Bistability of mitotic entry and exit switches ... - Wiley Online Library

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Bistability of mitotic entry and exit switches during open mitosis in mammalian cells Nadia Hegarat1), Scott Rata2) and Helfrid Hochegger1) Mitotic entry and exit are switch-like transitions that are driven by the activation and inactivation of Cdk1 and mitotic cyclins. This simple on/off reaction turns out to be a complex interplay of various reversible reactions, feedback loops, and thresholds that involve both the direct regulators of Cdk1 and its counteracting phosphatases. In this review, we summarize the interplay of the major components of the system and discuss how they work together to generate robustness, bistability, and irreversibility. We propose that it may be beneficial to regard the entry and exit reactions as two separate reversible switches that are distinguished by differences in the state of phosphatase activity, mitotic proteolysis, and a dramatic rearrangement of cellular components after nuclear envelope breakdown, and discuss how the major Cdk1 activity thresholds could be determined for these transitions.

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Keywords: Cdc25; Greatwall; mammalian cells; mitosis; MPF; Wee1

Introduction Cell cycle transitions cause rapid and irreversible changes that ensure a unidirectional sequence of events. This is exemplified by the dramatic changes that cells undergo during mitotic entry and exit. In higher eucaryotes, triggering mitotic entry results DOI 10.1002/bies.201600057 1) 2)

Genome Damage and Stability Centre, University of Sussex, Brighton, UK Department of Biochemistry, Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK

*Corresponding author: Helfrid Hochegger E-mail: [email protected]

in changes in every cellular compartment. Within minutes, the cell rounds up, centrosomes separate and microtubule growth and shrinkage accelerate, the nuclear envelope breaks down and fuses with the endoplasmic reticulum, the Golgi apparatus fragments, and chromosomes condense and are captured by microtubules at the emerging kinetochores [1]. Mitotic exit starts with the segregation of sister chromatids and elongation of the central spindle powered by motor proteins of the kinesin family. This is followed by the execution of cytokinesis and establishment of the interphase membrane systems and chromosome decondensation [2]. These two switch-like transitions are orchestrated predominantly by phosphorylation and dephosphorylation of thousands of proteins [3–5] by a cohort of mitotic kinases and phosphatases. The switch itself is triggered by activation of the major mitotic kinase Cyclin-dependent kinase 1 (Cdk1) and inactivation of its counteracting phosphatases during mitotic entry [6]. Conversely, mitotic exit is triggered by Cdk1 inactivation and ordered reactivation of the phosphatases [7, 8]. Since the regulatory proteins participating in these switches are themselves subjected to control by kinase and phosphatase activity, a complex feedback system emerges that ensures a robust, rapid and irreversible response. Mitotic entry and exit are both all or none decisions [9]. Once they are triggered, a series of pre-programmed events take place, taking the cell toward metaphase and G1, respectively, without permitting delays in intermediate conditions. This is achieved mainly because thresholds for forward and reverse reactions differ so that directionality of the system is ensured. These properties of the switch systems can be summarized by the term bistability [10–12]. At the heart of mitotic entry and exit switches lies the regulation of Cdk1 and mitotic cyclins. Activation of Cdk1 depends on the synthesis of cyclin B1 and removal of inhibitory phosphorylations on Cdk1 by the dual specificity Cdc25 phosphatase, while inactivation of Cdk1 depends on degradation of cyclin B1, and rephosphorylation of the inhibitory T14/Y15 residues by Wee1/Myt1 once new cyclin B1 is re-synthesized. The Wee1/Cdc25 switch is further subjected to positive and negative feedback since Cdk1 acts as an activator of Cdc25 and inhibits Wee1. Moreover, the

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Prospects & Overviews

phosphatases that counteract these feedback reactions play a critical role in preventing mitotic entry. Phosphatase inactivation is thus a critical requirement for full Cdk1 activation. Based on these observations, Novak and Tyson [13] modeled the forward and reverse reactions of Cdk1 activation and proposed that this feedback system is sufficient to generate a bistable switch with a higher entry than exit threshold for cyclin B levels. Moreover, the system is designed to oscillate because cyclin B destruction is a consequence of full Cdk1 activation. Ten years later, bistability of the G2/M switch was confirmed experimentally by two studies in cell-free Xenopus egg extracts [14, 15]. The extracts were depleted of endogenous cyclin B1, and reconstituted with recombinant stable cyclin B1 to measure how much cyclin B1 is required to enter mitosis, and to maintain the mitotic state of the extracts. As predicted by the bistability model, the system required 2–3 fold higher cyclin concentrations to enter mitosis than to maintain the mitotic state. The bistability model also predicts an increasing time-lag in mitotic entry with decreasing cyclin B levels at concentrations close to the threshold, which was again experimentally confirmed in the Xenopus studies [14, 15]. Finally, the model predicted that Cdk1 activation propagates in space in the form of a trigger wave. This has been recently confirmed in elegant experiments analyzing the spatial dynamics of Cdk1 activation in CSF extracts stretched out in teflon tubes [16]. In principle, a simple feedback system between Cdk1, Wee1/Cdc25, and the Cdk1 counteracting phosphatases is sufficient to generate a bistable switch that gives mitosis its robust directionality and prevents cells from going back and forth between G2 and M phases. Accordingly, the Xenopus experiments directly compared the thresholds of Cdk1 Y15 dephosphorylation and rephosphorylation that are the basis for the original mathematical model [13]. However, in intact mammalian cells, the G2/M switch is more complex, and irreversibility is built into the system at various points. Here, we would like to propose a model that splits the mitotic switch system in two halves (Fig. 1). This is based on experimental evidence by Potapova et al. [17] that the effect of Cdk1 inhibition is dramatically different before and after nuclear envelope breakdown (NEBD). If Cdk1 activity is lost in prophase, cells will simply revert to G2 phase without activating mitotic proteolysis, chromosome segregation, and cytokinesis. This constitutes a first reversible switch, namely G2/prophase, and it remains to be determined if this transition is bistable. It will also be important to determine what precisely sets the Cdk1 activity thresholds that prevent cells to flip back and forth between G2 and early prophase. The next step is the commitment to forward progression that occurs as cells move toward prometaphase following further Cdk1 activation. Phosphatase inactivation [18] and NEBD [19] are major determinants of irreversibility in this transition. Cyclin B and securin degradation then trigger the second switch during mitotic exit. This transition is in principle also reversible [20], but in normal circumstances cyclin B proteolysis ensures that cells cannot flip back into mitosis until enough cyclin B has been remade in the next cell cycle [11]. A major determinant of Cdk1 thresholds in this mitotic exit switch is the re-balancing of kinase and phosphatase activity that is currently only poorly understood. In this article we would like to

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systematically review the mitotic entry and exit reactions and speculate on determinants of bistability and irreversibility. Table 1 gives a brief overview of the major players that take part in this complex regulatory network.

