Abstract
BackgroundMembers of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.Methodology/Principal FindingsHere, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.Conclusions/SignificancehMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.
Highlights
The purpose of mitosis is to distribute the duplicated genome amongst dividing cells
In this study we examined in vivo Human Mps1 (hMps1) phosphorylation by mass spectrometry and investigated candidate upstream kinases for their possible involvement in hMps1 phosphorylation
A number of the hMps1 phosphorylation sites analyzed here were identified independently by other groups [29,30,32,33,47], we emphasize that our study provides in vivo evidence for phosphorylation of several sites that had previously been identified only in vitro
Summary
The purpose of mitosis is to distribute the duplicated genome amongst dividing cells. The purpose of the SAC signaling cascade is to delay the onset of anaphase until all chromosomes have undergone stable, bi-oriented attachments [6,7]. This is achieved through inhibition of the ubiquitin ligase APC/C (anaphase promoting complex/cyclosome), in part through sequestration of Cdc, an activator protein of the APC/C [7,8]. Members of the Mps kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. The upstream kinases responsible for these phosphorylations are not presently known
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