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Molecular Cell

Article The BEG (PP2A-B55/ENSA/Greatwall) Pathway Ensures Cytokinesis follows Chromosome Separation Michael J. Cundell,1 Ricardo Nunes Bastos,1 Tongli Zhang,1 James Holder,1 Ulrike Gruneberg,1 Bela Novak,1 and Francis A. Barr1,* 1University of Oxford, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.09.005 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

SUMMARY

Cytokinesis follows separase activation and chromosome segregation. This order is ensured in budding yeast by the mitotic exit network (MEN), where Cdc14p dephosphorylates key conserved Cdk1substrates exemplified by the anaphase spindleelongation protein Ase1p. However, in metazoans, MEN and Cdc14 function is not conserved. Instead, the PP2A-B55a/ENSA/Greatwall (BEG) pathway controls the human Ase1p ortholog PRC1. In this pathway, PP2A-B55 inhibition is coupled to Cdk1-cyclin B activity, whereas separase inhibition is maintained by cyclin B concentration. This creates two cyclin B thresholds during mitotic exit. Simulation and experiments using PRC1 as a model substrate show that the first threshold permits separase activation and chromosome segregation, and the second permits PP2A-B55 activation and initiation of cytokinesis. Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. Therefore, temporal order during mitotic exit is promoted by the metazoan BEG pathway. INTRODUCTION Entry into and exit from mitosis is driven by oscillation in the activity of the cyclin-dependent kinases (Cdks) (Morgan, 1997). The accumulation of cyclin B activates Cdk1 and drives cells into mitosis. Cdk1-cyclin B phosphorylates target proteins playing key roles in nuclear envelope breakdown, chromosome condensation, and remodelling the actin and microtubule cytoskeletons. As a consequence, a bipolar mitotic spindle structure is formed, upon which the chromosomes become captured (Foley and Kapoor, 2013; Tanenbaum and Medema, 2010). Cdk1-cyclin B also phosphorylates and inhibits the activity of proteins required in anaphase in order to promote central spindle formation and

cytokinesis (Jiang et al., 1998; Mishima et al., 2004; Mollinari et al., 2002; Potapova et al., 2006). As cells enter mitosis complex, regulatory networks ensure the rapid and timely accumulation of these phosphorylations. Primarily, Cdk1 is controlled by the Wee1 and Myt1 kinases that inhibit its activation and the Cdc25 phosphatases that relieve this inhibition (O’Farrell, 2001). In metazoans, there is an additional level of regulation involving the PP2A-B55 and PP1 phosphatases that remove Cdk1 phosphorylations (Bollen et al., 2009; Glover, 2012; Mochida and Hunt, 2012; Wurzenberger and Gerlich, 2011). These phosphatases are subject to Cdk1-dependent inhibition. PP1 is directly phosphorylated at T320, resulting in the inhibition of its activity and binding to phosphorylated inhibitor 1 (Wu et al., 2009). In the case of PP2A, Cdk1-cyclin B activates the Greatwall/MASTL protein kinase (Yu et al., 2006), which phosphorylates and activates endosulfine a (ENSA) and ARPP19, small heat-stable inhibitors of PP2A-B55 (Archambault et al., 2007; Gharbi-Ayachi et al., 2010; Mochida et al., 2010; Rangone et al., 2011). Together, these mechanisms ensure that Cdk1 activity undergoes a rapid switch-like transition at the onset of mitosis (Krasinska et al., 2011), and Cdk1 phosphorylation events are no longer opposed by counteracting phosphatases and, therefore, also rapidly accumulate. Exit from mitosis is triggered by anaphase promoting complex/cyclosome (APC/C)dependent ubiquitylation and proteasomal degradation of cyclin B and securin (Peters, 2006). This results in a drop in cyclin B and securin levels and the activation of the protease separase that cleaves the cohesin rings linking sister chromatids (Gorr et al., 2005; Holland et al., 2007; Holland and Taylor, 2006; Stemmann et al., 2001). As the chromosomes segregate, there are large changes in spindle architecture and a microtubule structure, referred to as the central spindle, which serves to direct the position of cell cleavage and cytokinesis by recruiting the Polo-like kinase 1 (Plk1), is created between them (Neef et al., 2007; Petronczki et al., 2007; Santamaria et al., 2007; Wolfe et al., 2009). PRC1 is a highly conserved component of the anaphase central spindle, required to promote anaphase spindle elongation, and stabilize and organize microtubules in an antiparallel overlapped fashion (Walczak and Shaw, 2010). This activity is inhibited in metaphase cells through phosphorylation at T481 by the Cdk1-cyclin B mitotic kinase (Jiang et al., 1998; Mollinari et al., 2002). In addition to this inhibitory control, PRC1 is subject to activating phosphorylation by Plk1 at T602 that accumulates

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Molecular Cell EG Timer: A Cytokinesis Control Network

