Cdc20: A WD40 Activator for a Cell Cycle Degradation Machine

Cdc20 is an essential cell-cycle regulator required for the completion of mitosis in organisms from yeast to man and contains at its C terminus a WD40 repeat domain that mediates protein-protein interactions. In mitosis, Cdc20 binds to and activates the ubiquitin ligase activity of a large molecular machine called the anaphase-promoting complex/cyclosome (APC/C) and enables the ubiquitination and degradation of securin and cyclin B, thus promoting the onset of anaphase and mitotic exit. APC/CCdc20 is temporally and spatially regulated during the somatic and embryonic cell cycle by numerous mechanisms, including the spindle checkpoint and the cytostatic factor (CSF). Therefore, Cdc20 serves as an integrator of multiple intracellular signaling cascades that regulate progression through mitosis. This review summarizes recent progress toward the understanding of the functions of Cdc20, the mechanisms by which it activates APC/C, and its regulation by phosphorylation and by association with its binding proteins. Cdc20 is an essential cell-cycle regulator required for the completion of mitosis in organisms from yeast to man and contains at its C terminus a WD40 repeat domain that mediates protein-protein interactions. In mitosis, Cdc20 binds to and activates the ubiquitin ligase activity of a large molecular machine called the anaphase-promoting complex/cyclosome (APC/C) and enables the ubiquitination and degradation of securin and cyclin B, thus promoting the onset of anaphase and mitotic exit. APC/CCdc20 is temporally and spatially regulated during the somatic and embryonic cell cycle by numerous mechanisms, including the spindle checkpoint and the cytostatic factor (CSF). Therefore, Cdc20 serves as an integrator of multiple intracellular signaling cascades that regulate progression through mitosis. This review summarizes recent progress toward the understanding of the functions of Cdc20, the mechanisms by which it activates APC/C, and its regulation by phosphorylation and by association with its binding proteins. The cell-division cycle consists of a series of complex, interdependent processes that are coordinated by core cell cycle regulatory proteins. The molecular understanding of the cell cycle began when Lee Hartwell and coworkers isolated Cell Division Cycle (CDC) mutants of S. cerevisiae that, despite continued cell growth, failed to execute or complete key cell-cycle events, such as DNA replication or mitosis (Hartwell et al., 1970Hartwell L.H. Culotti J. Reid B. Genetic control of the cell-division cycle in yeast. I. Detection of mutants.Proc. Natl. Acad. Sci. USA. 1970; 66: 352-359Crossref PubMed Google Scholar). Among the original Hartwell mutant collection were the cdc20 mutants that arrest cell division in mitosis and fail to initiate anaphase and chromosome segregation (Hartwell et al., 1973Hartwell L.H. Mortimer R.K. Culotti J. Culotti M. Genetic control of the cell division cycle in yeast: V. genetic analysis of cdc mutants.Genetics. 1973; 74: 267-286Crossref PubMed Google Scholar). Molecular cloning of the CDC20 gene revealed that it encodes a protein related to the β subunit of trimeric G proteins. Both Cdc20 and Gβ contain seven WD40 repeats that form a seven-bladed β propeller structure ideally suited for mediating protein-protein interactions (Figure 1). Orthologs of Cdc20 were then discovered in various organisms, including mammals and the fruit fly (Weinstein et al., 1994Weinstein J. Jacobsen F.W. Hsu-Chen J. Wu T. Baum L.G. A novel mammalian protein, p55CDC, present in dividing cells is associated with protein kinase activity and has homology to the Saccharomyces cerevisiae cell division cycle proteins Cdc20 and Cdc4.Mol. Cell. Biol. 1994; 14: 3350-3363Crossref PubMed Google Scholar, Dawson et al., 1995Dawson I.A. Roth S. Artavanis-Tsakonas S. The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae.J. Cell Biol. 1995; 129: 725-737Crossref PubMed Google Scholar). In Drosophila, the Cdc20 ortholog, Fizzy, was shown to be required for the degradation of mitotic cyclins A and B (Dawson et al., 1995Dawson I.A. Roth S. Artavanis-Tsakonas S. The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae.J. Cell Biol. 1995; 129: 725-737Crossref PubMed Google Scholar). However, the biochemical functions of Cdc20 remained obscure until the discovery of the anaphase-promoting complex/cyclosome (APC/C) (King et al., 1995King R.W. Peters J.M. Tugendreich S. Rolfe M. Hieter P. Kirschner M.W. