Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests
Judith Zich1, Karen May1, Konstantinos Paraskevopoulos1
1Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
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. Mps1(Mph1) 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 Mps1(Mph1) 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 Cdc20(Slp1)-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1(Mph1) kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.
Insights
The spindle checkpoint ensures accurate cell division by inhibiting the anaphase-promoting complex/cyclosome (APC/C). This study reveals Mad3 is a key target of Mps1 kinase, crucial for APC/C inhibition during mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The spindle checkpoint prevents errors in chromosome segregation during cell division.
- It functions by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a critical E3 ubiquitin ligase.
- Mad3 (BubR1) is a component of the mitotic checkpoint complex (MCC) that inhibits APC/C.
Purpose of the Study:
- To identify substrates of Mps1 (Mph1) kinase involved in spindle checkpoint signaling.
- To investigate the role of Mad3 phosphorylation by Mps1 in regulating APC/C activity.
- To elucidate the mechanism by which Mps1-dependent modifications maintain mitotic arrest.
Main Methods:
- Genetic analysis of fission yeast Mad3 mutants.
- Site-directed mutagenesis to map phosphorylation sites on Mad3.
- Biochemical assays to assess APC/C inhibition in vitro.
Main Results:
- Mad3 was identified as a direct substrate of Mps1 kinase.
- Mutants of Mad3 with altered phosphorylation sites showed reduced APC/C binding and failed to maintain checkpoint arrest.
- Phosphorylated Mad3 mimics effectively inhibited APC/C activity in vitro.
Conclusions:
- Mps1 kinase-dependent phosphorylation of Mad3 is essential for robust spindle checkpoint function.
- Mad3 phosphorylation directly modulates the inhibitory interaction with Cdc20 (Slp1)-APC/C.
- Concerted modifications of Mad3 and Mad2 by Mps1 maintain spindle checkpoint-mediated mitotic arrest.
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