in anaphase human cells (Neef et al., 2007). In budding yeast, the dephosphorylation of the PRC1 ortholog Ase1p is mediated by the phosphatase Cdc14p (Khmelinskii et al., 2007, 2009). Given that mitotic kinase activity falls during mitotic exit, Cdc14p substrates become dephosphorylated at different kinase activity thresholds in an ordered series (Bouchoux and Uhlmann, 2011). Although there is some evidence supporting a role for Cdc14 family phosphatases in metazoan mitosis (Gruneberg et al., 2002; Mishima et al., 2004; Tumurbaatar et al., 2011), other studies have indicated that it is not essential for cell division and, instead, functions in the DNA damage checkpoint signaling pathway (Mocciaro et al., 2010; Mocciaro and Schiebel, 2010). Therefore, alternative phosphatases must regulate mitotic exit in human cells, and various lines of evidence indicate members of the PPP family of enzymes fulfill this role (Bollen et al., 2009; Glover, 2012). Mitotic exit in mammalian cells can be induced by Cdk1 inhibition when cyclin B degradation is prevented (Potapova et al., 2006) yet persists when both proteolysis and phosphatase activity are blocked (Skoufias et al., 2007), showing that phosphatase regulation is a central determinant of mitotic exit control. While cyclin B is degraded in anaphase, PP1 autodephosphorylates at T320 and subsequently dephosphorylates bound inhibitor 1 to become fully active (Wu et al., 2009). PP1 has generally been implicated in the removal of Cdk-phosphorylations at mitotic exit (Wu et al., 2009), although the precise targets remain, with a few exceptions (Qian et al., 2011; Trinkle-Mulcahy et al., 2006; Vagnarelli et al., 2006), poorly defined. Other studies have shown that PP2A-B55 is required for the removal of some Cdk phosphorylations at the metaphaseanaphase transition (Mochida et al., 2009) specifically for the reassembly of the Golgi apparatus and nuclear envelope in telophase (Lowe et al., 2000; Schmitz et al., 2010). The other major isoform of PP2A in mitosis, PP2A-B56, has not been implicated in anaphase regulation but, instead, protects centromeric cohesion from premature release in prophase through to metaphase (Kitajima et al., 2006; Riedel et al., 2006) and promotes the formation of stable microtubule-kinetochore attachments (Foley et al., 2011). To date, the metazoan phosphatase required for the regulation of PRC1 with the analogous function to budding yeast Cdc14p has not been found. The correct timing of PRC1 dephosphorylation with respect to separase activation is critical for allowing chromosome segregation to complete before central spindle formation is triggered. Therefore, the characteristics of a candidate PRC1 phosphatase would need to comply with these timing requirements. As described previously, the literature provides two alternative pathways centered about PP1 and PP2A that could carry out this function, and this is investigated here. RESULTS Identification of PRC1 Phosphatases in Mitotic Cell Extracts To identify PRC1 phosphatases active during mitosis, we fractioned extracts of nocodazole-arrested cells by ion exchange chromatography. Then, these fractions were incubated at 4 C with recombinant PRC1 phosphorylated at the T481 Cdk site or the T602 Plk1 site (Figure 1). This analysis revealed a single

peak of PRC1-pT481 phosphatase activity from fractions E13E17 (Figure 1A), matching the elution profile of PP2A catalytic and regulatory subunits (Figure S1 available online). Little or no activity toward PRC1-pT602 was detected (Figure 1B). Previous work has found that T602 phosphorylation does not accumulate on PRC1 phosphorylated by Cdk1 (Neef et al., 2007), suggesting that the phosphatase may recognize this dual phosphorylated form of PRC1 and remove the T602 phosphorylation. When a PRC1 pT481-pT602 substrate was used, pT602 phosphatase activity was observed in a peak spreading from E11 onward (Figure 1C), matching the profile of PP2A-B56 (Figure S1). These findings indicated that one of the PP2A subtypes is the pT481 Cdk site phosphatase. Furthermore, they suggested that a second form of PP2A, possibly B56, is a pT481-dependent pT602 phosphatase. When taken together, these two activities provide a means for ensuring the switch in PRC1 phosphorylation seen at the metaphase-anaphase transition (Figure 1D). Then, a two-stage small interfering (siRNA) screen was performed in order to identify the specific phosphatases acting on PRC1. The subclass of PPP enzyme was defined by first screening catalytic subunits, and then a secondary screen was performed with only the regulatory subunits for that enzyme class in order to define the specific holoenzyme complex. The PP6 catalytic subunit PPP6C provided a positive control, given that this is known to act as a specific phosphatase for AuroraA pT288 (Hammond et al., 2013; Zeng et al., 2010). This approach showed that PP2A was the phosphatase acting on both the pT481 and pT602 sites, given that the removal of PPP2CA and PPP2CB, but not other catalytic subunits, stabilized these phosphorylations (Figures 2A and 2B). The addition of okadaic acid to equivalent samples at cell lysis showed that samples all start out with the same level of PRC1 phosphorylation (Figure 2A). In the case of pT481, it was possible to define a PP2A-B55a holoenzyme comprised of the PPP2R1A scaffold, PPP2R2A regulatory, and the PPP2CA or PPP2CB catalytic subunit (Figure 2C). For pT602, the further definition of specific subunits proved not to be possible, suggesting there may be redundancy between two or more forms of PP2A. Altogether, these results support a model where PP2A-B55a is the major phosphatase opposing the Cdk1 phosphorylation of PRC1, whereas additional forms of PP2A act on pT602 (Figure 2D). Direct confirmation of this model was achieved with purified PP2A-B55a and PP2A-B56g holoenzymes (Figure S2A). Purified PP2A-B55a was titrated in order to yield the same level of activity as mitotic cell extract at 4 C and 30 C (Figure S2B); then, the amount of PP2A-B56g matched to this (Figures S2C–S2D). This analysis indicates that 7.4 ng of PP2A-B55 and B56 holoenzyme are present per mg of cell extract. Dephosphorylation time courses showed that this matched amount of purified PP2AB55a (Figures S2B–S2D) had the same level of activity toward PRC1 pT481 at 4 C as mitotic lysate, and this was greatly increased at 30 C (Figures 3A and 3B). An equivalent amount of purified PP2A-B56g (Figure S2D) was unable to dephosphorylate pT481 (Figures 3A and 3B). However, this treatment downshifted PRC1, indicating that PP2A-B56g acts at another site on PRC1. Then, the activity of both PP2A-B55a and PP2-B56g was tested on the other forms of PRC1. Although neither enzyme showed high activity toward PRC1 pT602, both enzymes were

