Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests.

Judith Zich,Ali Sarkeshik,Heather M Syred,Konstantinos Paraskevopoulos,Kevin G Hardwick,Onur Sen,Juri Rappsilber,Hitesh Patel,Karen May,Sjaak Van Der Sar,James J Moresco,John R Yates

doi:10.1371/journal.pgen.1005834

Abstract

The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.

Highlights

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1,2,3]
We propose that Mps1Mph1 kinase phosphorylates multiple components of the fission yeast mitotic checkpoint complex (MCC) to stabilise its interaction with the anaphase promoting complex or cyclosome (APC/C) and thereby maintain spindle checkpoint arrests
In the absence of Mps1Mph1 kinase activity the mitotic checkpoint complex (MCC) is not tightly associated with APC/C [39], so we tested whether fission yeast Mad3p is a substrate of Mps1Mph1 kinase

Summary

Introduction

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1,2,3]. One of the major controls is the spindle checkpoint which acts as a surveillance system monitoring kinetochore-microtubule attachments. It delays anaphase onset until all sister-chromatid pairs are bi-oriented on the mitotic spindle [4,5,6]. Anaphase onset is initiated by an E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), and its activating co-factor Cdc20Slp1 [7,8]. Cdc20Slp1-APC/C targets the separase inhibitor securin and the CDK1 activating subunit cyclin B for destruction by the 26S proteasome [9,10,11]. Cohesin is cleaved and sister chromatids separate and segregate in anaphase [12]

Methods

Results

Conclusion