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The metaphase-to-anaphase transition: avoiding a mid-life crisis
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The metaphase-to-anaphase transition: avoiding a mid-life crisis Orna Cohen-Fix* and Douglas Koshland The metaphase-to-anaphase transition is a highly regulated process, which is governed by the activity of the anaphase-promoting complex (APC). The APC promotes the degradation of several proteins, including mitotic cyclins and newly identified anaphase inhibitors, Several discoveries made this year shed invaluable light on the regulation of APC activation and its substrate specificity. Addresses Howard Hughes Medical Institute, The Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210,
USA "e-mail: [email protected] re-mail: [email protected] Current Opinion in Cell Biology 1997, 9:800-806
http:/Ibiomednet.comlelecref10955067400900800 © Current Biology Ltd ISSN 0955-0674 Abbreviations APC anaphase-promoting complex CDK cyclin-dependent-kinase-cyclin complex
Introduction T h e metaphase-to-anaphase transition in mitosis involves a fascinating and complex sequence of biochemical events that initiate the segregation of replicated chromosomes (sister chromatids) (Figure 1). Following DNA replication, the sister chromatids are associated with each other along their length by a putative cohesion factor(s) (reviewed in [1]). By metaphase, sister chromatids have made stable attachments to spindle microtubules emanating from opposite spindle poles, and usually have congressed to the metaphase plate (Figure 1; for the definition of metaphase in budding yeast, see Figure 1 legend and [2,3]). T h e spindle attachments are mediated through the kinetochore, a specialized domain on each chromatid with microtubule-binding and microtubule-dependent motor activities. At the onset of anaphase, inactivation of the cohesion factor(s) leads to synchronous sister chromatid separation. Sister chromatids can separate in the absence of attachment to the spindle (reviewed in [4]), and anaphase spindle dynamics occur in the absence of chromosomes [5°]. Therefore, the metaphase-to-anaphase transition involves the coordinated execution of independent processes. T h e loss of this coordination could result in chromosome missegregation which is almost always deleterious to the cell. Because of the irreversible nature of this transition, cells have evolved regulatory mechanisms that couple the initiation of anaphase to the proper completion of prior events.
The a n a p h a s e - p r o m o t i n g complex T h e transition from metaphase to anaphase is controlled by the ubiquitin-dependent proteolysis of a specific subset of proteins [6-9]. Recently, a complex that promotes cell cycle specific ubiquitination has been identified. This complex, known as the anaphase-promoting complex (APC) [10,11] or cyclosome [12], was initially identified as the ubiquitin ligase required for the degradation of mitotic cyclins (reviewed in [13}). Subsequent studies showed that the APC is composed of at least eight subunits [14°,15], several of which are conserved among many organisms (see [13]). Ubiquitination by the APC requires the presence of a nine amino acid destruction box motif on the target protein [16]. A role for the APC in the metaphase-to-anaphase transition was inferred from the failure of cells to initiate anaphase when defective for APC function due to either mutations in APC subunits [10,17-21], the injection of antibodies directed against particular APC subunits [22], or the overexpression of a peptide containing a destruction box motif [23°]. These observations suggest that anaphase initiation requires the APC-mediated degradation of at least one, if not several, proteins. These proteins are likely to be distinct from the mitotic cyclins, because mitotic cyclin degradation is required for the exit from mitosis rather than for anaphase initiation [6-9]. T h e identification of the APC as a regulator of the metaphase-to-anaphase transition has led to three interesting questions that are the focus of this review. First, does the APC degrade a master regulator of the metaphaseto-anaphase transition that initiates a cascade of events, or does it degrade a series of targets that independently regulate processes such as cohesion, microtubule motors and microtubule dynamics? Second, as APC-mediated degradation is required for the metaphase-to-anaphase transition and for the exit from mitosis, how is the temporal order of degradation, and thus the temporal order of events, ensured? Finally, what is the mechanism that activates the APC only at the appropriate time for anaphase initiation? Recent findings, mostly from the budding and fission yeasts, provide insights into these questions.
A P C targets at the m e t a p h a s e - t o - a n a p h a s e transition In the past year, two proteins have been identified as important targets of the APC at the metaphase-to-anaphase transition, namely, Cut2 in the fission yeast [24 °°] and P d s l p in the budding yeast [25"°,26°°]. Both proteins are stabilized in the absence of APC function; they are degraded at the metaphase-to-anaphase transition; and
The metaphase-to-anaphase transition: avoiding a mid-life crisis Cohen-Fix and Koshtand
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Figure 1
Metaphase
Anaphase A
Anaphase B
Current Opinion in Cell Biology
The metaphase-to-anaphase transition. At metaphase, the duplicated chromosomes align on the metaphase plate. In the budding yeast, as well as in various other fungi, there appears to be no classical metaphase state in that the duplicated chromosomes do not align on a metaphase plate [2,3]. However, there does appear to be a stable stage at which the sister chromatids are associated, condensed and, under certain conditions, are inhibited from initiating anaphase by the activity of the spindle assembly checkpoint (see text). For our purposes, we define this state as a physiological metaphase, thereby classifying the budding yeast as an organism that undergoes a metaphase-to-anaphase transition. During anaphase A, the chromatids separate synchronously and move away from each other by microtubule-dependent movement toward the spindle poles. In anaphase B, the spindle poles move away from each other by microtubule-dependent migration, thereby further moving the chromosomes away from the plane of division. Chromatids are indicated as bi-tobed structures; kinetochores are white; microtubules are light gray; spindle poles are dark gray circles.