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Figure 1. PRC1 pT481 Phosphorylation Promotes pT602 Dephosphorylation in a Fractionated Mitotic Cell Extract (A–C) Phosphatase assays with recombinant PRC1 substrates phosphorylated at pT481 (A), pT602 (B), or pT481 and pT602 (C) were performed with mitotic extract separated by anion exchange chromatography into 27 eluate (E1–E27) and five flow-through (FT1– FT5) fractions. Assays were incubated with each fraction for 35 min on ice. Samples were western blotted for PRC1, PRC1 pT481, and PRC1 pT602 or subunits of PPP enzymes (see Figure S1). (D) In metaphase, Cdk1-cyclin B phosphorylates PRC1-pT481, preventing its associate with microtubules and Plk1 and promoting PP2A-dependent dephosphorylation of the activating pT602 Plk1 phosphorylation, thus preventing its precocious accumulation in metaphase. Entry into anaphase results in APCdependent cyclin B destruction and pT481 dephosphorylation. PRC1 can now bind to and crosslink antiparallel microtubules at the central spindle. In turn, Plk1 phosphorylates pT602, creating its own docking site, allowing it to accumulate at the central spindle, where it promotes the initiation of cytokinesis.

able to dephosphorylate T602 in the dual-phosphorylated PRC1 pT481-pT602 substrate (Figures 3C and 3D). Therefore, PP2AB55a is both necessary and sufficient for the dephosphorylation of the PRC1 Cdk1 site at T481, whereas the T602 Plk1 phosphorylation can be removed by either PP2A-B55a or B56g contingent on the presence of phosphorylation by Cdk1 at T481. Regulation of PP2A-B55a Activity toward PRC1 by ENSA and Greatwall It is known that PP2A-B55 is inhibited in mitosis through the action of the ENSA pathway (Castilho et al., 2009; Mochida et al., 2009; Vigneron et al., 2009), so the results described thus far with mitotic cell extract create an apparent paradox. During mitosis Cdk1-cyclin B activates the Greatwall/MASTL kinase (Yu et al., 2006), which phosphorylates its substrate ENSA. In turn, phosphorylated ENSA interacts with PP2A-B55 and inhibits its activity (Gharbi-Ayachi et al., 2010; Mochida et al., 2010). Therefore, this inhibitory pathway must decay in vitro in order for PP2A-B55 activity toward PRC1 to develop. Consequently, PRC1 pT481 dephosphorylation only occurred after a lag period of 15 min (Figure 4A), whereas the dephosphorylation of AuroraA pT288 by PP6 commenced immediately. During this initial 15 min of incubation, ENSA underwent a downshift (Figure 4A) due to the dephosphorylation at the Greatwall/MASTL site at

S67 (Figure S3A). This could explain the delayed dephosphorylation of PRC1 pT481, given that the initial inhibition of PP2AB55 by phosphorylated ENSA would be lost after 15 min of incubation. To test this idea, extracts were prepared from mitotic cells depleted of ENSA, the closely related factor ARPP19, or Greatwall/MASTL (Figures S3B and S3C). As expected, the depletion of Greatwall/MASTL resulted in the loss of phosphorylated ENSA (Figure S3D). These ENSA- and Greatwall/ MASTL-depleted extracts also failed to show the delay in PRC1 pT481 dephosphorylation observed in control extracts (Figure 4B). Furthermore, the removal of the PP2A regulatory subunits PPP2R2A–PPP2R2D stabilized both PRC1 pT481 and ENSA phosphorylation (Figure 4B), indicating that PP2A-B55 is the ENSA phosphatase in this system. Given that ARPP19 was not detected (Figure S3E) and that ARPP19 siRNA had little effect (Figure 4B), ENSA is the major target of Greatwall/MASTL in these cells. Studies using Xenopus egg extracts have shown that, at high extract concentrations, PP2A-B55 inhibition is maintained (Mochida et al., 2010), and the human cell extract system used here displayed similar properties. Only at extract concentrations lower than 20 mg/ml was there measurable PRC1 pT481 phosphatase activity, which became maximal at 10 mg/ml (Figures 4C and 4D). Importantly, this effect was not seen for all Cdk



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(A) To identify PRC1 pT481 phosphatases, catalytic subunits for each PPP family member were depleted. Cells were arrested in mitosis, lysed on ice for 1 hr as described in the phosphatase library screening method, and western blotted for endogenous PRC1, PRC1 pT481, and subunits of PPP enzymes. Aurora-A and Aurora-A pT288, the known target of PPP6C, were used as positive controls. Tubulin was used as a loading control. (B) An exogenous PRC1 substrate phosphorylated at both pT481 and pT602 was added to cell lysates depleted of catalytic subunits. Samples were incubated for 1hr and western blotted as indicated in the figure. (C) Cells depleted of PP2A regulatory and scaffold subunits were arrested in mitosis, lysed on ice for 1 hr as described in the phosphatase library screening method, and western blotted as indicated in the figure. PP2A-B55b antibodies crossreacted with a 50 kD protein marked by an asterisk, and the lower band is the B55b subunit. (D) In this iteration of the model, PP2A-B55a antagonizes Cdk1-cyclin B-dependent PRC1pT481 phosphorylation. Phosphorylation at pT481 also promotes the dephosphorylation of pT602 via a PP2A holoenzyme.