Mitotic entry The maturation promoting factor In the early 1970s, Masui and co-workers discovered a biochemical activity termed maturation promoting factor (MPF) that triggered meiotic maturation in frog oocytes [21, 22]. It took almost 20 years to purify this activity but it was eventually identified as a Cdk/cyclin complex [23, 24]. These experiments demonstrated that Cdks constitute the core cell cycle engine that drives mitotic entry by phosphorylating its substrates. The Cdk paralogue in the complex was clearly Cdk1 (then called Cdc2). The question regarding the identity of mitotic cyclin is more complex. Both Sea-Urchin cyclin B and Clam cyclin A can induce Xenopus oocyte maturation and could thus constitute MPF. Using cell-free extracts of parthenogenetically-activated Xenopus eggs, the Kirschner and Hunt labs could show that both cyclin B1 and B2 are sufficient and essential to trigger mitotic entry in early embryonic cell cycles. Similarly, cyclin A1 showed MPF activity in these assays [25–28]. In somatic mammalian cells the family of mitotic cyclins consists of four members of cyclins: A2, B1, 2, and 3. Among these, only cyclin A2 and B1 appear to be essential for mouse development [29, 30]. The precise function of cyclin A2 during mitotic entry is unclear but it appears to act upstream of the cyclin B1/Cdk1 activation [31]. Thus, cyclin B1/Cdk1 seems to be the major driver of mitotic entry in mammalian cells. This idea is, however, challenged by the lack of phenotypes following Cyclin B1 depletion in human cells [32]. The “cyclin B1-centric” view needs to be reassessed using more precise genetic tools in somatic human cells. Another important question that should be further assessed is the apparent inability of Cdk2 to trigger mitotic entry. In Xenopus egg extracts cyclinE/Cdk2 is not capable of triggering mitotic entry even at biochemical activity levels comparable to mitotic Cdk1 [33]. On the other hand, microinjection of purified cyclin A2/Cdk2 can trigger mitotic entry in G2 HeLa cells, although it is not clear if the injected cyclin A2 does associate with Cdk1 as well [34]. From genetic experiments in various somatic mouse cell lines it is clear that Cdk1 is absolutely essential [35], while Cdk2 is dispensable for cell cycle progression and cellular survival [36, 37]. The reason for this distinct difference between the highly similar Cdk paralogues requires further exploration.

Basic elements of Cdk1 regulation The first essential step in the activation of Cdk1 is cyclin A and B mRNA transcription and protein synthesis, because of their proteolysis during and after mitosis. There are excellent reviews on the structure and function of the cyclin destruction system [38–40] and the regulation of cyclin transcription [12, 41] and we will not further discuss these subjects here.

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Figure 1. Mitotic entry and exit switches (Images show mitotic stages in a HeLa cell expressing GFP-tubulin and mCherry-H2B). This review is based on the assumption that the mitotic switch system is not a binary on/off switch but consists of two different forward and reverse reactions. These are separated by an irreversible transition that is marked by NEBD. Mitotic entry is triggered by Cdc25 dependent dephosphorylation of the inhibitory residues T14/ Y15 of Cdk1. At a critical threshold of Cdk1 activity this will induce cyclin B translocation into the nucleus and trigger prophase. This reaction is reversible, but we do not know if the Cdk1 activity threshold for the forward and reverse reaction differ significantly. Cyclin B translocation is followed by further Cdk1 activation, APC/C activation and NEBD resulting in an irreversible change that commits the cell to exit into G1 rather than back to G2 phase. Inactivation of PP2A/B55, potentially PP1, and other potential Cdk1 counteracting phosphatases is also a critical component of this commitment step, but the precise timing and mechanism of this regulatory network is poorly understood. Exit from M-phase is initiated by cyclin B degradation and triggered by phosphatase reactivation. This is in principle also a rapidly reversible reaction, but is kept unidirectional by cyclin B degradation (dashed arrow).

Newly made Cdk/cyclin complexes are not yet active. It requires a phosphorylation at a threonine residue in its activation loop segment, T160 in human Cdk1 [42]. This phosphorylation is required to free the entrance to the active site and to stabilise ATP binding by changing the conformation of the PSTAIR loop [43]. It is catalyzed by the Cdk-activating kinase (CAK) Cdk7 in complex with cyclin H [44, 45]. It is generally believed that this

activity is constitutive and does not contribute to cell cycle regulation. The regulation of Cdk1 that generates the switch-like mitotic entry occurs via inhibitory phosphorylation at T14 and Y15 [46] mediated by the kinases Wee1 and Myt1. Both kinases can phosphorylate Cdk1 at Y15, but only Myt1 can also phosphorylate T14. The two related kinases show distinctly different localization in interphase, with Wee1 being nuclear and Myt1 bound to the membrane [47–49]. The functional differences and the precise impact on G2/M regulation of these two kinases are not yet determined. Wee1 knock-out mice are embryonic lethal [50] and Wee1 siRNA depletion in human cells causes severe mitotic phenotypes [51]. Moreover, Wee1-specific inhibitors cause abrupt and premature mitotic entry in S and even G1 cells, suggesting that this kinase plays a critical role in controlling the G2/M switch [17]. Much less is known about the functions of Myt1. Knock-out mice have, to our knowledge, not been characterized and siRNA depletion does not appear to cause significant phenotypes [51]. However, Myt1 has been reported to contribute to recovery from the DNA damage checkpoint [52]. Further genetic studies will be required to determine the importance of Myt1 in the regulation of the G2/M transition.

Triggering the switch: the Cdc25 phosphatases As soon as Cdk1/cyclin B complexes form, they are shut down by T14/Y15 phosphorylation. Cdk1 activation then occurs in


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Table 1. Overview of main players involved in mitotic entry and exit Players Kinases Cdk1/cyclin B

Cdk/cyclin A Greatwall Wee1 Myt1 Phosphatases Cdc25A/B/C PP2A/B55 PP1 Fcp1 Others Ensa/ARPP19 Inhibitor-1

Role in mitotic entry Phosphorylates mitotic substrates Activating Cdc25 Inhibits Wee1/Myt1 Activates Greatwall Inhibits PP1 Required for Cdk1/cyclin B activation Inhibits PP2A/B55 via Ensa/ARPP19 activation Inhibits Cdk1/cyclin B (Y15 residue) Inhibits Cdk1/cyclin B (Y15 and T14 residues)

Dephosphorylates mitotic substrates Inactivates Greatwall Dephosphorylates mitotic substrates Inactivates Greatwall Inactivates PP2A/B55 Inactivates PP1

References [4] [59] [59] [93, 94] [128, 129] [31] [87–92] [49] [48]

Activates Cdk1/cyclin B (Y15 and T14 residues)

late G2 by the removal of these inhibitory phosphates by the dual specificity Cdc25 phosphatases that can dephosphorylate both on T14 and Y15. In mammalian cells there are three different Cdc25 paralogues: Cdc25 A, B, and C. The B and C paralogues are thought to play a prominent role in the G2/M switch due to their strict regulation by mitotic kinases and centrosomal localization [53]. Surprisingly, double deletion of Cdc25B and C does not affect mouse development and does not show any phenotype in adult mice, except for sterility, which already occurs in Cdc25B single knock out mice [54–56]. Thus, Cdc25A appears to be able to step in and take over the mitotic control functions of its relatives, despite its more prominent role in the G1/S transition [53]. Similar gene deletion studies in human cells have, to our knowledge, not yet been performed, and the role of the human Cdc25 family of proteins needs to be further analyzed in somatic cells. Even though the Y15 dephosphorylation switch is generally accepted to constitute the core trigger of mitosis, there is conflicting evidence regarding its actual importance for mitotic control. As expected, over-expression of a mutant Cdk1 that cannot be phosphorylated by Wee1 and Myt1 (Cdk1AF: T14A and Y15F) causes premature mitotic entry, but interestingly only after one round of normal mitotic progression [57]. Conversely, preventing Y15 phosphorylation using Wee1 inhibitors has a more dramatic and immediate effect, causing premature mitotic entry in G1 and S-phase cells [17], presumably after a rate-limiting concentration of cyclin A and/ or B has been reached. In a recent study, Gravells et al. [58] engineered cell lines that have lost the endogenous Cdk1 alleles and stably express a Xenopus laevis version of Cdk1AF that also carries an analogue sensitive mutation in the gatekeeper residue. Unexpectedly, these cells are viable and appear to have a normal cell cycle profile. This constitutes a proof of principle that the Cdc25/Wee1 control system can be bypassed without losing the basic features of mitotic entry control, and suggests that other control elements exist that contribute to switch-like Cdk1 activation. The results

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[53] [104, 105] [122–124] [7–8] [122] [92–93] [130]

presented so far are partly contradictory and need to be further clarified. Why, for example, does Wee1 inhibition have such an immediate and dramatic effect, compared to expression of Cdk1AF? How can cells survive with a Cdk1AF mutation? What other control elements contribute to the G2/M switch? Does Wee1 have other targets apart from the Cdk1 Y15 phosphorylation site? A detailed genetic analysis of the Wee1/Cdc25 control system in human cell lines will be critical to address these questions and clarify the importance of this regulatory mechanism and the contributions of the various components of this control system.