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B.Cell. 1995; 81: 279-288Abstract Full Text PDF PubMed Google Scholar, Sudakin et al., 1995Sudakin V. Ganoth D. Dahan A. Heller H. Hershko J. Luca F.C. Ruderman J.V. Hershko A. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.Mol. Biol. Cell. 1995; 6: 185-197Crossref PubMed Google Scholar). (A) Domain structure of human Cdc20 with its seven WD40 repeats, C box, KEN box, Mad2-interacting motif (MIM), CRY box, IR motif, and in vivo phosphorylation sites indicated. (B) A structure model of the WD40 domain of human Cdc20 (left, top view; right, side view). Residues on the top face of the β propeller of Cdc20 homolog 1 (Cdh1) can be crosslinked to D box-containing peptides, suggesting that the top face of the WD40 domain of Cdc20 might be involved in substrate binding. The C box and IR motif have been implicated in binding to APC/C. They are predicted to lie at the bottom face of the propeller. Progression through the cell cycle relies on the periodic fluctuations of the activities of cyclin-dependent kinases (CDKs). Activation of Cdk1 is required for the execution of many if not all events during mitosis whereas inactivation of Cdk1 is a prerequisite for the exit from mitosis. An important mechanism for the inactivation of Cdk1 is the degradation of its positive regulatory subunits, mitotic cyclins A and B, through the ubiquitin-proteasome pathway (Glotzer et al., 1991Glotzer M. Murray A.W. Kirschner M.W. Cyclin is degraded by the ubiquitin pathway.Nature. 1991; 349: 132-138Crossref PubMed Scopus (1290) Google Scholar). In addition, the same ubiquitin-dependent degradation machinery that degrades mitotic cyclins is also responsible for the degradation of an anaphase inhibitor that blocks the onset of sister-chromatid separation (Holloway et al., 1993Holloway S.L. Glotzer M. King R.W. Murray A.W. Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor.Cell. 1993; 73: 1393-1402Abstract Full Text PDF PubMed Scopus (379) Google Scholar). The convergence of genetic and biochemical studies in yeast and clam and frog egg extracts then identified APC/C as the ubiquitin ligase in this mitotic degradation system (King et al., 1995King R.W. Peters J.M. Tugendreich S. Rolfe M. Hieter P. Kirschner M.W. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B.Cell. 1995; 81: 279-288Abstract Full Text PDF PubMed Google Scholar, Sudakin et al., 1995Sudakin V. Ganoth D. Dahan A. Heller H. Hershko J. Luca F.C. Ruderman J.V. Hershko A. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.Mol. Biol. Cell. 1995; 6: 185-197Crossref PubMed Google Scholar). APC/C is a multisubunit enzyme conserved from yeast to man and contains at least 12 core subunits (reviewed in Peters, 2006Peters J.M. The anaphase promoting complex/cyclosome: a machine designed to destroy.Nat. Rev. Mol. Cell Biol. 2006; 7: 644-656Crossref PubMed Scopus (602) Google Scholar and Thornton and Toczyski, 2006Thornton B.R. Toczyski D.P. Precise destruction: an emerging picture of the APC.Genes Dev. 2006; 20: 3069-3078Crossref PubMed Scopus (99) Google Scholar). APC/C becomes active during mitosis and remains active through much of G1. During mitosis, APC/C mediates the ubiquitination of the anaphase inhibitor securin (known as Cut2 in fission yeast and Pds1 in budding yeast) that is an inhibitory chaperone of separase (reviewed in Nasmyth, 2002Nasmyth K. Segregating sister genomes: the molecular biology of chromosome separation.Science. 