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

ARTICLE Received 14 Dec 2016 | Accepted 3 May 2017 | Published 12 June 2017 DOI: 10.1038/ncomms15822 OPEN Bub1 positions Mad1 close to KNL1 MELT repeats to promote checkpoint signalling Gang Zhang1, Thomas Kruse1, Blanca López-Méndez1, Kathrine Beck Sylvestersen1, Dimitriya H. Garvanska1, Simone Schopper1, Michael Lund Nielsen1 & Jakob Nilsson1 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. 1 The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Correspondence and requests for materials should be addressed to G.Z. (email: ) or to J.N. (email: ). NATURE COMMUNICATIONS | 8:15822 | DOI: 10.1038/ncomms15822 | www.nature.com/naturecommunications 1 ARTICLE A NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15822 prerequisite for life is the equal distribution of genetic information to the new daughter cells and this requires that the genome is accurately duplicated and subsequently distributed to new cells. Accurate segregation of sister chromatids during cell division depends on the spindle assembly checkpoint (SAC), which in response to improper attachments between kinetochores and microtubules generates a diffusible ‘wait anaphase’ inhibitor1–3. This inhibitor is the mitotic checkpoint complex (MCC) composed of the Mad2 and BubR1–Bub3 checkpoint proteins bound to Cdc20, the mitotic co-activator of the anaphase-promoting complex (APC/C)4. The MCC potently inhibits the APC/C-Cdc20 complex and this prevents the entry into anaphase, hereby providing time for proper biorientation of all sister chromatids. Once all kinetochores are attached to microtubules, the production of the MCC ceases and, in addition, the existing MCC is disassembled resulting in active APC/C-Cdc20 and the progression into anaphase. The essential components of the SAC were identified in the early 1990s and include the mitotic arrest deficient (Mad) proteins Mad1, Mad2 and Mad3 (BubR1 in humans) as well as the budding uninhibited by benzimidazole proteins (Bub) Bub1 and Bub3 (refs 5,6). Bub1 and BubR1 are in a stable complex with Bub3, while Mad2 exists in a stable complex with Mad1 and as free Mad2 (refs 7,8). In addition to these structural components, checkpoint signalling also depends on at least three kinases namely cyclin-dependent kinase 1 (Cdk1), Aurora B and monopolar spindle 1 (Mps1)9–16. However, only a few phosphosites on kinetochore and checkpoint proteins that directly regulate the SAC have been identified. A major question in the field is how the checkpoint proteins are recruited to kinetochores and how this stimulates the generation of the MCC. It is clear that all checkpoint proteins localize dynamically to unattached kinetochores, as does Cdc20. This localization depends on a large outer kinetochore complex composed of the KNL1-Zwint complex, the Mis12 complex and the Ndc80 complex (collectively referred to as the KMN network)17–21. The KNL1 protein is a direct receptor for the Bub1–Bub3 and BubR1–Bub3 complexes because the phosphorylation of so-called Met–Glu–Leu– Thr (MELT) repeats in KNL1 by the Mps1 kinase generates binding sites for Bub3 (refs 22–30). Bub1 and BubR1 both contain short linear motifs referred to as ABBA motifs (also known as Phe-box or A-box) that act to localize Cdc20 to kinetochores with the removal of the ABBA motif in Bub1 having a more pronounced effect on SAC signalling31–36. The exact mechanism behind recruitment of the Mad1/Mad2 complex to kinetochores in humans has not been established, but given the central role of this complex for SAC signalling this is crucial to understand. This contrasts the situation in budding yeast and worms where a direct interaction between Mad1 and Bub1 has been shown to localize Mad1/Mad2 to kinetochores37–39. In budding yeast, this Mad1–Bub1 interaction is stimulated by Mps1 phosphorylation of multiple sites in a central unstructured region in Bub1 spanning residues 369–608 (ref. 37). Interestingly, in worms it is the kinase domain of Bub1 that binds Mad1 directly suggesting that the Mad1/Mad2 complex can be recruited to kinetochores by different mechanisms38. In human cells a direct interaction between Mad1 and Bub1 stimulated by Mps1 was recently described40,41 and consistently Bub1 has been proposed to scaffold the assembly of SAC complexes on MELT repeats30. In addition the Rod-Zwilch-ZW10 (RZZ) complex is required for Mad1/Mad2 kinetochore localization and checkpoint signalling in higher eukaryotes33,42–44. The exact contribution of Bub1 and the RZZ complex is still to be fully dissected. To further our understanding of Mad1/Mad2 kinetochore recruitment and the function of Bub1 in human cells, we here 2 focus on the molecular function of conserved domain (CD1) in Bub1 that is essential for SAC signalling in both humans and yeast45,46. We show here that CD1 is required for a phosphoregulated direct interaction between human Bub1 and Mad1 and that disturbance of this interaction is detrimental to the SAC. In line with this we can bypass the requirement for CD1 by fusing minimal kinetochore targeting Bub1 fragments to Mad1, suggesting that the main function of Bub1 in SAC signalling is to localize Mad1 correctly at kinetochores. Interestingly, residues in a C-terminal globular domain of Mad1 are still required for SAC signalling when Bub1 and Mad1 are fused arguing that Mad1 has additional functions in the SAC. In conclusion, we establish a direct interaction between human Bub1 and Mad1, identify a critical phosphorylation site required for this interaction and identify the key function of the interaction. We hereby provide novel insight into Mad1/Mad2 kinetochore localization in human cells by Bub1 and propose that precise positioning of Mad1/Mad2 close to KNL1 MELT repeats is critical for checkpoint signalling. Results Bub1 conserved domain 1 is essential for SAC signalling. The Bub1 checkpoint protein contains different motifs that have been implicated in its function including CD1 and the ABBA m (...truncated)