The Mitotic Checkpoint Complex Requires an Evolutionary Conserved Cassette to Bind and Inhibit Active APC/C.

The Spindle Assembly Checkpoint (SAC) ensures genomic stability by preventing sister chromatid separation until all chromosomes are attached to the spindle. It catalyzes the production of the Mitotic Checkpoint Complex (MCC), which inhibits Cdc20 to inactivate the Anaphase Promoting Complex/Cyclosome (APC/C). Here we show that two Cdc20-binding motifs in BubR1 of the recently identified ABBA motif class are crucial for the MCC to recognize active APC/C-Cdc20. Mutating these motifs eliminates MCC binding to the APC/C, thereby abolishing the SAC and preventing cells from arresting in response to microtubule poisons. These ABBA motifs flank a KEN box to form a cassette that is highly conserved through evolution, both in the arrangement and spacing of the ABBA-KEN-ABBA motifs, and association with the amino-terminal KEN box required to form the MCC. We propose that the ABBA-KEN-ABBA cassette holds the MCC onto the APC/C by binding the two Cdc20 molecules in the MCC-APC/C complex.

Spindle assembly checkpoint activation and silencing at kinetochores.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that promotes accurate chromosome segregation in mitosis. The checkpoint senses the attachment state of kinetochores, the proteinaceous structures that assemble onto chromosomes in mitosis in order to mediate their interaction with spindle microtubules. When unattached, kinetochores generate a diffusible inhibitor that blocks the activity of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase required for sister chromatid separation and exit from mitosis. Work from the past decade has greatly illuminated our understanding of the mechanisms by which the diffusible inhibitor is assembled and how it inhibits the APC/C. However, less is understood about how SAC proteins are recruited to kinetochores in the absence of microtubule attachment, how the kinetochore catalyzes formation of the diffusible inhibitor, and how attachments silence the SAC at the kinetochore. Here, we summarize current understanding of the mechanisms that activate and silence the SAC at kinetochores and highlight open questions for future investigation.

Cubism and the cell cycle: the many faces of the APC/C.

One does not often look to analytic cubism for insights into the control of the cell cycle, but Pablo Picasso beautifully encapsulated the fundamentals when he said that "every act of creation is, first of all, an act of destruction". The rapid destruction of specific cell cycle regulators at just the right moment in the cell cycle ensures that daughter cells receive an equal and identical set of chromosomes from their mother and that DNA replication always follows mitosis. Remarkably, one protein complex is responsible for this surgical precision, the APC/C (anaphase-promoting complex, also known as the cyclosome). The APC/C is tightly regulated by its co-activators and by the spindle assembly checkpoint.

The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C.

The spindle assembly checkpoint (SAC) maintains genomic stability by delaying chromosome segregation until the last chromosome has attached to the mitotic spindle. The SAC prevents the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase from recognizing cyclin B and securin by catalysing the incorporation of the APC/C co-activator, CDC20, into a complex called the mitotic checkpoint complex (MCC). The SAC works through unattached kinetochores generating a diffusible 'wait anaphase' signal that inhibits the APC/C in the cytoplasm, but the nature of this signal remains a key unsolved problem. Moreover, the SAC and the APC/C are highly responsive to each other: the APC/C quickly targets cyclin B and securin once all the chromosomes attach in metaphase, but is rapidly inhibited should kinetochore attachment be perturbed. How this is achieved is also unknown. Here, we show that the MCC can inhibit a second CDC20 that has already bound and activated the APC/C. We show how the MCC inhibits active APC/C and that this is essential for the SAC. Moreover, this mechanism can prevent anaphase in the absence of kinetochore signalling. Thus, we propose that the diffusible 'wait anaphase' signal could be the MCC itself, and explain how reactivating the SAC can rapidly inhibit active APC/C.

A PP1-PP2A phosphatase relay controls mitotic progression.

The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1-cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.

Quantitative proteomics reveals the basis for the biochemical specificity of the cell-cycle machinery.

Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.

Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex.

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.

The APC/C: a smörgåsbord for proteolysis.

In a recent issue of Molecular Cell, Matyskiela and Morgan (2009) identify the sites on the APC/C that are required for activation and substrate binding, providing insights into how the APC/C works, with implications for the spindle assembly checkpoint that regulates it.