2002; 297: 559-565Crossref PubMed Scopus (377) Google Scholar). Degradation of securin activates separase, which cleaves the cohesin complex and allows sister-chromatid separation. APC/C also mediates the degradation of mitotic cyclins, resulting in the activation of Cdk1 and mitotic exit. APC/C-dependent degradation of mitotic cyclins and other mitotic regulators persists in G1, preventing their premature accumulation during the cell cycle. The S. cerevisiae cdc20 mutants fail to initiate anaphase, suggesting that Cdc20 might be required for the activation of APC/C at the metaphase-anaphase transition. Furthermore, Cdc20 and Cdc20 homolog 1 (Cdh1; also known as Hct1 for homolog of Cdc twenty) apparently mediate degradation of distinct subsets of APC/C substrates in budding yeast (Schwab et al., 1997Schwab M. Lutum A.S. Seufert W. Yeast Hct1 is a regulator of Clb2 cyclin proteolysis.Cell. 1997; 90: 683-693Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, Visintin et al., 1997Visintin R. Prinz S. Amon A. CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis.Science. 1997; 278: 460-463Crossref PubMed Scopus (567) Google Scholar). This finding suggests that the Cdc20/Cdh1 family of proteins serve as substrate-specific activators of APC/C. In Drosophila, fizzy is required for the degradation of mitotic cyclins and for anaphase onset whereas the fizzy-related (the ortholog of Cdh1) gene is responsible for the continued degradation of these cyclins in G1 (Sigrist and Lehner, 1997Sigrist S.J. Lehner C.F. Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles.Cell. 1997; 90: 671-681Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). This suggests that Cdc20 and Cdh1 regulate APC/C during different phases of the cell cycle. It was later shown that Cdc20 and Cdh1 directly bind to APC/C and activate its ubiquitin ligase activity toward cyclin B in vitro (Fang et al., 1998bFang G. Yu H. Kirschner M.W. Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1.Mol. Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Google Scholar). Binding of Cdc20 to APC/C requires phosphorylation of core APC/C subunits by Cdk1 and other mitotic kinases (Fang et al., 1998bFang G. Yu H. Kirschner M.W. Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1.Mol. Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Google Scholar, Kraft et al., 2003Kraft C. Herzog F. Gieffers C. Mechtler K. Hagting A. Pines J. Peters J.M. Mitotic regulation of the human anaphase-promoting complex by phosphorylation.EMBO J. 2003; 22: 6598-6609Crossref PubMed Scopus (237) Google Scholar). In contrast, CDK-dependent phosphorylation of Cdh1 prevents its binding to APC/C (Zachariae et al., 1998aZachariae W. Schwab M. Nasmyth K. Seufert W. Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex.Science. 1998; 282: 1721-1724Crossref PubMed Google Scholar). Thus, Cdc20 activates APC/C predominantly in mitosis when CDK activities are high whereas Cdh1 mainly interacts with APC/C in telophase/G1 when CDK activities are low. Finally, ubiquitination in vitro by APC/CCdc20 and APC/CCdh1 requires different sequence motifs within the APC/C substrates (Fang et al., 1998bFang G. Yu H. Kirschner M.W. Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1.Mol. Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Google Scholar, Pfleger and Kirschner, 2000Pfleger C.M. Kirschner M.W. The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1.Genes Dev. 2000; 14: 655-665PubMed Google Scholar). Collectively, these findings demonstrate that Cdc20 and Cdh1 are activators of APC/C that function during different phases of the cell cycle and contribute to the substrate specificity of APC/C. This topic has been covered by several excellent recent reviews (Peters, 2006Peters J.M. The anaphase promoting complex/cyclosome: a machine designed to destroy.Nat. Rev. Mol. Cell Biol. 2006; 7: 644-656Crossref PubMed Scopus (602) Google Scholar, Thornton and Toczyski, 2006Thornton B.R. Toczyski D.P. Precise destruction: an emerging picture of the APC.Genes Dev. 2006; 20: 3069-3078Crossref PubMed Scopus (99) Google