Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex

Abstract

The specificity of ubiquitin-mediated protein degradation with regards to the selection of substrates to be polyubiquitinated has only been determined rather recently. Substrate targeting by the N-end rule and HECT (homology to E6AP carboxyl terminus) domain ubiquitin ligases occurs through substrate-specific binding domains. In contrast, the SCF complex recruits substrates through a substrate adaptor protein, the F-box subunit. Despite evidence showing that Cdc20 and Cdh1 bind and activate the anaphase-promoting complex (APC) in a substrate-specific manner, there is no evidence that the activating protein and substrate interact directly; hence, no clear model exists for the mechanism of APC activation or recruitment of substrates. We show here that the activators Cdc20 and Cdh1 can associate with substrates via their N termini. In the absence of APC, Cdc20 and Cdh1 bind substrates reflecting Cdc20-APC and Cdh1-APC specificity. The N termini of Cdc20 and Cdh1 provide specificity functionally, as demonstrated by the generation of active chimeras that display the specificity corresponding to their N termini. Thus, Cdc20 and Cdh1 act as both substrate recognition and activating modules for APC. Ubiquitin-mediated protein degradation regulates important cellular processes from cell cycle progression to Wnt signaling (Peters 1998; for reviews, see Zachariae and Nasmyth 1999;Jackson et al. 2000). Attachment of a polyubiquitin chain to a protein targets that protein for degradation by the 26S proteasome (Coux et al. 1996;Baumeister et al. 1998). Ubiquitin becomes covalently attached to a substrate by interaction with a cascade of enzymes. The last in the series, a ubiquitin protein ligase (E3), transfers the ubiquitin to a lysine on the substrate protein (for review, see Hershko and Ciechanover 1998).In the N-end rule pathway, extending from bacteria to humans, a destabilizing residue at the N terminus directs proteins for ubiquitination via the E3 N-recognin/ Ubr1p (Saccharomyces cerevisiae) or E3␣ (in mammals). Direct binding of substrates to N-recognin/E3␣ is observed (Reiss et al. 1988;Gonda et al. 1989;Bartel et al. 1990; for review, see Varshavsky 1996). Similarly, HECT (homology to E6AP carboxyl terminus) domain E3s are highly conserved from yeast to vertebrates. They share homology in their C termini to E6AP and contain a conserved C-terminal cysteine residue that forms a thioester with ubiquitin; they bind substrates via their N termini (for review, see Jackson et al. 2000). E6AP targets the src kinases (Oda et al. 1999), HHR23A and HHR23B (Kumar et al. 1999). The HECT domain RSP5 in yeast targets Gap1 and Fur4 by interaction with its N-terminal WW domains (Hein et al. 1995;Rotin 1998;Wang et al. 1999). The fission yeast pub1 targets Cdc25 (Nefsky and Beach 1996). Xsmurf1 binds and targets Smad1 for degradation (Zhu et al. 1999).Two ubiquitin protein ligases play a role in the cell cycle: the SCF (Skp1/Cullin/F-box) complex and the anaphase-promoting complex (APC) (Peters 1998; for reviews, see Zachariae and ...

APC2 Cullin Protein and APC11 RING Protein Comprise the Minimal Ubiquitin Ligase Module of the Anaphase-promoting Complex

“…The association between VHL and the elongin BC complex is mediated by the SOCS-box motif of VHL (Kamura et al, 1998). The substrate recognition factors of APC are the WD40 repeat-containing proteins Cdc20 and Cdh1 (Burton et al, 2001;Hilioti et al, 2001;Pfleger et al, 2001). We have observed direct binding between Cdc20 or Cdh1 and the APC2/11 ubiquitin ligase core (our unpublished data).…”

“…Binding of either Cdc20 or Cdh1 to APC increases the activity of APC drastically (Fang et al, 1998;Kramer et al, 1998). Although the exact mechanism by which Cdc20 and Cdh1 contribute to the ubiquitination reactions is unknown, several recent observations suggest that they might be directly involved in substrate recruitment (Schwab et al, 1997;Sigrist and Lehner, 1997;Visintin et al, 1997;Burton et al, 2001;Hilioti et al, 2001;Pfleger et al, 2001).…”

“…Nearly all APC substrates contain the destruction box (D-box) or the KENbox motifs, which are required for the efficient ubiquitination and degradation of these substrates (King et al, 1996b;Pfleger and Kirschner, 2000). Cdc20 and Cdh1 have been shown to confer the D-box and KEN-box specificity of APC (Burton et al, 2001;Hilioti et al, 2001;Pfleger et al, 2001). The activities of the intact APC Cdc20 and APC Cdh1 from Xenopus were assayed with cyclin B1 as the substrate.…”

The anaphase-promoting complex: a key factor in the regulation of cell cycle

“…Further specific and direct interactions of Cdc20 with Pds1 and of Cdh1/Hct1 with Clb2, Clb3 and Cdc5 were also demonstrated in budding yeast (Burton and Solomon, 2001;Schwab et al, 2001). Finally, Pfleger et al (2001a) showed that in the absence of APC, Cdc20 and Cdh1/Hct1 can directly and specifically bind a large number of APC substrates including Xkid, securin, geminin, cyclin A and cyclin B.…”

“…Comparison between structures of the SCF and APC suggests a putative role of these activators in substrate recognition. In this regard, recent results have shown that different APC substrates can directly and specifically bind to Cdc20 and Cdh1/Hct1 (Fang et al, 1998b;Burton and Solomon, 2001;Sorensen et al, 2001;Pfleger et al, 2001a).…”