nondegradable forms of either protein block anaphase initiation. T h e observation that anaphase initiation requires protein degradation prompted the suggestion that one of the APC's targets might be a factor(s) mediating sister chromatid cohesion [6]. However, at this point it seems unlikely that either Cut2 or P d s l p plays such a role. T h e Cut2 protein localizes to spindle microtubules and cut2 null mutants fail to separate sister chromatids [24"']. Pdslp shows a general nuclear localization and pdsl null mutants do exhibit precocious dissociation of sister chromatids, as expected for a cohesion factor [25°']. However, the deletion of PDS1 alleviates the requirement for the APC in all aspects of anaphase initiation, including spindle elongation and the dissolution of cohesion. Given the evidence that these two processes are independent, Pdslp is unlikely to be the cohesion factor. Rather, Pdslp may be a master regulator of anaphase initiation that is inactivated by the APC. Whether or not the APC controls cohesion directly or indirectly is still unknown. Consistent with Pdslp's role as a master regulator, nondegradable Pdslp derivatives block all aspects of cell cycle progression [26"']. Nondegradable forms of Cut2, on the other hand, block anaphase initiation but not exit from mitosis [24"']. This, together with the phenotypic differences of the pdsl and cut2 null mutants, suggests that Cut2 plays a more limited rote in anaphase inhibition than Pdslp. Furthermore, the Pdslp and Cut2 proteins share no striking sequence similarity. These differences may suggest that the regulation of anaphase initiation by the APC differs among eukaryotes. In Saccharomyces cerevisiae, the APC-mediated degradation of a master
inhibitor (i.e. Pdslp) allows the initiation of multiple processes, some of which may require the degradation of additional APC substrates. In other organisms, such as Schizosaccharomycespombe, different aspects of anaphase initiation may be regulated individually by specific inhibitors, and therefore the absence of one inhibitor (i.e. Cut2) is not sufficient to allow complete anaphase initiation.
D o i n g mitosis o n e step at a t i m e Although the discovery of the APC's role in anaphase initiation provided tremendous insight into the process, it also generated a dilemma. T h e APC is required not only at the initiation of anaphase for the degradation of P d s l p and Cut2, but also at the exit of mitosis for the degradation of the mitotic cyclins and S. cerevisiae Aselp, a microtubule-binding protein that is required for proper spindle function [7,23",27,28"']. How is APC activity controlled such that at a given time some substrates are degraded while others are not? A possible mechanism is that the APC's accessibility to different substrates is cell cycle regulated. Alternatively, the substrate specificity of the APC may be modulated during the cell cycle. Recently, two related proteins that may determine the substrate specificity of the APC have been identified, namely, S. cerevisiae Cdc20p and Hctlp. Defects in the yeast Cdc20p or its Drosophila homolog Fizzy lead to a metaphase arrest [29-31], raising the possibility that they might be involved in APC-mediated degradation of anaphase inhibitors. Indeed, in S. cerevisiae, pdsl cdc20 double mutants bypass the mitotic arrest observed in a cdc20 mutant, suggesting that this arrest stems from the
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inability to degrade Pdslp [25°°]. Furthermore, Pdslp stability is sensitive to Cdc20p levels: Pdslp is stabilized in cdc20 mutants and it is destabilized when Cdc20p is overexpressed (A Amon, personal communication; EJ Schott, MA Hoyt, personal communication; see Note added in proof). Under these same conditions, the stability of the mitotic cyclins and Aselp is largely unaffected (A Amon, personal communicatiow, see Note added in proof). Similarly, the ubiquitination of the mitotic cyclins in vitro was independent of Cdc20p [32°].
In S. cerevisiae cells mutated in HCTI, a gene homologous to S. cerevisiae CDC20 [33"°], mitotic cyclins are not degraded at the exit of mitosis. Rather, they are stable throughout the cell cycle, hctl mutants display only a mild growth defect, and the persisting mitotic cyclins are counteracted in late mitosis by the activity of the kinase inhibitor Siclp. Overexpression of Hctlp causes a rapid APC-mediated degradation of the mitotic cyclins and Aselp ([33°°]; A Amon, personal communication; see Note added in proof). The stability of Pdslp is not affected by hctl mutations or by overexpressing Hctlp ([33"]; A Amon, personal communication; see Note added in proof). Taken together, these results suggest that Cdc20p and Hctlp act as APC substrate specificity factors, with Cdc20p being responsible for the APC-mediated degradation of Pdslp and Hctlp being responsible for the APC-mediated degradation of the mitotic cyclins and Aselp. This interpretation would predict that the APC physically interacts with Cdc20p and Hctlp. In this regard, it is noteworthy that some APC subunits contain tetratricopeptide repeat motifs whereas Cdc20p and Hctlp belong to a family of proteins with WD40 repeats [19,34,35], and there is precedence for a physical interaction between proteins having these two types of motifs [36]. Given the complex pattern of mitotic cyclin degradation in other eukaryotes (see below), we expect that additional Cdc20p-related proteins that control APC substrate specificity will be identified.
It remains unclear how only APC-Cdc20p complexes, but not APC-Hctlp complexes, are active at anaphase initiation. Interestingly, when Pdslp is rendered nondegradable, none of the postmetaphase events, including mitotic cyclin degradation, take place ([26°°]; O CohenFix, D Koshland, unpublished data). This kind of interdependence also occurs in the degradation of mitotic cyclins of other organisms. For example, in Drosophila, cyclin A is normally degraded before cyclin B and cyclin B3 [37]. Although all three proteins are present simultaneously, the presence of nondegradable cyclin A inhibits the degradation of cyclins B and B3 [30]. These findings suggest that cells ensure the sequential degradation of specific APC substrates by coupling a change in APC substrate specificity with the degradation of prior substrates. This, in turn, may be one of the
mechanisms by which the orderly execution of events in mitosis is controlled.