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substrates. Many of the phosphoproteins detected by a panCdk site antibody showed either no change on extract dilution or increased signals at low extract concentration (Figure 4C, arrows). Notably, the Cdk1 site on PPP1C T320 showed little turnover at all extract concentrations tested (Figures 4C and 4D). Furthermore, mass spectrometry indicated that the pS23 Cdk site on ENSA was not dephosphorylated (Figure S3A). These results agree with other evidence that PP2A-B55 is not the sole Cdk1-counteracting phosphatase during mitotic exit (Mochida et al., 2009; Wu et al., 2009). In addition, they demonstrate that PRC1 is a specific target for PP2A-B55 and suggest that PRC1 dephosphorylation can only commence once the ENSA/Greatwall (EG) pathway has been inactivated through a feedback loop where PP2A-B55 promotes ENSA dephosphorylation (Figure 4E). This raises the interesting possibility that the EG pathway may impart specific timing properties to PP2A-B55 activation during exit from mitosis. A Model for the PP2A-B55/ENSA/Greatwall Pathway in Mitotic Exit To establish the consequences of the PP2A-B55/ENSA/Greatwall (BEG) pathway for mitotic exit, we created a model of the known network of regulatory interactions (Figures 5A and S4). In this model, both PP2A-B55 and separase, the protease responsible for cohesin cleavage and anaphase initiation, are inhibited by the action of Cdk1-cyclin B. For PP2A-B55, this is due to the activation of the Greatwall/MASTL kinase, which phos-

phorylates the ENSA inhibitor (GharbiAyachi et al., 2010; Mochida et al., 2010; Rangone et al., 2011). Separase is subject to dual inhibition by cyclin B (Boos et al., 2008; Holland et al., 2007; Holland and Taylor, 2006) and securin (Shindo et al., 2012; Waizenegger et al., 2002). Once the spindle assembly checkpoint is silenced, the Cdc20 form of APC/C (APC/CCdc20) promotes the ubiquitin-dependent degradation of both securin and cyclin B with similar kinetics (Hagting et al., 2002). Because of their shared properties, these two APC/CCdc20 substrates are combined in the model under the name of anaphase inhibitors (Ana I). This creates a situation where PP2A-B55 inhibition depends on Cdk1-cyclin B activity, whereas separase inhibition is dependent on cyclin B and securin concentration (Figure 5A). Stoichiometric inhibition of separase defines a cyclin B threshold below which separase becomes activated (Figure 5B). The correct temporal order of anaphase and central spindle formation requires that the cyclin B threshold of PP2A-B55 activation is lower than that for separase. This is achieved if a low level of Cdk1-cyclin B activity can maintain Greatwall/MASTL and, hence, ENSA in the phosphorylated active state. Furthermore, because PP2A-B55 is inactive in M phase and feeds back onto Greatwall/MASTL and ENSA (Figure 4B) (Gharbi-Ayachi et al., 2010), a double-negative feedback loop in the BEG pathway is created (Figure 5A). Therefore, the dependence of PP2A-B55 steady-state activity on cyclin B levels follows a Z-shaped curve with low activation and high inactivation thresholds (Figure 5B). Mitotic cells occupy the lower right region of this plot, where both cyclin B level and Cdk1 activity are maximal, therefore sustaining the inhibition of separase



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and PP2A-B55 (dot in the green area in Figure 5B). The release of the spindle checkpoint and APC/C activation causes a decrease in both cyclin B and securin levels and, along with separase, reduces Cdk1 activity. Given that the separase activation threshold is crossed first, anaphase onset takes place while PP2A-B55 is still inhibited by phosphorylated ENSA through Greatwall/MASTL (the yellow area in Figure 5B). This is because Cdk1 still drives Greatwall/MASTL activity. Only once Cdk1cyclin B activity drops below the PP2A-B55 activation threshold does the dephosphorylation of key PP2A-B55 substrates such as PRC1 commence (the red area in Figure 5B). The difference in the two thresholds ensures a time delay between the onset of chromosome segregation and other events during mitotic exit, including PRC1 dependent central spindle formation (Mollinari et al., 2002; Neef et al., 2007). The model also accounts for PP2A-B55 activity in interphase and mitotic entry. In low Cdk1 activity interphase cells, PP2A-B55 occupies its high-activity steady state on the upper branch (G1, S, and G2 populate the red area in Figure 5B). Given that Cdk1 activity lags behind cyclin B levels in G2 phase, the inactivation threshold is crossed only during G2/M transition, when Cdk1 activity rises abruptly (the yellow area in Figure 5B). Therefore, the EG timer module provides a mechanism for delaying the removal of Cdk1 phosphorylations during mitotic exit. This conclusion was confirmed by the numerical simulation of normal mitotic progression (Figure 5C).

Experimental Validation of the BEG Pathway A series of experimentally testable predictions arise from this model and the accompanying simulations. These are based on the manipulation of the activity of Cdk1 independently from the level of cyclin B. First, preventing cyclin B degradation will stabilize the system (Figure 5D), whereas the shutdown of Cdk1cyclin B activity with chemical inhibition will create a state where Cdk1 activity is lost, but cyclin B levels initially remain high (Figure 5E). The predicted outcome of this latter perturbation is that separase activation will be slightly delayed, whereas PP2A-B55 activation will initiate prematurely in comparison to normal mitotic exit. In this scenario, the model predicts accelerated PRC1 pT481 dephosphorylation with respect to separase activation. To test this proposal, cells were arrested in mitosis with nocodazole. Then, nocodazole was removed, and mitotic spindle formation was allowed to proceed at 37 C for 25 min. By this stage, most cells had built a bipolar mitotic spindle with attached chromosomes. Then, synchronous passage into anaphase was triggered by the addition of an inhibitor for the MPS1 spindle assembly checkpoint kinase (Hewitt et al., 2010). This resulted in the degradation of the anaphase inhibitors cyclin B and securin followed by separase activation and PRC1 pT481 dephosphorylation and then the accumulation of the anaphase pT602 Plk1 phosphorylation (Figure 5G). As predicted by the simulation (Figure 5D), these changes were prevented by proteasome inhibition in order to stabilize securin and cyclin B


