Spindle checkpoint: trapped by the corona, cyclin B1 goes MAD
2020; Springer Nature; Volume: 39; Issue: 12 Linguagem: Inglês
10.15252/embj.2020105279
ISSN1460-2075
Autores Tópico(s)Cancer-related Molecular Pathways
ResumoNews & Views18 May 2020free access Spindle checkpoint: trapped by the corona, cyclin B1 goes MAD Carlos Conde Corresponding Author [email protected] orcid.org/0000-0002-4177-8519 Instituto de Investigação e Inovação em Saúde—i3S, Universidade do Porto, Porto, Portugal Search for more papers by this author Reto Gassmann Corresponding Author [email protected] orcid.org/0000-0002-0360-2977 Instituto de Investigação e Inovação em Saúde—i3S, Universidade do Porto, Porto, Portugal Search for more papers by this author Carlos Conde Corresponding Author [email protected] orcid.org/0000-0002-4177-8519 Instituto de Investigação e Inovação em Saúde—i3S, Universidade do Porto, Porto, Portugal Search for more papers by this author Reto Gassmann Corresponding Author [email protected] orcid.org/0000-0002-0360-2977 Instituto de Investigação e Inovação em Saúde—i3S, Universidade do Porto, Porto, Portugal Search for more papers by this author Author Information Carlos Conde *,1 and Reto Gassmann *,1 1Instituto de Investigação e Inovação em Saúde—i3S, Universidade do Porto, Porto, Portugal EMBO J (2020)39:e105279https://doi.org/10.15252/embj.2020105279 See also: LA Allan et al (June 2020) PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The spindle checkpoint protects against aneuploidy by ensuring that dividing cells only proceed with chromosome segregation once all kinetochores are stably attached to spindle microtubules. The checkpoint protein MAD1 localizes to the corona, a structural expansion of the kinetochore forming in the absence of microtubule attachment, but molecular mechanism or functional significance of this localization remains unknown. Recent results now show that cyclin B1 recruits MAD1 to the corona and that this MAD1 pool is required for robust checkpoint signaling. Accurate cell division requires that the dramatic structural re-organization of the cell upon mitotic entry proceeds in an ordered and coordinated manner. In prophase, the master mitotic kinase complex cyclin B1-CDK1 is imported into the nucleus to orchestrate chromosome condensation, nuclear envelope breakdown (NEB), and assembly of kinetochores, the supra-molecular protein structures that attach sister chromatids to spindle microtubules in prometaphase for subsequent segregation of sisters to opposite spindle poles during anaphase. Cyclin B1-CDK1 is recruited to different subcellular locations at different times, including kinetochores, but the underlying mechanisms of this spatial regulation remain poorly understood. One key function of cyclin B1-CDK1 is to activate the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that in turn targets cyclin B1 and other mitotic substrates for proteasomal degradation, thereby triggering anaphase and mitotic exit. APC/C activity is antagonized by the spindle assembly checkpoint (SAC), a kinetochore-based signaling pathway whose effector is a soluble APC/C inhibitor, the mitotic checkpoint complex (MCC). Kinetochores produce MCC until their stable attachment to microtubules, and a single unattached kinetochore can delay anaphase onset for hours. Cyclin B1-CDK1 is implicated in SAC signaling, but the precise contribution has been difficult to tease out from the multitude of its other functions. SAC signaling strength correlates with the amount of kinetochore-localized MAD1, a coiled-coil protein that together with its binding partner MAD2 constitutes the catalytic center of MCC production. MAD1 is recruited to the microtubule-proximal (outer) kinetochore region by BUB1, and this requires phosphorylation of BUB1 by the checkpoint kinase MPS1. MAD1-MAD2 also localize to the fibrous corona, a transient structural expansion that assembles peripherally to the outer kinetochore and aids in microtubule capture. How MAD1-MAD2 are recruited to the corona and the implications for SAC signaling are unclear. Recent studies from the Gruneberg/Barr, Pines, and Saurin groups describe a direct interaction between the MAD1 N-terminus and cyclin B1 (Alfonso-Pérez et al, 2019; Allan et al, 2020; Jackman et al, 2020). Allan et al (2020), writing in The EMBO Journal, use purified recombinant proteins to define a minimal binding site for cyclin B1 at the start of MAD1′s coiled-coil (residues 41–62), and a triple charge substitution in an acidic helical segment (E52K/E53K/E56K; hereafter 3EK) abolishes the