The regulation of A P C activation Understanding how the APC is activated is crucial to elucidating the mechanism of anaphase initiation. Any model for the activation of the APC must take into account the spindle assembly checkpoint pathway and the activity of the mitotic cyclin-dependent-kinase-cyclin complexes (henceforth known as mitotic CDKs). The spindle assembly checkpoint is a surveillance system that inhibits the metaphase-to-anaphase transition until all sister chromatids have achieved a bipolar spindle attachment (reviewed in [38,39]). The name of this checkpoint should be used with the cautionary note that the 'spindle' in this case includes the kinetochore, as kinetochores may be a prime source for the checkpoint signal. Indeed, anaphase initiation is delayed in the presence of defective or unattached kinetochores [5",40 a,A.]. Recent data further suggest that evolutionarily conserved components of the spindle assembly checkpoints localize to unattached kinetochores [45°-47°]. The notion that the spindle assembly checkpoint could inhibit the APC stems from the observations that cells with an activated spindle assembly checkpoint fail to degrade APC substrates [26°°] and extracts from these cells do not ubiquitinate mitotic cyclins in vitro [32°]. Mitotic CDKs, which are activated during G z phase [27,48], also play a role in controlling APC activity. Conflicting observations suggest that these kinases can activate as well as inhibit the APC. In vitro, the APC is activated by mitotic CDK activity and is inactivated by phosphatase treatment [14",49]. However, cells arrested by the spindle assembly checkpoint initiate anaphase when mitotic CDK activity is inhibited by the kinase inhibitor Siclp [50°°], or through mutations in the kinase subunit [51°]. These latter observations suggest that inhibition of mitotic CDK activity causes APC activation and subsequent anaphase initiation. With these observations in mind, we propose the following 'primed APC' model: the APC is first primed by the kinase activity of the mitotic CDKs. The primed APC is inhibited from degrading the anaphase inhibitor until the cell has completed metaphase. This inhibition is mediated by the spindle assembly checkpoint pathway. The completion of metaphase turns the spindle assembly checkpoint off, allowing for the transition of primed APC to active APC, thereby promoting the degradation of the anaphase inhibitor. According to this model, the requirement for high kinase activity is twofold: it is required for APC activation and it is also required for maintaining the spindle assembly checkpoint pathway (see above). Therefore, inhibiting mitotic CDK activity in checkpoint-arrested cells, either by kinase inhibitors
The metaphase-to-anaphase transition: avoiding a mid-life crisis Cohen-Fix and Koshland
or though mutations as described above, will lead to a collapse of the checkpoint pathway. This will lead to a premature transition form primed to active APC and hence to the initiation of anaphase. How the spindle assembly checkpoint inhibits the transition from primed APC to active APC is currently unknown. It has been proposed previously that the APC could be activated by a drop in the mitotic CDK activity [50",52]. In this regard, it is possible that when the mitotic CDKs prime the APC, they add not only activating phosphates but inhibitory phosphates as well (either on the APC itself or on an APC regulator). The removal of these inhibitory phosphates would be required for the primed to active APC transition, and this process may be efficient only when the activity of the mitotic CDKs drops. In this case, the spindle assembly checkpoint could be regulating the APC indirectly by preventing the drop in the mitotic CDK activity (Figure 2, large shaded rectangle). Thisis consistent with the involvement of protein phosphatase activity in the spindle assembly checkpoint pathway [53-56], where it could be required to maintain high kinase activity by removing inhibitory phosphorylation. If this drop in kinase activity takes place, one would have to argue that it is a localized event, as cells which maintain globally high mitotic CDK activity due to the presence of nondegradable mitotic cyclins can nonetheless initiate anaphase [7].
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It should be noted that the target of regulation by the spindle assembly checkpoint may not be the APC itself but rather a protein that determines APC's substrate recognition, such as Cdc20p. In this case, primed APC could be considered as APC lacking its substrate recognition factor. It has recently been demonstrated that overexpression of Cdc20p overrides a spindle assembly checkpoint arrest (A Amon, personal communication; EJ Schott, MA Hoyt, personal communication; see Note added in proof). The high levels of Cdc20p could overcome the inhibitory effect of the checkpoint pathway, thereby converting primed APC to active APC and promoting anaphase initiation. Would the primed APC model predict that the spindle assembly checkpoint is essential? Not necessarily. According to our model, there are multiple potential inputs that could influence the timing of the metaphase-to-anaphase transition, only one of which is the spindle assembly checkpoint. Even in the absence of the checkpoint pathway, there may be a finite time period required to convert the primed APC to the active APC. Moreover, anaphase initiation may require a reduction in the concentration of the anaphase inhibitor below some threshold level, a process which may be affected by the rate of APC-mediated ubiquitination or degradation. In certain cell types, these time intervals may be sufficient to achieve bipolar spindle attachments prior to the initiation of anaphase. Consistent with this possibility, in the
Figure 2
High mitotic CDK, activity
> £
Degradation of the anaphase ~ - - ~ inhibitor
Anaphase initiation
Current Opinion Jn Cell Biology
Regulation of APC activation: the primed APC model. According to this model, the APC can exist in the following three states: inactive APC, primed APC and active APC. Inactive APC is converted to primed APC by the kinase activity of the mitotic CDKs. Primed APE; is inhibited from degrading the anaphase inhibitor(s) prematurely by the spindle assembly checkpoint pathway, which is also activated by the mitotic CDKs. This checkpoint is active until all duplicated chromosomes form bipolar spindle attachments. The inhibitory effect of the checkpoint on the transition from primed APC to active APC could be direct or indirect (via inhibition of other kinases). In the latter case (large shaded rectangle), the transition from primed APC to active APC could be brought about by a drop in the mitotic CDK activity (see text), in this case, the spindle assembly checkpoint inhibits APC activation by preventing the drop in the kinase activity of the mitotic CDKs.
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budding yeast the spindle assembly checkpoint is not essential for viability, although mutants in components of this checkpoint show elevated levels of chromosome loss ([41,57]; MA Hoyt, personal communication). However, in at least some vertebrate cell types, the spindle assembly checkpoint appears to be active in each and every cell cycle: checkpoint components are localized to the kinetochore in prophase, [45"-47°~58] and microinjection of antibodies against one of the checkpoint components (Mad2p) results in premature initiation of anaphase and a mitotic catastrophe (G Gorbsky, personal communication). The relative contributions of various processes that control anaphase initiation may differ in different cell types. Under normal growth conditions the spindle assembly checkpoint may be dispensable in yeast, whereas other cell types may rely more heavily on the checkpoint in every cell division.
