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• W1980766960 abstract "The Fizzy/Cdc20 family of proteins are essential activators of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase. However, apart from the well-established role of the C-terminal WD40 domain in substrate recognition, the precise roles of the activators remain elusive. Here we show that Nek2A, which directly binds the APC/C, can be ubiquitylated and destroyed in Fizzy/Cdc20-depleted Xenopus egg extracts when only the N-terminal domain of Fizzy/Cdc20 (N-Cdc20) is added. This activity is dependent upon the C box and is conserved in the alternative activator, Fizzy-related/Cdh1. In contrast, canonical substrates such as cyclin B and securin require both the N-terminal and WD40 domains, unless N-Cdc20 is fused to substrates when the WD40 domain becomes dispensable. Furthermore, in Cdc20-depleted cells, N-Cdc20 can facilitate Nek2A destruction in a C box-dependent manner. Our results reveal a role for the N-terminal domain of the Fizzy/Cdc20 family of activators in triggering substrate ubiquitylation by the APC/C. The Fizzy/Cdc20 family of proteins are essential activators of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase. However, apart from the well-established role of the C-terminal WD40 domain in substrate recognition, the precise roles of the activators remain elusive. Here we show that Nek2A, which directly binds the APC/C, can be ubiquitylated and destroyed in Fizzy/Cdc20-depleted Xenopus egg extracts when only the N-terminal domain of Fizzy/Cdc20 (N-Cdc20) is added. This activity is dependent upon the C box and is conserved in the alternative activator, Fizzy-related/Cdh1. In contrast, canonical substrates such as cyclin B and securin require both the N-terminal and WD40 domains, unless N-Cdc20 is fused to substrates when the WD40 domain becomes dispensable. Furthermore, in Cdc20-depleted cells, N-Cdc20 can facilitate Nek2A destruction in a C box-dependent manner. Our results reveal a role for the N-terminal domain of the Fizzy/Cdc20 family of activators in triggering substrate ubiquitylation by the APC/C. Ubiquitin is a small protein and serves as molecular tag for protein degradation. This tagging process, ubiquitylation, is regulated by a cascade of enzymes comprising a ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s) (Hershko and Ciechanover, 1998Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6467) Google Scholar). The APC/C is an E3 ubiquitin ligase that controls destruction of numerous proteins at specific times in the cell cycle including the metaphase-to-anaphase transition and exit from mitosis by degrading securin/Cut2/Pds1 and cyclin B, respectively (Morgan, 2007Morgan D.O. The Cell Cycle: Principles of Control. New Science Press, London2007Google Scholar, 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 (962) 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 (128) Google Scholar). However, the molecular mechanisms by which the APC/C recognizes so many different proteins at the correct time and place are still unclear. The primary candidate for such determinants is the Fizzy/Cdc20 family of APC/C activator proteins, which have a characteristic conserved WD40 repeat domain at their C termini (Yu, 2007Yu H. Cdc20: a WD40 activator for a cell cycle degradation machine.Mol. Cell. 2007; 27: 3-16Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Although they are not stoichiometric components of the APC/C, genetic and biochemical evidence suggests that these activators are essential for the APC/C to function. First, Fizzy/Cdc20/Slp1 is required for the APC/C activity in anaphase, whereas Fizzy-related/Cdh1/Ste9 maintains activity during late mitosis and G1 phase (Morgan, 2007Morgan D.O. The Cell Cycle: Principles of Control. New Science Press, London2007Google Scholar, 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 (962) 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 (128) Google Scholar). Second, the Fizzy/Cdc20 family of proteins have been shown to bind several substrates (Burton and Solomon, 2001Burton J.L. Solomon M.J. D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p.Genes Dev. 2001; 15: 2381-2395Crossref PubMed Scopus (145) Google Scholar, Hilioti et al., 2001Hilioti Z. Chung Y.S. Mochizuki Y. Hardy C.F. Cohen-Fix O. The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner.Curr. Biol. 2001; 11: 1347-1352Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, Pfleger et al., 2001Pfleger C.M. Lee E. Kirschner M.W. Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex.Genes Dev. 2001; 15: 2396-2407Crossref PubMed Scopus (196) Google Scholar, Schwab et al., 2001Schwab M. Neutzner M. Mocker D. Seufert W. Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC.EMBO J. 2001; 20: 5165-5175Crossref PubMed Scopus (162) Google Scholar), and recent photocrosslinking experiments have demonstrated that APC/C substrates directly bind to the WD40 repeat domain of the Fizzy/Cdc20 family of activators (Kimata et al., 2008Kimata Y. Trickey M. Izawa D. Gannon J. Yamamoto M. Yamano H. A mutual inhibition between APC/C and its substrate Mes1 required for meiotic progression in fission yeast.Dev. Cell. 2008; 14: 446-454Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Kraft et al., 2005Kraft C. Vodermaier H.C. Maurer-Stroh S. Eisenhaber F. Peters J.M. The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C substrates.Mol. Cell. 2005; 18: 543-553Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Thus, an accepted view in the field is that the APC/C activators achieve their essential function by recruiting substrates to the APC/C, which would be equivalent to the role of F box proteins in the SCF (Skp1-Cullin 1-F box protein) complex (Deshaies, 1999Deshaies R.J. SCF and Cullin/Ring H2-based ubiquitin ligases.Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1058) Google Scholar). Yet, compared to the number of the F box proteins identified (Jin et al., 2004Jin J. Cardozo T. Lovering R.C. Elledge S.J. Pagano M. Harper J.W. Systematic analysis and nomenclature of mammalian F-box proteins.Genes Dev. 2004; 18: 2573-2580Crossref PubMed Scopus (507) Google Scholar), so far only a few Fizzy family activators have been identified (Morgan, 2007Morgan D.O. The Cell Cycle: Principles of Control. New Science Press, London2007Google Scholar, 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 (962) 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 (128) Google Scholar). This suggests that it is unlikely that substrate specificity is determined by individual members of the Fizzy/Cdc20 family, since such a wide range of substrates are degraded at different cell-cycle stages. In addition, core APC/C subunit or subunits have also been shown to recognize substrates (Carroll et al., 2005Carroll C.W. Enquist-Newman M. Morgan D.O. The APC subunit Doc1 promotes recognition of the substrate destruction box.Curr. Biol. 2005; 15: 11-18Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Hayes et al., 2006Hayes M.J. Kimata Y. Wattam S.L. Lindon C. Mao G. Yamano H. Fry A.M. Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.Nat. Cell Biol. 2006; 8: 607-614Crossref PubMed Scopus (125) Google Scholar, Passmore et al., 2003Passmore L.A. McCormack E.A. Au S.W. Paul A. Willison K.R. Harper J.W. Barford D. Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition.EMBO J. 2003; 22: 786-796Crossref PubMed Scopus (159) Google Scholar, Yamano et al., 2004Yamano H. Gannon J. Mahbubani H. Hunt T. Cell-cycle-regulated recognition of the destruction box of cyclin B by the APC/C in Xenopus egg extracts.Mol. Cell. 2004; 13: 137-147Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Therefore, it remains unclear how APC/C substrates are recognized and what are the exact roles of the Fizzy family of proteins in APC/C-dependent ubiquitylation. Nek2A is a vertebrate NIMA-related kinase involved in regulation of the centrosome duplication cycle (Fry et al., 1998Fry A.M. Meraldi P. Nigg E.A. A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators.EMBO J. 1998; 17: 470-481Crossref PubMed Scopus (322) Google Scholar). We have shown that Nek2A is an APC/C substrate that is strikingly different from other canonical APC/C substrates. Nek2A stably binds the APC/C core complex via its unique MR tail even in the absence of the Fizzy family of activators in Xenopus egg extracts and is degraded in prometaphase when the spindle checkpoint inhibits destruction of other APC/C substrates in HeLa cells (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 (161) Google Scholar, Hayes et al., 2006Hayes M.J. Kimata Y. Wattam S.L. Lindon C. Mao G. Yamano H. Fry A.M. Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.Nat. Cell Biol. 2006; 8: 607-614Crossref PubMed Scopus (125) Google Scholar). These unique characteristics have enabled us to investigate previously unidentified roles of the Fizzy/Cdc20 family. Here we report that the activators are essential not only for recognizing substrates but also for a postrecognition step, in stimulating the ubiquitin ligase activity of the APC/C. This activity is dependent upon the C box, a short motif conserved among members of the Fizzy/Cdc20 family, in the N-terminal domain. In the case of proteins such as cyclin B or securin, Fizzy/Cdc20 is thought to be required for substrate recognition and recruitment, a prerequisite step for the subsequent ubiquitylation by the APC/C. However, we wanted to establish whether or not Fizzy/Cdc20 had additional roles in the control of substrate degradation. We hypothesized that such a role(s) might only be discovered by investigating a substrate, which directly binds the APC/C independently of the Fizzy/Cdc20 activator, such as Nek2A (Figure 1A) (Hayes et al., 2006Hayes M.J. Kimata Y. Wattam S.L. Lindon C. Mao G. Yamano H. Fry A.M. Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.Nat. Cell Biol. 2006; 8: 607-614Crossref PubMed Scopus (125) Google Scholar). First, we examined whether Fizzy/Cdc20 is indeed required for Nek2A destruction in cell-free Xenopus egg extracts. Not only Nek2A but also Cdc13/cyclin B and cyclin A were stabilized by Fizzy/Cdc20 depletion from egg extracts, whereas they are efficiently destroyed in mock-treated extracts (Figure 1B). These results indicate that Fizzy/Cdc20 is required for APC/C-dependent destruction even after a substrate such as Nek2A is recruited onto the APC/C. We next sought to investigate this additional role by first depleting Fizzy/Cdc20 from Xenopus egg extracts and adding back in vitro-translated wild-type (WT) Cdc20 or mutated versions of Fizzy/Cdc20 (Figure 1C). Three different APC/C substrates, Nek2A, Cdc13/cyclin B, and Mes1, were used as model substrates (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 (161) Google Scholar, Kimata et al., 2008Kimata Y. Trickey M. Izawa D. Gannon J. Yamamoto M. Yamano H. A mutual inhibition between APC/C and its substrate Mes1 required for meiotic progression in fission yeast.Dev. Cell. 2008; 14: 446-454Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Yamano et al., 1996Yamano H. Gannon J. Hunt T. The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.EMBO J. 1996; 15: 5268-5279Crossref PubMed Scopus (133) Google Scholar). As expected, full-length Fizzy/Cdc20 (WT) restored the destruction of all the substrates (Figure 1D, lanes 5–8). Surprisingly, destruction of Nek2A, but not Cdc13 or Mes1, was supported by a fragment of Fizzy/Cdc20 that lacks the entire WD40 domain (N159) (Figure 1D, lanes 25–28) as well as by Fizzy/Cdc20 constructs with mutations in the C-terminal IR motif (ΔIR) and the WD40 domain (WD2A and WD5A; Figure 1D, lanes 13–24). In contrast, the C-terminal domain alone (C348) did not rescue Nek2A destruction, suggesting that only the N-terminal domain is essential for the destruction of Nek2A. As predicted for substrates that require Fizzy/Cdc20 for recruitment of the APC/C, neither Cdc13 nor Mes1 was destroyed when the truncated Fizzy/Cdc20 fragments were used. To further investigate this activity of the N-terminal domain, we prepared bacterially purified GST-fused N159 (GST-N159) and MBP-fused N159 (MBP-N159) and used these recombinant proteins in place of WT Fizzy/Cdc20 (Figure 1E). Both recombinant proteins were able to facilitate destruction of Nek2A, but not that of Cdc13/cyclin B in Fizzy/Cdc20-depleted egg extracts. Moreover, this destruction was dependent upon the MR tail of Nek2A (data not shown). These results indicate that the N-terminal 159 residues of Fizzy/Cdc20 have an additional