Unattached Kinetochores Catalyze Production of an Anaphase Inhibitor that Requires a Mad2 Template to Prime Cdc20 for BubR1 Binding

Abstract

Summary Premature anaphase onset is prevented by the mitotic checkpoint through production of a “wait anaphase” inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase which targets them for destruction. Using physiologically-relevant levels of Mad2, Bub3, BubR1, and Cdc20, we demonstrate that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. We show that purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. Our results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1.

The Spindle Assembly Checkpoint: Clock or Domino?

“…This complex can already be detected during interphase in human and yeast cells (Sudakin et al 2001), at a time where functional kinetochores are not yet assembled, indicating that it acts outside of kinetochores. The same is true if BubR1 is the effector that ultimately inhibits APC/C Cdc20 , as both in a reconstituted functional Xenopus laevis assay, human cell extracts or mouse embryo fibroblasts, BubR1 appears to act as a cytoplasmic APC/C inhibitor (Kulukian et al 2009;Malureanu et al 2009;Nilsson et al 2008). These data are also consistent with functional data in human cells, which are based on the RNAi-mediated depletion of Mad2 and BubR1 or on the depletion of the landing pads of these proteins on kinetochores (Meraldi et al 2004).…”

“…Finally, a third attractive model has emerged recently, which proposes that the MCC is only a transient step in the activation of the spindle assembly checkpoint (Nilsson et al 2008). In this model Mad2 bound to Cdc20 acts as a catalyst for the binding of Mad3/BubR1 to Cdc20 (Kulukian et al 2009;Nilsson et al 2008). Mad3/BubR1 not only acts a pseudosubstrate for Cdc20-APC/C, it can also target Cdc20 itself for ubiquitination by the APC/C, leading to the degradation of Cdc20 (Burton and Solomon 2007;Nilsson et al 2008).…”

Systems Biology Modeling of Five Pathways for Regulation and Potent Inhibition of the Anaphase-Promoting Complex (APC/C): Pivotal Roles for MCC and BubR1

“…However, the rate of its uncatalyzed formation in the cytosol is slow (Sudakin et al, 2001). The formation of Bub3:BubR1:Cdc20 is accelerated in the presence of unattached chromosomes (Kulukian et al, 2009a) and it may be that MCC forms as an intermediate complex from which OMad2 rapidly dissociates (Kulukian et al, 2009a;Malureanu et al, 2009;Medema, 2009). Microtubule attachment depletes the template-complexes from the respective kinetochore, thereby silencing SAC-signaling locally (Buffin et al, 2005;Sivaram et al, 2009).…”

“…The APC can be inhibited in multiple ways, and complexes of APC together with either Cdc20:C-Mad2, Bub3:BubR1, Bub3:BubR1:Cdc20, MCC, or MCF2 have been found to be inactive (Eytan et al, 2008;Herzog et al, 2009;Kulukian et al, 2009a;Malureanu et al, 2009;Medema, 2009;Sudakin et al, 2001). However, the demand mechanisms for binding the inhibitory complexes to the APC are subject to current research.…”

“…It has been proposed that Mad2 may act as a catalyst for the assembly of a complex of Bub3:BubR1 with Cdc20 (Kulukian et al, 2009b;Overlack et al, 2014). Furthermore, BubR1 was suggested to interact with APC/C (Han et al, 2013).…”