Feedback control during Cdk1 activation Similar to Cdk1 itself, both Wee1/Myt1 and Cdc25 A, B, and C are regulated by phosphorylation and dephosphorylation, including positive and negative feedback with Cdk1 [59]. Activation of Cdc25 and inactivation of Wee1 lies at the root of the bistable mitotic entry system. In Xenopus oocytes and egg extracts, the classic G2/M model system, Cdc25C is thought to regulate meiotic and mitotic entry. Regulation of this phosphatase is complex and involves phosphorylation of various inhibitory and activating residues that integrate signals from Chk1/2 kinases, PKA and cTAK1, Cdks, Plk1 and Cdk1 itself. The major inhibitory mechanism of this phosphatase involves S287 phosphorylation and 14-3-3 binding [60], which appear to be interdependent [61]. Release from 14-3-3 binding is thought to be mediated by Cdk2 and Plk1 activities [61–63]. Cdk1 initiates a positive feedback loop by further activating Cdc25 [64–66] by recruiting PP1 [67] and maintaining an active conformation of Cdc25 via the prolyl isomerase Pin1 [68, 69]. Moreover, since Cdk1 further stimulates Plk1 activity, further signal amplification takes place via this additional feedback loop. The precise activation mechanism of mammalian Cdc25B and C is likely to involve the same elements that have been characterized in Xenopus. A major conundrum in this Cdc25


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firm block of mitotic entry that can be rescued by further inhibition or depletion of PP2A/B55. Likewise, Xenopus Greatwall depletion from mitotic egg extract results in rapid mitotic exit, which is dependent on the reactivation of the PP2A/B55 phosphatase [88]. Greatwall kinase itself is activated by Cdk1 phosphorylation at various residues in its large activation loop domain, and also stimulates its own activity via autophosphorylation [94, 95]. The precise activation mechanism of this unusual AGC kinase remains to be determined. Cdk1 also directly phosphorylates Ensa/ ARPP19, and this appears to be critical for starfish oocyte maturation [96]. However, this phosphorylation has only a modest impact on the inhibitory activity of Xenopus Ensa [97]. The data discussed so far concerning the role for Greatwall kinase in the G2/M transition are mostly derived from work in Xenopus egg extracts. Greatwall kinases and ARPP19/Ensalike inhibitors of PP2A/B55 are conserved from yeast to humans, but their functions seem to vary considerably. In yeast, the Greatwall pathway seems to be involved in metabolic growth responses [98–100]. The major effects of Greatwall depletion in mouse embryonic fibroblasts and human cells appears to be a mitotic delay due to spindle assembly checkpoint activation, mitotic collapse, and cytokinesis defects, but only show modest delays in the G2/M transition [101–103]. Likewise B55 depletion does not seem to have a major effect on mitotic entry, but causes predominantly mitotic exit phenotypes [104, 105]. It is possible that the depletion experiments mentioned above did not sufficiently reduce the protein levels of Greatwall and B55 subunits. Alternatively, other OA sensitive phosphatases that are inhibited in a Greatwall-independent manner could be involved in Cdk1 activation.

Control of Cdk1-counteracting phosphatases The first experiments that probed Cdk1 activation in Xenopus egg extracts already suggested that phosphatases play a major role in this mechanism. In their pioneering experiments Cyert, Solomon and Kirschner found that a major determinant of Cdk1 activation dynamics was a phosphatase of type 2A that they named INH [81, 82]. This notion was further supported by mammalian cell experiments using Okadaic Acid (OA), a potent phosphatase inhibitor that targets among others PP2A and PP1. Surprisingly, OA triggers mitotic entry even in the presence of Cdk1 inhibitors [83]. This strongly suggests that an OA-sensitive phosphatase plays a major role in preventing premature mitotic entry in mammalian cells, since even low levels of this phosphatase inhibitor results in Cdk1 activation. The identity and the mechanisms of regulation of this phosphatase remained unclear until a novel mitotic kinase, Greatwall, was discovered and characterized in Drosophila and Xenopus [84–86]. Work from the Goldberg, Castro&Lorca, and Hunt&Mochida labs uncovered the role of this new player in Cdk1 activation in Xenopus egg extracts and found that it acts as a major inhibitor of the PP2A/B55 phosphatase [87–91]. Greatwall acts via two highly related small and unstructured proteins, Ensa and ARPP19, that become potent PP2A/B55 inhibitors once they are phosphorylated by Greatwall at a conserved serine residue [92, 93]. Accordingly, depletion of Xenopus Greatwall from interphase egg extract results in a

Spatial control of Cdk1 activation Changes in the localization of many of the major players in the Cdk1 amplification loop add another layer of complexity to the switch system. Cyclin B1 shuttles between nucleus and cytoplasm during interphase, but has only a very brief retention phase within the nucleus [106]. Other components of the feedback system, such as cyclin A, Cdc25A, Wee1, and Greatwall are concentrated in the nucleus, while Cdc25B/C and Myt1 are cytoplasmic [6]. Finally, the dynamic localization of other important players such as Ensa/ARPP19, PP2A/ B55, and various PP1 complexes needs to be firmly established in mammalian cells. Current models of spatial regulation of mitotic entry suggest that the earliest event in the Cdk1 amplification loop is the activation of Cdk1/cyclin B1 at the centrosome [107]. This is thought to trigger signal amplification and translocation of cyclin B1 together with Cdc25C into the nucleus [108, 109]. Following cyclin B1 translocation, Greatwall kinase is activated by Cdk1, and active Greatwall is exported into the cytoplasm, presumably to phosphorylate Ensa/ARPP19 and inhibit cytoplasmic PP2A/B55 [102]. All of these events happen before NEBD, which is initiated by Cdk1dependent phosphorylation of nuclear lamins [110]. We do not fully understand the significance of most of these translocation events, but several studies have addressed the importance of controlled localization during mitotic entry. The


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activation model is Cdc25A, which has been primarily ascribed a role in the G1/S transition [53]. However, since mice lacking both Cdc25B and C are viable, Cdc25A must be able to step in and perform the critical regulation of Cdk1 activation. Few of the regulatory elements described above are present in Cdc25A, which appears to be regulated primarily via protection from proteolysis by Cdk1 [70]. Strikingly, the Wee1 kinase is subjected to astonishingly similar control elements that appear almost as a mirror image of Cdc25 control [59]. Thus, the major negative control of Cdc25, phosphorylation dependent binding of 14-3-3 proteins, is also used in Wee1 but in this case as a stimulator of interphase Wee1 activity [71–73]. Another major control element appears to be phosphorylation-dependent degradation of Wee1 at the G2/M transition [74, 75]. Several phosphodegrons have been identified in frog and human Wee1 that are regulated by Cdk1 itself, Plk1 and Casein Kinase 1 [76–78]. Lastly, Cdk1 appears to stimulate Pin1 binding by phosphorylating a conserved threonine residue in a N-terminal domain of Wee1 [79]. In summary, Cdk1 initiates its burst-like activation by positive feedback with Cdc25 and negative feedback with Wee1 both directly (inner feedback loops), or indirectly (outer feedback loops) via auxiliary kinases such as Plk1, Casein Kinase 1, and Aurora-A. The fine tuning of the balance of Wee1 and Cdc25 activity has a crucial impact on the cell cycle control of mitotic entry. This has been demonstrated both by experimental evidence and modeling in the early embryonic cell cycles of Xenopus leavis, where a shift from Cdc25 toward Wee1 leads to a shortening of cell cycles 2–11, after a longer first division cycle [80].