Scholar). In this article, I focus on the recent progress on the functions, mechanism, and regulation of Cdc20, highlighting results that have altered or refined our views in these areas. A typical cell-division cycle of a human cell lasts about 24 hr. Though mitosis only occupies about 2 hr of this 24 hr cycle, a series of complicated interdependent cellular events are accomplished during this relatively short period of time, including nuclear envelope breakdown, fragmentation of cellular organelles such as the endoplasmic reticulum (ER) and the Golgi apparatus, chromosome condensation, formation of the mitotic spindle, chromosome congression and segregation, and finally cytokinesis. Many of these events involve global changes in the cytoskeleton and membranes of the cell, and yet they are completed on the time scale of minutes. Due to its relatively fast kinetics and its irreversibility, ubiquitin-dependent proteolysis is ideally suited for the regulation of the rapid and orderly progression through mitosis. The best-characterized biochemical activity of Cdc20 is to activate APC/C during mitosis. Therefore, the major function of Cdc20 is to coordinate mitotic progression by facilitating the orderly degradation of mitotic APC/C substrates. CDC20 and most genes that encode core APC/C subunits are essential for viability in yeast (Zachariae et al., 1998bZachariae W. Shevchenko A. Andrews P.D. Ciosk R. Galova M. Stark M.J. Mann M. Nasmyth K. Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins.Science. 1998; 279: 1216-1219Crossref PubMed Scopus (259) Google Scholar). Their corresponding orthologs in mammals are also essential for the completion of the cell-division cycle (Wirth et al., 2004Wirth K.G. Ricci R. Gimenez-Abian J.F. Taghybeeglu S. Kudo N.R. Jochum W. Vasseur-Cognet M. Nasmyth K. Loss of the anaphase-promoting complex in quiescent cells causes unscheduled hepatocyte proliferation.Genes Dev. 2004; 18: 88-98Crossref PubMed Scopus (54) Google Scholar). In contrast, CDH1 is not essential for viability in yeast (Schwab et al., 1997Schwab M. Lutum A.S. Seufert W. Yeast Hct1 is a regulator of Clb2 cyclin proteolysis.Cell. 1997; 90: 683-693Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, Visintin et al., 1997Visintin R. Prinz S. Amon A. CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis.Science. 1997; 278: 460-463Crossref PubMed Scopus (567) Google Scholar). Depletion of Cdh1 by RNA interference (RNAi) from human cells does not significantly perturb the cell cycle (Qi and Yu, 2007Qi W. Yu H. KEN-box-dependent degradation of the bub1 spindle checkpoint kinase by the anaphase-promoting complex/cyclosome.J. Biol. Chem. 2007; 282: 3672-3679Crossref PubMed Scopus (33) Google Scholar). Therefore, APC/CCdc20 is capable of mediating the degradation of critical mitotic APC/C substrates. Thornton and Toczyski reasoned that if the only essential functions of APC/C were to mediate the degradation of mitotic regulators, genetic ablation of such regulators would then rescue the lethality of APC/C mutants (Thornton and Toczyski, 2003Thornton B.R. Toczyski D.P. Securin and B-cyclin/CDK are the only essential targets of the APC.Nat. Cell Biol. 2003; 5: 1090-1094Crossref PubMed Scopus (90) Google Scholar). Indeed, deletion of the CLB5 (a mitotic cyclin) and PDS1 (yeast securin) genes and integration of about ten copies of the SIC1 gene (a mitotic CDK inhibitor) renders the essential APC/C subunits dispensable for the viability of budding yeast cells (Thornton and Toczyski, 2003Thornton B.R. Toczyski D.P. Securin and B-cyclin/CDK are the only essential targets of the APC.Nat. Cell Biol. 2003; 5: 1090-1094Crossref PubMed Scopus (90) Google Scholar). This result suggests that securin and mitotic cyclins are the only essential substrates of APC/C in budding yeast. In mammalian cells, expression of nondegradable securin causes incomplete sister-chromatid separation (Zur and Brandeis, 2001Zur A. Brandeis M. Securin degradation is mediated by fzy and fzr, and is required for complete chromatid separation but not for cytokinesis.EMBO J. 2001; 20: 792-801Crossref PubMed Scopus (141) Google Scholar). Elevated levels of cyclin A delay the metaphase-anaphase transition (Geley et al., 2001Geley S. Kramer E. Gieffers C. Gannon J. Peters J.M. Hunt T. Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint.J. Cell Biol. 2001; 153: 137-148Crossref PubMed Scopus (241) Google Scholar). Depending on the levels of expression, nondegradable cyclin B arrests cells at metaphase or in anaphase (Wolf et al., 2006Wolf F. Wandke C. Isenberg N. Geley S. Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells.EMBO J. 2006; 25: 2802-2813Crossref PubMed Scopus (50) Google Scholar). Thus, degradation of securin and mitotic cyclins appears to be the essential task of APC/CCdc20 in organisms from yeast to man. APC/CCdc20 degrades various mitotic regulators with the correct temporal order to trigger the successive execution of mitotic events. The current available evidence is consistent with the following framework that explains the functions of APC/CCdc20 and APC/CCdh1 during mitosis and G1 (Figure 2). During early mitosis, the core APC/C subunits become phosphorylated by Cdk1 and other mitotic kinases, enabling efficient binding of Cdc20 to APC/C (Kraft et al., 2003Kraft C. Herzog F. Gieffers C. Mechtler K. Hagting A. Pines J. Peters J.M. Mitotic regulation of the human anaphase-promoting complex by phosphorylation.EMBO J. 2003; 22: 6598-6609Crossref PubMed Scopus (237) Google Scholar). Phosphorylation of Cdh1 by CDKs (Zachariae et al., 1998aZachariae W. Schwab M. Nasmyth K. Seufert W. Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex.Science. 1998; 282: 1721-1724Crossref PubMed Google Scholar) and binding of nucleoporins to Cdh1 (Jeganathan et al., 2005Jeganathan K.B. Malureanu L. van Deursen J.M. The Rae1-Nup98 complex prevents aneuploidy by inhibiting securin degradation.Nature. 2005; 438: 1036-1039Crossref PubMed Scopus (95) Google Scholar) prevent the activation of APC/CCdh1, which ensures that APC/CCdc20 is the major form of active APC/C in mitosis. The activity of APC/CCdc20 is further restricted by Emi1 and the spindle checkpoint in early mitosis (see below). APC/CCdc20 mediates degradation of cyclin A and Nek2A prior to the metaphase-anaphase transition (Geley et al., 2001Geley S. Kramer E. Gieffers C. Gannon J. Peters J.M. Hunt T. Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint.J. Cell Biol. 2001; 153: 137-148Crossref PubMed Scopus (241) Google Scholar, Hames et al., 2001Hames R.S. Wattam S.L. Yamano H. Bacchieri R. Fry A.M. APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box.EMBO J. 2001; 20: 7117-7127Crossref PubMed Scopus (50) Google Scholar). At the metaphase-anaphase transition, APC/CCdc20 then degrades securin to relieve one layer of inhibition of separase. APC/CCdc20 also initiates the degradation of cyclin B at this juncture, lowering the activity of Cdk1 and thus relieving the phosphorylation-mediated inhibition of separase (Stemmann et al., 2001Stemmann O. Zou H. Gerber S.A. Gygi S.P. Kirschner M.W. Dual inhibition of sister chromatid separation at metaphase.Cell. 2001; 107: 715-726Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Active separase then cleaves the Scc1 subunit of the cohesin complex and triggers sister-chromatid separation. Separase that is free of securin is also capable of binding directly to cyclin B and inhibits the activity of Cdk1 (Gorr et al., 2005Gorr I.H. Boos D. Stemmann O. Mutual inhibition of separase and Cdk1 by two-step complex formation.Mol. Cell. 2005; 19: 135-141Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In late anaphase, APC/CCdc20-mediated degradation of mitotic cyclins and the direct inhibition of Cdk1 by separase reduce the activities of CDKs to a level that is insufficient to prevent Cdh1 from binding to APC/C, resulting in the activation of APC/CCdh1. APC/CCdh1 then mediates the degradation of a wider range of APC/C substrates, including Cdc20 (Fang et al., 1998bFang G. Yu H. Kirschner M.W. Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1.Mol. Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Google Scholar, Prinz et al., 1998Prinz S. Hwang E.S. Visintin R. Amon A. The regulation of Cdc20 proteolysis reveals a role for APC components Cdc23 and Cdc27 during S phase and early mitosis.Curr. Biol. 1998; 8: 750-760Abstract Full Text Full Text PDF PubMed Google Scholar), as cells undergo exit from mitosis. In G1, APC/CCdc20 is rendered inactive by the dephosphorylation of APC/C core subunits and by APC/CCdh1-mediated degradation of Cdc20. In contrast, APC/CCdh1 remains active in G1 and prevents the premature accumulation of mitotic regulators. APC/CCdh1 is also active in certain terminally differentiated cells and has substrates that are not core cell-cycle regulators (reviewed in Peters, 2006Peters J.M. The anaphase promoting complex/cyclosome: a machine designed to destroy.Nat. Rev. Mol. Cell Biol. 2006; 7: 644-656Crossref PubMed Scopus (602) Google Scholar). At the G1-S transition, E2F activates the transcription of Emi1. The Emi1 protein is stabilized by Evi5. Accumulation of Emi1 inhibits APC/CCdh1 and stabilizes cyclin A, which activates Cdk2 and promotes entry into S phase. Cdk2 also phosphorylates Cdh1 and further inhibits APC/CCdh1. In early mitosis, Cdk1 and other mitotic kinases phosphorylate the APC/C core, promoting its binding to Cdc20. Emi1 inhibits APC/CCdc20 and prevents the premature destruction of cyclin B. APC/CCdc20 is also blocked by the spindle checkpoint in response to misaligned chromatids. Plk1-mediated degradation of Emi1 and inactivation of the spindle checkpoint allow activation of APC/CCdc20, which promotes the degradation of securin and cyclin B. This in turn leads to the activation of separase, cleavage of cohesin, and anaphase onset. APC/CCdc20-dependent degradation of cyclin B and separase binding to cyclin B reduce the Cdk1 activity, resulting in the dephosphorylation of Cdh1 and activation of APC/CCdh1 in late anaphase. APC/CCdh1 then orchestrates the degradation of a wide spectrum of substrates, facilitating mitotic exit and cytokinesis. Several studies in the budding yeast suggest that Cdc20 might have APC/C-independent functions. For example, Cdc20 promotes spindle elongation and premature chromosome segregation in mec1 or rad53 mutants that are defective for the DNA damage checkpoint and are under replication stress (Clarke et al., 2003Clarke D.J. Segal M. Andrews C.A. Rudyak S.G. Jensen S. Smith K. Reed S.I. S-phase checkpoint controls mitosis via an APC-independent Cdc20p function.Nat. Cell Biol. 2003; 5: 928-935Crossref PubMed Scopus (28) Google Scholar). This function of Cdc20 appears to be independent of APC/C, as forced accumulation of Cdc20 induces chromosome segregation even in strains harboring mutations in APC/C subunits. A subsequent study confirmed that the spindle elongation and abnormal chromosome segregation seen in mec1 or rad53 mutants are independent of APC/C and identified two microtubule-associated proteins, Cin8 and Stu2, as additional targets of Mec1 and Rad53 in the S phase checkpoint (Krishnan et al., 2004Krishnan V. Nirantar S. Crasta K. Cheng A.Y. Surana U. DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior.Mol. Cell. 2004; 16: 687-700Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The detailed molecular mechanisms for APC/C-independent functions of Cdc20 have not been established. It is also unclear whether these non-APC/C functions of Cdc20 are conserved in other organisms. On the basis of their catalytic mechanisms, ubiquitin ligases can be divided into two major classes: the homology to E6AP C terminus (HECT) domain-containing proteins and the RING-finger-containing proteins or protein complexes (reviewed in Pickart, 2001Pickart C.M. Mechanisms underlying ubiquitination.Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (1936) Google Scholar). The HECT ligases catalyze ubiquitination reactions by transiently accepting ubiquitin as a thioester through their active-site cysteines and then transferring the ubiquitin to substrates. In contrast, the RING-containing ligases do not form ubiquitin-thioester intermediates. Instead, they are capable of binding to ubiquitin-conjugating enzymes (UBCs) and substrates simultaneously, and thus act as scaffolds to bridge the interactions between UBCs and substrates. Binding of RING to UBCs also stimulates the release of ubiquitin from the UBC-ubiquitin thioesters. Therefore, in the presence of RING ligases, the combined proximity effect and the allosteric activation of UBCs allow more efficient ubiquitin transfer from UBCs to substrates. Among the RING ligases, the so-called Skp1-cullin-F box (SCF) complexes have been characterized extensively (Petroski and Deshaies, 2005Petroski M.D. Deshaies R.J. Function and regulation of cullin-RING ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (818) Google Scholar). The SCF ligases contain a cullin-RING module that binds to and activates UBCs and an F box-containing adaptor that recruits substrates for ubiquitination. Many F box-containing adaptors contain WD40 repeats that, in the cases of βTrCP and Cdc4, contact substrates through the top face of their β propeller structures (Orlicky et al., 2003Orlicky S. Tang X. Willems A. Tyers M. Sicheri F. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase.Cell. 2003; 112: 243-256Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, Wu et al., 2003Wu G. Xu G. Schulman B.A. Jeffrey P.D. Harper J.W. Pavletich N.P. Structure of a β-TrCP1-Skp1-β-catenin complex: destruction motif binding and lysine specificity of the SCFβ-TrCP1 ubiquitin ligase.Mol. Cell. 2003; 11: 1445-1456Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Because the APC2 and APC11 subunits of APC/C contain a cullin homology domain and a RING-finger domain, respectively, it has been anticipated that APC/C catalyzes the ubiquitination of its substrates using a mechanism similar to the SCF type of ubiquitin ligases (Figure 3A). Indeed, a binary complex containing only APC2 and APC11 is capable of binding to and activating UbcH10 (the APC/C-specific UBC) in vitro (Tang et al., 2001bTang Z. Li B. Bharadwaj R. Zhu H. Ozkan E. Hakala K. Deisenhofer J. Yu H. APC2 Cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex.Mol. Biol. Cell. 2001; 12: 3839-3851Crossref PubMed Google Scholar), indicating that APC/C contains a functional cullin-RING module for binding to UBCs, similar to SCF ligases. Like βTrCP and Cdc4, the two APC/C activators Cdc20 and Cdh1 contain WD40 repeats, hinting at a role for Cdc20 and Cdh1 in substrate binding (Figure 3A). Though there is little doubt that Cdc20 and Cdh1 facilitate APC/C-mediated ubiquitination through contributing to substrate binding, recent studies paint a complicated picture of how Cdc20 and Cdh1 facilitate the recruitment of substrates to APC/C. (A) Mechanisms of ubiquitination reactions catalyzed by SCF and APC/C. APC/C and the SCF type of ubiquitin ligases are thought to facilitate the transfer of ubiquitin to substrates by primarily acting as scaffolds that bring together ubiquitin-charged UBCs through their RING-finger subunits and substrates through adaptors (Cdc20 and Cdh1 in the case of APC/C and F box proteins in the case of SCF complexes, such as Cdc4 and βTrCP). The ubiquitination reactions are processive, leading to the formation of ubiquitin chains that target the substrates to the proteasome for degradation. To achieve processivity, APC/C and SCF need to support ubiquitin transfer from the UBCs to continuously elongating ubiquitin chains, which requires conformational flexibility of the substrates and/or the ligases in these systems. Along this vein, the degrons of several APC/C substrates are located in regions that adopt flexible conformations (unpublished data). The architecture of cullins and possibly APC2 resembles that of karyopherins and the scaffolding subunit of protein phosphatase 2A, both of which are capable of undergoing large-scale conformational changes. (B) Four different models that ex