oncogene


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different F-box proteins, which will directly associate with phosphorylated substrates (Bai et al., 1996; Patton et al., 1998). Like the SCF components Cdc53 and Hrt1/Rbx1, the APC subunits Apc2 and Apc11 contain a cullin and a RING-H2 finger domain, respectively. This sequence homology suggests that Apc2 and Apc11 might fulfill in the APC complex a similar role to that developed by Cdc53 and Hrt1/Rbx1 in the SCF enzyme (Figure 3). Recent results demonstrate that this is the case. Tang et al. (2001b) have shown that the Apc11 RING-H2 protein directly interacts with the Ubc4 E2 enzyme and induces E2-dependent ubiquitination of protein substrates, whereas binding of Apc11 to the Ubch10 E2 enzyme requires the C-terminal cullin domain of Apc2. Thus, at least in the UbcH10-mediated ubiquitination, Apc2 and Apc11 are required to form the minimal ubiquitin-ligase module of the APC. Accordingly, a heterodimeric Apc2/Apc11 complex purified from baculoviral-coinfected insect cells is sufficient to catalyse securin and cyclin B1 ubiquitination in presence of the UbcH10 enzyme, although this ubiquitination does not possess substrate specificity (Leverson et al., 2000). Apc10/Doc1 Apc10/Doc1 was first identified as an APC subunit in budding yeast in a genetic screen for mutants defective in the degradation of mitotic cyclins (Hwang and Murray, 1997). Orthologs of this protein have also been found in fission yeast and human (Kominami et al., 1998; Grossberger et al., 1999). This APC subunit is characterized by the presence of a ‘Doc’ domain in its amino-acid sequence. This sequence is also found in several proteins containing other motifs linked to ubiquitination such as cullin and HECT motifs (Kominami et al., 1998; Grossberger et al., 1999). No differences in the subunit composition of the APC purified from temperature-sensitive alleles of the DOC1 gene have been observed in budding yeast, indicating that Apc10/Doc1 binding to the APC is not required to stabilize this complex (Kominami et al., 1998; Grossberger et al., 1999). Nevertheless, this APC subunit directly associates to Apc11 (Tang et al., 2001b), Apc3/ Cdc27 (Wendt et al., 2001) and Apc7 (see above) (Vodermaier and Peters, 2004). The physiological role of these direct associations is not clear. However, a recent report shows that Apc10/Doc1 could act as a processivity factor for the APC (Carroll and Morgan, 2002). Moreover, a role of Apc10/Doc1 in the recognition of APC substrates has also been proposed. In this regard, binding of the APC activator Cdh1 with APC substrates is prevented in APC complexes purified from Doc1 mutants (Passmore et al., 2003). These results imply that Apc10/Doc1 mediates substrate binding to Cdh1 and to the APC either direct or indirectly. On the basis of these results, it would be interesting to determine if this binding is mediated by a conformational change of APC or through direct binding of Apc10/Doc1 to the substrate. If Doc1/Apc10 bound the APC substrates directly, it would suggest that a Doc1 recognition motif is present in these proteins. In this case, two different Oncogene