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Figure 4. PP2A-B55a Activity toward PRC1 pT481 Is under the Control of the Greatwall/ENSA Pathway (A) Dephosphorylation time course assays were carried out on ice with 0.8 mg/ml mitotic cell extracts. Dephosphorylation of endogenous PRC1 and Aurora-A was followed with phosphospecific antibodies. Phos-tag reagent was used to detect the MASTL phosphorylated form of ENSA. Blots were quantified (right graph), and the half-life of PRC1 pT481, Aurora A pT288, and ENSA pS67 were calculated. (B) The dephosphorylation of endogenous PRC1 pT481 and ENSA in 0.8 mg/ml extracts made from control, ARPP19, ENSA, MASTL, and combined PPP2R2A– PPP2R2D-depleted HeLa cells. Blots were quantified (right graphs), and the half-life of PRC1 pT481 was calculated (mean ± SEM, n = 2). See also Figure S3. (C) Equal protein amounts from mitotic cells lysed on ice for 1 hr were western blotted in order to determine the phosphorylation status of endogenous phosphoproteins as a function of extract concentration. Arrows mark Cdk substrates stabilized by extract dilution. (D) PRC1 pT481 and PPP1CA pT320 Cdk1 phosphorylation were quantified and plotted as a function of extract concentration (mean ± SEM, n = 2). (E) Data presented thus far allow us to build further complexity into the model. Cdk1-cyclin B not only phosphorylates pT481 but also inhibits the phosphatase that removes this posttranslational modification via the activation of the EG pathway. This inhibits PP2A-B55 in mitosis, increasing the half-life of pT481, and, in turn, promotes the PP2A-B56-dependent dephosphorylation of the Plk1 pT602 site. As cells enter anaphase, the EG pathway must decay before PP2A-B55a can become fully active and act on PRC1-pT481, thus creating a delay. At a critical threshold when there is not enough Cdk1-cyclin B to sustain this pathway, PP2AB55a becomes active and dephosphorylates pT481, promoting microtubule binding and Plk1 recruitment.

and, thus, prevent Cdk1 inactivation (Figure 5H). In contrast, the addition of a chemical inhibitor of Cdk resulted in complete PRC1 pT481 dephosphorylation before cyclin B destruction (Figure 5I). As a consequence, the PRC1 pT602 anaphase Plk1 phosphorylation accumulated prematurely and, therefore, overlapped with separase activation (Figure 5I). Modeling and experimental data both showed that, under conditions where Cdk activity is removed, proteasome inhibition to prevent cyclin B destruction uncoupled PP2A-B55 activation and subsequent PRC1 pT481 dephosphorylation from separase activation (Fig-

ures 5F and 5J). A plot of the experimentally determined PP2AB55 activity toward PRC1 pT481 and active separase as a function of cyclin B level demonstrated different thresholds for these activities (Figure 5K). Importantly, the lower cyclin B threshold for PP2A-B55 activation was dependent on Cdk activity rather than cyclin B concentration, given that chemical inhibition of Cdk resulted in maximal PP2A-B55 activity at all cyclin B concentrations (Figure 5K). Altogether, these results show that PP2A-B55 inhibition depends on Cdk1-cyclin B activity, whereas separase inhibition is dependent on Cdk1-cyclin B level.



Ordering of Events in Mitotic Exit Requires the BEG Pathway Live-cell imaging was performed to examine the order of chromosome segregation and cytokinesis and the timing of PRC1 and Plk1 recruitment. This revealed that, although anaphase cell elongation and furrowing follow chromosome segregation in control cells (Figure 6A), Cdk inhibition perturbed this order (Figure 6B). The recruitment of PRC1 to the central spindle occurred prematurely in Cdk-inhibitor-treated cells and was followed by Plk1 immediately prior to the initiation of furrowing (Figures 6C and 6D). This was in agreement with the accelerated removal of pT481 and the creation of the Plk1 docking site on PRC1 at T602 (Figure 5H). Cell elongation and contraction also commenced prior to the movement of the chromosomes after Cdk inhibition (Figure 6B). Furthermore, lagging chromatin in the furrow region indicated that chromosome separation was incomplete (Figure 6B), consistent with the idea that other anaphase events had been accelerated with respect to separase activation under these conditions. To demonstrate the generality of the model presented here, we simulated the effects of introducing nondegradable cyclin B or cyclin B binding mutant separase. Previous work has investigated the effects of expressing different levels of stable cyclin B on chromosome separation and mitotic exit (Potapova et al., 2006; Wolf et al., 2006). These studies reported that cells arrest in metaphase with cohesed sister chromatids when the level of nondegradable cyclin B is high, whereas moderate levels allow a pseudometaphase state with sister chromatid separation. Only at low levels of nondegradable cyclin B did cells progress into an anaphase- and telophase-like state. Simulations with the model presented here show that the ‘‘high’’ condition does not allow separase or PP2A-B55 activation, whereas the ‘‘moderate’’ condition permits separase, but not PP2A-B55, activation and cytokinesis (Figure S5A). Only once cyclin B falls to low levels are separase and PP2A-B55 active and are cells predicted to enter a true anaphase state (Figure S5A), which is in agreement with previous observations (Wolf et al., 2006). A related study has shown that the removal of the cyclin B dependent separase inhibitory pathway has little effect on the kinetics of separase activation when securin is present but results in delayed anaphase spindle elongation. The model presented here suggests this may be due to altered PP2A-B55 activation and dephosphorylation of the anaphase spindle elongation factor PRC1. Simulations of separase and PP2A-B55 activity were performed with either wild-type separase or a cyclin B-binding mutant (SA-separase) to test this idea (Figure S5B). In agreement with experimental data (Shindo et al., 2012), SA-separase behaved essentially like the wild-type enzyme because of the securin component in the anaphase inhibitor term (Ana I), but separase inhibition of Cdk1-cyclin B was lost, potentiating PP2A-B55 inhibition by the BEG pathway. This delays the dephosphorylation and activation of PRC1, thus explaining the anaphase spindle elongation defect observed previously (see Figure 5A in Shindo et al., 2012). Therefore, the proposed model for separase and PP2A-B55 activation recapitulates the published experimental data on nondegradable cyclin B and cyclin B-binding-defective separase (Shindo et al., 2012; Wolf et al., 2006).