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Conclusions The metaphase-to-anaphase transition is a big step in the cell's life cycle: having gone through it, the cell will have to live (or die) with the consequences. It is, therefore, not surprising that this transition is controlled at multiple levels. In the past year, we have witnessed the discovery of anaphase inhibitors and factors that may mediate APe substrate specificity. These and other findings suggest that the APe is regulated at the level of its activation and its substrate specificity. An additional level of regulation comes from the spindle assembly checkpoint pathway. Different cell types are likely to have common as well as distinct strategies in controlling anaphase initiation. We anticipate that the coming years will prove to be an exciting time for this field.
5. Zhang D, Nicklas R: 'Anaphase' and cytokinesis in the absence U of chromosomes. Nature 1996, 382:466-468. sing the powerful tool of micromanipulation, all chromosomes were removed from grasshopper sparmatocytes. However, the time of anaphase initiation, as detected by the appearance of a gap in the spindle, was comparable to that of cells with chromosomes. This study further demonstrates that dissolution of sister chromatid cohesion does not serve as a positive signal for anaphase initiation. 6.
Holloway S, Glotzer M, King R, Murray A: Anaphase is initiated by proteolysis rather than by the inactivation of maturationpromoting factor. Cell 1993, 73:1393-1402.
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Note added in proof T h e papers referred to as '(A Amon, personal communication)' and '(EJ Schott, MA Hoyt, personal communication)' have now been accepted for publication [59,60]. In addition, Sirgit and Lehner have recently reported the identification of a Drosophila protein called Fizzy related, which is similar in structure and perhaps function to the S. cerevisiae protein Hctl [61].
Acknowledgements We are grateful to Angelica Amon, Wolfgang Seufert, Gary Gorbsky, Eric Schott and Andy Hoyt for sharing results prior to publication. We also wish to thank Paul Megee, Pare Meluh, Shikha Laloraya, Mary Lilly, Andy Hoyt, Terry Orr-Weaver and Gary Gorbsky for their excellent comments and Christine Norman for help in preparing this manuscript. O Cohen-Fix is supported by a National Institutes of Health (NIH) grant (GM18382-02). D Koshland is supported by an NIH grant (GMI718) and is an investigator with the Howard Htighes Medical Institute.
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Yamamoto A, Guacci V, Koshland D: Pdslp, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s). J Cell Bio/1996, 133:99-110. The identification of Pdslp as a negative regulator of anaphase initiation is described. This study provides the first link between the anaphase-promoting complex and a potential anaphase inhibitor. Cohen-Fix O, Peters J, Kirschner MW, Koshland D: Anaphase initiation in Saccheromyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pdslp. Genes Dev 1996, 10:3081-3093. This study provides evidence that Pdslp is the formerly hypothetical anaphase inhibitor: Pdslp is an APC substrate both in vivo and in vitro; it is degraded at the time of anaphase initiation; and nondegradabie forms of Pdslp inhibit anaphase initiation as well as all other aspects of cell cycle progression.
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Zachariae W, Nasmyth K: TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast. Mo/Biol Cell 1996, 7:791-801. The development of an in vitro system for anaphase-promoting complex (APC)-dependent ubiquitination is described, in this system, protein extracts from wild-type as well as mutant S. cerevisiae strains are utilized, thereby examining the involvement of specific proteins in mitotic cyclin ubiquitination.
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Chen R-H, Waters JC, Salmon ED, Murray AW: Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science 1996, 274:242-246. This paper, together with [46"], describes the identification of Xenopus and human homologs of the S. cerevisiae Mad2p spindle assembly checkpoint protein, indicating that this checkpoint has been conserved throughout evolution. In both organisms, the Mad2p homolog localizes to unattached kinetochores as early as prophase. These findings substantiate the notion that unattached kinetochores are a source for the spindle assembly checkpoint signal, and suggest that the checkpoint may be active in every cell cycle. 46. his
Li Y, Banezra R: Identification of a human mitotic checkpoint gene: hsMAD2. Science 1996, 274:246-248.. paper, together with [45"], describes the identification of Xenopus and human homologs of the S. cerevisiae Mad2p spindle assembly checkpoint protein, indicating that this checkpoint has been conserved throughout evolution. In both organisms, the Mad2p homolog localizes to unattached kinetochores as early as prophase. These findings substantiate the notion that unattached kinetochores are a source for the spindle assembly checkpoint signal, and suggest that the checkpoint may b e active in every cell cycle. 47. •
Taylor SS, McKeon F: Kinetochore localization of murine Bubl is required for normal mitotic timing and checkpoint response to spindle damage. Cell 1997, 89:727-735. The murine Bubl p homolog localizes to kinetochores prior to the formation of stable bipolar spindle attachments. Microinjection of anti-Bubl antibodies led to the premature initiation of anaphase, either under normal conditions or under conditions in which the spindle assembly checkpoint was activated. As in [45°,46"], these results suggest that this checkpoint is conserved throughout evolution and may be active in every cell cycle. 48.
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Lahav-BaratzS, Sudakin V, Ruderman JV, Hershko A: Reversible phosphorytation controls the activity of cyclosome-associated cyclin ubiquitin ligase. Proc Nat/Acad Sci USA 1995, 92:93039307.
33. Schwab M, Schulze Lutum A, Seufert W: The yeast Hctl is a •• regulator of CIb2 cyclin proteolysis. Cell 1997, 90:683-693. Hctl p, an S. cerevisiae homolog of Cdc20p, appears to be involved in the anaphase-promoting complex (APC)-mediated degradation of the mitotic cyclins but not of Pdsl p. This finding, in conjunction with the possible involvement of Cdc20p in Pdslp degradation (A Amon, personal communication), shed invaluable light on how the substrate specificity of the APC may be determined.
