[Cell Cycle 6:12, 1408-1411, 15 June 2007]; 2007 Landes Bioscience

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The End of the Beginning

Cdk1 Thresholds and Exit from Mitosis
Frank Wolf Reinhard Sigl Stephan Geley*
Biocenter; Division of Molecular Pathophysiology; Innsbruck Medical University; Innsbruck, Austria *Correspondence to: Stephan Geley; Biocenter, Division of Molecular Pathophysiology; Innsbruck Medical University; Fritz-Pregl-Str.3; 6020 Innsbruck, Austria; Tel: +43.512.9003.70365; Fax: +43.512.9003.73960; Email: stephan. geley@i-med.ac.at Original manuscript submitted: 04/25/07 Manuscript accepted: 04/27/07 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=4361

Abstract
Exit from mitosis requires the proteolytic degradation of mitotic cyclins, which is insti gated by the APC/C ubiquitin ligase. The coincidence of mitotic cyclin B1 degradation with the onset of anaphase intuitively suggested a requirement of cyclin degradation for sister chromatid separation. While this hypothesis has originally been refuted, evidence that cyclin B1 degradation is required for anaphase during meiosis has been obtained, while its requirement for anaphase during mitosis is still more controversial. By studying human cells engineered to express nondegradable cyclin B1, we have recently shown that stable cyclin B1 affects progression through mitosis at various steps in a dosedependent manner. These experiments suggest that controlled exit from mitosis might involve CDK activity thresholds for important late mitotic events, such as the onset of anaphase, forma tion of the spindle midzone, the onset of cytokinesis, cellular abscission and chromosome decondensation.

cyclin, mitosis, proteolysis, ubiquitin, APC/C Acknowledgements We are grateful to Drs. R. Kofler and C. Ploner for their help with lentiviral transduction and the members of the lab for their help and stimulating discussion. This work was supported by grants from the Austrian Science Funds (FWF) P16400-B10, SFB021 Cell proliferation and cell death in tumors and EU grant LSHS-CT-2004-503438 (TRANSFOG).

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Finishing a project successfully may sometimes be as, or even more, difficult than initiating one. This daytoday experience may also hold true for certain biological processes, which involve large scale, yet reversible, cellular changes to achieve a certain biological outcome. For cellular division, cells have to reorganise their cytoskeleton, their chromatin state, membrane dynamics and signalling interfaces to the extracellular compartment. All these changes occur during the dramatic events that happen when cells enter mitosis. To revert to an interphase state, however, cells have to revert all of these changes in order to regain the ability to perform their specific physiological tasks or for another round of cellular replication. Exit from mitosis is a complex transition involving many cellular changes to occur in a timely coordinated manner.1 In addition, different organisms and cell types might have to organise exit from mitosis slightly differently, in order to be successful. Thus, although the regulation of exit from mitosis involves conserved signalling proteins, these signalling pathways might be used in a cell type specific manner to coordinate the nuclear division cycle and cytokinesis. In all eukaryotes exit from mitosis is controlled by proteolysis. The conserved multisubunit ubiquitin ligase APC/C controls sister chromatid separation and exit from mitosis by targeting securin and mitotic cyclins for proteasomedependent degradation.2 Linking securin and cyclin degradation ensures that sister chromatid separation is not uncoupled from exit from mitosis (and vice versa). Anaphase and exit from mitosis are not only coupled by being dependent on the APC/C, but separase and cyclin B1 activities mutually inhibit each other.3 Cyclin B1 degradation and CDK inactivation are required for the onset of anaphase in some systems (e.g., during mouse oocyte meiosis, ref. 4 ) but are essential for exit from mitosis in all eukaryotes (see ref. 5 and references therein). In budding yeast, Pds1 (securin) and the mitotic Clb cyclins are the only essential targets for the APC/C.6-8 In vertebrate cells (near complete) exit from mitosis can be triggered in cells arrested at metaphase by the proteasome inhibitor MG132 or due to the lack of E1 enzyme activity by using chemical inhibition of CDK activity.9,10 This suggests that after anaphase, mitotic cyclins are the only targets that need to be degraded for initiation of cytokinesis. Whether completion of cytokinesis requires proteolysis, be it APC/C dependent or not, is, however, a still unresolved question. At the meta to anaphase transition, the substrate specificity of the APC/C expands due to replacement of the activator and substrate binding factor Cdc20 by its homolog Cdh1/Fzr1.11,12 Fzr1activated APC/C continues to polyubiquitylate Dbox containing substrates but also recognises KENbox substrates as targets for polyubiquitylation and subsequent degradation13 but, in contrast to Cdc20,14,15 Cdh1/Fzr is not essential for exit from mitosis in any organism studied thus far.16,17,18 These data suggest that, among the many mitotic APC/C substrates, only securin and cyclinB1 are essential targets for mitotic proteolysis. 2007; Vol. 6 Issue 12

