Sequential Multisite Phospho-Regulation of KNL1-BUB3 Interfaces at Mitotic Kinetochores

Vleugel, Mathijs; Omerzu, Manja; Groenewold, Vincent; Hadders, Michael A.; Lens, Susanne M.A.; Kops, Geert J. P. L.

doi:10.1016/j.molcel.2014.12.036

Cited by 113 publications

(191 citation statements)

References 41 publications

(84 reference statements)

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“…The SAC response is initiated at the KMN network through phosphorylation of multiple motifs in repeat sequences of KNL1 (also known as CASC5), which is mediated by MPS1 (also known as TTK) (London et al, 2012;Shepperd et al, 2012;Vleugel et al, 2015;Yamagishi et al, 2012). When phosphorylated, these motifs recruit BUB1-BUB3 dimers that are essential for SAC signaling and chromosome bi-orientation (Krenn et al, 2014;Primorac et al, 2013;Vleugel et al, 2013Vleugel et al, , 2015Zhang et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…When phosphorylated, these motifs recruit BUB1-BUB3 dimers that are essential for SAC signaling and chromosome bi-orientation (Krenn et al, 2014;Primorac et al, 2013;Vleugel et al, 2013Vleugel et al, , 2015Zhang et al, 2014). Since the discovery of BUB1, the molecular mechanism by which it participates in SAC activation has been a matter of controversy.…”

Section: Introductionmentioning

confidence: 99%

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Dissecting the roles of human BUB1 in the spindle assembly checkpoint

Vleugel

Hoek

Tromer

et al. 2015

Journal of Cell Science

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Mitotic chromosome segregation is initiated by the anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 (forming APC/C CDC20 ). APC/C CDC20 is inhibited by the spindle assembly checkpoint (SAC) when chromosomes have not attached to spindle microtubules. Unattached kinetochores catalyze the formation of a diffusible APC/C CDC20 inhibitor that comprises BUBR1 (also known as BUB1B), BUB3, MAD2 (also known as MAD2L1) and a second molecule of CDC20. Recruitment of these proteins to the kinetochore, as well as SAC activation, rely on the mitotic kinase BUB1, but the molecular mechanism by which BUB1 accomplishes this in human cells is unknown. We show that kinetochore recruitment of BUBR1 and BUB3 by BUB1 is dispensable for SAC activation. Unlike its yeast and nematode orthologs, human BUB1 does not associate stably with the MAD2 activator MAD1 (also known as MAD1L1) and, although required for accelerating the loading of MAD1 onto kinetochores, BUB1 is dispensable for the maintenance of steady-state levels of MAD1 there. Instead, we identify a 50-amino-acid segment that harbors the recently reported ABBA motif close to a KEN box as being crucial for the role of BUB1 in SAC signaling. The presence of this segment correlates with SAC activity and efficient binding of CDC20 but not of MAD1 to kinetochores.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dissecting the roles of human BUB1 in the spindle assembly checkpoint

Vleugel

Hoek

Tromer

et al. 2015

Journal of Cell Science

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“…How Mps1 activates the SAC is now becoming clear. Mps1 recruits Bub1/Bub3 and BubR1/Bub3 to kinetochores through phosphorylation of KNL1, the kinetochore receptor protein of Bub1 and BubR1 (25)(26)(27)(28)(29)(30).…”

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confidence: 99%

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

Dou

Liu

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C-MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.Mps1 kinase | spindle assembly checkpoint | chromosome alignment | kinetochore-microtubule attachment | reversine

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“…To detect Mps1 kinase activity in vitro, the myelin basic protein (MBP) is widely employed as a substrate, using radiolabelled ATP or specific phospho-Mps1 antibodies [16][17][18]. Alternatively, the KNL1 protein is used as substrate [11], which is a well-documented Mps1 substrate of kinetochore components [19,20]. These assays are highly specific for Mps1, but hard to quantify.…”

Section: Cc-by-nc-nd 40 International License Not Peer-reviewed) Is mentioning

confidence: 99%

Understanding inhibitor resistance in Mps1 kinase through novel biophysical assays and structures

Hiruma

Koch

Hazraty

et al. 2017

Preprint

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Monopolar spindle 1 (Mps1/TTK) is a protein kinase essential in mitotic checkpoint signalling, preventing anaphase until all chromosomes are properly attached to spindle microtubules. Mps1 has emerged as a potential target for cancer therapy, and a variety of compounds have been developed to inhibit its kinase activity. Mutations in the catalytic domain of Mps1 that give rise to inhibitor resistance, but retain catalytic activity and do not display cross-resistance to other Mps1 inhibitors, have been described. Here we characterize the interactions of two such mutants, Mps1 C604Y and C604W, which raise resistance to two closely related compounds, NMS-P715 and its derivative Cpd-5, but not to the well-characterised Mps1 inhibitor, reversine. We show that estimates of the IC50 (employing a novel specific and efficient assay that utilizes a fluorescently labelled substrate) and of the binding affinity (KD) indicate that in both mutants, Cpd-5 should be better tolerated than the closely related NMS-P715. To gain further insight, we determined the crystal structure of the Mps1 kinase mutants bound to Cpd-5 and NMS-P715, and compare the binding modes of Cpd-5, NMS-P715 and reversine. The difference in steric hindrance between Tyr/Trp604 and the trifluoromethoxy moiety of NMS-P715, the methoxy moiety of Cpd-5, and complete absence of such a group in reversine, account for differences we observe in vitro. Our analysis enforces the notion that inhibitors targeting Mps1 drug-resistant mutations can emerge as a feasible intervention strategy based on existing scaffolds, if the clinical need arises.Summary statementThe inhibition of specific Mps1 kinase inhibitors towards the wild-type protein and inhibitor-resistant mutants is explained by a novel specific activity assay, biophysical characterisation, and X-ray structures.AbbreviationsATPadenosine triphosphateBub1/Bub3budding uninhibited by benzimidazoles 1 / budding uninhibited by benzimidazoles 3Cpd-5Compound-5 (N-(2,6-diethylphenyl)-8-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide)FPfluorescence polarizationGSTGlutathione S-transferaseIC50half maximal inhibitory concentrationKDdissociation constantΔGcalccalculated Gibbs energy difference for ligand bindingKNL1kinetochore null protein 1KPi(inorganic) potassium phosphateMps1monopolar spindle 1MSTmicroscale thermophoresisNMS-P715N-(2,6-diethylphenyl)-1-methyl-8-({4-[(1-methylpiperidin-4-yl)carbamoyl]-2-(trifluoromethoxy)phenyl}amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamideTMRtetramethylrhodamineWTwild-type

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Sequential Multisite Phospho-Regulation of KNL1-BUB3 Interfaces at Mitotic Kinetochores

Cited by 113 publications

References 41 publications

Dissecting the roles of human BUB1 in the spindle assembly checkpoint

Dissecting the roles of human BUB1 in the spindle assembly checkpoint

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

Understanding inhibitor resistance in Mps1 kinase through novel biophysical assays and structures

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