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significance of the centrosome as a signaling platform that triggers mitotic entry has been cast in doubt because both physical destruction and genetic ablation of the centrosome has little effect on mitotic progression [111, 112]. Thus, it seems unlikely that the early activation of centrosomal Cdk1/cyclin B1, or the centrosomal localization of Cdc25B and C, play a critical role in the amplification loop. Cyclin B1 enrichment in the nucleus, on the other hand, appears to be a critical step in Cdk1 activation, because forced nuclear enrichment of Cdk1/ cyclin B1 can cause premature mitotic entry [113]. Likewise, the translocation of Greatwall into the cytoplasm seems to be important because tethering Greatwall to chromatin and thereby preventing nuclear export prevents its mitotic functions [102]. However, this defect could also be a consequence of the tethering to chromatin during mitosis. It will be interesting to repeat these experiments by manipulating the localization of endogenous components of the Cdk1 amplification loop and studying the effects of changed localization on mitotic entry. This will provide more definite answers to the question of how changes in localization and spatial separation of different players contributes to the G2/M switch.

Triggers of Cdk1 activation A critical question in the G2/M transition that remains to be answered concerns the initiation of the feedback systems, and we know very little about the actual trigger of Cdk1 activation. The answer to this question is most likely closely linked to the release from the DNA replication checkpoint system. Chk1 and Chk2 kinases inhibit Cdc25 and activate Wee1 by controlling interactions with 14-3-3 proteins, and their activity may be reduced once DNA replication is complete, although it is far from clear how Chk1/2 activity and DNA replication progression are actually coupled. An important question relating to this proposition is the actual timing of Cdk1 activation with regards to DNA replication. A pioneering study in human cells using a FRET probe for Cdk1 found a fairly precise time lag of 30 minutes between the first Cdk1 activation and NEBD [114]. This would leave a good 2–3 hours between the end of S-phase and the onset of the Cdk1 activation loop, suggesting that other factors apart from checkpoint signaling, such as cyclin accumulation, cell growth or metabolic signals may play a role in the decision point. However, the reported FRET signal is extremely weak and may not be sensitive enough to pick up slow initial activation dynamics. Another quantification of Cdk1 activation was recently performed using fixed cells that were ordered within their cell cycle status using various quantitative immuno-fluorescence markers [115]. This study concludes that Cdk1-dependent phosphorylation events become detectable as soon as DNA replication diminishes, as judged by DAPI intensity and PCNA foci dispersal. This suggests that the classic Cdk1 amplification loop may take much longer than previously expected. However, the currently available detection tools do not allow a differentiation between Cdk2/cyclin A and Cdk1/cyclin B activity. It is possible that Cdk2/cyclin A activity starts to rise in late Sphase, but that Cdk1/cyclin B activation constitutes the real

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Cdk1 activation loop that occurs briefly before mitotic entry. The role of cyclin A in mitotic entry definitely requires further attention and is likely to be a central element in the Cdk1 amplification network [31]. Overall the search for a single trigger is probably a hopeless endeavor because the Cdk1 activation loop can be kick-started via various entry points, such as phosphatase inhibition, Wee1 inhibition, or cyclin B1 nuclear accumulation. Conceivably, there is no fixed linear series of events that lead to mitotic entry, but there are various redundant pathways that ensure a robust and stable system response to integrate upstream signaling events. To make sense of this complex system of parallel feedback loops, a first step will have to be to simplify and to remove as much of the redundant components as possible to uncover the most basic essential elements of the G2/M control system and their hierarchical relationship.

Mitotic exit At the onset of mitotic exit, segregation of the sister chromatids takes place followed by the activation of the cytokinesis network that ultimately leads to two equal daughter cells with re-established G1 nuclei. This series of events is triggered by inactivation of Cdk1 and the ordered removal of Cdk1-dependent mitotic phosphorylations. It is important to note that this is not simply the reverse reaction of mitotic entry but a linear progression into a new cell cycle phase due to the activation of the chromosome segregation machinery, the splitting of the daughter cells during cytokinesis and the degradation of several essential factors required for mitotic entry. Cdk1 inactivation is initiated by cyclin B ubiquitylation and proteolysis once the spindle assembly checkpoint is satisfied. Mitotic proteolysis, anaphase promoting complex (APC) dependent ubiquitylation and spindle assembly checkpoint signaling have all been covered in excellent recent reviews [38–40, 116, 117] and are beyond the scope of this article. In the next paragraphs we will focus instead on the reactivation of the Cdk1 counteracting phosphatases that constitutes a critical and poorly understood trigger of mitotic exit. In budding yeast this process is well described and depends primarily on the Cdc14 phosphatase. Surprisingly, genetic analysis of the two mammalian Cdc14 phosphatase homologues have so far not revealed any notable cell cycle phenotypes that would suggest a critical role for these enzymes similar to the major mitotic exit functions in yeast [118, 119]. Higher eukaryotes appear to employ PP1 and PP2A/B55 and possibly other phosphatases for this critical transition.

PP2A/B55 reactivation PP2A/B55 is inhibited during mitotic entry by Ensa and ARPP19 that have been phosphorylated at a conserved serine residue by Greatwall kinase (see above). This inhibition depends indirectly on Cdk1 because of the Cdk1-dependent activation mechanism of Greatwall kinase. A critical question


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trigger that causes an initial drop in Greatwall activity. This results in partial activation of PP2A/B55 which then takes over and accelerates Greatwall inactivation via a negative feedback loop. Finally, Della Monica et al. [125] published a study that suggests an essential role for Fcp1 in the Greatwall dephosphorylation network, which could explain the effects of Fcp1 depletion on Ensa/ARPP dephosphorylation that Hegarat et al observed. In summary, there appears to be a complex interplay of PP1, Fcp1, and PP2A/B55 that generates the mitotic exit switch. The precise sequence of events in this switch system remains to be further validated by experimental evidence and mathematical modeling.

