Abstract
Mps1 is a dual specificity protein kinase that regulates the spindle assembly checkpoint and mediates proper microtubule attachment to chromosomes during mitosis. However, the molecular mechanism that controls Mps1 protein level and its activity during the cell cycle remains unclear. Given that sumoylation plays an important role in mitotic progression, we investigated whether Mps1 was SUMO-modified and whether sumoylation affects its activity in mitosis. Our results showed that Mps1 was sumoylated in both asynchronized and mitotic cell populations. Mps1 was modified by both SUMO-1 and SUMO-2. Our further studies revealed that lysine residues including K71, K287, K367 and K471 were essential for Mps1 sumoylation. Sumoylation appeared to play a role in mediating kinetochore localization of Mps1, thus affecting normal mitotic progression. Furthermore, SUMO-resistant mutants of Mps1 interacted with BubR1 more efficiently than it did with the wild-type control. Combined, our results indicate that Mps1 is SUMO-modified that plays an essential role in regulating Mps1 functions during mitosis.
Highlights
The spindle assembly checkpoint (SAC) is a conserved surveillance mechanism that regulates partitioning of duplicated genome into two daughter cells during mitosis [1]
To further confirm the slow mobility shifts were sumoylated Mps1, HEK293T cells were transfected with plasmids expressing Myc-tagged Mps1 alone or along with constructs either expressing His6-SUMO-1 and FLAG-tagged wild-type SENP1 or His6-SUMO-1 and FLAG-tagged enzymatically defective SENP1 (SENP1-mut)
We show evidence that Mps1 is modified by both SUMO-1 and SUMO-2 during the cell cycle and that this post-translational modification occurs on multiple lysine residues including K71, K287, K367 and K471
Summary
The spindle assembly checkpoint (SAC) is a conserved surveillance mechanism that regulates partitioning of duplicated genome into two daughter cells during mitosis [1]. Extensive research in the past has revealed that protein kinases including Aurora A/B, Mps, Bub, and BubR1 positively regulate SAC activities, arresting cell at metaphase until all condensed chromosomes are correctly orientated [2, 3]. The key molecular target of SAC is Cdc, a substrate-specific activator of anaphase promoting complex/cyclosome (APC/C) [4]. Mps (mono-polar spindle 1) is an evolutionarily conserved protein that functions as a key component of SAC [9, 10]. Mps plays an essential role in recruiting Mad and Mad to unattached kinetochores, mediating proper chromosome congression and accurate chromosome segregation [11,12,13,14,15,16,17]. Given its essential role in SAC functions, Mps undergoes a dynamic distribution during mitosis [10, 18].
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