role besides substrate recruitment. Consistent with this idea, the fragment containing the C-terminal WD40 domain (C348), which is able to bind Mes1 (Kimata et al., 2008Kimata Y. Trickey M. Izawa D. Gannon J. Yamamoto M. Yamano H. A mutual inhibition between APC/C and its substrate Mes1 required for meiotic progression in fission yeast.Dev. Cell. 2008; 14: 446-454Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), was unable to support Mes1 destruction in these assays (Figure 1D, lanes 29–32), underscoring the importance of the N-terminal domain of Fizzy/Cdc20 for a postsubstrate recognition event in the APC/C-dependent proteolysis. In contrast to the conserved C-terminal WD40 repeat domain, the N-terminal regions of the Fizzy/Cdc20 family are divergent both in primary sequence and length. To identify the important elements within the N-terminal domain (N-Cdc20), we made several truncated versions of N159 and asked which were able to activate Nek2A destruction in Fizzy/Cdc20-depleted Xenopus egg extracts (see Figure S1 available online). We found that truncation of the first 36 residues (37–159) still facilitated Nek2A destruction, whereas further deletions of the N-terminal 82 residues (83–159) or the C-terminal 60 residues (N98) completely abolished the activity. Within this N-terminal region there is a C box. This is a highly conserved motif present in all members of the Fizzy/Cdc20 family that is required for the association with the APC/C (Schwab et al., 2001Schwab M. Neutzner M. Mocker D. Seufert W. Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC.EMBO J. 2001; 20: 5165-5175Crossref PubMed Scopus (162) Google Scholar). We therefore investigated whether the C box had a role in the activity of the N159 fragment. We generated a version of GST-N159 with mutation of the C box (DRFIP to AAAAA; GST-N159ΔC box) and examined the ability of this protein to support destruction of Nek2A and Cdc13 in Fizzy/Cdc20-depleted extracts. As shown in Figure 2A, this mutation abolished the ability of N159 to activate Nek2A destruction. As the C box is required for association with the APC/C, we investigated whether the C box in the N159 protein was responsible for APC/C binding. When GST-N159 was added into Fizzy/Cdc20-depleted Xenopus egg extracts, APC/C was copurified with GST-N159 in a C box- and an N159-dose-dependent manner (Figure 2B), suggesting that N159 indeed interacts with the APC/C. To evaluate whether we had used physiological amounts of GST-N159 fragments in the add-back experiments in Fizzy/Cdc20-depleted extracts, we immunoprecipitated APC/C from mock- or Fizzy/Cdc20-depleted extracts supplemented with GST-N159 and quantified the amounts of Fizzy/Cdc20 associated with APC/C. The amount of recombinant N159 protein associated with the APC/C was equal to or slightly less than that of endogenous Fizzy/Cdc20 (Figure S2). As all members of the Fizzy/Cdc20 family of activators contain a C box, we next sought to investigate whether this N-terminal activity is conserved. We used bacterially purified N-terminal Xenopus Fizzy-related/Cdh1 (GST-FzrN186) and human Cdc20 (GST-Hs Cdc20N151), both of which were capable of activating the APC/C (Figure 2C). Interestingly, GST-FzrN186 could destroy Nek2A in Fizzy/Cdc20-depleted interphase extracts in a C box-dependent manner, but not Cdc13/cyclin B (Figure 2D). We next examined whether the N-terminal domain of Fizzy/Cdc20 could promote APC/C-dependent ubiquitylation in a cell-free assay. We purified APC/C from Fizzy/Cdc20-depleted extracts and analyzed Nek2A ubiquitylation supplemented with Fizzy/Cdc20 or N159 protein. Consistent with the above finding using cell-free destruction assays, N159 was able to support ubiquitylation of Nek2A as efficiently as the full-length Fizzy/Cdc20 (FzyWT) protein, whereas the N159 C box mutant failed (Figure 3A, lanes 1–16). In contrast, N159 could not activate APC/C-dependent ubiquitylation of Cdc13 or securin (Figure 3A, lanes 17–28, and Figure 3B). These results imply that N159 directly interacts with the APC/C and promotes the ubiquitin ligase activity of the APC/C. We further asked whether the ubiquitylation of Nek2A bound to the APC/C is