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Amon A: Regulation of B-type cyclin proteolysis by Cdc28associated kinases in budding yeast. EMBO J 1997, 16:26932702. Overexpression of the Cdo28 kinase inhibitor Sic1 results in untimely degradation of the mitotic cyclins. On the basis of this observation, the author suggests that activation of the anaphase-promoting complex requires a drop in the kinase activity of the mitotic C,dc28-cyclin complex. 51.
Li X, Cai M: Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae. Mol Cell Bio11997. 17:2723-2734.
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A new allele of cdc28 that causes a failure to arrest following the activation of either the DNA damage or the spindle assembly checkpoints is described. Until now, the role of the Cdc28p cyclin-dependent kinase was downplayed because of the inability to observe a DNA damage checkpoint defect in the available cdc28 mutants. 52. Nasmyth K: At the heart of the budding yeast cell cycle. Trends Genet 1996, 12:405-412. 53.
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Mayer-Jaekel R, Ohkura H, Gomes R, Sunkel CE, Baumgartner S, Hemmings BA, Glover DM: The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase. Cell 1993, 72:621-633. Minshull J, Straight A, Rudner AD, Dernburg AF, Belmont A, Murray AW: Protein phosphatese 2A regulates MPF activity and sister chromatid cohesion in budding yeast. Curr Biol 1996, 6:1609-1620. Wang Y, Burke D: Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae. Mol Cell Biol 1997, 17:620-626.
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Kinoshita N, Yamano H, Niwa H, Yoshida T, Yanagida M: Negative regulation of mitosis by the fission yeast protein phosphatese ppa2. Genes Dev 1993, 7:1059-1071.
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Gorbsky G, Ricketts W: Differential expression of a phosphoepitope at the kinetochores of moving chromosomes. J Cell Biol 1993, 122:1311-1321.
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Visintin R, Susanne P, Amon A: CDC20 and CDH1, a family of substrate-specific activators of APC-dependent proteolysis. Science 1997, in press.
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Schott EJ, Hoyt MA: Dominant alleles of Saccharomyces cerevisiae CDC2O reveal its role in promoting anaphase. Genetics 1997, in press.
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The metaphase-to-anaphase transition: avoiding a mid-life crisis Orna Cohen-Fix* and Douglas Koshland The metaphase-to-anaphase transition is a highly regulated process, which is governed by the activity of the anaphase-promoting complex (APC). The APC promotes the degradation of several proteins, including mitotic cyclins and newly identified anaphase inhibitors, Several discoveries made this year shed invaluable light on the regulation of APC activation and its substrate specificity. Addresses Howard Hughes Medical Institute, The Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210,
USA "e-mail: [email protected] re-mail: [email protected] Current Opinion in Cell Biology 1997, 9:800-806
http:/Ibiomednet.comlelecref10955067400900800 © Current Biology Ltd ISSN 0955-0674 Abbreviations APC anaphase-promoting complex CDK cyclin-dependent-kinase-cyclin complex
Introduction T h e metaphase-to-anaphase transition in mitosis involves a fascinating and complex sequence of biochemical events that initiate the segregation of replicated chromosomes (sister chromatids) (Figure 1). Following DNA replication, the sister chromatids are associated with each other along their length by a putative cohesion factor(s) (reviewed in [1]). By metaphase, sister chromatids have made stable attachments to spindle microtubules emanating from opposite spindle poles, and usually have congressed to the metaphase plate (Figure 1; for the definition of metaphase in budding yeast, see Figure 1 legend and [2,3]). T h e spindle attachments are mediated through the kinetochore, a specialized domain on each chromatid with microtubule-binding and microtubule-dependent motor activities. At the onset of anaphase, inactivation of the cohesion factor(s) leads to synchronous sister chromatid separation. Sister chromatids can separate in the absence of attachment to the spindle (reviewed in [4]), and anaphase spindle dynamics occur in the absence of chromosomes [5°]. Therefore, the metaphase-to-anaphase transition involves the coordinated execution of independent processes. T h e loss of this coordination could result in chromosome missegregation which is almost always deleterious to the cell. Because of the irreversible nature of this transition, cells have evolved regulatory mechanisms that couple the initiation of anaphase to the proper completion of prior events.
The a n a p h a s e - p r o m o t i n g complex T h e transition from metaphase to anaphase is controlled by the ubiquitin-dependent proteolysis of a specific subset of proteins [6-9]. Recently, a complex that promotes cell cycle specific ubiquitination has been identified. This complex, known as the anaphase-promoting complex (APC) [10,11] or cyclosome [12], was initially identified as the ubiquitin ligase required for the degradation of mitotic cyclins (reviewed in [13}). Subsequent studies showed that the APC is composed of at least eight subunits [14°,15], several of which are conserved among many organisms (see [13]). Ubiquitination by the APC requires the presence of a nine amino acid destruction box motif on the target protein [16]. A role for the APC in the metaphase-to-anaphase transition was inferred from the failure of cells to initiate anaphase when defective for APC function due to either mutations in APC subunits [10,17-21], the injection of antibodies directed against particular APC subunits [22], or the overexpression of a peptide containing a destruction box motif [23°]. These observations suggest that anaphase initiation requires the APC-mediated degradation of at least one, if not several, proteins. These proteins are likely to be distinct from the mitotic cyclins, because mitotic cyclin degradation is required for the exit from mitosis rather than for anaphase initiation [6-9]. T h e identification of the APC as a regulator of the metaphase-to-anaphase transition has led to three interesting questions that are the focus of this review. First, does the APC degrade a master regulator of the metaphaseto-anaphase transition that initiates a cascade of events, or does it degrade a series of targets that independently regulate processes such as cohesion, microtubule motors and microtubule dynamics? Second, as APC-mediated degradation is required for the metaphase-to-anaphase transition and for the exit from mitosis, how is the temporal order of degradation, and thus the temporal order of events, ensured? Finally, what is the mechanism that activates the APC only at the appropriate time for anaphase initiation? Recent findings, mostly from the budding and fission yeasts, provide insights into these questions.
