Catalytic Assembly of the Mitotic Checkpoint Inhibitor BubR1-Cdc20 by a Mad2-Induced Functional Switch in Cdc20 (original) (raw)
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Checkpoint Signalling: Mad2 Conformers and Signal Propagation
Current Biology, 2005
The spindle checkpoint is one of the key self-monitoring systems of the eukaryotic cell cycle, acting to delay anaphase until all the sister chromatids are appropriately lined up so that the replicated genome is correctly segregated with one copy going to each daughter cell. Mad2 is a central player in the spindle checkpoint's regulation of anaphase onset [1,2]. Mad2 interacts with Mad1, which recruits it to unattached kinetochores [3], and with the spindle checkpoint effector Cdc20 [4,5]. The latter interaction can take place either in a Mad2-Cdc20 complex or in the mitotic checkpoint complex (MCC):Mad2-Mad3/BubR1-Bub3-Cdc20 [6,7]. There is much controversy over the site(s) of assembly of these complexes, their mode of action and their importance as in vivo 'anaphase inhibitors'. Mad2 multimers have been studied for some time, although their physiological relevance was uncertain [8,9]. NMR and crystal structures of Mad2 alone or with binding peptides from Mad1 or Cdc20 have shown how the respective complexes are formed [10-13]. A 'safety-belt' structure formed by the carboxy-terminal tail of Mad2 explains why they are so stable [13].
BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex
Journal of Biological Chemistry, 2011
The mitotic checkpoint maintains genomic stability by ensuring that chromosomes are accurately segregated during mitosis. When the checkpoint is activated, the mitotic checkpoint complex (MCC), assembled from BUBR1, BUB3, CDC20, and MAD2, directly binds and inhibits the anaphase-promoting complex/cyclosome (APC/C) until all chromosomes are properly attached and aligned. The mechanisms underlying MCC assembly and MCC-APC/C interaction are not well characterized. Here, we show that a novel interaction between BUBR1 and closed MAD2 (C-MAD2) is essential for MCC-mediated inhibition of APC/C. Intriguingly, Arg 133 and Gln 134 in C-MAD2 are required for BUBR1 interaction. The same residues are also critical for MAD2 dimerization and MAD2 binding to p31 comet , a mitotic checkpoint silencing protein. Along with previously characterized BUBR1-CDC20 and C-MAD2-CDC20 interactions, our results underscore the integrity of the MCC for its activity and suggest the fundamental importance of the MAD2 ␣C helix in modulating mitotic checkpoint activation and silencing.
The Mad1/Mad2 Complex as a Template for Mad2 Activation in the Spindle Assembly Checkpoint
Current Biology, 2005
The SAC monitors this process and delays anaphase until all chromosomes have attained bipolar attachment [1,. Spindle microtubules attach on kinetochores, prothe tension between sister chromatids building up during this process [1, 2]. European Institute of Oncology Via Ripamonti 435 The SAC is conserved in all eukaryotes and includes mitotic arrest deficient (MAD) and budding uninhibited 20141 Milano Italy by benzimidazole (BUB) genes [1, 2]. Their products temporarily sequester Cdc20, an activator of the ana-Hill phase-promoting complex/cyclosome, the E3 ubiquitin ligase targeting securin and cyclin B for proteasome-607 Fordham Hall Chapel Hill, North Carolina 27599 mediated degradation. Destruction of securin activates separase, which triggers anaphase by cleaving the complex linking the sister chromatids, named Cohesin [4, 5]. The sequestration of Cdc20 requires Mad2 and Summary BubR1 [1, 6]. Mad2 is a 002ف residue protein containing a Horma domain [7]. BubR1 consist of an N-terminal Background: The spindle assembly checkpoint (SAC) domain containing Bub3 and Cdc20 binding sites and a imparts fidelity to chromosome segregation by delaying C-terminal kinase domain (missing in the budding yeast anaphase until all sister chromatid pairs have become ortholog, Mad3) [1, 6]. Both Mad2 and BubR1 bind bipolarly attached. Mad2 is a component of the SAC Cdc20 tightly, and their effects are synergic [8-13]. Coneffector complex that sequesters Cdc20 to halt anasistently, Mad2, BubR1 (or Mad3), Bub3, and Cdc20 phase. In prometaphase, Mad2 is recruited to kinetoenter a single complex known as mitotic checkpoint chores with the help of Mad1, and it is activated to bind complex (MCC) [11, 14-16]. Cdc20. These events are linked to the existence of two Unattached kinetochores establish and maintain the distinct conformers of Mad2: a closed conformer bound SAC, and all SAC proteins show kinetochore localization to its kinetochore receptor Mad1 or its target in the in prometaphase [1, 3, 17]. Fluorescence recovery after checkpoint Cdc20 and an open conformer unbound to photobleaching (FRAP) revealed stable kinetochore resthese ligands.
