Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding

Abstract

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase that targets for degradation cell-cycle regulatory proteins during exit from mitosis and in the G 1 phase of the cell cycle. The activity of APC/C in mitosis and in G 1 requires interaction with the activator proteins Cdc20 and Cdh1, respectively. Substrates of APC/C–Cdc20 contain a recognition motif called the “destruction box” (D-box). The mode of the action of APC/C activators and their possible role in substrate binding remain poorly understood. Several investigators suggested that Cdc20 and Cdh1 mediate substrate recognition, whereas others proposed that substrates bind to APC/C or to APC/C–activator complexes. All these studies used binding assays, which do not necessarily indicate that substrate binding is functional and leads to product formation. In the present investigation we examined this problem by an “isotope-trapping” approach that directly demonstrates productive substrate binding. With this method we found that the simultaneous presence of both APC/C and Cdc20 is required for functional substrate binding. By contrast, with conventional binding assays we found that either Cdc20 or APC/C can bind substrate by itself, but only at low affinity and relaxed selectivity for D-box. Our results are consistent with models in which interaction of substrate with specific binding sites on both APC/C and Cdc20 is involved in selective and productive substrate binding.

Cell cycle regulation of the human Six1 homeoprotein is mediated by APCCdh1

“…Our experiments demonstrate that deletion of either the N-terminus or the C-terminus stabilizes Six1 to degradation by APC Cdh1 . A double recognition motif such as this has been found in other APC Cdh1 substrates (Castro et al, 2005) and may be due to the fact that the core APC, as well as the activators Cdh1 and Cdc20, participate in substrate-binding interactions (Yamano et al, 2004;Passmore and Barford, 2005;Eytan et al, 2006). It is thus possible that the N-terminus of Six1 contains a novel motif important for Cdh1 binding, whereas the C-terminus contains an additional element that binds to the core APC.…”

“…Recent evidence indicates that Cdh1 and Cdc20 interact specifically with the D-box and KEN box destruction motifs of their substrates (Burton et al, 2005;Kraft et al, 2005). Thus, it is has been suggested that substrates lacking canonical D-boxes may contain a three-dimensional interaction surface similar to a D-box but with differing primary amino acid sequence (Eytan et al, 2006). Our experiments suggest that the Nterminus of Six1 contains a novel motif mediating interaction with Cdh1 and directing APC Cdh1 -mediated degradation of Six1, as deletion of the N-terminus stabilizes Six1 to degradation mediated by APC Cdh1 and GST pull-down experiments indicate that the N-terminus alone is sufficient for binding to Cdh1.…”

Cyclin A Degradation Employs Preferentially Used Lysines and a Cyclin Box Function other than Cdk1 Binding

“…The occurrence of multiple targeting signals on Cyclin A is consistent with a model wherein both core APC/C subunits and coactivators contribute to substrate recognition. 16,17 Further work is needed to decipher how the cyclin box functions as a destruction signal.…”

“…14,15 As a compromise between these two opposing hypotheses, new studies indicate that both the APC/C and its coactivators contribute to substrate recognition. 16,17 Drosophila has three mitotic cyclins: Cyclin A (CycA), Cyclin B (CycB) and Cyclin B3 (CycB3). Their sequential degradation facilitates mitotic exit.…”

Cell Cycle-dependent Expression of Centrosomal Ninein-like Protein in Human Cells Is Regulated by the Anaphase-promoting Complex

“…Cdh1 or Cdc20 Can Stimulate Nlp Degradation-It has been demonstrated that Cdc20 (Fizzy in Drosophila) and a related protein Cdh1 (Fizzy-related in Drosophila) activate APC through giving temporal control and substrate specificity to the APC/c E3 ligase (25,26). Each of these two activators physically associates with APC in a cell cycle-dependent manner and stimulates substrate degradation.…”