PP2A/B55 function Another critical question concerns the actual role that PP2A/ B55 plays in mitotic exit dephosphorylation. There are four highly similar B55 paralogues (a-d) in vertebrates. Depletion of B55d from Xenopus interphase extracts completely blocks the subsequent mitotic exit, triggered by Cdk1 inhibition, arguing that PP2A/B55d is the critical phosphatase that removes the bulk of Cdk1-dependent phosphosites. Conversely, removal of B55d from extracts that are already arrested in mitosis does not have the same effect, arguing against this hypothesis [91]. Several groups have reported the effects of OA on mitotic exit. If PP2A/B55 is the major “heavy lifting” anti-Cdk phosphatase, one would expect that 1 mM OA should block mitotic substrate dephosphorylation. According to several reports OA pretreatment of mitotic cells before Cdk1 inhibition results in a decreased dephosphorylation of substrates (see for example [126, 127]). However, when we measured the dynamics of dephosphorylation in similar experiments [121], we found that OA treated mitotic extracts started off with an increased amount of dephosphorylation, but also observed that the actual dynamics of dephosphorylation for the majority of substrates following Cdk1 inhibition are unchanged. This suggests that an OA sensitive phosphatase is actually active in mitosis, but OA insensitive phosphatases significantly contribute to Cdk substrate dephosphorylation during mitotic exit. The discrepancy to the earlier studies could thus lie in the difference between endpoint versus dynamic measurements. Moreover, the earlier studies used Roscovitine, a relatively poor Cdk1 inhibitor, while we used a more potent and specific inhibitor RO3306. The idea that PP2A/B55 represents the major anti-Cdk1 phosphatase during mitotic exit is also undermined by the observations that depletion of B55a in mammalian cells, or even all four B55 paralogues, affects only the timing of the cytokinesis furrow formation and nuclear envelope reformation, but does not completely block global mitotic exit [104, 105]. To determine the precise functions of PP2A/B55 it will be important to define its precise targets and characterize the effects of their delayed or abolished dephosphorylation. One such example is Prc1, which has been recently identified as a major substrate of PP2A/B55 [105]. Prc1 is a highly conserved component of the anaphase central spindle that organizes antiparallel microtubules during the furrow formation in cytokinesis. It is inhibited by


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that needs to be answered in order to understand the mitotic exit mechanism in higher vertebrates is: how is PP2A/B55 reactivated after the initial drop of Cdk1 activity that is triggered by cyclin B proteolysis? A biochemical study by the Goldberg lab and a genetic study from our lab have recently addressed this question and come to surprisingly different conclusions [120, 121]. Williams et al. propose a simple and convincing model for PP2A/B55 reactivation. Based on careful measurements of Ensa/PP2A/B55 interactions and enzymatic kinetics, they demonstrate that phosphorylated Ensa works as a substrate as well as an inhibitor with a very high binding affinity for the active site, but very slow enzymatic kinetics for the dephosphorylation reaction. Thus, phosphorylated Ensa/ARPP19 act as poor substrates that block the active site but are eventually dephosphorylated. As long as Greatwall kinase is active the inhibitors are quickly rephosphorylated and recapture the phosphatase again. The real trigger of PP2A/B55 reactivation is thus Greatwall dephosphorylation and deactivation that has to be achieved by a different, earlier, phosphatase. Conversely, we use a mitotic block and release assay and phospho-specific antibodies to monitor the dephosphorylation dynamics of Ensa/ARPP19 and Greatwall kinase. We observed that phosphatase inhibition by moderate OA concentrations blocks dephosphorylation of an Cdk1-dependent phosphosite (T194) in Greatwall, but does not affect Ensa/ARPP19 dephosphorylation. The fact that OA does not block PP2A/ B55 dependent Ensa/ARPP19 dephosphorylation is easily reconcilable with the unfair competition model since Ensa/ ARPP19 could simply outcompete OA for access to PP2A/B55. However, under these circumstances Greatwall retains at least 60% of its activity, while Ensa/ARPP19 are still dephosphorylated. Conversely, the unfair competition model would predict that at this level of kinase activity, Ensa/ ARPP19 should remain phosphorylated. Our study then finds that PP2A/B55 depletion has a moderate effect on Greatwall T194 dephosphorylation, while Ensa/ARPP19 dephosphorylation is only inhibited either by the depletion of another phosphatase Fcp1, or combined phosphatase inhibition by OA and Tautomycetin. These genetic experiments do not allow a firm conclusion on which phosphatase is directly involved in the biochemical dephosphorylation mechanism and it is indeed unlikely that Fcp1 is the actual Ensa/ARPP19 phosphatase given the poor phosphatase activity it shows in vitro against these proteins [120]. Nevertheless, it remains a challenge to reconcile the data of these two studies. The simplest explanation would be that various phosphatases including PP2A, PP1, and Fcp1 are involved in regulating Greatwall kinase dephosphorylation and that the effects seen with the PP2A/PP1 inhibition and Fcp1 siRNA treatments in the Hegarat paper are due to maintaining Greatwall activity rather than blocking Ensa dephosphorylation directly. Two recent Xenopus studies [122, 123], indeed demonstrate that PP1 regulates a C-terminal residue that is most likely an autoactivation site in an active-site tether motif [95]. Moreover, another new study in mammalian cells suggests that PP1 regulates the initial dephosphorylation of other activating sites in Greatwall, while the C-terminal phosphorylation remains constitutive [124]. Using simulation of the mitotic exit network they propose that PP1 constitutes the

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a Cdk1-dependent phosphorylation and Cundell et al. demonstrate that the phosphatase that acts on this site is PP2A/B55. This study gives a satisfactory explanation of the observed cytokinesis defects in Greatwall-depleted mammalian cells. In these cells, Prc1 is dephosphorylated too early before the sister chromatids had enough time to segregate and make space for the cell division furrow. Thus, a major function of the Greatwall pathway in mammalian cells is to delay reactivation of PP2A/B55 during mitotic exit, and this is reflected by the phenotypes following loss of Greatwall in human and mouse fibroblasts. In summary, the available literature suggests that PP2A/ B55 is a major exit phosphatase, but that other phosphates significantly contribute to Cdk1 substrate dephosphorylation. Thus, phosphatases are likely to act in parallel to, and even upstream of PP2A/B55 during mitotic exit to trigger the coordinated dephosphorylation of Cdk1 substrates.

PP1 regulation and function

challenge in determining specific functions of PP1 at a given time. Functions for this phosphatase have also been reported for nuclear envelope reassembly, kinetochore disassembly, and cytokinesis but the precise substrates and mode of interaction with PP1 remain in many cases unsolved [7, 8]. A recent study by Grallert et al. [133] in fission yeast reported that PP1 directly interacts with PP2A/B55 and PP2A/B56 via a conserved PP1 docking motif. The data suggest that PP1 is initially recruited to PP2A/B55 following the first drop of Cdk1 activity due to cyclin proteolysis. This allows PP2A/B55 to dephosphorylate PP2A/B56 and generate access for PP1 binding and full activation of the phosphatase relay. These PP1 docking sites in B55 and B56 subunits are conserved across species but, given the available structural data [134], seem to be buried within the phosphatase holoenzyme complexes. The presence of this phosphatase relay in higher eukaryotes and the structural discrepancies remain to be resolved.

Fcp1 and other phosphatases

PP1 is another mitotic exit phosphatase, but similar to PP2A/ B55 its precise function remains poorly understood. PP1 is phosphorylated by Cdk1 at an inhibitory site, T320 [128, 129]. Work in Xenopus egg extracts suggests that it is also kept inactive by the binding of a small inhibitory protein, Inhibitor1, and that this interaction is mediated by a PKA-dependent phosphorylation site, analogous to the interaction of Ensa/ ARPP19 and PP2A/B55 [130]. The drop of Cdk1 activity during mitotic exit is sufficient to allow autodephosphorylation of T320, followed by dephosphorylation of Inhibitor-1 by PP1 itself. It remains to be shown that this follows a similar inhibition by the unfair competition mechanism that has been proposed for Ensa/ARPP19. The same study suggests that PP1 is the major Cdk1 counteracting phosphatase and that continuous inhibition of PP1 prevents the dephosphorylation of the bulk of Cdk1 substrates. As discussed above, the crucial role of PP1 in mitotic exit in Xenopus egg extracts has recently been re-emphasized by the Xenopus and mammalian studies that place PP1 at the top of the mitotic exit dephosphorylation cascade [122–124]. However, the role of mammalian PP1 in mitotic entry and exit needs to be further substantiated by experimental evidence. The Rogers et al. study does not achieve a significant inhibition of Greatwall dephosphorylation using siRNA against catalytic PP1 subunits. This may be due to toxic effects in substantially PP1 depleted cells. However, the PP1 inhibitor Tautomycetin also does not affect Greatwall inactivation and Ensa dephosphorylation in human cells [121], but the actual penetration of this inhibitor and the amount of PP1 inhibition in this assay are difficult to verify. A better way of inhibiting PP1 in mammalian cells has recently been established by Winkler et al. [131] who engineered an inducible expression of nuclear inhibitor of PP1 (NIPP1) in HeLa cells. This study suggests that the major function of PP1 in mitosis lies in spindle checkpoint signaling, and can be bypassed by concomitant inhibition of Aurora-B, arguing against a major role of PP1 in counteracting Cdk1 during mitotic exit. The catalytic subunits of PP1 are regulated in their substrate specificity, localization and activity by over 200 PP1 interacting proteins (PIPs) [132] and this poses a major