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Cyclin Degradation and Exit from Mitosis

Exit from mitosis in different organisms is not a uniform process because it has to meet specific requirements, e.g., in budding yeast where the daughter bud (and thus the site of cytokinesis) is determined very early in the cell cycle and the mitotic spindle has to be positioned accordingly for proper chromosome segregation. It is therefore not surprising, that in this organism exit from mitosis, i.e., events after the onset of anaphase, is controlled by an intricate signalling complex, called the mitotic exit network (MEN), that controls the post anaphase activity of CDK in order to prevent premature cytokinesis.19 Basically, this pathway consists of a GTPase controlled kinase pathway, whose components appear to be conserved throughout eukaryotes. In budding yeast, this pathway controls CDK activity during late mitosis by regulating Cdc14 phosphatase, which dephosphorylates CDK substrates and is essential for exit from mitosis in this organism.20 As cells of other organisms face rather different requirements (compared to finding the way into a bud for chromosome deposition) these conserved signalling proteins are used for different purposes during exit from mitosis. Whether vertebrate cells are able to delay exit from mitosis, e.g., to allow adjustment of the spindle axis, in relation to the polarity of the cell, is currently not known. It has been shown, however, that during exit from mitosis APC/C activity can be turned off, if microtubule function is blocked in anaphase cells.21 In addition, although Cdc14 homologs in other organisms are not essential for exit from mitosis, they are required for cytokinesis.22 In human cells, very little is known about the function of mitotic phosphatases and the balance of kinase and phosphatase activities during exit from mitosis. Our observations that different levels of nondegradable cyclin B1 arrest cells in different stages of mitosis suggest a model where exit from mitosis might be controlled by timed dephosphorylation of key regulatory proteins. Whether this dephosphorylation is primarily determined by the decay rate of cylin levels or the activation and/or recruitment of (specific) phosphatases is currently not known. Controlling exit from mitosis by regulating the rate of cyclin B1 proteolysis would only make sense if late mitotic Cdk1 substrates acted in a sequential manner. During early mitosis, Cdk1 not only activates proteins by phosphorylation, but also inhibits proteins that function after anaphase and require CDK inactivation. Whether all these Cdk1 substrates are dephosphorylated precipitously by nonspecific phosphatases as soon as Cdk1 activity drops at the onset of anaphase, or whether there is a temporal order of substrate dephosphorylation is poorly understood but several lines of investigation suggest that it might be.

of Aurora B kinase but also helps to recruit Plk1 to kinetochores. Dephosphorylation of INCENP might thus be an important event for the switch from a metaphase to an anaphase spindle. The relocalisation of the Aurora B kinase complex to the central spindle also requires the dephosphorylation of Mklp2, a kinesin that is phosphorylated (and inhibited) by CDK in early mitosis.27 The centralspindlin complex, which consists of Mklp1, MgcRacGAP, Ect2 and other proteins then determines the site of cytokinesis by controlling the activity of the cytokinesis regulator RhoA.28 Like the chromosome passenger proteins, several components for the centralspindlin complex are regulated by CDK activity, such as Mklp1, whose phosphorylation by Cdk1 reduces its kinesin motor activity.29 Finally, the spindle midzone and the formation of the midbody require the function of the microtubule crosslinking protein PRC1, which is phosphorylated during early mitosis and whose dephosphorylation is required for proper localisation and function.30,31 In cells expressing nondegradable cyclin B1, a spindle midzone is not formed,32 and chromosome passenger proteins remain at kinetochores, suggesting that cyclin B1 proteolysis triggers the formation of an anaphase spindle.25 Thus, in the presence of CDK activity formation of cytoskeletal structures important for cytokinesis is inhibited.

