CDK-Dependent Potentiation of MPS1 Kinase Activity Is Essential to the Mitotic Checkpoint

Abstract

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.

Dynamic Autophosphorylation of Mps1 Kinase Is Required for Faithful Mitotic Progression

“…S5B), suggesting that autophosphorylation of the N-terminal region (at least at the sites described here) does not affect Mps1 kinase activity. Consistent with the previous report that CDK1 potentiate Mps1 activity [43],the positive control phospho-mimetic mutant Mps1 5D (5 CDK1 phosphorylation sites S281, S436, T453, T468, S821) used in these assays displayed enhanced kinase activity.…”

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores

“…In line with this speculation, two kinases, Cdk1 and Aurora B, have recently been reported to potentiate Mps1 activation in mitosis. 40,57 Importantly, inhibiting Aurora B kinase activity prevents Mps1 kinetochore recruitment not only in prometaphase both also during metaphase upon UV irradiation, suggesting that Aurora B is an indispensable mediator of UV irradiation that promotes Mps1 association with the kinetochore.…”

Bub1 targeting to centromeres is sufficient for Sgo1 recruitment in the absence of kinetochores

“…Other antibodies used in this study have been described before: Xenopus CENP-C and CENP-T (Krizaic et al 2015); embryonic histone H1, RbAp48 (Bernad et al 2011); Xenopus Bub1, Sgo1, CENP-A (Rivera and Losada 2009); Mps1 (Morin et al 2012); Mis12 and Ndc80 (a generous gift of P.T. Stukenberg; Emanuele et al 2005); anti-myc (clone 9E10).…”