A P C targets at the m e t a p h a s e - t o - a n a p h a s e transition In the past year, two proteins have been identified as important targets of the APC at the metaphase-to-anaphase transition, namely, Cut2 in the fission yeast [24 °°] and P d s l p in the budding yeast [25"°,26°°]. Both proteins are stabilized in the absence of APC function; they are degraded at the metaphase-to-anaphase transition; and
The metaphase-to-anaphase transition: avoiding a mid-life crisis Cohen-Fix and Koshtand
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Figure 1
Metaphase
Anaphase A
Anaphase B
Current Opinion in Cell Biology
The metaphase-to-anaphase transition. At metaphase, the duplicated chromosomes align on the metaphase plate. In the budding yeast, as well as in various other fungi, there appears to be no classical metaphase state in that the duplicated chromosomes do not align on a metaphase plate [2,3]. However, there does appear to be a stable stage at which the sister chromatids are associated, condensed and, under certain conditions, are inhibited from initiating anaphase by the activity of the spindle assembly checkpoint (see text). For our purposes, we define this state as a physiological metaphase, thereby classifying the budding yeast as an organism that undergoes a metaphase-to-anaphase transition. During anaphase A, the chromatids separate synchronously and move away from each other by microtubule-dependent movement toward the spindle poles. In anaphase B, the spindle poles move away from each other by microtubule-dependent migration, thereby further moving the chromosomes away from the plane of division. Chromatids are indicated as bi-tobed structures; kinetochores are white; microtubules are light gray; spindle poles are dark gray circles.
nondegradable forms of either protein block anaphase initiation. T h e observation that anaphase initiation requires protein degradation prompted the suggestion that one of the APC's targets might be a factor(s) mediating sister chromatid cohesion [6]. However, at this point it seems unlikely that either Cut2 or P d s l p plays such a role. T h e Cut2 protein localizes to spindle microtubules and cut2 null mutants fail to separate sister chromatids [24"']. Pdslp shows a general nuclear localization and pdsl null mutants do exhibit precocious dissociation of sister chromatids, as expected for a cohesion factor [25°']. However, the deletion of PDS1 alleviates the requirement for the APC in all aspects of anaphase initiation, including spindle elongation and the dissolution of cohesion. Given the evidence that these two processes are independent, Pdslp is unlikely to be the cohesion factor. Rather, Pdslp may be a master regulator of anaphase initiation that is inactivated by the APC. Whether or not the APC controls cohesion directly or indirectly is still unknown. Consistent with Pdslp's role as a master regulator, nondegradable Pdslp derivatives block all aspects of cell cycle progression [26"']. Nondegradable forms of Cut2, on the other hand, block anaphase initiation but not exit from mitosis [24"']. This, together with the phenotypic differences of the pdsl and cut2 null mutants, suggests that Cut2 plays a more limited rote in anaphase inhibition than Pdslp. Furthermore, the Pdslp and Cut2 proteins share no striking sequence similarity. These differences may suggest that the regulation of anaphase initiation by the APC differs among eukaryotes. In Saccharomyces cerevisiae, the APC-mediated degradation of a master
inhibitor (i.e. Pdslp) allows the initiation of multiple processes, some of which may require the degradation of additional APC substrates. In other organisms, such as Schizosaccharomycespombe, different aspects of anaphase initiation may be regulated individually by specific inhibitors, and therefore the absence of one inhibitor (i.e. Cut2) is not sufficient to allow complete anaphase initiation.
D o i n g mitosis o n e step at a t i m e Although the discovery of the APC's role in anaphase initiation provided tremendous insight into the process, it also generated a dilemma. T h e APC is required not only at the initiation of anaphase for the degradation of P d s l p and Cut2, but also at the exit of mitosis for the degradation of the mitotic cyclins and S. cerevisiae Aselp, a microtubule-binding protein that is required for proper spindle function [7,23",27,28"']. How is APC activity controlled such that at a given time some substrates are degraded while others are not? A possible mechanism is that the APC's accessibility to different substrates is cell cycle regulated. Alternatively, the substrate specificity of the APC may be modulated during the cell cycle. Recently, two related proteins that may determine the substrate specificity of the APC have been identified, namely, S. cerevisiae Cdc20p and Hctlp. Defects in the yeast Cdc20p or its Drosophila homolog Fizzy lead to a metaphase arrest [29-31], raising the possibility that they might be involved in APC-mediated degradation of anaphase inhibitors. Indeed, in S. cerevisiae, pdsl cdc20 double mutants bypass the mitotic arrest observed in a cdc20 mutant, suggesting that this arrest stems from the
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inability to degrade Pdslp [25°°]. Furthermore, Pdslp stability is sensitive to Cdc20p levels: Pdslp is stabilized in cdc20 mutants and it is destabilized when Cdc20p is overexpressed (A Amon, personal communication; EJ Schott, MA Hoyt, personal communication; see Note added in proof). Under these same conditions, the stability of the mitotic cyclins and Aselp is largely unaffected (A Amon, personal communicatiow, see Note added in proof). Similarly, the ubiquitination of the mitotic cyclins in vitro was independent of Cdc20p [32°].