CDKDependent Control of Cytokinesis Regulators

Second, in mitotically arrested cells, e.g., by expression of stable Btype cyclins, cytokinesis can rapidly be triggered by chemical inhibition of CDK activity.5,33,34 Thus, cytokinesis can be triggered in the absence of instructive or permissive signals originating from the central spindle.33 Rather, the onset of cytokinesis seems to depend only on a decline of CDK activity, suggesting that a central cytokinesis regulator might be a direct target of CDK activity.

CDK Activity Beyond Anaphase

Third, in fly embryos, exit from mitosis is controlled by the sequential degradation of the mitotic cyclins A, B and B3, and expression of stable variants of these cyclins arrest early embryonic cell divisions at metaphase, early anaphase and telophase, respectively.35,36,37 This sequential cyclin degradation strongly suggests that CDK activity is involved in the regulation of exit from mitosis. Cyclin B3 is conserved in mice and humans but its expression appears to be restricted to testes and ovaries.38 In chicken B cells, however, cyclin B3 is expressed and may be involved in regulating events during exit from mitosis (e.g., chromosome decondensation).39

CDKDependent Control of Structures Required for Cytokinesis

First, progression into anaphase and formation of the central spindle requiresamong many other thingsthe action of chromosome passenger proteins, mitotic motor proteins and other microtubule binding and regulatory proteins. Chromosome passenger proteins reside at kinetochores until anaphase upon which they translocate to, and help to form, the central spindle.23 In budding yeast, release of the INCENP homolog Sli15 is mediated by Cdc14 phosphatase24 and cells arrested by non-degradable cyclin B1 do not release INCENP from kinetochores25 (and unpublished observations). Recently two CDK phosphorylation sites have been mapped on INCENP, one of which generates a Polobox binding domain.26 INCENP is thus not only an important regulator www.landesbioscience.com

Phosphatases Controlling Exit from Mitosis

Fourth, Cdc14 phosphatase homologs play important functions in various species, including human cells.20 Dephosphorylation is a key event in the completion of cytokinesis, but whether human Cdc14 is specific for CDK substrates remains to be determined. In order to coordinate anaphase with the formation of the central spindle, the onset and completion of cytokinesis, sequential dephosphorylation of key regulators can be envisaged to play an important role.40

Evidence for CDK Control of Exit from Mitosis

If progression beyond metaphase is controlled by the phosphorylation state of CDK substrates, then this model predicts that maintaining Cdk1 activity at different levels during mitosis 1409

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Cyclin Degradation and Exit from Mitosis

