Research Article: The mitotic checkpoint complex (MCC): looking back and forth after 15 years

Date Published: January 25, 2017

Publisher:

Author(s): Song-Tao Liu, Hang Zhang.

http://doi.org/10.3934/molsci.2016.4.597

Abstract

The mitotic checkpoint is a specialized signal transduction pathway that contributes to the fidelity of chromosome segregation. The signaling of the checkpoint originates from defective kinetochore-microtubule interactions and leads to formation of the mitotic checkpoint complex (MCC), a highly potent inhibitor of the Anaphase Promoting Complex/Cyclosome (APC/C)—the E3 ubiquitin ligase essential for anaphase onset. Many important questions concerning the MCC and its interaction with APC/C have been intensively investigated and debated in the past 15 years, such as the exact composition of the MCC, how it is assembled during a cell cycle, how it inhibits APC/C, and how the MCC is disassembled to allow APC/C activation. These efforts have culminated in recently reported structure models for human MCC:APC/C supra-complexes at near-atomic resolution that shed light on multiple aspects of the mitotic checkpoint mechanisms. However, confusing statements regarding the MCC are still scattered in the literature, making it difficult for students and scientists alike to obtain a clear picture of MCC composition, structure, function and dynamics. This review will comb through some of the most popular concepts or misconceptions about the MCC, discuss our current understandings, present a synthesized model on regulation of CDC20 ubiquitination, and suggest a few future endeavors and cautions for next phase of MCC research.

Partial Text

Feedback control is a common regulatory mechanism in biological processes. At the end of last century, through a series of landmark discoveries the mitotic checkpoint (or spindle assembly checkpoint or spindle checkpoint) was established as a critical feedback signal transduction pathway to protect cells from chromosomal instability. These results included at least the following events: In 1989, Hartwell and Weinert proposed the concept of cell cycle checkpoint to explain DNA damage responses in Rad9 mutants in S. cerevisiae [1]. In 1991, Li and Murray, Hoyt and colleagues carried out genetic screening and respectively reported mitotic arrest-deficient (MAD) and budding uninhibited by benzimidazole (BUB) checkpoint genes that are responsible for faithful chromosome segregation [2,3]. In 1995 and 1996, the multi-subunit E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C) was found to be critical for initiating metaphase-to-anaphase transition through promoting cyclin B and securin degradation [4–9]. Later, it was shown that CDC20, an APC/C substrate-specifying subunit, is the target of the mitotic checkpoint [10–13].

As early as in 1950s, scientists have realized that cells have dedicated mechanisms to ensure the well-known order of events during mitosis (for review see [28]). In 1995 Rieder et al. used laser microbeam to ablate an unattached kinetochore and found that anaphase would initiate even if the chromosome with destroyed kinetochore was not aligned [29]. Around the same time Li and Nicklas used a microneedle to apply tension to an unaligned chromosome and found that the cell proceeded into anaphase [30]. These classical experiments consolidated cell biological concepts that dividing cells check either microtubule occupancy or tension development at kinetochores to determine the timing of anaphase onset. Together with progress in genetic dissection of mitotic checkpoint genes in yeast and subsequent molecular and biochemical studies, a framework of understanding about the mitotic checkpoint has been established (Figure 1).

As mentioned, the MCC is regarded as the effector of the mitotic checkpoint signal transduction pathway. The target of the MCC is the APC/C E3 ubiquitin ligase, or more specifically the APC/CCDC20 holoenzyme. The core of human APC/C (apo-APC/C) is a 1.2 megadalton complex composed of 19 subunits of 14 distinct proteins [76–78]. Apo-APC/C is stable throughout the cell cycle though many subunits are heavily phosphorylated during mitosis [79–82]. To function properly as a holoenzyme, apo-APC/C associates with either CDC20 or CDH1, two related proteins that act as both a substrate binding subunit and an activator protein [76,79,80,82,83]. Two common motifs (called degrons) in substrates recognized by CDC20 or CDH1 are destruction box (or D box, RxxLXXXXN) and Lys-Glu-Asn (KEN) box [84,85]. Some other degrons have also been reported such as “ABBA” motif (or A motif, Phe box, IC20BD) or “CRY” box [86–90]. The APC/CCDC20 holoenzyme is responsible for ubiquitination of cyclin B and securin, driving them for proteasome-mediated degradation [77]. Cyclin B is the activating subunit of Cdk1, whose activity is indispensable for mitosis progression. Securin can inhibit separase, which is able to cleave the cohesin ring structures and cause separation of sister chromatids. When APC/CCDC20 is inhibited by the MCC, cyclin B and securin are spared and anaphase onset is delayed (Figure 1) [23,24,27].

