Quantitative Mass Spectrometry Analysis Reveals Similar Substrate Consensus Motif for Human Mps1 Kinase and Plk1

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.

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

“…Although the molecular mechanism remains unclear, Mps1 is required to recruit Mad1 and Mad2 to unattached kinetochores, supporting its essential role in SAC activity (15)(16)(17)(18). It also is clear that aurora B kinase activity and the outer-layer kinetochore protein nuclear division cycle 80 (Ndc80)/Hec1 are required for Mps1 localization to kinetochores, as evidenced by recent work, including ours (17,(19)(20)(21)(22)(23)(24). How Mps1 activates the SAC is now becoming clear.…”

“…nih.gov/ij/) on nondeconvoluted images. The levels of kinetochore-associated proteins were quantified as described previously (19). In brief, the average pixel intensities from at least 100 kinetochore pairs from five cells were measured, and background pixel intensities were subtracted.…”

“…The kinetochore localization of Mps1 WT depends on aurora B kinase activity (19,20,22,23). We next asked whether the recruitment of inactive Mps1 to the kinetochore also depends on aurora B.…”

Mitotic Regulator SKAP Forms a Link between Kinetochore Core Complex KMN and Dynamic Spindle Microtubules

“…Measurement of kinetochore intensities was performed in ZEN software (Carl Zeiss) on non-deconvolved images. Quantification of kinetochore intensities was performed as described previously (23). Essentially, a circular region with fixed diameter was centered on each kinetochore, and unless indicated otherwise, ACA intensity was measured in the same region and used for normalization after subtraction of background intensity measured outside the cell.…”

The Renaissance or the cuckoo clock

“…Many of the substrates of the Plks are important regulators of different aspects of mitosis and their phosphorylation is often coordinated with the actions of the Cdks through recruitment of the Plk to a particular substrate after it has been phosphorylated by a Cdk to create a polo-box-binding domain [6]. The importance of recruitment to a phospho-primed substrate may help to explain how Plk1 and the Mps1 kinases have very different roles in mitosis and yet phosphorylate a similar consensus sequence at the primary amino acid level [7]. In some circumstances, notably recovery from DNA damage, Aurora A is required to activate Polo [8,9], thereby creating the potential to generate higher levels of spatial control by demanding coordination between two independent recruitment events.…”