Releasing the brake: how kinetochore localized phosphatases allow silencing of the spindle-assembly checkpoint and consequently mitotic exit upon chromosome bi-orientation.
Final Report Abstract
The equal distribution of chromosomes during mitosis is essential to maintain genomic stability, as missegregations lead to aneuploidy, a deviation from the diploid chromosome set. Besides occurring in 70% of solid tumours, aneuploidy is also one of the main causes for miscarriages during early pregnancies. One of the safeguard mechanisms preventing the faulty distribution of chromosomes during mitosis is called the spindle assembly checkpoint (SAC). The SAC employs an elaborate signalling cascade preventing mitotic progression until all chromosomes are properly attached to kinetochore microtubules. Once this is the case, the SAC is silenced and the cell completes mitosis. Whereas engaging the SAC depends on the localized activity of kinetochore kinases, such as MPS1, its silencing is less-well understood. Although it is well-established that the delocalization of MPS1 itself and the counteracting activity of the phosphatases PP2A-B56 and PP1 contribute to SAC silencing, the factors/proteins undergoing changes in their posttranslational modification pattern and the significance of these (de)phosphorylation events are poorly studied. In this project, we set out to identify (new) mitotic PP2A-B56 and PP1 substrates using proximity-labelling based methods such as BioID and APEX. However, as we ran into technical problems regarding the MS-based identification of potential substrates of the two phosphatases, we decided to address the general SAC phospho-proteome instead. We were particularly interested in revealing timely changes in the phosphorylation pattern of (kinetochore) proteins occurring during the transition from an active SAC (SAC ON) to a silenced SAC (SAC OFF). To this end, we compared the phospho-proteome of HeLa cells either arrested in prometaphase (SAC ON) or metaphase (SAC OFF). We quantified more than 15 000 phosphosites and revealed 218 sites to be differentially phosphorylated. Strikingly, most of these changes were at proteins with known functions in transcriptional control and RNA processing, whereas less than one third occurred at known mitotic regulators. Furthermore, only 3% of the identified differentially regulated sites were present on kinetochore proteins. To specifically address phospho-sites on kinetochore proteins, we performed SILAC-MS experiments on various kinetochore complexes purified from synchronized HeLa BAC cell lines either displaying an active or silenced SAC. We thereby revealed that the E3 ligase APC/C that is required to degrade Cyclin B to allow mitotic progression is preferentially more phosphorylated upon SAC silencing. On the other hand, components of the KMN network that mediates microtubule binding display increased phosphorylation while the SAC is still engaged. Our results considerably contribute to a deeper understanding of the prometaphase/metaphase phosphorylation pattern that alters upon SAC silencing. Consequently, our hit lists could form the basis of follow-up studies addressing the functional relevance of carefully selected specific sites.
Publications
- Phylogenomics-guided discovery of a novel conserved cassette of short linear motifs in BubR1 essential for the spindle checkpoint. Open Biology. (2016) 6: 160315
Tromer E, Bade D, Snel B, Kops GJ
(See online at https://doi.org/10.1098/rsob.160315) - Regulation of Centromeric Chromatin. In Göndör A (Editor), Chromatin Regulation and Dynamics. (2016) 1: 303-319. Elsevier. ISBN: 9780128033951
Bade D, Erhardt S