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As discussed above, PP1 and PP2A/B55 have each been proposed to be the major mitotic exit phosphatase that removes the bulk of Cdk1 substrate phosphorylations. However, the effect of knocking down or inhibiting these enzymes does not appear to prevent cells from exiting mitosis. Mitotic cell cycle control involves a cohort of other phosphatases and new players are likely to be discovered. Fcp1 is an interesting example of a phosphatase that has only recently been implicated in mitotic exit control [135]. As discussed above, the major function of Fcp1 may lie in the inactivation of Greatwall kinase [125]. Fcp1 appears to dephosphorylate Greatwall kinase at critical activating residues, and this contributes to its mitotic exit phenotypes. This leaves us with a complex interplay of three phosphatases, PP2A/B55, PP1, and Fcp1 that regulate Greatwall kinase at different residues, and raises a critical question: how is this complex interplay between PP1, Fcp1, and PP2A/B55 regulated and which phosphatase ultimately kicks off the mitotic exit cascade? Apart from the above-mentioned phosphatases, there may well be other enzymes that contribute directly, or indirectly, to the removal of Cdk1 dependent phospho-sites. A search in the mitocheck database [51] flags up a variety of phosphatase enzymes and regulatory subunits that show mitotic phenotypes upon depletion (Table 2). This warrants further investigation. A major problem in this field is to distinguish between interphase and specific mitotic function for individual phosphatase complexes. This will require much more precise genetic tools than siRNA depletion, or broad chemical inhibition to inactivate a specific phosphatase complex in space and time. Chemical genetic inhibition or degron tags could provide such a possibility and need to be further optimized.

Cdk1 thresholds and bistability Differences of Cdk1 activity thresholds that trigger forward and prevent reverse reactions lie at the heart of the Cdk1


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Table 2. Mitotic phenotypes of phosphatases (from mitocheck database [51])

Regulatory subunits showing mitotic phenotypes

Gene name PPP1CA PPP1CB

Alternative names PP1a PP1b

PPP1CC PPP2CA PPP2CB PPP3CA PPP3CB PPP3CC PPM1B PPP4C PPP5C PPP6C PPEF1 PPEF2 CDC25A CDC25B CDC25C CDC14A CDC14B CTDP1 CDKN3 DUSP8

PP1g PP2Aa PP2Ab PP2Ba PP2Bb PP2Bg PP2Cb PP4 PP5 PP6 PP7 PPEF-2

LHPP

PHPT1

phospholysine phosphohistidine inorganic pyrophosphate phosphatase phosphohistidine phosphatase 1

PPM1F PPM1L

Protein phosphatase 1F PP2Ce

PPP1R1B

DARPP-32

PPP2R1A PPP2R2A

PR65a B55a

PPP2R2B PPP4R1L

B55b Protein phosphatase 4, regulatory subunit 1-like

FCP1 dual specificity phosphatase 8; hVH-5

activation switch. The observation that approximately three times more cyclin B is required for mitotic entry than to stay in mitosis in Xenopus egg extracts provided compelling experimental support for this notion and suggested that the mitotic switch is bistable [14, 15]. What remains unclear is, what determines these thresholds for Cdk1 activity? What are the major obstacles that need to be overcome to push cells into mitosis? Why do they no longer matter once cells have reached a stable mitotic state? Several studies have tried to correlate the levels of Cdk1 activity with mitotic entry events. These were based either on quantitative immunofluorescence [115, 136], or FRET measurements in live cells [114]. One can conclude from these studies that Cdk1 is first activated in the cytoplasm with relatively slow dynamics. This triggers nuclear translocation of cyclin B,

siRNA mitotic phenotypes [50] None Binuclear, nuclei stay close together, segregation problems, metaphase alignment problems None Nuclei stay close together, segregation problems None None None None None None Segregation problems None None None None None None None None None None Nuclei stay close together, segregation problems, metaphase alignment problems, strange nuclear shape Polylobed

Nuclei stay close together, segregation problems, strange nuclear shape, polylobed Binuclear Nuclei stay close together, segregation problems, strange nuclear shape Metaphase delay, mitotic delay, metaphase alignment problems, strange nuclear shape Metaphase delay, metaphase alignment problems Nuclei stay close together, segregation problems, strange nuclear shape Nuclei stay close together, binuclear Metaphase alignment problems

which causes a transient drop in Cdk1 concentration in the cytoplasm. The translocation itself appears to be subjected to positive feedback because it is triggered by Cdk1-dependent cyclin B phosphorylation and stimulated by cyclin B nuclear translocation [108, 113]. Nuclear Cdk1 then rapidly continues to rise in activity and finally causes NEBD, which happens at about the time when Cdk1 activity peaks. However, there appears to be a threshold for maximal Cdk1 activity after NEBD, because cells with decreased Cdk1 activity can enter prometaphase, but cannot satisfy the spindle assembly checkpoint and delay mitotic exit [136, 137]. Early events of mitotic entry such as cell rounding, centrosome separation and cyclin B nuclear translocation are thus likely to occur at a time before full Cdk1 activation and feedback-driven autoamplification. It is tempting to speculate that nuclear


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Phosphatase subunits Catalytic subunits

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translocation of cyclin B is in fact a pre-requisite for the autoamplification loop, which may involve inactivation of nuclear Wee1, activation of nuclear Cdc25 and inhibition of PP2A/B55 via Greatwall kinase. Once the positive and double negative feedback loops trigger full Cdk1 activation, cells quickly reach the threshold for forward progression and undergo NEBD and other mitotic rearrangements of the nucleus and cytoskeleton. This step causes a profound change in the cellular environment due to the mixing of cytoplasm and nucleoplasm and the activation of the mitotic proteolysis mechanisms. Progression through this transition is a true watershed that irreversibly commits the cell to move into G1 phase. This is exemplified by experiments by Potapova et al. [17] that show how Cdk1 inactivation before NEBD results in a reversion to G2, while the same treatment after NEBD results in forward progression into G1 that is accompanied by chromosome segregation and cytokinesis. Mitotic entry and exit are thus separated into two forward and reverse reactions, G2/prophase and M/G1 phases. These two switches are separated by a watershed that constitutes a true irreversible step due to the drastic change in the intracellular compartments (Fig. 1).

