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Coupling of Cdc20 inhibition and activation by BubR1.

ABSTRACT: Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.

SUBMITTER: Hein JB

PROVIDER: S-EPMC8025235 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Coupling of Cdc20 inhibition and activation by BubR1

Project description:Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.

2021-02-18 | MSV000086910 | MassIVE

Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition.

Project description:BubR1 is essential for the mitotic checkpoint that prevents aneuploidy in cellular progeny by triggering anaphase delay in response to kinetochores incorrectly/not attached to the mitotic spindle. Here, we define the molecular architecture of the functionally significant N-terminal region of human BubR1 and present the 1.8 A crystal structure of its tetratricopeptide repeat (TPR) domain. The structure reveals divergence from the classical TPR fold and is highly similar to the TPR domain of budding yeast Bub1. Shared distinctive features include a disordered loop insertion, a 3(10)-helix, a tight turn involving glycine positive Phi angles, and noncanonical packing of and between the TPR motifs. We also define the molecular determinants of the interaction between BubR1 and kinetochore protein Blinkin. We identify a shallow groove on the concave surface of the BubR1 TPR domain that forms multiple discrete and potentially cooperative interactions with Blinkin. Finally, we present evidence for a direct interaction between BubR1 and Bub1 mediated by regions C-terminal to their TPR domains. This interaction provides a mechanism for Bub1-dependent kinetochore recruitment of BubR1. We thus present novel molecular insights into the structure of BubR1 and its interactions at the kinetochore-microtubule interface. Our studies pave the way for future structure-directed engineering aimed at dissecting the roles of kinetochore-bound and other pools of BubR1 in vivo.

| S-EPMC2863239 | biostudies-literature

Catalytic assembly of the mitotic checkpoint inhibitor BubR1-Cdc20 by a Mad2-induced functional switch in Cdc20.

Project description:The mitotic checkpoint acts to maintain chromosome content by generation of a diffusible anaphase inhibitor. Unattached kinetochores catalyze a conformational shift in Mad2, converting an inactive open form into a closed form that can capture Cdc20, the mitotic activator of the APC/C ubiquitin ligase. Mad2 binding is now shown to promote a functional switch in Cdc20, exposing a previously inaccessible site for binding to BubR1's conserved Mad3 homology domain. BubR1, but not Mad2, binding to APC/C(Cdc20) is demonstrated to inhibit ubiquitination of cyclin B. Closed Mad2 is further shown to catalytically amplify production of BubR1-Cdc20 without necessarily being part of the complex. Thus, the mitotic checkpoint is produced by a cascade of two catalytic steps: an initial step acting at unattached kinetochores to produce a diffusible Mad2-Cdc20 intermediate and a diffusible step in which that intermediate amplifies production of BubR1-Cdc20, the inhibitor of cyclin B ubiquitination, by APC/C(Cdc20).

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Recruitment of Cdc20 to the kinetochore requires BubR1 but not Mad2 in Drosophila melanogaster.

Project description:To prevent aneuploidy, cells require a mitotic surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC prevents metaphase/anaphase transition by blocking the ubiquitylation and destruction of cyclin B and securin via the Cdc20-activated anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway. This checkpoint involves the kinetochore proteins Mad2, BubR1, and Cdc20. Mad2 and BubR1 are inhibitors of the APC/C, but Cdc20 is an activator. Exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear; in vertebrates, most current models suggest that kinetochore-bound Mad2 is required for initial binding to Cdc20 to form a stable complex that includes BubR1. Here, we show that the Mad2 kinetochore dimerization recruitment mechanism is conserved and that the recruitment of Cdc20 to kinetochores in Drosophila requires BubR1 but not Mad2. BubR1 and Mad2 can bind to Cdc20 independently, and the interactions are enhanced after cells are arrested at mitosis by the depletion of Cdc27 using RNA interference (RNAi) in S2 cells or by MG132 treatment in syncytial embryos. These findings offer an explanation of why BubR1 is more important than Mad2 for SAC function in flies. These findings could lead to a better understanding of vertebrate SAC mechanisms.

| S-EPMC2897573 | biostudies-literature

BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase.

Project description:BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control, as an active kinase at unattached kinetochores, and as a cytosolic inhibitor of APC/C(Cdc20) activity, as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, we demonstrate that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. We find that the BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G(2) phase prior to mitosis onset. Together, our results suggest that kinetochore-bound BubR1 is nonessential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/C(Cdc20) during interphase to prevent unscheduled degradation of specific APC/C substrates.

| S-EPMC2659634 | biostudies-literature

Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding.

Project description:Premature anaphase onset is prevented by the mitotic checkpoint through production of a "wait anaphase" inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, we demonstrate that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. We show that purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. Our results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1.

| S-EPMC2655205 | biostudies-literature

Bimodal activation of BubR1 by Bub3 sustains mitotic checkpoint signaling.

