The metaphase to anaphase transition

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1 Eur. J. Biochem. 263, 14±19 (1999) q FEBS 1999 MINIREVIEW The metaphase to anaphase transition A case of productive destruction Katie A. Farr and Orna Cohen-Fix The Laboratory of Molecular and Cellular Biology, NIDDK, NIH, Bethesda, MD, USA The metaphase to anaphase transition is a point of no return; the duplicated sister chromatids segregate to the future daughter cells, and any mistake in this process may be deleterious to both progeny. At the heart of this process lies the anaphase inhibitor, which must be degraded in order for this transition to take place. The degradation of the anaphase inhibitor occurs via the ubiquitin-degradation pathway, and it involves the activity of the cyclosome/anaphase promoting complex (APC). The fidelity of the metaphase to anaphase transition is ensured by several different regulatory mechanisms that modulate the activity of the cyclosome/apc. Great advancements have been made in this field in the past few years, but many questions still remain to be answered. Keywords: cell cycle; mitosis; anaphase promoting complex; cyclosome; mitotic checkpoint; protein degradation. One of the more demanding processes during the course of a cell cycle is that of the accurate transmission of genetic material from mother to daughter cells. The genetic material, which appears in the form of one or more chromosomes, must be duplicated with high fidelity prior to division to form two identical sister chromatids. The sister chromatids remain attached to each other until they separate at mitosis. This attachment allows the cell to identify these two DNA segments as identical chromosomes, and eventually to segregate them away from each other to the two daughter cells. Finally, there must be a mechanism to segregate one, and only one, copy of every chromosome to each of the daughter cells. Upon entry into mitosis, the duplicated chromosomes, or sister chromatids, attach in a bipolar fashion to spindle microtubules that emanate from opposite poles of the cell (Fig. 1A). These associations occur through a highly specialized structure, the kinetochore, of which there is one on each chromatid. At metaphase, all the microtubules that are bound to a specific kinetochore emanate from the same pole, and the microtubules that are bound to kinetochores of sister chromatids emanate from opposite poles (Fig. 1A, Metaphase). The microtubules exert pulling forces on the chromosomes in the direction of the poles from which they originate, forces which are counteracted by the cohesion between the sister chromatids. At the time of the metaphase to anaphase transition, the cohesion between sister chromatids is dissolved, thereby allowing the chromosomes to be pulled by the spindle microtubules towards opposite poles (Fig. 1A, Anaphase A). Further segregation is achieved by the movement of the spindle poles away from each other (Fig. 1A, Anaphase B). The dissolution of cohesion does not depend on the pulling forces of the microtubules [1], but it must be correlated to the state of microtubule binding by the kinetochores. Premature dissociation of the sister chromatids, prior to the formation of stable bipolar Correspondence to O. Cohen-Fix, The Laboratory of Molecular and Cellular Biology, NIDDK, NIH, 8 Center Drive, Building 8, Room 319, Bethesda, MD , USA. Fax: , Tel.: , ornacf@helix.nih.gov Abbreviations: APC, anaphase promoting complex. (Received 4 February 1999, accepted 26 April 1999) spindle attachments, will result in the loss of sister chromatid identity (Fig. 1B). As a result, unattached sister chromatids may bind microtubules emanating from the same pole, leading to chromosome missegregation at anaphase. Thus, one of the crucial steps in accurate chromosome segregation is the precise execution of the metaphase to anaphase transition. THE METAPHASE TO ANAPHASE TRANSITION: THE KEY PLAYERS The degradation machinery (I) The metaphase to anaphase transition is controlled by a ubiquitin-mediated degradation process. Many cellular proteins are degraded by this pathway, where a small ubiquitous protein, ubiquitin, is transferred in a `bucket-brigade' manner from a ubiquitin-activating enzyme (also known as E1) to a ubiquitinconjugating enzyme (E2), and finally to the substrate itself [2]. In some cases, the final transfer of ubiquitin to the substrate is mediated by a ubiquitin ligase (E3) [2]. Polyubiquitination