The APC Subunit Doc1 Promotes Recognition of the Substrate Destruction Box

Departments of Physiology and Biochemistry and Biophysics, University of California-San Francisco, San Francisco, CA 94143-2200, USA.
Current Biology (Impact Factor: 9.92). 02/2005; 15(1):11-8. DOI: 10.1016/j.cub.2004.12.066
Source: PubMed

ABSTRACT Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition.
To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates.
Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

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    • "Under these conditions, we were able to efficiently pull down the APC/C complex together with cyclin B1. Hyper-phosphorylated APC3 mediates cyclin B1 recruitment to the APC/C while the spindle checkpoint is still active, which becomes effective after checkpoint release, when APC10 and Cdc20 direct cyclin B1 to the catalytic site of the APC/ C (Carroll et al., 2005; Chang et al., 2014). Our results reveal that the stability of geminin and securin, two other known APC/C Cdc20 substrates (Clijsters et al., 2013; Hagting et al., 2002), is less dependent on the phosphorylation state of the APC/C. "
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    ABSTRACT: When cells enter mitosis, the anaphase-promoting complex/cyclosome (APC/C) is activated by phosphorylation and binding of Cdc20. The RXXL destruction box (D-box) of cyclin B1 only binds Cdc20 after release of the spindle checkpoint in metaphase, initiating cyclin B1 ubiquitination upon chromosome bi-orientation. However, we found that cyclin B1, through Cdk1 and Cks, is targeted to the phosphorylated APC/C(Cdc20) at the start of prometaphase, when the spindle checkpoint is still active. Here, we show that MASTL is essential for cyclin B1 recruitment to the mitotic APC/C and that this occurs entirely independently of Cdc20. Importantly, MASTL-directed binding of cyclin B1 to spindle checkpoint-inhibited APC/C(Cdc20) critically supports efficient cyclin B1 destruction after checkpoint release. A high incidence of anaphase bridges observed in response to MASTL RNAi may result from cyclin B1 remaining after securin destruction, which is insufficient to keep MASTL-depleted cells in mitosis but delays the activation of separase. © 2015. Published by The Company of Biologists Ltd.
    Biology Open 03/2015; 4(4). DOI:10.1242/bio.201410793 · 2.42 Impact Factor
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    • "Other APC/C subunits assemble onto this minimal module in order to confer specificity to the E3 ligase. The substrate recognition and poly-ubiquitination requires the additional function of APC10/DOC1 (Carroll et al., 2005). This selective target recognition is activated during the cell cycle by co-activators such as CDC20 (cell division cycle protein 20) and Cdh1 (cadherin 1) (Vodermaier et al., 2003). "
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    • "Coactivators function as adaptors to recruit substrates to the APC/C by their capacity to recognize two conserved APC/C degrons present in most APC/C substrates: the nine-residue D (destruction) box (Glotzer et al., 1991; King et al., 1996) and KEN box (Pfleger and Kirschner, 2000). Coactivators are solely responsible for KEN box recognition (Chao et al., 2012); however , the D box degron is engaged by a bipartite coreceptor on the APC/C-coactivator complex generated from both the coactivator and the core APC/C subunit Apc10 (Carroll et al., 2005; Chao et al., 2012; da Fonseca et al., 2011; Hilioti et al., 2001; Kraft et al., 2005). The coactivator's WD40 b-propeller domain interacts with D box and KEN box degrons (Chao et al., 2012; Kraft et al., 2005), whereas its N-terminal C box stimulates APC/C catalytic activity (Kimata et al., 2008a; Labit et al., 2012). "
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