Torin1-mediated TOR kinase inhibition reduces Wee1 levels and advances mitotic commitment in fission yeast and HeLa cells.

The target of rapamycin (TOR) kinase regulates cell growth and division. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell division, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in Schizosaccharomyces pombe. A mutation of the Tor2 glycine residue (G2040D) that lies adjacent to the key Torin-interacting tryptophan provides Torin1 resistance, confirming the specificity of Torin1 for TOR. Using this mutation, we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1-induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in Wee1 levels. Experiments in human cell lines recapitulated these yeast observations: mammalian TOR (mTOR) was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls.

Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A.

Successful mitosis requires the right protein be degraded at the right time. Central to this is the spindle checkpoint that prevents the destruction of securin and cyclin B1 when there are improperly attached chromosomes. The principal target of the checkpoint is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C). A Drosophila Cdc20/fizzy mutant arrests in mitosis with high levels of cyclins A and B, but paradoxically the spindle checkpoint does not stabilize cyclin A. Here, we investigated this paradox and found that Cdc20 is rate limiting for cyclin A destruction. Indeed, Cdc20 binds efficiently to cyclin A before and in mitosis, and this complex has little associated Mad2. Furthermore, the cyclin A complex must bind to a Cks protein to be degraded independently of the checkpoint. Thus, we identify a crucial role for the Cks proteins in mitosis and one mechanism by which the APC/C can target substrates independently of the spindle checkpoint.

Human Mob1 proteins are required for cytokinesis by controlling microtubule stability.

The completion of cytokinesis requires abscission of the midbody, a microtubule-rich cytoplasmic bridge that connects the daughter cells before their final separation. Although it has been established that both the midbody structure and membrane fusion are essential for abscission, the biochemical machinery and the cellular processes of abscission remain ill-defined. Here we report that human Mob1A and Mob1B proteins are involved in the regulation of abscission of the intercellular bridge. The Mob family is a group of highly conserved proteins in eukaryotes, described as binding partners as well as co-activators of protein kinases of the Ndr family, and as members of the Hippo pathway. We show that depletion of Mob1A and Mob1B by RNAi causes abscission failure as a consequence of hyper-stabilization of microtubules in the midbody region. Interestingly, depleting Mob1 also increases cell motility after cytokinesis, and induces prolonged centriole separation in G1 phase. In contrast, centrosomes fail to split when either Mob1A or Mob1B is overexpressed. Our findings indicate that human Mob1 proteins are involved in the regulation of microtubule stability at the midbody. We conclude that Mob1A and Mob1B are needed for cell abscission and centriole re-joining after telophase and cytokinesis.

Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins.

Post-translational modification (PTM) of proteins plays an important part in mediating protein interactions and/or the recruitment of specific protein targets. PTM can be mediated by the addition of functional groups (for example, acetylation or phosphorylation), peptides (for example, ubiquitylation or sumoylation), or nucleotides (for example, poly(ADP-ribosyl)ation). Poly(ADP-ribosyl)ation often involves the addition of long chains of ADP-ribose units, linked by glycosidic ribose-ribose bonds, and is critical for a wide range of processes, including DNA repair, regulation of chromosome structure, transcriptional regulation, mitosis and apoptosis. Here we identify a novel poly(ADP-ribose)-binding zinc finger (PBZ) motif in a number of eukaryotic proteins involved in the DNA damage response and checkpoint regulation. The PBZ motif is also required for post-translational poly(ADP-ribosyl)ation. We demonstrate interaction of poly(ADP-ribose) with this motif in two representative human proteins, APLF (aprataxin PNK-like factor) and CHFR (checkpoint protein with FHA and RING domains), and show that the actions of CHFR in the antephase checkpoint are abrogated by mutations in PBZ or by inhibition of poly(ADP-ribose) synthesis.

The centrosome opens the way to mitosis.

During mitosis, the interaction between chromosomes and microtubules requires nuclear envelope disassembly in prophase. Two articles in this issue of Developmental Cell show that centrosomes have a role in promoting nuclear envelope breakdown (Hachet et al., 2007; Portier et al., 2007). Surprisingly, the role of the centrosome in this process is independent of its role as a microtubule nucleation organelle. Instead, the centrosome seems to act as a spatial regulator for the activation of the Aurora A kinase.