targets proteins for degradation by the proteosome, a multisubunit complex that catalyzes the breakdown of the ubiquitinated proteins. Not all proteins are ubiquitinated by the same ubiquitin-conjugating enzymes or ubiquitin ligases. In the degradation process at the metaphase to anaphase transition, the activity of at least one component, the ubiquitin ligase, is cellcycle regulated. As will be described below, this ubiquitin ligase is involved in regulating progression though mitosis via selective degradation of different proteins. A key driving force of mitosis is that of the mitotic cyclindependent kinase (mitotic Cdk). There are different forms of this enzyme, each of which is composed of a catalytic subunit (the Cdk) and a regulatory subunit (the cyclin), and they each act at a specific stage of the cell cycle. The mitotic Cdk is inactivated in part by the ubiquitin-dependent degradation of its cyclin moiety (the mitotic cyclin). Mitotic cyclin degradation starts at around the time of the metaphase to anaphase transition [3], and it requires the presence of a nine amino-acid motif, the destruction box, in the cyclin [4]. Removal of the destruction box results in the stabilization of the cyclin. Biochemical

2 q FEBS 1999 Regulation of the metaphase to anaphase transition (Eur. J. Biochem. 263) 15 Fig. 1. The metaphase to anaphase transition (A) and premature sister chromatid separation (B). At metaphase, the duplicated chromosomes (sister chromatids, gray), form bipolar attachments with spindle microtubules (green) that emanate from centrosomes or spindle pole bodies (yellow) located on opposite poles of the cells. The attachment between the chromatids and the microtubules occurs at the kinetochore (blue). At the metaphase to anaphase transition, the sister chromatids separate and move closer to the spindle poles (Anaphase A). During Anaphase B, the spindle poles move away from each other, thereby moving the chromosomes even further away from the plane of division, which will be at the spindle midzone. (B) A failure to properly regulate the metaphase to anaphase transition may lead to the premature separation of sister chromatids, prior to the establishment of bipolar spindle attachments. In this case, sister chromatids will bind microtubules independently of each other, and as a result both sisters may segregate to the same daughter cell. analysis of cyclin degradation in Xenopus, clam and yeast led to the identification of a cyclin-specific ubiquitin ligase. This ligase, termed the cyclosome [5] or the anaphase promoting complex (APC) [6], contains at least 12 subunits, many of which are conserved throughout evolution [7,8]. The name anaphase promoting complex stems from the observation that inactivation of any of several subunits in this complex leads to a cell cycle arrest at metaphase [8±11]. This raised the possibility that the metaphase to anaphase transition requires the cyclosome/apc-mediated degradation of an anaphase inhibitor. If the mitotic cyclins were acting as anaphase inhibitors, one would predict that nondegradable forms of the mitotic cyclins should block cell cycle progression at metaphase. However, abolishing mitotic cyclin degradation by mutations in the destruction box motif did not lead to a metaphase arrest, but rather to an arrest late in anaphase, just before the exit of mitosis [12,13]. This finding suggested that the metaphase to anaphase transition requires the degradation of a cyclosome/apc substrate other than the mitotic cyclins. The anaphase inhibitor To date, two anaphase inhibitors have been identified: the Pds1 protein (Pds1p) of budding yeast [14,15], and the Cut2 protein (Cut2p) of fission yeast [16]. Both proteins are degraded at the metaphase to anaphase transition, contain a destruction box motif, and in both, mutations in the destruction box cause resistance to cyclosome/apc mediated degradation. The presence of nondegradable forms of Pds1p or Cut2p lead to a metaphase arrest, suggesting that the cyclosome/apc-dependent degradation of these inhibitory proteins is essential for anaphase initiation. In budding yeast, Pds1p is the only cyclosome/apc substrate that must be degraded at the metaphase to anaphase transition, as the inactivation of Pds1p completely abolished the requirement for cyclosome/apc activity in this step [15,17]. Interestingly, Pds1p and Cut2 share no significant sequence homology, although structural similarities cannot be ruled out.

