Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling.
Stefan Kemmler1, Manuel Stach, Maria Knapp
1Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, Heidelberg, Germany.
The EMBO Journal
|March 21, 2009
Summary
The spindle assembly checkpoint (SAC) relies on protein kinase Mps1. This study shows Mps1-dependent phosphorylation of Ndc80 is crucial for SAC activation at kinetochores.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation.
- Protein kinase Mps1 is essential for SAC function.
- The Ndc80 complex is a key kinetochore component involved in microtubule attachment.
Purpose of the Study:
- To investigate the interaction between Mps1 and Ndc80.
- To determine the role of Mps1-mediated Ndc80 phosphorylation in SAC regulation.
- To elucidate the functional consequences of Ndc80 phosphorylation sites.
Main Methods:
- Co-immunoprecipitation to study protein interactions.
- In vitro kinase assays to assess Mps1 activity on Ndc80.
- Site-directed mutagenesis of Ndc80 phosphorylation sites.
- Analysis of checkpoint signaling and kinetochore-microtubule attachment in yeast mutants.
Main Results:
- Saccharomyces cerevisiae Mps1 physically interacts with the N-terminal domain of Ndc80.
- Mps1 phosphorylates Ndc80 in vitro and in vivo.
- Mutations of 14 phosphorylation sites in Ndc80 compromise checkpoint signaling.
- Simulating constitutive phosphorylation of Ndc80 leads to metaphase arrest with proper kinetochore-microtubule attachment, dependent on SAC activity.
Conclusions:
- Mps1-dependent phosphorylation of Ndc80 is critical for SAC activation.
- This phosphorylation event plays a vital role in kinetochore function during cell division.
- Targeting Mps1-Ndc80 interactions could offer insights into cell cycle regulation.
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