degradation motifs could be present in the substrates to be recognized by the APC: a first Cdh1-recognition sequence and a second Apc10/Doc1-recognition motif. Apc4 and Apc5 Little is known about the role of these two subunits in APC structure and activity. Neither Apc4 nor Apc5 present any known motif in their sequence that could hint to their function. However, these two subunits, in addition to Apc1, are tightly associated to the Apc2/Apc11 ubiquitin-ligase module and could therefore connect Apc2/Apc11 with the TPR subunits (Vodermaier et al., 2003). Apc9 and Cdc26 Apc9 was identified by biochemical purification of yeast APC and no orthologs in other species have been identified so far (Zachariae et al., 1998b). Unlike Apc9, Cdc26 has been described in both yeast (Yamada et al., 1997; Zachariae et al., 1998b) and vertebrates (Gmachl et al., 2000). The analysis of the amino-acid sequences of these two proteins has not revealed any known motif. Both subunits seem to be required to maintain APC structure. Thus, association of Apc3/Cdc27 with the APC is drastically reduced in Apc9 mutants, whereas APC purified from Cdc26 mutants 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 the APC that have been recently described in Saccharomyces pombe (Yoon et al., 2002). Two orthologs of these subunits Apc13/Swm1 and Apc15/Mnd2 have also been described 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 be important in the stabilization of the APC structure (Hall et al., 2003). The role of these subunits is unknown so far. However, it has been suggested that Apc13/Swm1 and Apc15/Mnd2 could provide an essential function for the APC during meiosis that is not required during mitosis (Hall et al., 2003). APC activators: Cdc20, Cdh1 and Ama1 A Cdc20 mutant was originally described in budding yeast as causing cells to arrest after DNA synthesis at the G2 stage of the cell cycle (Hartwell and Smith, 1985). However, the first direct connection between this protein and APC-dependent degradation of the mitotic cyclins was described in Drosophila in which a mutation in the gene encoding the Cdc20 ortholog, Fizzy, blocked mitotic degradation of cyclin A, B and B3 (Sigrist et al., 1995). A subsequent study in this species described the presence of a new protein with significant homology to Fizzy that was named Fizzy-Related, and that corresponds to the Drosophila Cdh1/Hct1 ortholog. Fizzy-Related is required for cyclin removal during G1 in epidermal embryo cells of Drosophila (Sigrist and Lehner, 1997). Two other studies simultaneously


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appeared in budding yeast in which similar roles were ascribed to the Cdc20 and Cdh1/Hct1 (Schwab et al., 1997; Visintin et al., 1997). Cdc20 and Cdh1/Hct1 belong to a multigene family that includes four Cdh1/ Hct1 homologs (Wan and Kirschner, 2001) and a yeast meiotic-specific APC activator named Ama1 (Cooper et al., 2000). The four Cdh1/Hct1 homologs described in chicken are differently localized in proliferating, differentiated and postmitotic tissues and they mediate ubiquitination of different APC substrates (Wan and Kirschner, 2001). Studies with purified APC from budding yeast and human 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 which Cdc20 function is blocked presents a clearly inhibited ubiquitination activity (Lorca et al., 1998). The capacity of these activators to stimulate ‘in vitro’ ubiquitination of mitotic cyclins is dependent on their own association to the APC. This interaction seems to be mediated through direct binding of the TPR subunits Apc3/Cdc27 and Apc7 with the IR C-terminal tails of Cdc20 and Cdh1/Hct1 (see above) (Vodermaier et al., 2003). In addition to the IR motif, another sequence in the N-terminus of these activators, the C-Box, has indeed been found necessary for their association with APC in yeast (Schwab et al., 2001). Thus, further studies are required to establish the real mechanism through which these proteins bind the APC. All these results suggest that Cdc20, Cdh1/Hct1 and Ama1 act as APC activators, however, the exact mechanism through which these proteins increases APC activity is not known. They are all members of a conserved subfamily of WD40 proteins. This motif has also been described for a subset of F-box subunits of the SCF ubiquitin-ligase. The different F-box proteins contain two domains: one required for substrate recognition, and another one, the F-box, essential for Skp1 association. Interestingly, in this subset of F-box proteins, substrate binding to the F-box protein is mediated by the WD40 domain (Patton et al., 1998). Comparison between structures of the SCF and APC suggests a putative role of these activators in substrate recognition. In this regard, recent results have shown that different APC substrates can directly and specifically 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 could mediate substrate recognition by the APC through direct binding come from the study of Sorensen et al. These authors demonstrated direct binding of cyclin A with the last Cdh1’s WD40 repeat. The mutation of this binding site in Cdh1/Hct1 inhibited cyclin A association to Cdh1/Hct1 and cyclin A degradation (Sorensen et al., 2001). A second study demonstrated that the APC