In S. cerevisiae cells mutated in HCTI, a gene homologous to S. cerevisiae CDC20 [33"°], mitotic cyclins are not degraded at the exit of mitosis. Rather, they are stable throughout the cell cycle, hctl mutants display only a mild growth defect, and the persisting mitotic cyclins are counteracted in late mitosis by the activity of the kinase inhibitor Siclp. Overexpression of Hctlp causes a rapid APC-mediated degradation of the mitotic cyclins and Aselp ([33°°]; A Amon, personal communication; see Note added in proof). The stability of Pdslp is not affected by hctl mutations or by overexpressing Hctlp ([33"]; A Amon, personal communication; see Note added in proof). Taken together, these results suggest that Cdc20p and Hctlp act as APC substrate specificity factors, with Cdc20p being responsible for the APC-mediated degradation of Pdslp and Hctlp being responsible for the APC-mediated degradation of the mitotic cyclins and Aselp. This interpretation would predict that the APC physically interacts with Cdc20p and Hctlp. In this regard, it is noteworthy that some APC subunits contain tetratricopeptide repeat motifs whereas Cdc20p and Hctlp belong to a family of proteins with WD40 repeats [19,34,35], and there is precedence for a physical interaction between proteins having these two types of motifs [36]. Given the complex pattern of mitotic cyclin degradation in other eukaryotes (see below), we expect that additional Cdc20p-related proteins that control APC substrate specificity will be identified.
It remains unclear how only APC-Cdc20p complexes, but not APC-Hctlp complexes, are active at anaphase initiation. Interestingly, when Pdslp is rendered nondegradable, none of the postmetaphase events, including mitotic cyclin degradation, take place ([26°°]; O CohenFix, D Koshland, unpublished data). This kind of interdependence also occurs in the degradation of mitotic cyclins of other organisms. For example, in Drosophila, cyclin A is normally degraded before cyclin B and cyclin B3 [37]. Although all three proteins are present simultaneously, the presence of nondegradable cyclin A inhibits the degradation of cyclins B and B3 [30]. These findings suggest that cells ensure the sequential degradation of specific APC substrates by coupling a change in APC substrate specificity with the degradation of prior substrates. This, in turn, may be one of the
mechanisms by which the orderly execution of events in mitosis is controlled.
The regulation of A P C activation Understanding how the APC is activated is crucial to elucidating the mechanism of anaphase initiation. Any model for the activation of the APC must take into account the spindle assembly checkpoint pathway and the activity of the mitotic cyclin-dependent-kinase-cyclin complexes (henceforth known as mitotic CDKs). The spindle assembly checkpoint is a surveillance system that inhibits the metaphase-to-anaphase transition until all sister chromatids have achieved a bipolar spindle attachment (reviewed in [38,39]). The name of this checkpoint should be used with the cautionary note that the 'spindle' in this case includes the kinetochore, as kinetochores may be a prime source for the checkpoint signal. Indeed, anaphase initiation is delayed in the presence of defective or unattached kinetochores [5",40 a,A.]. Recent data further suggest that evolutionarily conserved components of the spindle assembly checkpoints localize to unattached kinetochores [45°-47°]. The notion that the spindle assembly checkpoint could inhibit the APC stems from the observations that cells with an activated spindle assembly checkpoint fail to degrade APC substrates [26°°] and extracts from these cells do not ubiquitinate mitotic cyclins in vitro [32°]. Mitotic CDKs, which are activated during G z phase [27,48], also play a role in controlling APC activity. Conflicting observations suggest that these kinases can activate as well as inhibit the APC. In vitro, the APC is activated by mitotic CDK activity and is inactivated by phosphatase treatment [14",49]. However, cells arrested by the spindle assembly checkpoint initiate anaphase when mitotic CDK activity is inhibited by the kinase inhibitor Siclp [50°°], or through mutations in the kinase subunit [51°]. These latter observations suggest that inhibition of mitotic CDK activity causes APC activation and subsequent anaphase initiation. With these observations in mind, we propose the following 'primed APC' model: the APC is first primed by the kinase activity of the mitotic CDKs. The primed APC is inhibited from degrading the anaphase inhibitor until the cell has completed metaphase. This inhibition is mediated by the spindle assembly checkpoint pathway. The completion of metaphase turns the spindle assembly checkpoint off, allowing for the transition of primed APC to active APC, thereby promoting the degradation of the anaphase inhibitor. According to this model, the requirement for high kinase activity is twofold: it is required for APC activation and it is also required for maintaining the spindle assembly checkpoint pathway (see above). Therefore, inhibiting mitotic CDK activity in checkpoint-arrested cells, either by kinase inhibitors
The metaphase-to-anaphase transition: avoiding a mid-life crisis Cohen-Fix and Koshland
or though mutations as described above, will lead to a collapse of the checkpoint pathway. This will lead to a premature transition form primed to active APC and hence to the initiation of anaphase. How the spindle assembly checkpoint inhibits the transition from primed APC to active APC is currently unknown. It has been proposed previously that the APC could be activated by a drop in the mitotic CDK activity [50",52]. In this regard, it is possible that when the mitotic CDKs prime the APC, they add not only activating phosphates but inhibitory phosphates as well (either on the APC itself or on an APC regulator). The removal of these inhibitory phosphates would be required for the primed to active APC transition, and this process may be efficient only when the activity of the mitotic CDKs drops. In this case, the spindle assembly checkpoint could be regulating the APC indirectly by preventing the drop in the mitotic CDK activity (Figure 2, large shaded rectangle). Thisis consistent with the involvement of protein phosphatase activity in the spindle assembly checkpoint pathway [53-56], where it could be required to maintain high kinase activity by removing inhibitory phosphorylation. If this drop in kinase activity takes place, one would have to argue that it is a localized event, as cells which maintain globally high mitotic CDK activity due to the presence of nondegradable mitotic cyclins can nonetheless initiate anaphase [7].