As the effector of the mitotic checkpoint, the MCC contains 4 types of components in most organisms studied: BUBR1, BUB3, CDC20 and MAD2. All four proteins are evolutionarily conserved; however, BUBR1 or its counterpart in lower eukaryotes can vary in size to a significant degree and BUB3 is absent from the fission yeast MCC [19,22]. We will focus our discussion on human proteins and cite other organisms when necessary.

The physiological function of the MCC is to inhibit APC/CCDC20, but the MCC is one of the many inhibitors of the APC/CCDC20. Its various components such as BUBR1 and MAD2 or sub-complexes such as BUBR1:BUB3, BUBR1:BUB3:CDC20 (called BBC) and MAD2:CDC20 all have been reported as inhibitors of APC/CCDC20, at least in vitro [13,137–141]. Emi1 is also a well characterized inhibitor of APC/CCDC20 especially during G2 and early mitosis [142]. Another protein complex, termed Mitotic Checkpoint Factor 2 (MCF2) was also suggested to antagonize APC/CCDC20, but its molecular nature remains unknown [143]. The presence of all these inhibitors or inhibitor complexes, together with historical course of events and different research methodology in various organisms, have confounded the understanding of how APC/CCDC20 is inhibited and when MCC is assembled, and sometimes challenged the very existence of the MCC. Again, the most recent models on the MCC:APC/CCDC20 structures have convincingly lifted lots of confusion [20,21]. In this section we will briefly summarize our view about temporal control of MCC assembly (Figure 4) and then discuss the origin and validity of some popular concepts or misconceptions in the field.

Despite many conflicting results in the past 15 years, the human MCC as an entity is now recognized as a complex of BUBR1:BUB3:CDC20:MAD2 at 1:1:1:1 ratio. The conclusion has been strongly supported by biochemical and structural studies reported in the past year [14,20,21,94]. Although Izawa and Pines showed that the MCC can bind to another CDC20 molecule in vitro, in vivo this second CDC20 is most likely the one associated with APC/C as revealed by the structural studies [14,20,21,94].

To better understand how the MCC inhibits APC/CCDC20, it is certainly helpful to first understand how APC/CCDC20 carries out its E3 ubiquitin ligase activity. In the ubiquitin-proteasome system, ubiquitin is normally first activated by covalently linking to E1 (ubiquitin activating enzyme) and then transferred to E2 (ubiquitin conjugating enzyme) through a thioester bond formed between the C-terminal carboxylic group of ubiquitin and a cysteine at the E2 active site. The multi-subunit E3 ubiquitin ligases usually interact simultaneously with and bring together an E2 and a substrate to facilitate transferring activated ubiquitin from E2 to the substrate. APC/CCDC20 works with two E2s: UbcH10 primarily responsible for initial ligation of ubiquitin to substrate lysine residues, and Ube2S for elongating the ubiquitin chain through conjugating ubiquitin polymers [53,167–171]. Note that UbcH10-mediated mono-ubiquitination at multiple sites works as degradation signals as well, but polyubiquitin or branched chains on individual sites of the target protein is more common [172,173]. To recruit E2s, APC/CCDC20 employs its catalytic core subunits APC2 and APC11, which tightly associate with each other [174]. APC2 has a cullin domain, while APC11 has a RING domain found in many other E3 ubiquitin ligases [174]. Recent results have shown that UbcH10 is recruited through interaction with APC11 RING domain, but Ube2S is recruited through APC2 [167,168,175]. To recruit substrates, APC/C requires association of CDC20 and CDH1 [102,103,176]. For the two key substrates of APC/CCDC20, the dominant degrons in cyclin B (including B1 and B2) and securin are D boxes. It has been established that CDC20 and APC10 work together to recognize D boxes [107,108].

When sister kinetochores are properly attached by spindle microtubules and aligned at metaphase plate, cells have ~15 min to silence the mitotic checkpoint for anaphase onset [29,66,67]. During this time, attached kinetochores stop generating checkpoint signals (C-MAD2 for example). However, cells still need disassemble the pre-formed MCC and MCC:APC/CCDC20 complexes to allow effective metaphase-to-anaphase transition. As both MCC and MCC:APC/CCDC20 complexes are very stable, the disassembly requires energy input and delicate regulation [45,179,180]. In recent years, it is understood that the tug of war between checkpoint activation and silencing events, or assembly and disassembly of the MCC and MCC:APC/CCDC20, may be ongoing throughout mitosis [23–27]. Nevertheless, there should be no doubt that the net output is that checkpoint activation dominates during prometaphase and checkpoint silencing prevails once the metaphase-to-anaphase transition is initiated. Currently two major mechanisms for disassembling the MCC have been established, mediated by CDC20 ubiquitination and TRIP13 AAA-ATPase respectively.