substrate, Pds1, whose degradation is exclusively induced by APCCdc20, binds directly Cdc20, whereas it is incapable of associating with Cdh1/Hct1, indicating that Cdc20 and Cdh1/Hct1 recognize and bind their substrates differently and selectively (Hilioti et al., 2001). Further specific and direct interactions of Cdc20 with Pds1 and of Cdh1/Hct1 with Clb2, Clb3 and Cdc5 were also demonstrated in budding yeast (Burton and Solomon, 2001; Schwab et al., 2001). Finally, Pfleger et al. (2001a) showed that in the absence of APC, Cdc20 and Cdh1/Hct1 can directly and specifically bind a large number of APC substrates including Xkid, securin, geminin, cyclin A and cyclin B. Compelling data indicate that the selective binding of the APC activators with each substrate is mediated by the presence in the substrate amino-acid sequence of different ‘degradation motifs’ specifically recognized by Cdc20 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 degradation and when fused to a foreign protein, was also sufficient to generate a similar proteolytic pattern to that observed for cyclin B (Glotzer et al., 1991). Subsequent studies demonstrated a Dbox-dependent degradation of other APC substrates such as cyclin A (Lorca et al., 1992; Sorensen et al., 2001), Nek2 (Hames et al., 2001) and Aurora 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 activator Cdc20 which is itself an APCCdh1 substrate. Cdc20 lacks a Dbox and its APCCdh1-dependent degradation is mediated by the KENbox. Like the Dbox, the KENbox sequence is a transposable motif that induces proteolysis of the hybrid protein and which acts as a specific recognition signal for APCCdh1. The authors also demonstrated that Cdh1/Hct1 specifically interacts with substrates containing a KENbox and/or a Dbox, whereas Cdc20 exclusively binds Dbox-containing substrates (Pfleger and Kirschner, 2000). A third degradation signal, the ‘GxENbox’, has recently been described (Castro et al., 2003). This motif was shown in the amino-acid sequence of the chromokinesin Xkid and mediates degradation of this protein by both APCCdc20 and APCCdh1. Deletion of this motif in Xkid prevents its APCCdc20 and APCCdh1-dependent degradation. Moreover, like other proteolytic sequences, when fused, it is capable to induce degradation of a foreign protein with a similar timing to that observed for Xkid. A particularly interesting case is degradation of Aurora A. Aurora A proteolysis is exclusively mediated by APCCdh1 and requires the presence of a double degradation motif: the Dbox and a new degradation signal named ‘DAD’ (for Dbox Activating Domain) or ‘Abox’. Mutation of either of these two domains prevents destruction of Aurora A (Castro et al., 2002a, b; Littlepage and Ruderman, 2002). A double recognition motif has also been described for other proteins such as cyclin A (Geley et al., 2001) and Nek2 (Hames et al., 2001). These results suggest that Oncogene


