Abstract

Progress of cell division is governed by the sequential degradation, mediated by the ubiquitination pathway, of proteins playing a key role in the cell cycle. There are two E3 ubiquitin protein ligase complexes which play a role in the cell cycle: the SCF (Skp1/Cullin/F-box) complex and the anaphase-promoting complex (APC/C) (Peters 2006, Castro et al. 2005). The APC/C complex contains at least a dozen different subunits, but it can ubiquitylate substrates only in the presence of the co-activator proteins Cdc20 (also known as fizzy) or Cdh1(also called fzr or Hct1). Cdh1 and Cdc20 are WD40-repeat proteins (well known linear motif-binding domains, folded as 7-blade beta-propellers), which recognise their target proteins via short "degradation motifs". Cdc20 and Cdh1 act at distinct phases of the cell cycle. Early in mitosis, during the metaphase-anaphase transition, APC/Cdc20 is mostly active, whereas Cdh1 is present, but as a phosphorylated inactive form which cannot bind to the APC/C. Later, in mitotic exit and further during the G1 phases, Cdh1 is activated by dephosphorylation, and binds to the APC/C by replacing Cdc20 and promoting its rapid degradation.
A number of degradation motifs have been identified within APC/C substrate proteins. These motifs are generally defined as short conserved sequences whose deletion or mutation promotes the stabilisation of the proteins where they are naturally found, and which can confer cell-cycle dependent degradation on unrelated proteins. The best characterised ones are the Destruction box (D-box) and KEN box.
The D-box was originally found in the cyclin B protein to be necessary to induce cyclin B degradation (Glotzer et al., 1991). Moreover, when fused to a foreign protein it is sufficient to generate a cycle-dependent proteolytic pattern similar to that observed for cyclin B. It is one of the earlier described linear motifs. Its short sequence is usually represented as a highly conserved RxxL motif, however the ELM annotators consider that the conservation is better described by the motif RxxLxx[LIVM]. Subsequent studies demonstrated a D-box-dependent degradation of other key cell-cycle players such as cyclin A (Lorca et al., 1992), geminin (McGarry and Kirchner, 1998), securin (Zou et al., 1999) and Plk1 (Lindon and Pines, 2004), all of which strictly obey the extended motif. Proposed D-boxes in Cdc6 and Nek2 do not match the extended motif while the motifs proposed in Aurora A and Aurora B are deeply buried in the kinase domain. Indeed, Nguyen et al. (2005) were not able to reproduce D-box function in Aurora B. It is of note that the problematic D-box proteins all have highly conserved KEN boxes.
The first KEN box (consensus KEN) was identified within Cdc20 itself (Pfleger and Kirschner 2000). Later, active KEN boxes were also reported within human CDC6, securin, Drosophila cyclin A, yeast Hsl1, Clb2, Aurora kinase B, BUB1 and CIN8 (see attached references). Candidate KEN boxes (without experimental verification) have been proposed in many other proteins including HipK2, Eg5, DNA Topo1 and Cdc27 (Michael et al., 2008).
Both D-box and KEN-box are recognised by Cdh1 and/or Cdc20, which subsequently recruit the APC/C complex, leading to the ubiquitination and proteasome-mediated degradation of the target protein. The D-box is recognized by both Cdc20 and Cdh1, whereas the KEN-box is preferentially recognized by Cdh1. Cdc20 itself contains a KEN box, which is therefore recognized by Cdh1, ensuring the temporal degradation of Cdc20 and its replacement by Cdh1 as a cofactor of the APC/C.
[Experimental studies of APC-target proteins have shown that some of them contain only D-box, others contain only KEN-box, some contain both. D-box and KEN can act as an independent entity or as a co-ordinate unit for protein degradation.
Finally, the presence of D-box or KEN-box motifs in a sequence does not always guarantee that they are active degradation signals for the proteins in which they are found. Indeed, there is always a possibility that the protein in which a potential destruction motif has been mutated becomes resistant to proteasome degradation due to serious misfolding and aggregation, and not because of losing a specific APC/C-targeting site. Putative destruction motifs found within areas predicted to be natively disordered are much more likely to be active than those found in known or predicted globular regions/domains.]