For a long time the MCC assembly and function was thought to be determined by phosphorylation of its subunits since all four MCC components can be phosphorylated during mitosis in human cells (Table S2). Take BUBR1 as an example. Early studies showed that BUBR1 is hyperphosphorylated upon entry into mitosis [223–225]. Several phosphorylation sites on BUBR1 have been attributed to Aurora B, Plk1, Cdk1 or MPS1 kinases, as slower migrating BUBR1 species on SDS-PAGE were reduced or phospho-signals disappeared in kinase assays or mass spectrometry when Cdk1, Plk1, Aurora B or MPS1 inhibitors were applied [132,223,224,226,227]. BUBR1 phosphorylation may thus be a consequence of regulation by multiple kinases. Moreover, the possibility of BUBR1 autophosphorylation via its own (pseudo)kinase domain cannot be completely excluded [34,70,227,228]. However, many experiments demonstrated that phosphorylation-deficient BUBR1 mutants, though leading to aberrant kinetochore-microtubule attachment, exerted normal mitotic checkpoint function [91,227,229,230]. Similarly, phosphatase treatment of BUBR1 immunoprecipitates did not change the levels of association with other MCC subunits, indicating that MCC assembly is uncoupled from BUBR1 phosphorylation [132]. In addition, multiple binary interactions between MCC components have been observed using recombinant proteins isolated from E. coli (lacking posttranslational modifications) [93,119,137,138]. Furthermore, MCC forms in G1/S cells expressing a C-conformer locked MAD2 mutant [136]. Recombinant MCCs isolated from insect cells are also competent in inhibiting mitotic or recombinant APC/CCDC20 (itself artificially phosphorylated either through treatment with phosphatase inhibitor okadaic acid or by mutating 100 serine/threonine residues into phosphomimic glutamic acid) [20,21,136]. Taken together, current results suggested that phosphorylation of BUBR1 or any MCC subunit is not essential for MCC assembly and its APC/C inhibitory activity.

Mathematical modeling or systems biology approach to understand the mitotic checkpoint signaling started from pioneering work by Doncic et al. [242] and Sear & Howard [243]. More complicated models have been developed over the years to evaluate both activation and silencing of the mitotic checkpoint [197,244–250]. Some results have provided certain guidance for experiments. For example, Doncic et al. captured the two most demanding features of the mitotic checkpoint: 1, A single unattached kinetochore could maintain inhibition of mitotic arrest of the cell; 2, Rapid inactivation of the checkpoint needs to happen once the last kinetochore is attached. Based on parameters for the closed mitosis in a budding yeast cell, their modeling supported the presence of a diffusible inhibitor catalyzed by an unattached kinetochore. They rejected a model that inhibition of the cell cycle activator (i.e. CDC20) only occurs at the kinetochore on the basis of inefficient inhibition; and rejected another autocatalysis model (i.e., CDC20:C-MAD2 in the cytoplasm could amplify O→C-MAD2 production) for inefficient inactivation of the checkpoint. Sear and Howard modeled open mitosis in metazoans and suggested a species of inhibited CDC20 (c*) that cannot amplify inhibitory signals [243]. Ibrahim suggested that MAD2 is insufficient to sequester CDC20 and proposed MCC might work through direct inhibition of the APC/C not sequestration of CDC20 [249,250]. Verdugo et al. showed that double negative feedback loop between MCC and APC/C could create good inhibition but does not allow timely inactivation of the checkpoint [197].

To maintain proper timing for chromosome segregation, delicate mechanisms have evolved to ensure the dynamic balance between mitotic checkpoint activation and silencing. At molecular level MCC assembly and disassembly happens throughout a cell cycle but the resultant output of signaling determines that checkpoint activation and silencing happens at a strict temporal order at cellular level. To better understand the control of the resultant output at each phase and transition between phases is going to be the major task in the next few years. Research at cellular level is still going to be important to provide the contextual information, but biochemical dissection that could isolate events for detailed mechanistic studies has demonstrated very impressive power and will be essential for pushing the field forward.

 

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http://doi.org/10.3934/molsci.2016.4.597

 

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