Stable MCC binding to the APC /C is required for a functional spindle assembly checkpoint

Abstract

The spindle assembly checkpoint (SAC) delays progression into anaphase until all chromosomes have aligned on the metaphase plate by inhibiting Cdc20, the mitotic co-activator of the APC/C. Mad2 and BubR1 bind and inhibit Cdc20, thereby forming the mitotic checkpoint complex (MCC), which can bind stably to the APC/C. Whether MCC formation per se is sufficient for a functional SAC or MCC association with the APC/C is required remains unclear. Here, we analyze the role of two conserved motifs in Cdc20, IR and C-Box, in binding of the MCC to the APC/C. Mutants in both motifs assemble the MCC normally, but IR motif integrity is particularly important for stable binding to the APC/C. Cells expressing Cdc20 with a mutated IR motif have a compromised SAC, as uninhibited Cdc20 can compete with the MCC for APC/C binding and activate it. We thus show that stable MCC association with the APC/C is critical for a functional SAC.

The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing

“…To this end, we generated a stable HeLa cell line expressing Venus-Cdc20 (for expression levels see Supplementary Fig. 1g), which has previously been shown to complement the Cdc20 RNAi phenotype 12,33 . This cell line and all other cell lines described here are stable isogenic inducible cell lines made using a HeLa FRT/TRex cell line.…”

Nek2A destruction marks APC/C activation at the prophase-to-prometaphase transition by spindle-checkpoint restricted Cdc20

“…The binding of Cdc20 to the APC/C is enforced by mitotic phosphorylation of the APC/C, but also by the spindle checkpoint: the Cdc20 C-box might be involved in stabilizing complexes between the APC/C and spindle checkpoint proteins (Hein and Nilsson, 2014). Hence, Cdc20, when incorporated in the MCC, effectively forms complexes with the APC/C at the start of prometaphase.…”

The Mitotic Checkpoint Complex Requires an Evolutionary Conserved Cassette to Bind and Inhibit Active APC/C

“…How the MCC binds stably to the APC/C is not known: it must involve APC/C-bound Cdc20 because mutating the latter’s isoleucine arginine (IR) tail destabilizes interaction with the MCC (Hein and Nilsson, 2014), but mutating the pseudo-substrate D-box and KEN box sites on BubR1 does not prevent MCC binding (Izawa and Pines, 2015). Thus, additional sites of interaction between the MCC and APC/C-Cdc20 must exist.…”