Journal of Biological Chemistry, 2008
Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.
Biophysical Chemistry, 2008
For successful mitosis, metaphase has to be arrested until all centromeres are properly attached. The onset of anaphase, which is initiated by activating the APC, is controlled by the spindle assembly checkpoint M SAC. Mad2, which is a constitutive member of the M SAC, is supposed to inhibit the activity of the APC by sequestering away its co-activator Cdc20. Mad1 recruits Mad2 to unattached kinetochores and is compulsory for the establishment of the Mad2 and Cdc20 complexes. Recently, based on results from in vivo and in vitro studies, two biochemical models were proposed: the Template and the Exchange model. Here, we derive a mathematical description to compare the dynamical behaviour of the two models. Our simulation analysis supports the Template model. Using experimentally determined values for the model parameters, the Cdc20 concentration is reduced down to only about half. Thus, although the Template model displays good metaphase-to-anaphase switching behaviour, it is not able to completely describe M SAC regulation. This situation is neither improved by amplification nor by p31 comet inhibition. We speculate that either additional reaction partners are required for total inhibition of Cdc20 or an extended mechanism has to be introduced for M SAC regulation.
Structure of the Mad2 spindle assembly checkpoint protein and its interaction with Cdc20
Nature structural biology, 2000
The checkpoint protein Mad2 inhibits the activity of the anaphase promoting complex by sequestering Cdc20 until all chromosomes are aligned at the metaphase plate. We report the solution structure of human Mad2 and its interaction with Cdc20. Mad2 possesses a novel three-layered alpha/beta fold with three alpha-helices packed between two beta-sheets. Using deletion mutants we identified the minimal Mad2-binding region of human Cdc20 as a 40-residue segment immediately N-terminal to the WD40 repeats. Mutagenesis and NMR titration experiments show that a C-terminal flexible region of Mad2 is required for binding to Cdc20. Mad2 and Cdc20 form a tight 1:1 heterodimeric complex in which the C-terminal segment of Mad2 becomes folded. These results provide the first structural insight into mechanisms of the spindle assembly checkpoint.
The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex
Nature Communications, 2023
The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly. During mitosis, a eukaryotic cell divides into two genetically identical daughter cells. To achieve this, the duplicated chromosomes in the parent cell must be equally distributed into the daughter cells. The spindle assembly checkpoint (SAC) serves as a surveillance mechanism to ensure that duplicated chromosomes are stably attached to spindle microtubules through an adapter structure named the kinetochore. Kinetochores lacking end-on microtubule attachment activate the SAC to prevent premature anaphase onset and avoid chromosome missegregation. The effector molecule generated upon SAC activation is the Mitotic Checkpoint Complex (MCC). The MCC consists of two subcomplexes: BUBR1:BUB3 and CDC20:MAD2 1,2. It inhibits the E3 ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) 3-5. APC/C ubiquitinates Cyclin B1, a key mitosis regulator, thereby targeting it for proteasome-mediated degradation 6-8. Inhibition of the APC/C suppresses the degradation of Cyclin B1, which in turn delays anaphase onset. The formation of the CDC20:MAD2 complex has been identified as the rate-limiting step in the assembly of the MCC 9,10. Other checkpoint proteins, including the MAD1:MAD2 complex and the BUB1:BUB3 complex, catalyze this reaction, by recruiting the MCC subunits at