Cdk1 activity thresholds during the G2/prophase transition During the early stage of Cdk1 activation, cells are not yet committed to progression into M-phase and can be pushed back into G2 by Cdk inhibitors. There may be a physiological relevance for this reversal in the form of an antephase checkpoint [138]. We know very little about this early phase of Cdk1 activation. According to estimations by Lindqvist et al. [136], Cdk1 Y15 phosphorylation drops to about 50% before cyclin B translocates to the nucleus. However, these data are based on fairly low sampling numbers and are not clearly correlated in time to NEBD. Moreover, this initial phase of Cdk1 dephosphorylation may not be instantly translated into substrate phosphorylation because of remaining phosphatase activity. In fact, the regulation of phosphatases is a critical question in this early phase of Cdk1 activation that needs to be addressed. An interesting idea by Alvarez-Fernandez et al. [6] proposes that cyclin B needs to move to the nucleus to switch off the phosphatases that counteract the feedback loops between Cdk1 and Cdc25/Wee1. This is exemplified by the first activation of nuclear Greatwall kinase and its nuclear export, which takes place after accumulation of cyclin B in the nucleus [102]. On the other hand, we know very little about the spatial and temporal regulation of other phosphatases such as PP1 and Fcp1, which may be critical to understand the system dynamics. High phosphatase activity during the initial phase of cytoplasmic Cdk1 activation could explain the relatively slow dynamics that have been reported to initiate already in early G2 [115]. Another important question arises from this observation: is the initial Cdk1 activation and prophase entry bistable? This is testable, if one can determine the Cdk1 activity thresholds for prophase entry and the reverse reaction from prophase to G2 using cyclin B localization as a marker. One can imagine that the Cdk1 activation system

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stabilises once PP2A/B55, PP1, and other potential phosphatases are inhibited. If this takes place only after nuclear cyclin B translocation one could expect that the G2 and prophase thresholds for Cdk1 differ significantly. This model could also explain the bistability of the cyclin B translocation reaction itself, as Santos et al. [113] have proposed. If the phosphatase activity that targets the cyclin B nuclear import signal is shut down, once the first wave of cyclin B concentrates in the nucleus, a feedback loop is established that results in rapid import of the remaining cytoplasmic cyclin B. It is important to re-emphasise here, that Greatwall depletion does not appear to affect the dynamics of the mammalian mitotic entry switch significantly [101–103]. It will be critical to investigate how the Cdk1 threshold that sets the reverse reaction from prophase back to G2 is changed in Greatwall depleted cells and if other phosphatases are involved to stabilise this switch. Figure 2 gives an overview of the G2/M switch and its major components.

Full Cdk1 activation and nuclear envelope breakdown Once cyclin B accumulates in the nucleus, further Cdk1 activation will commit cells to mitotic progression (Fig. 3) that will eventually result in chromosome segregation, cell division and cell cycle reset at G1 phase. This commitment involves several aspects such as the mitotic proteolysis system, sister-chromatid segregation, cytokinesis, and relicensing of origins of replication. Several major changes take place at this time. Firstly, as discussed above, Cdk1 counteracting phosphatases are inactivated due to Greatwall activation, PP1 T320 phosphorylation, and potentially inhibitor 1 activation. This is likely to significantly contribute to the establishment of a stable mitotic state. Secondly, Cdk1 activity initiates bTrCP-dependent degradation of Wee1 and Emi1 and at the same time activates the APC/C. This does result in irreversible changes because subsequent Cdk1 inactivation will not cause the immediate rephosphorylation of Y15, but result in the destruction of major components of the mitotic entry switch. Finally, NEBD causes the mixing of the nucleoplasm and cytoplasm and destines the cell to move on to G1 phase. One can imagine that NEBD generates novel combinations of protein complexes and thereby changes various cellular activities. This could be especially important for the regulation of the cytoskeleton to set the cell up for chromosome segregation and cytokinesis. Indeed, many of the relevant factors are sequestered in the nucleus and will only make contact with microtubules when the nuclear and cytoplasmic compartments are fused. Thus, NEBD dramatically alters cytoskeletal dynamics and also allows the establishment of the kinetochore/K-fiber connection required for chromosome segregation. As a result of these irreversible changes, inactivation of Cdk1 after NEBD will cause the execution of a pre-programmed mitotic exit sequence involving sister chromatid segregation and the establishment of cytokinesis. Perturbation of the feedback loops can cause surprising changes in the behavior of the system. Bypassing the Cdk1


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Review essays Secondly, it demonstrates that this inactivation of mitotic phosphatases is an essential requirement to commit cells to the mitotic exit reaction following NEBD. One could speculate that the mitotic collapse state is a result of loss of bistability in the system which allows the maintenance of an otherwise unstable steady state in between interphase and mitosis. Greatwall depletion in mammalian cells also causes a prometaphase delay due to incomplete PP2A/B55 inactivation [101–103]. However, cyclin degradation and cytokinesis cleavage furrow formation do proceed in Greatwall depleted cells, which ultimately fail to complete cytokinesis. This comparison suggests that sustained PP2A/B55 activity alone is unlikely to cause a mitotic collapse state without triggering the mitotic exit program. Other OA sensitive phosphatases must play a critical role in parallel to PP2A/B55.

Figure 2. G2/prophase transition. Mitotic entry is thought to start with dephosphorylation of Cdk1 pY15 in the cytoplasm. However, cytoplasmic Cyclin B/Cdk1 shuttles continuously between nucleus and cytoplasm and Cyclin A bound to either Cdk1 or 2 is concentrated in the nucleus. Feedback in the form of Cdk1 dependent Cdc25 activation and Wee1/Myt1 inhibition plays a crucial role in this activation mechanism, but it is unclear how the cytoplasmic and nuclear forms of Cdc25 and Wee1/Myt1 contribute to this initial Cdk1 activation phase. It is also unclear if Cdk1 counteracting phosphatases are already inactivated at this point. The model presented here assumes that this is not the case, but that Cdk1 activity is slowly but progressively increased against a steady state dephosphorylation activity until a critical threshold that triggers Cyclin B nuclear translocation. This sets the mitotic progression in motion, by further activating Cdc25 and inhibiting Wee1 and concomitantly inhibiting PP2A/B55 by Greatwall activation and nuclear export. Inhibition of Cdk1 at this stage causes reversion of the reaction, nuclear export of Cyclin B and rephosphorylation of Cdk1 at T14/Y15. Critical questions for these transitions are the different roles of Cyclin A and B in triggering and maintaining the feedback, contribution of the different Cdc25 paralogues and Wee1/Myt1, identity of counteracting phosphatases and finally timing and mechanisms of phosphatase inactivation. Regulation of nuclear cytoplasmic localization of various components of the system such as Greatwall, Cdc25, and other as yet unidentified localization changes are critical for this switch system.

activation feedback loop by over-expressing a non-inhibitable Cdk1AF mutant results in repeated, rapid rounds of prometaphase entry followed by partial cyclin B destruction and reversal into an interphase state without cytokinesis [57]. Likewise, dual inhibition of Cdc25 and Wee1 causes Cdk1 activation at sub-threshold levels for mitotic completion and results in a “collapsed mitosis” [17]. Interestingly this appears to be a result of incomplete inactivation of mitotic phosphatases and can be reversed by OA. This is a crucial experiment that bears two major implications. Firstly, it suggests that the Cdk1 threshold for NEBD can be reached despite continuous phosphatase activity.

