Article

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

ABSTRACT

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.

Download full-text

Full-text

Available from: Hiro Yamano
  • Source
    • "After attachment, checkpoint silencing enables progression into anaphase (Sacristan and Kops, 2014). Phosphorylation of Cdc20 by Cdk1 inhibits its ability to bind and activate APC/C, which suggests that reversal of these phosphorylation events is important for anaphase onset (Kramer et al., 2000; Yudkovsky et al., 2000; Labit et al., 2012). Phosphatase activities are also important for reversing Cdk1 phosphorylation but their control is less well understood. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The conserved Bub1/Bub3 complex is recruited to the kinetochore region of mitotic chromosomes, where it initiates spindle checkpoint signaling and promotes chromosome alignment. Here we show that, in contrast to the expectation for a checkpoint pathway component, the BUB-1/BUB-3 complex promotes timely anaphase onset in Caenorhabditis elegans embryos. This activity of BUB-1/BUB-3 was independent of spindle checkpoint signaling but required kinetochore localization. BUB-1/BUB-3 inhibition equivalently delayed separase activation and other events occurring during mitotic exit. The anaphase promotion function required BUB-1's kinase domain, but not its kinase activity, and this function was independent of the role of BUB-1/BUB-3 in chromosome alignment. These results reveal an unexpected role for the BUB-1/BUB-3 complex in promoting anaphase onset that is distinct from its well-studied functions in checkpoint signaling and chromosome alignment, and suggest a new mechanism contributing to the coordination of the metaphase-to-anaphase transition. © 2015 Kim et al.
    Preview · Article · May 2015 · The Journal of Cell Biology
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Nov 2013 · Development
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jul 2013 · The EMBO Journal
Show more