Article

A Role for the Fizzy/Cdc20 Family of Proteins in Activation of the APC/C Distinct from Substrate Recruitment

Cell Cycle Control Laboratory, Marie Curie Research Institute, The Chart, Oxted, RH8 0TL Surrey, UK.
Molecular cell (Impact Factor: 14.46). 12/2008; 32(4):576-83. DOI: 10.1016/j.molcel.2008.09.023
Source: PubMed

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.

Download full-text

Full-text

Available from: Hiro Yamano, Aug 28, 2015
0 Followers
 · 
213 Views
  • Source
    • "We think that the simplest model explaining our data is that rapid Nek2A disappearance marks the point when Cdc20, regardless of its incorporation into or inhibition by the MCC, activates the APC/C at the prophase-to-prometaphase transition. At mitotic entry, Cdc20 binds the APC/C by means of its Cterminal tail (Vodermaier et al., 2003), the KILR motif (Izawa and Pines, 2012) and by its N-terminal C-box (Kimata et al., 2008). The binding of Cdc20 to the APC/C is enforced by mitotic phosphorylation of the APC/C, but also by the spindle checkpoint: the Cdc20 C-box might be involved in stabilizing complexes between the APC/C and spindle checkpoint proteins (Hein and Nilsson, 2014). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nek2A is a presumed APC/C(Cdc20) substrate, which, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid stabilization by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A while Nek2A disappeared at normal rate. Secondly, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction critically depended on Cdc20 binding to the APC/C. Thirdly, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/C(Cdc20) catalytic activity.
    Journal of Cell Science 02/2015; 128(8). DOI:10.1242/jcs.163279 · 5.33 Impact Factor
  • Source
    • "The active APC/Cs comprise at least 16 individual subunits, including the coactivator proteins Cdc20 and Cdh1 [6] [7] [8]. The APC/C coactivators (Cdc20 and Cdh1) contain the C-box, KILR, and IR motifs, which binding to the core APC/C [9] [13]. Nevertheless, Emi1 does not impair the substrate binding of APC/C [48] and the location of the ZBR domain of Emi1 is distant from that of Cdh1 on APC/C [41]. "
  • Source
    • "The active APC/Cs comprise at least 16 individual subunits, including the coactivator proteins Cdc20 and Cdh1 [6] [7] [8]. The APC/C coactivators (Cdc20 and Cdh1) contain the C-box, KILR, and IR motifs, which binding to the core APC/C [9] [13]. Nevertheless, Emi1 does not impair the substrate binding of APC/C [48] and the location of the ZBR domain of Emi1 is distant from that of Cdh1 on APC/C [41]. "
Show more