processive. Using a specific anti-Apc3 antibody, we first isolated APC/C prebound Nek2A from Fizzy/Cdc20-depleted extracts where 35S-labeled-Nek2A had been incubated and used it for the ubiquitylation assay supplemented with N159. As shown in Figure 3C, more than 80% of Nek2A was converted into ubiquitylated forms by addition of N159 (Figure 3C, lanes 5–8), suggesting that Nek2A bound to the APC/C via its MR tail can be ubiquitylated in a processive manner in response to the interaction between N159 and the APC/C. We then hypothesized that if the WD40 domain of Fizzy/Cdc20 was only required for substrate binding in the case of proteins such as cyclin B and Mes1, the WD40 domain would be dispensable when the N-terminal APC/C-activating domain (N159) was directly fused to substrates, thereby bypassing the function of substrate recruitment. To test this, we fused N159 to Mes1WT (N159-Mes1) and a nondegradable version of Mes1 with mutations in the D box and KEN box (N159-Mes1DK). Surprisingly, not only N159-Mes1 but also N159-Mes1DK was strongly ubiquitylated in a cell-free ubiquitylation assay without the addition of either full-length or fragments of Fizzy/Cdc20 (Figure 3D, lanes 7–12), whereas the same fusion construct with the C box mutation (N159ΔC box-Mes1) was not ubiquitylated (Figure 3D, lanes 4–6). These data suggest that the N-terminal domain of Fizzy/Cdc20 has an as-yet-unidentified activity to stimulate APC/C-dependent ubiquitylation and proteolysis. It should also be noted that the N159 protein itself was not ubiquitylated, although it was loaded onto the APC/C (Figure 3D, lanes 1–3; Figure S3). Finally, we asked whether the N-terminal domain of Fizzy/Cdc20 (N-Cdc20) was also sufficient for degradation of Nek2A in vivo. We first demonstrated that RNAi-mediated depletion of Cdc20 led to stabilization of GFP-Nek2A in nocodazole-arrested mitotic cells (Figure 4A). Thus, Fizzy/Cdc20 is required for the destruction of Nek2A in prometaphase cells, despite not being required to recruit Nek2A to the APC/C, in agreement with our previous data (Hayes et al., 2006Hayes M.J. Kimata Y. Wattam S.L. Lindon C. Mao G. Yamano H. Fry A.M. Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.Nat. Cell Biol. 2006; 8: 607-614Crossref PubMed Scopus (125) Google Scholar). We then generated Flag-tagged constructs expressing N-Cdc20 with or without an intact C box and confirmed that the expression of both of these constructs is equivalent in the Cdc20 knockdown cells (Figure 4B). We then assayed Nek2A destruction in those cells by immunofluorescence microscopy using anti-GFP (GFP-Nek2A) antibodies. Following control depletion, only 15% of mitotic cells contained detectable Nek2A. In contrast, 54% of cells which had been depleted of Cdc20 showed a positive Nek2A signal during mitosis. Importantly, in cells depleted of Cdc20, expression of N-Cdc20 triggered Nek2A destruction and reduced Nek2A-positive cells to 31%, whereas the percentage of cells expressing Nek2A did not decrease when the N-Cdc20 protein with the C box mutation was expressed (Figures 4C and 4D). This indicates that the WT N-Cdc20, but not the construct lacking the C box, was capable of rescuing the degradation of GFP-Nek2A. Taken together, we conclude that the C box-dependent activation of the APC/C is an additional and essential role of the Fizzy/Cdc20 family of proteins. The Fizzy/Cdc20 family of APC/C activators are conserved proteins that are required for the activation of the APC/C. However, previous work had focused on their role in recruiting substrates to the APC/C through their C-terminal WD40 repeats domain (Yu, 2007Yu H. Cdc20: a WD40 activator for a cell cycle degradation machine.Mol. Cell. 2007; 27: 3-16Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). The results presented here demonstrate that the N-terminal domain of the activators has an essential role to initiate ubiquitylation of APC/C substrates in a C box-dependent manner. The C box was originally identified in budding yeast Cdc20 and is highly conserved among members of the Fizzy/Cdc20 family. It appears to be required for association of the activators with the APC/C (Schwab et al., 2001Schwab M. Neutzner M. Mocker D. Seufert W. Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC.EMBO J. 2001; 20: 5165-5175Crossref PubMed Scopus (162) Google Scholar, Vodermaier et al., 2003Vodermaier H.C. Gieffers C. Maurer-Stroh S. Eisenhaber F. Peters J.M. TPR subunits of the anaphase-promoting complex mediate binding to the activator protein CDH1.Curr. Biol. 2003; 13: 1459-1468Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Yet, it remains elusive whether it is required for recruitment of APC/C substrates in conjunction with the C-terminal WD40 domain and the IR (isoleucine-arginine) motif at the C terminus. Nek2A, which does not need Fizzy/Cdc20 for its recruitment to the APC/C, is ubiquitylated and destroyed in Fizzy/Cdc20-depleted egg extracts or cultured cells when the N-terminal region with an intact C box is present. Thus, the interaction between the C box and the APC/C seems to allow activation of the ubiquitin ligase activity by Fizzy/Cdc20. The N terminus may promote a conformational change of the APC/C core complex, allowing movement of recruited substrates relative to the catalytic center, thereby triggering their ubiquitylation (see Figure 4E). This idea is consistent with the recent finding that the main structural domain of the APC/C is rotated upon binding of Fizzy-related/Cdh1 (Dube et al., 2005Dube P. Herzog F. Gieffers C. Sander B. Riedel D. Muller S.A. Engel A. Peters J.M. Stark H. Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C.Mol. Cell. 2005; 20: 867-879Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is also possible that the C box interaction directly stimulates the intrinsic catalytic activity of the APC/C ubiquitin ligase. Our data also highlight that the Fizzy/Cdc20 family of activators have at least two functionally distinct roles: “APC/C activation” and “substrate recognition” via the N- and C-terminal domains, respectively. Importantly, the N-terminal domain (e.g., the C box) seems to be vital for ubiquitylation of all the substrates, but the C-terminal WD40 domain is dispensable once the substrates are recruited to the APC/C (Figures 3D and 4E). In the context of our present study, it is intriguing to note that the Apc10/Doc1 subunit of the APC/C has a C box-like (CL) motif in the Doc domain (Figure S4) that is found in a variety of proteins involved in ubiquitylation reactions (Au et al., 2002Au S.W. Leng X. Harper J.W. Barford D. Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit.J. Mol. Biol. 2002; 316: 955-968Crossref PubMed Scopus (38) Google Scholar, Grossberger et al., 1999Grossberger R. Gieffers C. Zachariae W. Podtelejnikov A.V. Schleiffer A. Nasmyth K. Mann M. Peters J.M. Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex.J. Biol. Chem. 1999; 274: 14500-14507Crossref PubMed Scopus (81) Google Scholar, Kominami et al., 1998Kominami K. Seth-Smith H. Toda T. Apc10 and Ste9/Srw1, two regulators of the APC-cyclosome, as well as the CDK inhibitor Rum1 are required for G1 cell-cycle arrest in fission yeast.EMBO J. 1998; 17: 5388-5399Crossref PubMed Scopus (85) Google Scholar, Wendt et al., 2001Wendt K.S. Vodermaier H.C. Jacob U. Gieffers C. Gmachl M. Peters J.M. Huber R. Sondermann P. Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex.Nat. Struct. Biol. 2001; 8: 784-788Crossref PubMed Scopus (76) Google Scholar). The Apc10/Doc1 subunit is a well-conserved subunit from human to the microsporidia Encephalitozoon cuniculi, and like the Fizzy/Cdc20 family of activators, it has been reported to bind Apc3/Cdc27 subunits via a C-terminal IR/LR tail region (Wendt et al., 2001Wendt K.S. Vodermaier H.C. Jacob U. Gieffers C. Gmachl M. Peters J.M. Huber R. Sondermann P. Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex.Nat. Struct. Biol. 2001; 8: 784-788Crossref PubMed Scopus (76) Google Scholar). In addition, Apc10/Doc1 has been shown to play important roles in promoting ubiquitylation as well as recognition of substrates (Carroll et al., 2005Carroll C.W. Enquist-Newman M. Morgan D.O. The APC subunit Doc1 promotes recognition of the substrate destruction box.Curr. Biol. 2005; 15: 11-18Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, Carroll and Morgan, 2002Carroll C.W. Morgan D.O. The Doc1 subunit is a processivity factor for the anaphase-promoting complex.Nat. Cell Biol. 2002; 4: 880-887Crossref PubMed Scopus (109) Google Scholar, Passmore et al., 2003Passmore L.A. McCormack E.A. Au S.W. Paul A. Willison K.R. Harper J.W. Barford D. Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition.EMBO J. 2003; 22: 786-796Crossref PubMed Scopus (159) Google Scholar). Notably, the CL motif is located in the ligand-binding interface where it may interact with other core APC/C subunits and promote APC/C ubiquitin ligase activity. This idea is in agreement with the recent report that mutations in the β sheet (β 11), which is structurally adjacent to the CL motif, prevented the Apc10/Doc1-dependent processing activity (Carroll et al., 2005Carroll C.W. Enquist-Newman M. Morgan D.O. The APC subunit Doc1 promotes recognition of the substrate destruction box.Curr. Biol. 2005; 15: 11-18Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Thus, the mechanism by which Apc10/Doc1 stimulates processive ubiquitylation may underlie this type of interaction. In more general terms, the activity of the APC/C might be controlled by an interaction between the APC/C and an individual C box or a CL motif or a combination of the two, depending on substrates or cell-cycle stages. Moreover, Fizzy/Cdc20 and Fizzy-related/Cdh1 are both phosphorylated in mitosis, and several phosphorylation sites lie within the N-terminal domain in close proximity to the C box (Yu, 2007Yu H. Cdc20: a WD40 activator for a cell cycle degradation machine.Mol. Cell. 2007; 27: 3-16Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Although the roles of phosphorylation are poorly understood, it is possible that protein kinases and phosphatases control the activity of the APC/C by regulating the patterns of phosphorylation around the C box. At present, we do not know the precise mechanism for the C box-dependent activation of the APC/C. Equally, whether the CL motif in the Apc10/Doc1 subunit works in a similar manner to the C box in Fizzy/Cdc20 remains to be clarified, but our results certainly provide a paradigm for understanding protein ubiquitylation by the APC/C ubiquitin ligase and the Fizzy/Cdc20 family of activators. See the Supplemental Data for additional experimental procedures. Destruction assays were performed essentially as described previously (Yamano et al., 1996Yamano H. Gannon J. Hunt T. The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.EMBO J. 1996; 15: 5268-5279Crossref PubMed Scopus (133) Google Scholar). Substrates were labeled with [35S]methionine (PerkinElmer) in a coupled in vitro transcription-translation (IVT) system (Promega). IVT-Fizzy/Cdc20 constructs or recombinant GST-FzyN159 proteins were added to Fizzy/Cdc20-depleted egg extracts, and after 5 min incubation at 23°C, the APC/C substrates were mixed. The samples were taken at the indicated time points after adding CaCl2 and were analyzed by SDS-PAGE and fluorography. For ubiquitylation assays, Xenopus APC/C was immunoprecipitated from 12.5 μl of Fizzy-depleted mitotic extracts using monoclonal anti-Apc3 antibody (mAB, AF3.1) immobilized on Dynabeads Protein A (Invitrogen). Reactions were performed at 23°C in 10 μl of the buffer (20 mM Tris-HCl [pH 7.5], 100 mM KCl, 2.5 mM MgCl2, 2 mM ATP, 0.3 mM DTT) containing 0.1 mg/ml E1, 0.1 mg/ml UbcH5 and UbcH10, 1.5 mg/ml ubiquitin, 1 μM ubiquitin-aldehyde, 150 μM MG132, and 1 μl in vitro-translated (IVT) Fizzy or bacterially purified GST-FzyN159 protein. Reactions were stopped at the indicated time points with SDS sample buffer, and mixtures were resolved by SDS-PAGE. The authors would like to thank T. Hunt, J. Gannon, and members of the Yamano laboratory for helpful discussions and critical reading of the manuscript. We would also like to thank T. Hunt and H. Mahbubani for access to the Cancer Research UK Clare Hall Laboratories Xenopus colony. This work was supported by Marie Curie Cancer Care and the Association for International Cancer Research (AICR). Download .pdf (.17 MB) Help with pdf files Document S1. Supplemental Experimental Procedures and Four Figures" @default.
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