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It should be noted that the target of regulation by the spindle assembly checkpoint may not be the APC itself but rather a protein that determines APC's substrate recognition, such as Cdc20p. In this case, primed APC could be considered as APC lacking its substrate recognition factor. It has recently been demonstrated that overexpression of Cdc20p overrides a spindle assembly checkpoint arrest (A Amon, personal communication; EJ Schott, MA Hoyt, personal communication; see Note added in proof). The high levels of Cdc20p could overcome the inhibitory effect of the checkpoint pathway, thereby converting primed APC to active APC and promoting anaphase initiation. Would the primed APC model predict that the spindle assembly checkpoint is essential? Not necessarily. According to our model, there are multiple potential inputs that could influence the timing of the metaphase-to-anaphase transition, only one of which is the spindle assembly checkpoint. Even in the absence of the checkpoint pathway, there may be a finite time period required to convert the primed APC to the active APC. Moreover, anaphase initiation may require a reduction in the concentration of the anaphase inhibitor below some threshold level, a process which may be affected by the rate of APC-mediated ubiquitination or degradation. In certain cell types, these time intervals may be sufficient to achieve bipolar spindle attachments prior to the initiation of anaphase. Consistent with this possibility, in the
Figure 2
High mitotic CDK, activity
> £
Degradation of the anaphase ~ - - ~ inhibitor
Anaphase initiation
Current Opinion Jn Cell Biology
Regulation of APC activation: the primed APC model. According to this model, the APC can exist in the following three states: inactive APC, primed APC and active APC. Inactive APC is converted to primed APC by the kinase activity of the mitotic CDKs. Primed APE; is inhibited from degrading the anaphase inhibitor(s) prematurely by the spindle assembly checkpoint pathway, which is also activated by the mitotic CDKs. This checkpoint is active until all duplicated chromosomes form bipolar spindle attachments. The inhibitory effect of the checkpoint on the transition from primed APC to active APC could be direct or indirect (via inhibition of other kinases). In the latter case (large shaded rectangle), the transition from primed APC to active APC could be brought about by a drop in the mitotic CDK activity (see text), in this case, the spindle assembly checkpoint inhibits APC activation by preventing the drop in the kinase activity of the mitotic CDKs.
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budding yeast the spindle assembly checkpoint is not essential for viability, although mutants in components of this checkpoint show elevated levels of chromosome loss ([41,57]; MA Hoyt, personal communication). However, in at least some vertebrate cell types, the spindle assembly checkpoint appears to be active in each and every cell cycle: checkpoint components are localized to the kinetochore in prophase, [45"-47°~58] and microinjection of antibodies against one of the checkpoint components (Mad2p) results in premature initiation of anaphase and a mitotic catastrophe (G Gorbsky, personal communication). The relative contributions of various processes that control anaphase initiation may differ in different cell types. Under normal growth conditions the spindle assembly checkpoint may be dispensable in yeast, whereas other cell types may rely more heavily on the checkpoint in every cell division.
2.
Guacci V, Hogan E, Koshland D: Centromere position in budding yeast: evidence for anaphase A. Mo/Biol Cell 1997, 8:957-972.
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Straight AF, Marshall WF, Sedat JW, Murray AW: Mitosis in living budding yeast: anaphase A but no metaphase plate. Science 1997, 277:574-579.
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Conclusions The metaphase-to-anaphase transition is a big step in the cell's life cycle: having gone through it, the cell will have to live (or die) with the consequences. It is, therefore, not surprising that this transition is controlled at multiple levels. In the past year, we have witnessed the discovery of anaphase inhibitors and factors that may mediate APe substrate specificity. These and other findings suggest that the APe is regulated at the level of its activation and its substrate specificity. An additional level of regulation comes from the spindle assembly checkpoint pathway. Different cell types are likely to have common as well as distinct strategies in controlling anaphase initiation. We anticipate that the coming years will prove to be an exciting time for this field.
5. Zhang D, Nicklas R: 'Anaphase' and cytokinesis in the absence U of chromosomes. Nature 1996, 382:466-468. sing the powerful tool of micromanipulation, all chromosomes were removed from grasshopper sparmatocytes. However, the time of anaphase initiation, as detected by the appearance of a gap in the spindle, was comparable to that of cells with chromosomes. This study further demonstrates that dissolution of sister chromatid cohesion does not serve as a positive signal for anaphase initiation. 6.
Holloway S, Glotzer M, King R, Murray A: Anaphase is initiated by proteolysis rather than by the inactivation of maturationpromoting factor. Cell 1993, 73:1393-1402.
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Surana U, Amon A, Dowzer C, McGrew J, Byers B, Nasmyth K: Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J 1993, 12:1969-1978.
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Van der Velden HMW, Lohka MJ: Mitotic arrest caused by the amino terminus of Xenopus cyclin B2. Mol Cell Biol 1993, 13:1480-1498.
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Stueland CS, Law DJ, Cismowski MJ, Reed Sh Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiee. Mol Cell Biol 1993, 13:3744-3755.
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Imiger S, Piatti S, Michaelis C, Nasmyth K: Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell 1995, 81:269-278.
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King RW, Peters JM, Tugendreich S, Rolfe M, Hieter P, Kirschner MW: A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell 1995, 81:279-288.
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Sudakin V, Ganoth D, Dahan A, Heller H, Hershko J, Luca FC, Ruderman JV, Hershko A: The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol Biol Cell 1995, 6:185-198.
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Note added in proof T h e papers referred to as '(A Amon, personal communication)' and '(EJ Schott, MA Hoyt, personal communication)' have now been accepted for publication [59,60]. In addition, Sirgit and Lehner have recently reported the identification of a Drosophila protein called Fizzy related, which is similar in structure and perhaps function to the S. cerevisiae protein Hctl [61].
Acknowledgements We are grateful to Angelica Amon, Wolfgang Seufert, Gary Gorbsky, Eric Schott and Andy Hoyt for sharing results prior to publication. We also wish to thank Paul Megee, Pare Meluh, Shikha Laloraya, Mary Lilly, Andy Hoyt, Terry Orr-Weaver and Gary Gorbsky for their excellent comments and Christine Norman for help in preparing this manuscript. O Cohen-Fix is supported by a National Institutes of Health (NIH) grant (GM18382-02). D Koshland is supported by an NIH grant (GMI718) and is an investigator with the Howard Htighes Medical Institute.
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Yamano H, Gannon J, Hunt T: The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe. EMBO J 1996, 15:5268-5279. Recapitulating an earlier in vitro observation [6], this study shows that, in S. pombe, overexpression of a peptide carrying the destruction box inhibits anaphase initiation.
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