Project description:The mitotic checkpoint (also known as the spindle assembly checkpoint) prevents premature anaphase onset through generation of an inhibitor of the E3 ubiquitin ligase APC/C, whose ubiquitination of cyclin B and securin targets them for degradation. Combining in vitro reconstitution and cell-based assays, we now identify dual mechanisms through which Bub3 promotes mitotic checkpoint signaling. Bub3 enhances signaling at unattached kinetochores not only by facilitating binding of BubR1 but also by enhancing Cdc20 recruitment to kinetochores mediated by BubR1's internal Cdc20 binding site. Downstream of kinetochore-produced complexes, Bub3 promotes binding of BubR1's conserved, amino terminal Cdc20 binding domain to a site in Cdc20 that becomes exposed by initial Mad2 binding. This latter Bub3-stimulated event generates the final mitotic checkpoint complex of Bub3-BubR1-Cdc20 that selectively inhibits ubiquitination of securin and cyclin B by APC/C(Cdc20). Thus, Bub3 promotes two distinct BubR1-Cdc20 interactions, involving each of the two Cdc20 binding sites of BubR1 and acting at unattached kinetochores or cytoplasmically, respectively, to facilitate production of the mitotic checkpoint inhibitor.

| S-EPMC4210015 | biostudies-literature

sFRP3 inhibition improves age-related cellular changes in BubR1 progeroid mice.

Project description:Wnt signaling is a well-known molecular pathway in age-related pathogenesis and therapy of disease. While prior studies have mainly focused on Wnt ligands or Wnt activators, the in vivo functions of naturally secreted Wnt inhibitors are not clear, especially in brain aging. Using BubR1H/H mice as a novel mouse model of accelerated aging, we report that genetic inhibition of sFRP3 restores the reduced body and brain size observed in BubR1H/H mice. Furthermore, sFRP3 inhibition ameliorates hypomyelination in the corpus callosum and rescues neural progenitor proliferation in the hippocampal dentate gyrus of BubR1H/H mice. Taken together, our study identifies sFRP3 as a new molecular factor that cooperates with BubR1 function to regulate brain development, myelination, and hippocampal neurogenesis.

| S-EPMC6413750 | biostudies-literature

BubR1 Promotes Bub3-Dependent APC/C Inhibition during Spindle Assembly Checkpoint Signaling.

Project description:The spindle assembly checkpoint (SAC) prevents premature sister chromatid separation during mitosis. Phosphorylation of unattached kinetochores by the Mps1 kinase promotes recruitment of SAC machinery that catalyzes assembly of the SAC effector mitotic checkpoint complex (MCC). The SAC protein Bub3 is a phospho-amino acid adaptor that forms structurally related stable complexes with functionally distinct paralogs named Bub1 and BubR1. A short motif ("loop") of Bub1, but not the equivalent loop of BubR1, enhances binding of Bub3 to kinetochore phospho-targets. Here, we asked whether the BubR1 loop directs Bub3 to different phospho-targets. The BubR1 loop is essential for SAC function and cannot be removed or replaced with the Bub1 loop. BubR1 loop mutants bind Bub3 and are normally incorporated in MCC in vitro but have reduced ability to inhibit the MCC target anaphase-promoting complex (APC/C), suggesting that BubR1:Bub3 recognition and inhibition of APC/C requires phosphorylation. Thus, small sequence differences in Bub1 and BubR1 direct Bub3 to different phosphorylated targets in the SAC signaling cascade.

| S-EPMC5640511 | biostudies-literature

BubR1 deficiency results in enhanced activation of MEK and ERKs upon microtubule stresses.

Project description:UNLABELLED:Disruption of microtubules activates the spindle checkpoint, of which BubR1 is a major component. Our early studies show that BubR1 haplo-insufficiency results in enhanced mitotic slippage in vitro and tumorigenesis in vivo. OBJECTIVE:Given that both MAPKs/ERKs and MEK play an important role during mitosis, we investigated whether there existed regulatory relationship between the MAPK signalling pathway and BubR1. METHOD AND RESULTS:Here, we have demonstrated that BubR1 deficiency is correlated with enhanced activation of MEK and ERKs after disruption of microtubule dynamics. Specifically, treatment with nocodazole and paclitaxel resulted in hyper-activation of ERKs and MEK in BubR1(+/-) murine embryonic fibroblasts (MEF) compared to that of wild-type MEFs. This enhanced activation of ERKs and MEK was at least partly responsible for more successful proliferation completion when cells were treated with nocodazole. BubR1 knockdown via RNAi resulted in enhanced activation of ERKs and MEK in HeLa cells, correlating with inhibition of PP1, a negative regulator of MEK. Moreover, when BubR1 was partially inactivated due to premature missegregation of chromosomes after Sgo1 depletion, phosphorylation of ERKs and MEK was enhanced in mitotic cells; in contrast, little, if any activated ERKs and MEK were detected in mitotic cells induced by nocodazole. Furthermore, BubR1, activated ERKs and activated MEK all localized to spindle poles during mitosis, and also, the proteins physically interacted with each other. CONCLUSION:Our studies suggest that there exists a cross-talk between spindle checkpoint components and ERKs and MEK and that BubR1 may play an important role in mediating the cross-talk.

| S-EPMC6495970 | biostudies-literature

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