Proteolysis: anytime, any place, anywhere?

Proteolysis via the ubiquitin-proteasome system (UPS) is a rapid and effective method of degrading a specific protein at a specific time, and in many cases a protein is degraded only in response to a particular cellular signal or event. However, an added dimension to the control of protein degradation is possible because the ubiquitin system can be spatially regulated. Controlling where a protein is degraded can enhance the specificity and timing of proteolysis, generate asymmetry and maintain sub-compartments even in the mitotic cell. Here, we discuss this aspect of the UPS.

Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C.

Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in this study, we show that the degradation of Emi1 is not required to activate the APC/C in mitosis. Instead, we uncover a key role for Emi1 in inhibiting the APC/C in interphase to stabilize the mitotic cyclins and geminin to promote mitosis and prevent rereplication. Thus, Emi1 plays a crucial role in the cell cycle to couple DNA replication with mitosis, and our results also question the current view that the APC/C has to be inactivated to allow DNA replication.

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy.

Measuring the dynamics with which the regulatory complexes assemble and disassemble is a crucial barrier to our understanding of how the cell cycle is controlled that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of the kinetics of protein-protein interaction in living cells. Here, we apply fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

APC/C Cdh1 targets aurora kinase to control reorganization of the mitotic spindle at anaphase.

BACKGROUND: Control of mitotic cell cycles by the anaphase-promoting complex or cyclosome (APC/C) ubiquitin ligase depends on its coactivators Cdc20 and Cdh1. APC/C(Cdc20) is active during mitosis and promotes anaphase onset by targeting mitotic cyclins and securin. APC/C(Cdh1) becomes active during mitotic exit and has essential targets in G1 phase. It is not known whether targeting of substrates by APC/C(Cdh1) plays any role in the final stages of mitosis. Here, we have investigated the role of APC/C(Cdh1) at this time in the cell cycle by using siRNA-mediated depletion of Cdh1 in human cells. RESULTS: In contrast to the current view that Cdh1 takes over from Cdc20 at anaphase, we show that reduced Cdh1 levels have no effect on destruction of many APC/C substrates during mitotic exit but strongly and specifically stabilize Aurora kinases. We find that APC/C(Cdh1) is required for assembly of a robust spindle midzone at anaphase and for normal timings of spindle elongation and cytokinesis. The effect of Cdh1 siRNA on anaphase spindle dynamics requires Aurora A, and its effect can be mimicked by nondegradable Aurora kinase. CONCLUSIONS: Targeting of Aurora kinases at anaphase by APC/C(Cdh1) participates in the control of mitotic exit and cytokinesis.

Mitosis: a matter of getting rid of the right protein at the right time.

There are two major problems for the cell to solve in mitosis: how to ensure that each daughter cell receives an equal and identical complement of the genome, and how to prevent cell separation before chromosome segregation. Both these problems are solved by controlling when two specific proteins are destroyed: securin, an inhibitor of chromosome segregation, and cyclin B, which inhibits cell separation (cytokinesis). It has recently become clear that several other proteins are degraded at specific points in mitosis. This review (which is part of the Chromosome Segregation and Aneuploidy series) focuses on how specific proteins are selected for proteolysis at defined points in mitosis and how this contributes to the proper coordination of chromosome segregation and cytokinesis.

The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint.

The anaphase promoting complex/cyclosome (APC/C) is crucial to the control of cell division (for a review, see ref. 1). It is a multi-subunit ubiquitin ligase that, at defined points during mitosis, targets specific proteins for proteasomal degradation. The APC/C is itself regulated by the spindle or kinetochore checkpoint, which has an important role in maintaining genomic stability by preventing sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle. The spindle checkpoint regulates the APC/C by inactivating Cdc20, an important co-activator of the APC/C. There is also evidence to indicate that the spindle checkpoint components and Cdc20 are spatially regulated by the mitotic apparatus, in particular they are recruited to improperly attached kinetochores. Here, we show that the APC/C itself co-localizes with components of the spindle checkpoint to improperly attached kinetochores. Indeed, we provide evidence that the spindle checkpoint machinery is required to recruit the APC/C to kinetochores. Our data indicate that the APC/C could be regulated directly by the spindle checkpoint.