Cdk1 threshold during mitotic exit Mitotic exit after NEBD is ultimately triggered by cyclin B degradation followed by the timely reactivation of the Cdk1 counteracting phosphatases and the ordered removal of Cdk1 dependent phospho-sites. Phosphatase reactivation is likely to be of critical importance to determine the Cdk1 activity threshold determinant during this cell cycle


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Figure 3. Prophase-NEBD-metaphase transition. Changes in localization and phosphatase inactivation give the Cdk1 activation loop further impetus resulting in a steep acceleration in activation dynamics. Cdk1 activity rapidly rises above the required threshold for APC/C activation, Wee1 degradation, and NEBD. In unperturbed conditions cells take less than 10 minutes from cyclin B translocation to NEBD. Proteolysis and NEBD then cause a dramatic change by mixing up nuclear and cytoplasmic proteins. This may result in a further push in Cdk1 activity that is required to reach metaphase. In intermediate Cdk1 inhibition levels cells can undergo NEBD, but are not able to establish a correctly aligned metaphase spindle suggesting that this requires the highest amount of Cdk1 activity. Cells that are driven into mitosis by concomitant Wee1 and Cdc25 inhibition will also be capable of undergoing NEBD, but do so without inactivation of phosphatases and remain in a mitotic collapse state. This suggests that phosphatase inactivation is only essential to reach the highest Cdk1 threshold for metaphase, but not for NEBD. Accordingly, Greatwall depleted cells also reach prometaphase and exhibit a similar collapsed mitosis phenotype.

transition. It is important to note that the original Novak/ Tyson model for the mitotic switch was primarily based on the dephosphorylation and rephosphorylation of Cdk1 at Y15. The equilibrium of this reaction is critical for the G2/ prophase switch (see above) but is unlikely to play an important role during the metaphase to G1 transition since both Wee1 and cyclin B are degraded and Cdk1 rephosphorylation will only occur at a much later stage when both proteins have been re-synthesized. Phosphatase re-activation is likely to play a critical role in determining the mitotic exit threshold (see Fig. 4). We do not know enough about

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mitotic regulation of Cdk1-counteracting phosphatases to fully understand how the Cdk1 threshold for their reactivation could be set. The two known major Cdk1-dependent mechanisms of phosphatase inactivation are T320 phosphorylation of PP1 and Greatwall kinase-dependent phosphorylation of Ensa/ARPP19 blocking PP2A/B55. As discussed above, PP1 is thought to reactivate itself once Cdk1 levels begin to fall due to cyclin B degradation. If the PP1-PP2A relay that Grallert et al [133] described in S.pombe is of general relevance, then one could easily imagine that this reactivation of PP1 is the critical event that sets the mitotic exit phosphatase network in motion. Moreover, the new observation that PP1 dephosphorylates Greatwall [122–124] could constitute another link between PP1 and PP2A/B55 reactivation. The Cdk1 threshold for triggering this network would then simply be set by the forward and reverse reaction of PP1 T320 dephosphorylation. In reality the threshold may be determined by more complex interactions, and other phosphatases such as Fcp1 need to be accounted for in this network. The precise interplay of PP1, PP2A/B55, Fcp1, and other phosphatases that may contribute to mitotic exit will need more quantitative work to be precisely understood and modeled. Conceivably, the mitotic state should be robust and allow for significant fluctuations in Cdk1 activity before triggering the exit reactions. This means that the threshold for phosphatase activation should be relatively low. The bistability models would even predict that it is lower than the initial Cdk1 activity threshold for mitotic entry. This appears to be the case in the Xenopus egg extracts, but this remains to be confirmed in an intact cell system.


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Figure 4. Mitotic exit. Cyclin B degradation results in a drop in Cdk1 activity, but not in the immediate reactivation of the Cdk1 counteracting phosphatases. Coordinated and timely dephosphorylation of the large number of mitotic phosphorylation sites is crucial for the execution of this complex transition through mitotic exit. The best understood examples of mitotic exit phosphatases are PP1 and PP2A/B55. PP1 is directly inhibited by Cdk1 by T320 phosphorylation, and its reactivation by auto-dephosporylation can be expected to play a critical role at the onset of mitotic exit. Since this site is targeted by PP1 itself T320 dephosphorylation could simply be achieved by changes in the kinase/phosphatase equilibrium due to a drop in cyclin B levels below a critical threshold. PP1 then inactivates Greatwall, which allows PP2A/B55 to dephosphorylate its inhibitors ARPP19 and Ensa and reactivate itself. PP1 may also directly contribute to PP2A/B55 activation, but this remains to be confirmed in mammalian cells. Fcp1 has also been implicated in this mechanism by directly contributing to Greatwall dephosphorylation. It is probable that other phosphatases also take part in the exit reaction (see Table 2). The investigation of the identity, regulation and interplay of these enzymes during the progression from metaphase to G1 remains an important challenge for cell biology.

Outlook: Assessing bistability in the mitotic switch system We have attempted to give a systematic outline of what we consider important features of the mitotic entry and exit reactions in higher eukaryotic cells. The complexity of this

regulatory system is staggering, but we are beginning to gain a critical understanding of some of its essential components. The pioneering work of Novak and Tyson to generate a mathematical model of this switch has also generated a conceptual framework on which to build a precise quantitative understanding of this cell cycle transition. As we propose here, it will be important to incorporate the various thresholds and potential bistable switches in the entry and exit reactions into a revised model (Fig. 5). In our view the most critical missing links are a functional understanding of some of the redundant components in the system such as the different Cdc25 paralogues, the Wee1/Myt1 kinases, and the different mitotic cyclins. Moreover, many of the key experiments on mitotic entry and exit are done in cell-free extracts of Xenopus eggs and oocytes. There could be major discrepancies between this system and mammalian cells. This is exemplified by the much more striking effects that Greatwall depletion has on mitotic entry in Xenopus extracts when compared to mammalian cells. Ultimately we need to establish how the entry and exit switches work in at least some un-transformed mammalian cells to generate a reference model that incorporates the basic principles of the system. This needs to include the phosphatase control as well as spatial and temporal order of events. Finally, we have to develop more precise tools to manipulate the system to test predictions that arise from the mathematical models. Overall, this cell cycle transition is an exciting example of how a complex interplay of protein phosphorylation, dephosphorylation and proteolysis generates a robust and irreversible switch system.


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Figure 5. Cdk1 thresholds. A major assumption of the bistability model for mitotic entry and exit is that the Cdk1 activity required for mitotic entry is higher than the activity required to maintain the mitotic state. Given the complexity and the various observed thresholds for mitotic progression we propose here a more detailed model for Cdk1 activity thresholds. Firstly, the G2/prophase transition based on Cdk1 Y15 dephosphorylation and Cyclin B nuclear accumulation could itself be a bistable switch system. If this were true Cdk1 Threshold 01F should be higher than 01R, but these thresholds have to our knowledge not been precisely tested. Nuclear envelope breakdown, phosphatase inactivation and APC/C activation introduce irreversibility and require a higher Cdk1 threshold 02 to reach prometaphase. An even higher threshold 03 for Cdk1 is required to reach a metaphase state with correctly aligned sister chromatids (Threshold 03). This is strictly dependent on phosphatase inactivation, while Threshold 01 and 02 can be reached even if counteracting phosphatases are still active. Cdk1 activity then drops due to Cyclin degradation. The threshold for mitotic exit (Threshold 04) is likely to be determined by phosphatase reactivation and should be lower than Threshold 01 based on the experimental evidence from Xenopus egg extracts.

Acknowledgments We would like to thank the BBSRC Bicycle consortium (http:// cellcycle.org.uk), including Chris Bakal, Francis Barr, Ulrike Gruneberg, and Bela Novak for helpful discussion and suggestions. HH is funded by a CRUK senior fellowship C28206/A14499, NH is funded by a BBSRC LoLa grant BB/ M00354X/1, SR is supported by an EPSRC studentship. No new data and materials were generated in this publication. The authors have declared no conflict of interest.

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