stable interaction between reconstituted MAD1-MAD2 and cyclin B1-CDK1 complexes. An interesting open question is whether this acidic MAD1 patch, also independently identified by Jackman et al (2020), could represent a general docking motif for cyclin B1. If so, structural analysis of the cyclin B1-MAD1 interaction may provide important insights into how cyclin B1-CDK1 recognizes it substrates. To probe the function of the MAD1-cyclin B1 interaction, Jackman et al (2020) employed genome editing to generate diploid RPE1 cells expressing the MAD1 mutant E53K/E56K (hereafter 2EK), which cannot be co-immunoprecipitated with cyclin B1, and Allan et al (2020) used HeLa cells to express transgene-encoded MAD1 wild-type (WT) or 3EK in a MAD1 null background. MAD1(2EK)-expressing RPE1 cells become aneuploid, show reduced capacity for mitotic arrest in the presence of microtubule poisons, and exhibit severe chromosome mis-segregation after partial MPS1 inhibition that correlates with accelerated cyclin B1 degradation (Jackman et al, 2020). Sensitivity to partial MPS1 inhibition is also observed in HeLa MAD1(3EK) cells (Allan et al, 2020). Together with SAC defects associated with an N-terminal MAD1 deletion (residues 1–100) in HeLa cells (Alfonso-Pérez et al, 2019), these results unequivocally establish cyclin B1 as a bona fide checkpoint protein. All three groups then use their MAD1 mutants to dissect how cyclin B1 may contribute to SAC signaling. In interphase, MAD1 binds the inner nuclear pore protein TPR, and the MAD1-MAD2 complex remains associated with nuclear pores until mitotic entry. Live-cell imaging shows that MAD2 recruitment to newly formed kinetochores in late prophase is delayed in RPE1 MAD1(2EK) cells, and MAD1(2EK) itself continues to co-localize with TPR on condensing chromosomes at a time when wild-type MAD1 has already relocated to kinetochores (Jackman et al, 2020). Cyclin B1 also partially co-localizes with TPR at the nuclear pore, but this is not observed in MAD1(2EK) cells. Restoring cyclin B1 to nuclear pores by artificial tethering facilitates MAD2 release and partially rescues defective SAC signaling in MAD1(2EK) cells. This suggests that MAD1 recruits cyclin B1-CDK1 to promote its own release from nuclear pores prior to NEB, which in turn allows MAD1-MAD2 to accumulate at prophase kinetochores to start MCC production. Interestingly, partial MPS1 inhibition further increases MAD1(2EK) co-localization with TPR. This is consistent with recent work by Cunha-Silva et al (2020), which shows that MPS1-mediated phosphorylation of Drosophila melanogaster TPR disrupts its ability to bind MAD1 and is essential for kinetochore localization of MAD1 at levels required for robust SAC signaling and faithful chromosome segregation. How cyclin B1-CDK1 cooperates with MPS1 in promoting MAD1 dissociation from TPR remains to be determined. One plausible scenario is that cyclin B1-CDK1 phosphorylates MPS1 to potentiate MPS1 activity at the inner nuclear pore (Morin et al, 2012). Overall, the findings by Jackman et al (2020) and Cunha-Silva et al (2020) reveal a mechanism that coordinates cyclin B1-CDK1-mediated activation of APC/C at mitotic entry with its immediate inhibition by kinetochore-based SAC signaling (Fig 1). Figure 1. The cyclin B1-MAD1 interaction promotes checkpoint signaling at kinetochoresCyclin B1-CDK1 is imported into the prophase nucleus, where cyclin B1 binds to the MAD1 N-terminus (i) and CDK1 phosphorylates the APC/C, which results in APC/C activation (ii). Cyclin B1 binding to MAD1 releases MAD1 along with MAD2 from the inner nuclear pore component TPR, perhaps by locally potentiating MPS1 activity toward TPR (i, iii). Free MAD1-MAD2 is then recruited to the outer kinetochore by BUB1 to catalyze assembly of the MCC (iv), which is needed to antagonize rising APC/C activity (v). An MPS1 phosphorylation cascade plays a key role in SAC signaling: Phospho-KNL1 recruits BUB1, phospho-BUB1 recruits MAD1, and phosphorylation of the MAD1 C-terminus is needed for MCC catalysis (iv). Cyclin B1-CDK1 in turn phosphorylates MPS1 to promote its kinetochore localization and BUB1 to prime it for MPS1 phosphorylation. Thus, feedback between MPS1 and cyclin B1-CDK1 activities ensures efficient SAC activation. In early prometaphase, kinetochores assemble an outermost domain, the corona, which facilitates microtubule capture (vi). Cyclin B1 anchors the MAD1 N-terminus in the corona, and the highly elongated shape of the MAD1 coiled-coil may allow its C-terminus (and the associated MAD2) to reach the outer kinetochore