When cells arrested by nondegradable cyclinB1 were treated with a chemical CDK inhibitor, they rapidly initiated cytokinesis, even if chromosomes did not segregate properly. In summary, these data suggest that different levels of Cdk1 activity are required to phosphorylate and inhibit key regulatory substrates during mitosis. With the exception of separase, these potential targets are, however, largely unknown. As evidence derived from overexpression experiments has to be considered with caution, we wondered whether a hypomorphic APC/C mutant might cause the same mitotic arrest phenotypes. We Figure 1. Cdc27 hypomorphic cells arrest in pseudometaphase. U2OS cells expressing the tetracyclinedependent transcriptional silencer tetR,42 were infected with a lentiviral construct have therefore used a tetracyclineinducible shRNA expressing a Cdc27 (APC2) specific shRNA and single clones isolated using puromycin expression system to generate human cell lines with selection. (A) Cells were left untreated or induced using 1mg/ml doxycyline for 48 hours stable, inducible RNAi to target the APC/C subunit before addition of nocodazole (500nM) for 16 hours. Mitotically arrested and released cells Cdc27. As can be seen in Figure 1, treatment with were harvested and total cell extracts probed for expression of Cdc27 (BD Transduction 1mg/ml doxycycline for 48 hours causes a signifiLaboratories), Cyclin B1 (V152) and GAPDH (Abcam). (B) 48 hours after induction with cant knockdown of Cdc27 levels, which interfered 1mg/ml. U2OS cells expressing H2BGFP were monitored for progression through mitosis. with cyclin B1 degradation in cells released from Time is shown in hours:minutes. The white arrow indicates onset of anaphase after a pro longed metaphase. a nocodazoleinduced mitotic block (Fig 1 A) and arrested cells in mitosis. Histone H2BGFP cells expressing TetR and infected with a lentivirus for inducible Cdc27 RNAi were treated with doxycyline and monitored by live cell fluorescence time lapse microscopy over a period of three days. Among cells that became arrested in mitosis, 85% arrested at metaphase, while 15% arrested like stable cyclinB1 expressing cells in anaphase (Fig. 1B), suggesting the presence of residual APC/C activity sufficient to eliminate securin but not cyclin B1 from these Cdc27 RNAi hypomorphic cells. Thus, when cyclin B1 levels are maintained by low APC/C activity in RNAi hypomorphs, cells arrest at different time points in mitosis. These experiments confirm our previous findings that complete cyclinB1 degradation is not required for the onset of anaphase but for exit from mitosis. Analysis of cells expressing low amounts of nondegradable cyclin B1 (or endogenous cyclin B1 in Cdc27 RNAi hypomorphs) progress well into anaphase but fail to complete cellular abscission, upon which cells often fuse and are able to reestablish a bipolar spindle, i.e., they reverse mitosis Figure 2. Control of exit from mitosis by timed dephosphorylation of CDK to arrest in a pseudometaphase state. The mitotic arrest phenotypes observed in cells expressing nondesubstrates. Exit from mitosis might be controlled by the specific dephosphor ylation of key regulators of late mitotic events, such as formation of the cen gradable cyclin B1 or Cdc27 knockdown are consistent with the idea tral spindle, cytokinesis and cellular abscission. Since key regulators of these that late mitotic events exhibit different sensitivities to remaining events have been identified (see text for details) to be CDK substrates that are CDK activity. While very high levels appear to block the onset of inhibited during early mitosis, a gradual decline of CDK activity or specific anaphase, lower levels suppress the onset of cytokinesis and are dephosphorylation of these key substrates might control the timed execution sufficient to maintain a bipolar spindle with chromosomes arranged of these events during exit from mitosis. P, prophase; PM, prometaphase; M, in a pseudometaphase plate, while the lowest levels are sufficient to metaphase; A, anaphase; T/C, telophase/cytokinesis; G1, G1 phase. block completion of cytokinesis and chromosome decondensation. A stepwise removal of CDK activity as a regulatory mechanism for coorshould arrest cells at different phases during late mitosis. To test dinated exit from mitosis has been suggested previously by OFarrell this hypothesis, we have generated human cell lines with inducible and coworkers who have studied the consequences of expressing nondegradable cyclin B1 to maintain Cdk1 activity in mitosis.5 By nondegradable versions of the three mitotic Drosophila cyclins (A, B using GFP fused to nondegradable cyclin B1, we were able to corre- and B3) in fly embryos.36 These cyclins become degraded at specific late expression levels with three distinct phenotypes. At high levels times in mitosis and expression of nondegradable mutants blocked (~4 times higher than endogenous cyclinB1) cells did not undergo progression through mitosis at or before metaphase (cyclin A), in visible anaphase as evaluated by live cell imaging of histone H2BRFP early anaphase (B) and in telophase (B3). In contrast to fly embryos, which express a mitotic cyclin until tagged cells. At physiological levels, nondegradable cyclin B1 arrested cells in anaphase. In these cells, cytokinesis was blocked, chromo- late in mitosis, human mitotic Btype cyclins in somatic cells become some passenger proteins remained at kinetochores and a bipolar unstable at the onset of anaphase. By quantitative monitoring of spindle with merotelically attached chromosomes was present. Cells cyclin B1 degradation in human cells using a cyclinB1GFP reporter expressing low levels of nondegradable cyclinB1 arrested in telophase, protein, Clute et al showed that cyclin B1GFP levels start to decline because completion of cytokinesis, i.e., abscission, was blocked, already during metaphase, and gradually decline during exit from and the chromatin remained condensed in these arrested cells. mitosis to become undetectable only at the onset of cytokinesis.21 1410 Cell Cycle 2007; Vol. 6 Issue 12

Cyclin Degradation and Exit from Mitosis

In summary, it is likely that specific dephosphorylation of key regulatory proteins is essential for coordinated exit from mitosis (Fig. 2). A gradual decline in Cdk1 activity might be an important contributor to this regulatory pathway which most likely also involves the recruitment of phosphatases in a timely coordinated manner as has been shown recently for CdcA2, a mitotic phosphoprotein which, upon dephosphorylation, is able to recruit PP1g to anaphase chromosomes.41 As little is known about mitotic phosphatases, it will be interesting to learn more about how these are controlled during exit from mitosis.
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