The Bub1-Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation.

Luying Jia,Hongtao Yu,Bing Li

doi:10.1038/ncomms10818

Abstract

The spindle checkpoint senses unattached kinetochores and inhibits the Cdc20-bound anaphase-promoting complex or cyclosome (APC/C), to delay anaphase, thereby preventing aneuploidy. A critical checkpoint inhibitor of APC/CCdc20 is the mitotic checkpoint complex (MCC). It is unclear whether MCC suffices to inhibit all cellular APC/C. Here we show that human checkpoint kinase Bub1 not only directly phosphorylates Cdc20, but also scaffolds Plk1-mediated phosphorylation of Cdc20. Phosphorylation of Cdc20 by Bub1–Plk1 inhibits APC/CCdc20 in vitro and is required for checkpoint signalling in human cells. Bub1–Plk1-dependent Cdc20 phosphorylation is regulated by upstream checkpoint signals and is dispensable for MCC assembly. A phospho-mimicking Cdc20 mutant restores nocodazole-induced mitotic arrest in cells depleted of Mad2 or BubR1. Thus, Bub1–Plk1-mediated phosphorylation of Cdc20 constitutes an APC/C-inhibitory mechanism that is parallel, but not redundant, to MCC formation. Both mechanisms are required to sustain mitotic arrest in response to spindle defects.

Highlights

The spindle checkpoint senses unattached kinetochores and inhibits the Cdc20-bound anaphase-promoting complex or cyclosome (APC/C), to delay anaphase, thereby preventing aneuploidy
BubR1 and Mad[2] can each independently inhibit APC/CCdc[20] using different mechanisms in vitro[15,16,17] and can collaborate to inhibit APC/CCdc[20] in vivo by forming the mitotic checkpoint complex (MCC) that consists of the constitutive BubR1–Bub[3] complex, Cdc[20] and Mad[2]
To explore the relationship between MCC and Plk1-dependent Cdc[20] phosphorylation in APC/CCdc[20] inhibition, we examined the phenotypes of Cdc[20] S92E-expressing cells depleted of Mad[2] or BubR1

Summary

Introduction

The spindle checkpoint senses unattached kinetochores and inhibits the Cdc20-bound anaphase-promoting complex or cyclosome (APC/C), to delay anaphase, thereby preventing aneuploidy. Phosphorylation of Cdc[20] by Bub1–Plk[1] inhibits APC/CCdc[20] in vitro and is required for checkpoint signalling in human cells. Bub1–Plk1-mediated phosphorylation of Cdc[20] constitutes an APC/C-inhibitory mechanism that is parallel, but not redundant, to MCC formation Both mechanisms are required to sustain mitotic arrest in response to spindle defects. Unattached kinetochores recruit and activate checkpoint proteins to produce diffusible anaphase inhibitors, which inhibit the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C) bound to Cdc[20] (refs 4,5). Plk[1] is a cell cycle kinase with myriad functions, including spindle assembly and chromosome alignment[31] Both Bub[1] and BubR1 contain an STP motif that, when phosphorylated by Cdk[1] in mitosis, binds to the polo-box domain of Plk[1] (refs 32,33). The Bub1–Plk[1] interaction recruits a population of Plk[1] to kinetochores[32], but the functional substrate of Bub1–Plk[1] at kinetochores remains to be identified

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Conclusion