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degradation of these proteins is mediated by either a double motif formed by a bipartite sequence placed in two distal parts of the substrate or by the presence of two different degradation signals that could be recognized by two different APC subunits. In this regard, a recent study demonstrated a cell cycle-dependent Dbox-binding activity of the APC in the absence of Cdc20 or Cdh1, indicating that another APC subunit besides Cdc20 and Cdh1 possesses Dbox receptor activity (Yamano et al., 2004). One possible candidate for the Dbox receptor of the APC is the core subunit Apc10/Doc1. As described above, binding of substrates with APC is prevented in APC complexes purified from Apc10/Doc1 mutants (Passmore et al., 2003). These results imply that Apc10/Doc1 directly or indirectly mediates substrate binding to Cdh1 and the APC. Thus, it is possible that two different APC receptors, Cdc20 or Cdh1/Hct1 and Apc10/Doc1, recognize two different degradation signals in the substrate and thereby inducing APC binding and ubiquitination. APC regulation Phosphorylation of APC is one of the mechanisms used by the cell to modulate APC activity. As described above, the core subunits of the APC, Apc1, Apc3/ Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23, are phosphorylated during mitosis (Peters et al., 1996). This phosphorylation modulates Cdc20 binding to the APC and APC activity. In this regard, three different kinases have been described to phosphorylate APC: cyclin B/cdk1, Plk1 and PKA (Kotani et al., 1998; Rudner and Murray, 2000; Golan et al., 2002; Kraft et al., 2003). Cyclin B/cdk1-mediated phosphorylation of APC in Xenopus egg extracts requires Suc1. This conclusion is based on the observation that the immunodepletion in interphase Xenopus egg extracts of the Suc1 ortholog, Xe-p9, at the beginning of mitosis, prevents phosphorylation of Apc3/Cdc27 by this kinase (Patra and Dunphy, 1998). Both, ‘in vitro’ and ‘in vivo’ phosphorylation of Apc3/Cdc27, Apc6/Cdc16 and Apc8/Cdc23 by 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 phosphorylation by Plk1 does not stimulate either Cdc20 binding or APC activity; however, when combined with cyclin B/cdk1, both kinases act synergistically to increase APC ubiquitination activity (Golan et al., 2002; Kraft et al., 2003). In vitro phosphorylation of APC by PKA inhibits ubiquitination of cyclin B even in the presence of the regulatory factors Cdc20 and Cdh1/Hct1 (Kotani et al., 1998). In addition, yeast APC mutants are suppressed by mutations in the PKA pathway that lower its kinase activity (Yamashita et al., 1996). Taken together, these evidence suggests that cyclin B/cdk1- and Plk1-mediated phosphorylations induce APC activation, whereas PKA phosphorylation inhibits its activity. Cell cycle-regulated phosphorylation of Cdc20 has also been observed (Weinstein, 1997; Lorca et al., 1998; Chung and Chen, 2003), however this phosphorylation Oncogene

seems not to be required to induce activation of the APC but probably to allow APC inhibition by the spindle checkpoint. Accordingly, recent results have demonstrated that this APC activator is phosphorylated in Thr 64 and Thr 68 by MAPK during mitosis and that a Thr-to-Ala mutation in these residues decreases spindle checkpoint-dependent inhibition of the APC by reducing its affinity for the spindle checkpoint proteins (Chung and Chen, 2003). Two proteins of the spindle checkpoint, Mad2 and BubR1, are capable of inhibiting APCCdc20. Different studies have demonstrated that this inhibition is mediated by direct binding of Mad2 and BubR1 to Cdc20. Thus, Fang et al. (1998a) have shown that the microinjection of recombinant Mad2 protein into Xenopus embryos arrests cells at mitosis with inactive APC. Moreover, despite the fact that the two different forms of Mad2, monomer and tetramer, bind Cdc20, only the tetramer is capable of inhibiting activation of APC (Fang et al., 1998a). Purified BubR1 also binds directly to recombinant Cdc20 and inhibits APC and this inhibition is 12-fold more potent than Mad2. However, both Mad2 and BubR1 mutually promote each other’s binding and act synergistically to decrease APC activity (Fang, 2002). The presence of BubR1-Bub3-Cdc20, Mad2-Cdc20 and BubR1-Bub3Mad2-Cdc20 APC-inhibitory complexes has also been demonstrated in vivo in spindle checkpoint-activated cells (Sudakin et al., 2001; Tang et al., 2001a). BubR1Bub3-Mad2-Cdc20 inhibitory activity has been determined 3000-fold greater than recombinant Mad2 oligomers (Sudakin et al., 2001). This complex is present in both interphase and mitotic cells, however in interphasic cells it is not associated to APC, thus its APC-inhibitory capacity is limited to mitosis (Sudakin et al., 2001). A third inhibitor, Mad2L2, specifically inhibits the APCCdh1 complex. Like Mad2, Mad2L2 forms monomers and oligomers; however, both are capable of inhibiting APCCdh1. ‘In vitro’ translated Mad2L2 selectively binds APCCdh1 but not APCCdc20. This protein does not affect either Cdh1 or substrate binding to the APC, but inhibits substrate ubiquitination. Finally, injection of this protein in Xenopus embryos causes a post-Mid Bastula Transition (MBT)gastrulation arrest. This is consistent with Mad2L2 inhibiting endogenous APCCdh1, since this ubiquitinligase is only present after MBT in Xenopus embryos (Pfleger et al., 2001b). Besides the spindle checkpoint-dependent inhibitors of the APC and Mad2L2, two other proteins, Emi1 and RASSF1A, have been recently described as negative regulators of this ubiquitin-ligase (Reimann et al., 2001; Song et al., 2004). Emi1 inhibits both APCCdh1 and APCCdc20 by directly binding to Cdc20 and Cdh1 and thereby preventing substrate-Cdc20/Cdh1-APC associations. In human cells, Emi1 accumulates in late G1 and is destroyed early in mitosis by the SCF (Hsu et al., 2002; MargottinGoguet et al., 2003). Its overexpression in these cells induces cyclin A accumulation and accelerates S phase entry. In addition, Emi1 immunodepletion from cycling Xenopus egg extracts strongly delays cyclin B