to participate in MCC assembly. Since kinetochore recruitment of corona-anchored MAD1-MAD2 no longer depends on the KNL1-BUB1 pathway, MAD1-MAD2 can persist at kinetochores to catalyze MCC assembly even when MPS1 activity is decreased with small molecule inhibitors ("low MPS1 activity"). Thus, corona-localized MAD1-MAD2 makes SAC signaling more robust. This may become physiologically relevant when a single unattached kinetochore in late prometaphase needs to generate a strong enough checkpoint signal to inhibit the APC/C until that last chromosome is incorporated into the spindle. Download figure Download PowerPoint Just like MAD1, cyclin B1 accumulates prominently at unattached kinetochores in early prometaphase and disappears from kinetochores following microtubule attachment. MAD1 mutants that cannot bind cyclin B1 abolish (Alfonso-Pérez et al, 2019; Jackman et al, 2020) or reduce (Allan et al, 2020) cyclin B1 levels at kinetochores, which establishes MAD1 as a major, albeit not the only, kinetochore receptor for cyclin B1-CDK1. Alfonso-Pérez et al (2019) additionally note that kinetochores without cyclin B1 have reduced MPS1 levels. Interplay between cyclin B1-CDK1 and MPS1 may explain their co-dependency for kinetochore localization: MPS1 phosphorylation of BUB1 facilitates MAD1 recruitment (Faesen et al, 2017; Ji et al, 2017), and cyclin B1-CDK1 brought in via MAD1 reinforces MPS1 recruitment by phosphorylating that kinase in its kinetochore-binding domain (Hayward et al, 2019). Additionally, cyclin B1-CDK1 phosphorylation of BUB1 primes BUB1 for phosphorylation by MPS1 (Ji et al, 2017; Zhang et al, 2017). This positive feedback loop is predicted to facilitate rapid and efficient SAC activation (Alfonso-Pérez et al, 2019; Fig 1). It will be interesting to determine whether MAD1-bound cyclin B1 drives phosphorylation of additional CDK1 substrates at the kinetochore. Allan et al (2020) find that cyclin B1 localizes to the corona along with MAD1 while the cyclin binding-site mutant MAD1(3EK) is preferentially lost from this domain, arguing that cyclin B1 is MAD1's long-sought-after corona receptor. Residual MAD1(3EK) localization at the outer kinetochore likely represents MAD1-MAD2 complexes recruited by phospho-BUB1, as the MAD1(3EK), but not MAD1(WT), kinetochore signal is lost when nocodazole-arrested cells are challenged with low doses of MPS1 inhibitor. Detection of a C-terminal MPS1-dependent MAD1 phosphoepitope (pT716) involved in MCC catalysis (Faesen et al, 2017; Ji et al, 2017) reveals that mitotic duration after MPS1 inhibition correlates with pT716 levels at kinetochores, which are higher in the presence of MAD1(WT) than MAD1(3EK). This suggests that by anchoring MAD1 in the corona, cyclin B1 allows MAD1-MAD2 to persist at kinetochores when MPS1 activity is low, thereby maintaining pT716 at levels needed for robust SAC signaling. During prometaphase, MPS1-mediated phosphorylation of BUB1 at unattached kinetochores is gradually antagonized by the protein phosphatase complex PP2A-B56 (Qian et al, 2017). Indeed, MAD1(3EK) levels at unattached kinetochores are normal at NEB but drop significantly thereafter (Allan et al, 2020). The corona-localized MAD1-MAD2 pool may therefore give unattached "lost" chromosomes in late prometaphase critical extra time to complete their incorporation into the spindle (Fig 1). Interestingly, while MAD1 itself is present throughout the corona, the MAD1 pT716 signal is restricted to the outer kinetochore, where MPS1 is localized along with BUB1. This is consistent with the view that BUB1 lies at the center of MCC production (Faesen et al, 2017; Ji et al, 2017; Rodriguez-Rodriguez et al, 2018; Zhang et al, 2019). If MCC is not produced in the corona, how does corona-localized MAD1-MAD2 participate in SAC signaling? Visualization of the reconstituted complex between cyclin B1-CDK1 and MAD1-MAD2 by electron microscopy suggests that cyclin B1 at the MAD1 N-terminus and pT716 at the MAD1 C-terminus are separated by about 66 nm of coiled-coil (Allan et al, 2020). MAD1's highly elongated shape may therefore enable its C-terminal region to interact with BUB1 at the outer kinetochore, while its N-terminus is anchored in the corona. Overall, the findings by Allan et al (2020) support an integrated view of SAC signaling, in which the BUB1 and corona pools of MAD1 converge on the same site of MCC production (Rodriguez-Rodriguez et al, 2018; Zhang et al, 2019). Clarifying the underlying mechanism remains an important future goal. 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