Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

Cell Cycle Control Group, UCL Cancer Institute, University College London, London, UK.
The EMBO Journal (Impact Factor: 10.43). 06/2012; 31(15):3351-62. DOI: 10.1038/emboj.2012.168
Source: PubMed


The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.

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    • "However, in mouse eggs the target of PP2A is Emi2, in order to maintain its interaction and inhibition of the APC/C (Wu et al., 2007), and so could not operate in ascidian eggs in which Emi2 is absent. It has been reported that cdc20 dephosphorylation is necessary before it can activate the APC/C in HeLa cells (Yudkovsky et al., 2000) and more recently in Xenopus egg extracts (Labit et al., 2012), and that cdc20 is a substrate of MAPK in Xenopus (Chung and Chen, 2003). We therefore propose a mechanism (Fig. 5) whereby in the absence of Emi2, the Mos/MAPK pathway mediates meiotic arrest in ascidian eggs. "
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    ABSTRACT: The fertilising sperm triggers a transient Ca(2+) increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/C(cdc20) inhibitor, links release from metaphase II arrest with the Ca(2+) transient and its degradation is triggered by the Ca(2+)-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca(2+) transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca(2+)-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca(2+) increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates.
    Development 11/2013; 140(22):4583-93. DOI:10.1242/dev.096578 · 6.46 Impact Factor
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    • "In one of them, activation of the phosphatase PP2B/calcineurin initiates global dephosphorylation of MPF substrates, such as the APC/C subunit Cdc27/Apc3, and its activator Cdc20 (Mochida and Hunt, 2007; Nishiyama et al, 2007b). As calcineurin inhibition delays calcium-triggered cyclin B degradation in Xenopus egg extract, the removal of inhibitory Cdc20 phosphorylation may directly contribute to APC/C activation (Labit et al, 2012). On the other hand, calcineurin appears to be dispensable for exit from the MIIarrest in mice (Suzuki et al, 2010) and, therefore, further studies are required to dissect how global dephosphorylation of MPF substrates is initiated in fertilized mouse oocytes. "
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    ABSTRACT: Ex ovo omnia-all animals come from eggs-this statement made in 1651 by the English physician William Harvey marks a seminal break with the doctrine that all essential characteristics of offspring are contributed by their fathers, while mothers contribute only a material substrate. More than 360 years later, we now have a comprehensive understanding of how haploid gametes are generated during meiosis to allow the formation of diploid offspring when sperm and egg cells fuse. In most species, immature oocytes are arrested in prophase I and this arrest is maintained for few days (fruit flies) or for decades (humans). After completion of the first meiotic division, most vertebrate eggs arrest again at metaphase of meiosis II. Upon fertilization, this second meiotic arrest point is released and embryos enter highly specialized early embryonic divisions. In this review, we discuss how the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages. Specifically, we focus on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII-arrest), the first mitotic cell cycle, and early embryonic divisions.
    The EMBO Journal 07/2013; 32(16). DOI:10.1038/emboj.2013.164 · 10.43 Impact Factor
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    • "These observations suggest that either APC/C inactivation is not required for the normal execution of meiosis and spore formation or that this ubiquitin ligase is disabled by redundant systems. In support of the latter possibility, several mechanisms are known to control APC/C function including inhibitory phosphorylation [41-44], APC/C specific inhibitors [45-52], or removal of the activator from the APC/C complex [53]. The roles these mechanisms play as cells exit the meiotic program are not well understood. "
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    ABSTRACT: The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/CAma1. In this study we investigated how Ama1p is down regulated at the completion of meiosis. Here we show that Ama1p is a substrate of APC/CCdc20 but not APC/CCdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/CCdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a "KEN-box" like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/CAma1-mediated Cdc20p degradation. Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.
    Cell Division 07/2013; 8(1):9. DOI:10.1186/1747-1028-8-9 · 3.53 Impact Factor
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