Bub3-Bub1 Binding to Spc7/KNL1 Toggles the Spindle Checkpoint Switch by Licensing the Interaction of Bub1 with Mad1-Mad2

Mora-Santos, Mar; Hervas-Aguilar, America; Sewart, Katharina; Lancaster, Theresa C.; Meadows, John C.; Millar, Jonathan

doi:10.1016/j.cub.2016.07.040

Cited by 46 publications

(48 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In normal cells, Bub3 would prevent early nucleoplasmic signaling, and this effect would later be overcome when Mad-Bub complexes assemble at kinetochores and Spc7-Bub3-Bub1 interactions induce conformational changes in the Bub proteins, thereby activating Bub1 for downstream signaling. These Bub3 findings from our synthetic arrest are entirely consistent with a recent study published while our manuscript was in revision [40]. …”

Section: Resultssupporting

confidence: 92%

Generation of a Spindle Checkpoint Arrest from Synthetic Signaling Assemblies

et al. 2017

View full text Add to dashboard Cite

SummaryThe spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules and ensuring high-fidelity chromosome segregation [1, 2, 3]. The checkpoint is activated by unattached kinetochores, and Mps1 kinase phosphorylates KNL1 on conserved MELT motifs to generate a binding site for the Bub3-Bub1 complex [4, 5, 6, 7]. This leads to dynamic kinetochore recruitment of Mad proteins [8, 9], a conformational change in Mad2 [10, 11, 12], and formation of the mitotic checkpoint complex (MCC: Cdc20-Mad3-Mad2 [13, 14, 15]). MCC formation inhibits the anaphase-promoting complex/cyclosome (Cdc20-APC/C), thereby preventing the proteolytic destruction of securin and cyclin and delaying anaphase onset. What happens at kinetochores after Mps1-dependent Bub3-Bub1 recruitment remains mechanistically unclear, and it is not known whether kinetochore proteins other than KNL1 have significant roles to play in checkpoint signaling and MCC generation. Here, we take a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1Spc7 and Mps1Mph1 is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest. We demonstrate that a Mad1-Bub1 complex is formed during synthetic checkpoint signaling. Analysis of bub3Δ mutants demonstrates that Bub3 acts to suppress premature checkpoint signaling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analyzing several mutants that affect localization of checkpoint complexes, we conclude that spindle checkpoint arrest can be independent of their kinetochore, spindle pole, and nuclear envelope localization.

show abstract

Section: Resultssupporting

confidence: 92%

Generation of a Spindle Checkpoint Arrest from Synthetic Signaling Assemblies

et al. 2017

View full text Add to dashboard Cite

show abstract

“…An interesting aspect uncovered here is that the CD1 needs to be phosphorylated to function in line with observations from budding and fission yeast374558. We show that this occurs at kinetochores in prometaphase and our in vitro phosphorylation assays implicate Cdk1 and Mps1 as possible kinases.…”

Section: Discussionsupporting

confidence: 85%

Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling

et al. 2017

View full text Add to dashboard Cite

Proper segregation of chromosomes depends on a functional spindle assembly checkpoint (SAC) and requires kinetochore localization of the Bub1 and Mad1/Mad2 checkpoint proteins. Several aspects of Mad1/Mad2 kinetochore recruitment in human cells are unclear and in particular the underlying direct interactions. Here we show that conserved domain 1 (CD1) in human Bub1 binds directly to Mad1 and a phosphorylation site exists in CD1 that stimulates Mad1 binding and SAC signalling. Importantly, fusion of minimal kinetochore-targeting Bub1 fragments to Mad1 bypasses the need for CD1, revealing that the main function of Bub1 is to position Mad1 close to KNL1 MELT repeats. Furthermore, we identify residues in Mad1 that are critical for Mad1 functionality, but not Bub1 binding, arguing for a direct role of Mad1 in the checkpoint. This work dissects functionally relevant molecular interactions required for spindle assembly checkpoint signalling at kinetochores in human cells.

show abstract

“…It is clear that phosphorylation of Met-Glu-Leu-Thr (MELT) repeats in the outer kinetochore protein KNL1 by the checkpoint kinase Mps1 generates binding sites for the checkpoint complexes Bub1/Bub3 and BubR1/Bub3 (London et al, 2012;Shepperd et al, 2012;Yamagishi et al, 2012;Vleugel et al, 2013Vleugel et al, , 2015bZhang et al, 2014Zhang et al, , 2016. Subsequent phosphorylation of Bub1 by Mps1 then facilitates an interaction between Mad1 and Bub1 a mechanism conserved from yeast to man (London & Biggins, 2014;Mora-Santos et al, 2016;Faesen et al, 2017;Ji et al, 2017;Qian et al, 2017;Zhang et al, 2017). Mad1 is in a stable complex with Mad2 and the recruitment of Mad1/Mad2 to kinetochores is essential because this complex catalyzes the first step in MCC formation by loading Mad2 onto Cdc20 (De Antoni et al, 2005;Faesen et al, 2017;Ji et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

et al. 2019

View full text Add to dashboard Cite

Kinetochore localized Mad1 is essential for generating a “wait anaphase” signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod‐Zw10‐Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1‐Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1‐Bub1 complex.

show abstract

Bub3-Bub1 Binding to Spc7/KNL1 Toggles the Spindle Checkpoint Switch by Licensing the Interaction of Bub1 with Mad1-Mad2

Abstract: (2016) Bub3-Bub1 binding to Spc7/KNL1 toggles the spindle checkpoint switch by licensing the interaction of Bub1 with Mad1-Mad2.

Cited by 46 publications

References 23 publications

Generation of a Spindle Checkpoint Arrest from Synthetic Signaling Assemblies

Generation of a Spindle Checkpoint Arrest from Synthetic Signaling Assemblies

Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

Contact Info

Product

Resources

About