Premature Recruitment of PRC1-Plk1 in the Absence of the EG-Timer The predicted functional consequences of removal of Greatwall/ MASTL or ENSA are precocious PP2A-B55 activity, and the premature generation of the pT602 Plk1 docking site required for initiating cytokinesis (Figure 7A). This generates a testable prediction that untimely recruitment of PRC1 and Plk1 should occur in the absence of the EG timer module and be accompanied by a cut phenotype (Bastos and Barr, 2010; Neef et al., 2007). The depletion of PP2A-B55 regulatory subunits resulted in premature entry into mitosis (Mochida et al., 2009; Vigneron et al., 2009), whereas the depletion of Greatwall/MASTL delayed mitotic entry (Figure S6A), as expected from the previous work (Voets and Wolthuis, 2010; Yu et al., 2004). In both cases, the cells formed normal mitotic spindles (Figures 7B–7D), suggesting that, although the kinetics of mitotic entry are altered, there are no major consequences (Figure 7F). The depletion of PP2A-B56 resulted in a protracted mitotic delay with abnormal spindle morphology (Figure 7E), consistent with its function in stabilizing microtubule-kinetochore interactions (Foley et al., 2011). When Greatwall/MASTL-depleted cells exited mitosis, central spindle microtubule bundles formed prematurely between the partially segregated sister chromatids, and the nucleus reformed around this structure (Figure 7C). These changes were accompanied by the premature onset of contractions, and the cut phenotype arising ultimately led to cytokinesis failure in over 75% of cells (Figures 7C and 7F). Checkpoint silencing and APC/C activation measured with a Mad2 and securin dual reporter cell line were not obviously altered (Figures S6B and S6C), supporting the view that the cut phenotype is not due to a separase activation defect. Importantly, securin removal paralleled that of securin and cyclin B measured independently (compare Figures S6B and S6C to Figure 5G). Conversely, the depletion of PP2A-B55 regulatory subunits delayed central spindle formation and contraction (Figures 7D and 7F) due to delayed PRC1 recruitment and increased segregation of the chromosomes due to these central spindle defects (Figures 7D and 7G). Imaging of stable cell lines expressing PRC1 and Plk1 showed that PRC1 and Plk1 are recruited to the central spindle from 4–6 min after cells enter anaphase under control conditions (Figures 7H and 7K). Depletion of Greatwall/MASTL resulted in the precocious recruitment of PRC1 and Plk1 to metaphase spindles and elevated levels of both components at early times in anaphase (Figures 7I and 7K). In contrast, the depletion of PP2A-B55 regulatory subunits resulted in slightly delayed PRC1 and Plk1 recruitment and reduced levels at later times in anaphase (Figures 7J and 7K). These findings are consistent with a model wherein the inhibition of PP2A-B55 in metaphase is important in order to maintain the full phosphorylation of PRC1 and prevent its binding to the spindle. The release of this inhibition ordinarily delays PRC1 dephosphorylation in anaphase until after sister chromatid separation has been initiated by separase activation. DISCUSSION BEG for Orderly Mitotic Exit and Cytokinesis A feature emerging from this work is the delay period between Cdk1 inactivation due to cyclin B degradation and the



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Figure 5. Timing and Ordering of Events in Metazoan Mitotic Exit (A) A schematic of the network of interactions between Cdk-cyclin B, the BEG pathway, separase, and PRC1 (see also Figure S4). Securin is assumed to play the equivalent role to cyclin B with respect to separase; hence, we simplify, given that both are comparable APC/C substrates. (B) Model-calculated steady-state activities of separase (green) and PP2A-B55 (red) as a function of cyclin B are plotted in the graph and normalized to their maximal values. Cyclin B is divided into three ranges (low-red, intermediate-yellow, and high-green). Anaphase and mitotic exit are driven by the decrease in cyclin B levels, causing the activation of separase followed by PP2A-B55. (C–F) Outcomes were simulated for normal mitotic exit or mitotic exit following proteasome inhibition and rapid chemical inhibition of Cdk alone and combined with proteasome inhibition. (legend continued on next page)



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Figure 6. Precocious PRC1-Plk1 Recruitment and Cytokinesis with Respect to Chromosome Separation Follow Cdk1 Inhibition (A and B) Cell lines expressing histone H2B-mCherry eGFP-tubulin were treated with 200 mM MPS1 inhibitor (AZ3146; A) or 5 mM flavopiridol (B) and followed as they progressed into anaphase and initiated cytokinesis. (C and D) An eGFP-PRC1 and mCherry-Plk1 cell line was imaged undergoing synchronous progression into anaphase triggered with 200 mM MPS1 inhibitor (AZ3146) in order to override the spindle checkpoint (C) or the addition of 5 mM flavopiridol to inhibit Cdk1 (D). Maximum projections of eGFP-PRC1 and mCherryPlk1 with a bright-field reference image are shown. The scale bar represents 10 mm. Spindle- and central-spindle-associated PRC1 and Plk1 were measured as a function of time and plotted in the line graphs (mean ± SEM, n = 5). See also Figure S5.