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accumulation and mitotic entry, whereas its addition stabilizes APC substrates and causes a mitotic block (Reimann et al., 2001). Thus, Emi1 may participate in S phase entry by promoting accumulation of cyclin A through APCCdh1 inhibition and in the subsequent entrance into mitosis by inducing cyclin B accumulation through APCCdc20 inhibition. RASSF1 is a tumour suppressor gene that is frequently silenced in different tumour types as a result of a hypermethylation of CpG islands in its promoter. Two major isoforms of RASSF1, A and C, are produced from the RASSF1 gene. RASSF1A contains a cysteinerich diacylglycerol-binding domain (C1 domain) and a Ras-association (RA) domain. RASSF1C contains the RA domain but the C1 domain is replaced by a distinct amino-acid sequence. RASSF1C is thought to play a role in RAS-mediated function, whereas RASSF1A is implicated in the regulation of cell cycle. A recent study by Song et al. has reported that RASSF1A acts at early prometaphase to prevent degradation of mitotic cyclins, and to delay progression beyond metaphase by inhibiting APCCdc20 complex. Accordingly, overexpression of this protein in HeLa cells induces stabilization of cyclin A and cyclin B and mitotic arrest at prometaphase, whereas, its depletion by RNA interference accelerates mitotic cyclin degradation and mitotic progression.

Moreover, RASSF1A interacts with Cdc20 and inhibits APC activity. Finally, they demonstrate that this inhibition is independent of the spindle checkpoint APC inhibitors and Emi1 (Song et al., 2004). In conclusion, APC activity is subjected to tight regulation to ensure the correct timing of protein degradation during the mitotic cell cycle (Figure 2). Thus, APC activity is negatively controlled by different inhibitors. First, Emi1 ensures S and M phase entries by allowing accumulation of cyclin A and cyclin B from S until prophase. Subsequently, from prophase until prometaphase, this role is developed by the RASSF1A APC inhibitor and finally inhibition of the APC at metaphase is taken over by the spindle checkpoint pathway. Besides this negative control, APC is also positively regulated first by phosphorylation of the different core subunits during early mitosis and subsequently by its association to the APC activators Cdc20 from prophase to anaphase, and Cdh1/Hct1 from anaphase through mitotic exit and into G1. Acknowledgements We are indebted to Dr Catherine Bonne-Andrea, Dr Carsten Janke and Dr May Morris for the critical reading of this manuscript. This work was supported by the Ligue Nationale Contre le Cancer (Equipe Labelliseé).

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