dephosphorylation of a subset Cdk sites by PP2A-B55. Previous work has hinted at functions for Greatwall/MASTL in the control of cytokinesis and exit from mitosis (Burgess et al., 2010; Manchado et al., 2010; Voets and Wolthuis, 2010); however, the molecular basis for such a requirement has been unclear, and this is explained here. The BEG pathway ensures a delay in the dephosphorylation of key anaphase targets exemplified by PRC1 until after the activation of separase. At the heart of this pathway lies the EG timer module, which delays PP2AB55 activation relative to separase-dependant cleavage of cohesion between sister chromatids. Therefore, PP2A-B55 adopts the equivalent role to Cdc14p in the budding yeast mitotic exit network or fission yeast septation initiation network (Bardin and Amon, 2001; McCollum and Gould, 2001). Without this delay period, cell division would be less robust or fail entirely because of the cell division plane cutting through

the chromatin. The model presented here is consistent with the observations that mitotic exit is freely reversible if cyclin B is stabilized by proteasome inhibition (Potapova et al., 2006; Potapova et al., 2011), given that the re-establishment of PP2A-B55 inhibition by the EG pathway and the stabilization of the mitotic state are possible under these conditions. Our model also accounts for the failure of sister chromatid separation under these conditions. Moreover, it explains why the partial inhibition of Cdk1 in Cdc20 knockout cells that cannot degrade cyclin B or securin, can be induced in order to exit mitosis when securin is removed and Greatwall/ MASTL is inactivated (Manchado et al., 2010). Under these conditions, sister chromatid separation is not observed, but our model shows that PP2A-B55 would become activated and drive the removal of crucial Cdk phosphorylations and, hence, mitotic exit.

(G–J) Synchronous progression of mitotic cells into anaphase was triggered with 200 mM MPS1 inhibitor (AZ3146) in order to override the spindle checkpoint in the absence (G) or presence (H) of 100 mM proteasome inhibitor MG132 or the addition of 5 mM flavopiridol (I) in order to inhibit Cdk1 in the absence or presence (J) of 100mM proteasome inhibitor MG132. MG132 was added 5 min prior to the kinase inhibitor. Graphs show the kinetics of PRC1 dephosphorylation and separase activation detected by autocleavage following MPS1 or Cdk1 inhibition. (K) Experimentally determined values for PP2A-B55 activity toward PRC1 pT481 and active separase are plotted as a function of cyclin B concentration during mitotic exit. The effect of Cdk inhibition on PP2A-B55 activity is also shown. Cyclin B thresholds for separase and PP2A-B55 activation (BEG) are signified by dotted lines. The separase level at which chromosome separation occurred is marked.



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Figure 7. The Temporal Delay between Chromosome Segregation and the Onset of Cytokinesis Requires the ENSA/Greatwall Timer Module (A) Simulation of normal mitotic exit and loss of the EG pathway. (B–E) HeLa cells stably expressing histone H2B-mCherry and eGFP-tubulin were treated with control (B), Greatwall/MASTL (C), PPP2R2A– PPP2R2D (B55; D), or PPP2R5A– PPP2R5E (B56; E) siRNA were imaged undergoing mitosis. Representative maximum projections of eGFP-tubulin and mCherry-histone H2B with a brightfield reference image are shown. The scale bar represents 10 mm. (F) Time from nuclear envelope breakdown (NEBD) to the formation of a metaphase plate and onset of anaphase was measured or from anaphase onset until the contraction of the cell cortex was observed were measured and plotted on the bar graphs (mean ± SEM, n = 10). (G) The distance of anaphase chromosome segregation was measured and plotted on the bar graph (mean ± SEM, n = 12). (H–J) An eGFP-PRC1 mCherry-Plk1 cell line was treated with control (H), Greatwall/MASTL (I), or PP2A-B55 (J) siRNA and imaged undergoing mitosis. Representative maximum projections of eGFP-PRC1 and mCherry-Plk1 with a bright-field reference image are shown. The scale bar represents 10 mm. (K) Spindle- and central-spindle-associated PRC1 and Plk1 were measured as a function of time for the conditions shown and plotted in the line graphs (mean ± SEM, n = 4). See also Figure S6.

It is important to mention that there are some key differences to the biochemical activity of Cdc14p in budding yeast. Careful structural analysis has shown that Cdc14p is a proline-directed

phosphatase with high specificity toward Cdk sites (Gray et al., 2003). Here, we find that PP2A-B55 is able to act on both Cdk1 and Plk1 consensus sites on PRC1 and, therefore, does



not have the same restricted specificity as Cdc14p. An additional key finding is that PP2A-B56 cannot remove Cdk phosphorylations, a view supported by other work (Lowe et al., 2000). Therefore, the unique property of PP2A-B55 may be its inhibition during mitosis by the EG pathway rather than its substrate selectivity. In turn, PP2A-B56, which is also required in mitosis and meiosis (Foley et al., 2011; Kitajima et al., 2006; Riedel et al., 2006), may therefore be under selection to show little activity toward Cdk sites. Linking a Binary Switch to an Orderly Series of Events It has been proposed that the change in Cdk1 activity at the metaphase-anaphase transition is too binary (i.e., either on or off) to explain the spatiotemporal pattern of activation described for key regulators of cytokinesis such as PRC1 and Plk1 (Hu et al., 2012). However, this suggestion does not allow for the balance of phosphatase to kinase activity during mitotic exit, which results in the dephosphorylation of Cdk substrates at distinct thresholds (Bouchoux and Uhlmann, 2011). An additional consideration is the temporal separation of cyclin B degradation, and, hence, Cdk1 inactivation, and the subsequent dephosphorylation of key Cdk1 substrates by mitotic phosphatases. In fact, an implicit assumption of the ‘‘too binary’’ argument is that the dephosphorylation of Cdk1 substrates such as PRC1 directly matches the inactivation of Cdk1, given that Cdk1-opposing phosphatases are unregulated and, therefore, continuously active. As shown here, this view is not correct—partly because phosphatases active during metaphase such as PP2A-B56 cannot remove Cdk phosphorylations (Figure 3). The separation of Cdk inactivation and dephosphorylation by PP2A-B55 is achieved in the case of PRC1, and most likely other key substrates, by means of the EG pathway. Multiple Phosphatases Regulating Mitotic Exit The picture developing is one where mitotic exit in mammalian cells is under the control of multiple phosphatases of the PPP family, although, as discussed elsewhere, many questions remain to be answered (Glover, 2012; Mochida and Hunt, 2012). Importantly, the data presented here suggest that PP2A-B55 removes only a subset of Cdk phosphorylations during mitotic exit, including those on PRC1. Although PP2AB55 becomes active in mitotic cell extracts due to the decay of the EG pathway, mitotic Cdk phosphorylations on PP1C at T320, and those detected by a pan-Cdk site antibody are not turned over (Figure 4C). Interestingly, in both cases, these phosphorylations were lost in cells at the metaphase-anaphase transition (Figure 5G). This is consistent with the view that additional phosphatases, including PP1, promote mitotic exit, as suggested previously (Wu et al., 2009). PP1 and PP2A-B55 are both under tight regulatory control, and understanding the properties of these regulatory networks will be necessary for explaining the ordered series of events in mitotic exit. EXPERIMENTAL PROCEDURES Microscopy, the purification of recombinant PRC1 substrates and PP2A holoenzymes, phosphatase assays, and details of the BEG pathway model and simulation are described in the Supplemental Information.

Cell Culture Procedures HeLa cells were cultured in growth medium (Dulbecco’s modified Eagle’s medium containing 10% [vol/vol] bovine calf serum [Invitrogen]) at 37 C and 5% CO2. Stable cell lines expressing eGFP-tubulin and mCherry-histone H2B, eGFP-Mad2/mCherry-securin, and eGFP-PRC1 and mCherry-Plk1 were produced and imaged as described previously (Bastos and Barr, 2010; Zeng et al., 2010). For synchronization, cells were treated for 18–20 hr with 2 mM thymidine and washed three times in PBS and twice with growth medium. Phosphatase Library Screening Assay HeLa cells were seeded at a density of 125,000 cells per 10 cm dish in 8 ml growth medium and left to adhere for 24 hr prior to siRNA transfection. Then, cells were incubated for 54 hr followed by 18 hr in 100 ng/ml nocodazole. Mitotic cells were isolated by shake off, washed three times in 5 ml ice-cold PBS, pelleted at 200 3 g for 3 min at 4 C, and lysed for 15 min on ice at 1,000 cells/ml ice-cold mitotic lysis buffer (50 mM Tris-HCl [pH7.35], 150 mM NaCl, 1% [vol/vol] IGEPAL, 1 mM dithiothreitol, and protease inhibitor cocktail [Sigma-Aldrich, P8340]) supplemented with 100 nM okadaic acid as required. Lysates were clarified at 20,817 3 g for 10 min at 4 C, and supernatants were incubated on ice for an additional 30 min before analysis by western blotting. Dephosphorylation Time Course Assays For dephosphorylation time course assays, 23 15 cm dishes seeded with 750,000 HeLa cells each were incubated for 48 hr and treated with 100 ng/ml nocodazole for 18 hr. In experiments testing the roles of MASTL, ENSA, and ARPP19, 600,000 HeLa cells were seeded per 15 cm plate then left for 24 hr prior to siRNA. After, 54 hr cells were incubated in the presence of nocodazole (100 ng/ml) for 18 hr. Cells were harvested by shake off. Pooled cells were split into two aliquots and washed twice in ice-cold PBS. Then, cells were lysed on ice in ice-cold mitotic lysis buffer at 1,000 cells/ml lysate either containing or lacking 100 nM okadaic acid. At the indicated time points, 123 ml of the minus okadaic acid lysate was added to tubes containing 2 ml 0.1 mM okadaic acid ([1.6 mM] final) in order to stop the reaction. After 60 min, all samples were clarified at 20,817 3 g for 15 min at 4 C, and supernatants were analyzed by western blotting. For detection of the MASTL phosphorylated form of ENSA, resolving gel buffer was supplemented with 12.5 mM Phos-tag (NARD Chemicals) and 100 mM MnCl2. In Vivo Dephosphorylation Time Course Two 80% confluent 15 cm dishes of HeLa cells were treated with 100 ng/ml nocodazole for 20 hr. Mitotic cells were harvested and washed from nocodazole with prewarmed 37 C solutions: three times in 25 ml PBS and once in 25 ml growth medium before resuspension in 7.5 ml growth medium. Cells were left for 25 min at 37 C in order to form bipolar metaphase spindles, and a sample was taken prior to the addition of 5 mM flavopiridol or 200 mM AZ3146. At each time point, 525 ml of sample was added to 800 ml of icecold PBS, spun at 200 3 g for 4 min at 4 C, and supernatant aspirated, and cell pellets were snap frozen with liquid nitrogen. These were subsequently lysed on ice for 25 min in 100 ml mitotic lysis buffer containing 100 nM okadaic acid. The lysate was clarified at 20,817 3 g for 15 min at 4 C, and supernatants were analyzed by western blotting. SUPPLEMENTAL INFORMATION Supplemental Information contains Supplemental Experimental Procedures and six figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.molcel.2013.09.005. ACKNOWLEDGMENTS A Cancer Research UK programme grant award (C20079/A15940) to F.A.B. funded this work. U.G. was supported by a Cancer Research UK career development fellowship (C24085/A8296). B.N. is supported by the European Community’s Seventh Framework Programme (MitoSys/241548). We thank Dean Hammond for ENSA mass spectrometry. Imaging was performed at the



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