3 16 K. A. Farr and O. Cohen-Fix (Eur. J. Biochem. 263) q FEBS 1999 The targets of the anaphase inhibitor For the most part, it is still unclear how Pds1p or Cut2p inhibit anaphase initiation. While there is no apparent homology between the two proteins, they physically interact with related proteins: Pds1p associates with the Esp1 protein [17], and Cut2p associates with the Cut1 protein [18]. Esp1p and Cut1p share regions of homology, they are conserved throughout evolution, and in the absence of either protein sister chromatids fail to separate and the spindle does not elongate [19,20]. Thus, Esp1p and Cut1p appear to be positive regulators of the metaphase to anaphase transition, and they are likely to be negatively regulated by Pds1p and Cut2p, respectively. It has been suggested that Esp1p is responsible for the dissociation of cohesion factors from the paired sister chromatids, resulting in sister chromatid separation [17]. The degradation machinery (II) The findings that have been presented suggest that the cyclosome/apc is involved in regulating progression through mitosis at two different stages: at the metaphase to anaphase transition (for Pds1p/Cut2p degradation), and at the exit from mitosis (for mitotic cyclin degradation). In budding yeast, the degradation of Pds1p precedes that of the Clb2p mitotic cyclin [21] (O. Cohen-Fix and D. Koshland, unpublished results). This, presumably, controls the orderly progression through mitosis; first the chromosomes separate at the metaphase to anaphase transition and only then can cells exit mitosis. However, this raises the question of what determines the specificity and temporal order of degradation of different cyclosome/apc substrates. Recently, two proteins likely to be involved in determining the substrate specificity of the cyclosome/apc have been identified: the Cdc20 protein (Cdc20p) and the Cdh1/Hct1 protein (Cdh1p) [22,23]. Both belong to a family of WD repeat proteins that are conserved throughout evolution. In budding yeast, Cdc20p is required for Pds1p degradation [22], and inactivation of Cdc20p blocks cell cycle progression at metaphase [24]. Cdh1p is required for cyclin degradation, but not for Pds1p degradation [23]. Cdh1p is also required for the degradation of other noncyclin cyclosome/apc substrates at the end of mitosis [23]. Both Cdc20p and Cdh1p can directly associate with the cyclosome/apc [25,26]. Thus, the cyclosome/apc exists in at least two forms (Fig. 2): cyclosome/ APC Cdc20 for Pds1p degradation at the metaphase to anaphase transition, and cyclosome/apc Cdh1 for the degradation of the mitotic cyclins and other substrates at the exit of mitosis. Cdc20p and Cdh1p may also determine the temporal order of substrate degradation. This is not accomplished by the differential expression of these proteins; although the expression of Cdc20p is cell-cycle regulated, the expression of Cdh1p is not, and it is present at an almost constant level throughout the cell cycle [27]. However, the association between the cyclosome/apc and either Cdc20p or Cdh1p may change throughout the cell cycle. Indeed, Cdc20p associates with the cyclosome/apc before Cdh1p does [25,26], and this, in turn, may direct the degradation of the anaphase inhibitor prior to the degradation of the mitotic cyclins. Still, what determines the timing of Cdc20p and Cdh1p association with the cyclosome/ APC is not known. It is possible that the binding of Cdc20p to the cyclosome/apc excludes the Cdh1p-cyclosome/APC association, or that Cdc20p binding is a prerequisite for Cdh1p binding. Interestingly, the presence of nondegradable forms of Pds1p in mitosis inhibits cyclin degradation, despite the fact that both the cyclosome/apc and Cdh1p are present (O. Cohen-Fix and D. Koshland, unpublished results). It is therefore possible that Pds1p affects the association or activation of the cyclosome/apc Cdh1 complex, and that the temporal order of cyclosome/apc substrate degradation is determined by the substrates themselves, such that early substrates inhibit the degradation of later ones (Fig. 2). THE METAPHASE TO ANAPHASE TRANSITION: LAYERS OF REGULATION UNFOLD The role of phosphorylation in cyclosome/apc regulation The involvement of phosphorylation in APC function is somewhat controversial. Several cyclosome/apc subunits are in a nonphosphorylated state when the cyclosome/apc is inactive (at interphase), and in a phosphorylated state when the cyclosome/apc is active (in mitosis) [11,28]. In-vitro work with clam and Xenopus cyclosome/apc suggests that the cyclosome/apc is activated by phosphorylation that is mediated by the mitotic Cdk [29,30]. However, Amon [31] found that inactivating the mitotic Cdk in metaphase led to the degradation of cyclosome/apc substrates, suggesting that phosphorylation may play an inhibitory role in cyclosome/ APC function. One possible explanation for this seeming contradiction is that different parts of the cyclosome/apc machinery respond differently to phosphorylation. For example, while the cyclosome/apc itself may be activated by phosphorylation, one of its auxiliary subunits, e.g. Cdh1p, may be inhibited by phosphorylation. In support of this model, Zachariae et al. [26] found that Cdh1p can be phosphorylated by the mitotic Cdk, and that only the dephosphorylated form of Cdh1p binds cyclosome/apc. Whether Cdc20p is also regulated by phosphorylation is currently unknown. Fig. 2. Regulation of cyclosome/apc function. The substrate specificity of the cyclosome/apc is determined by Cdc20p and Cdh1p. These proteins may also direct the sequential degradation of Pds1p and the mitotic cyclins. A possible mode of regulation is shown.? indicates that the APC-mediated degradation of Pds1p affects the timing of mitotic cyclin degradation by a yet unknown mechanism. See text for more details.

4 q FEBS 1999 Regulation of the metaphase to anaphase transition (Eur. J. Biochem. 263) 17 The mitotic Cdk is not the only kinase that can phosphorylate the cyclosome/apc. Several groups have demonstrated that polo-like kinases have the ability to phosphorylate the cyclosome/apc, and there is evidence to suggest that this phosphorylation activates the cyclosome/apc [32,33]. Protein kinase A can also phosphorylate the cyclosome/apc, but this phosphorylation appears to be inhibitory [32]. As none of the phosphorylation sites have been mapped, the exact contribution of phosphorylation to cyclosome/apc activity remains to be determined. The mitotic checkpoint Failure to properly segregate chromosomes at the metaphase to anaphase transition can have drastic consequences. Indeed, this transition is governed by regulatory mechanisms, defects in which may result in cellular abnormalities. One regulatory mechanism that controls anaphase initiation is the mitotic, or spindle assembly, checkpoint. This checkpoint mechanism prevents the premature initiation of anaphase until all chromosomes have established stable bipolar spindle attachments. Recently, a defect in one component of the mitotic checkpoint was correlated with the appearance of neoplasia that manifest gross chromosomal instability [34]. To date, seven proteins have been shown to be directly involved in the mitotic checkpoint: the Bub proteins (Bub1p, Bub2p and Bub3p [35]), the Mad proteins (Mad1p, Mad2p and Mad3p [36]), and the Mps1 protein (Mps1p [37]). These proteins were initially identified in budding yeast, but homologs have also been found in fission yeast, nematode, frog, mouse, and human [38]. In budding yeast, most of the checkpoint proteins are not essential for viability, although mutants lacking mitotic checkpoint function show an enhanced rate of chromosome loss [36,39]. In mammalian cells, at least one of the checkpoint proteins, Mad2p, may be essential, as microinjection of antibodies against Mad2p caused the premature initiation of anaphase, with a catastrophic outcome [40], indicating that the mitotic checkpoint machinery may be required during each and every cell cycle. The mitotic checkpoint proteins may constitute a signaling complex. The pair-wise associations that have been reported include Bub1p and Bub3p, Mad3p and Bub3p, and Mad1p and Mad2p [41±43]. Bub1p, a serine/threonine protein kinase, is able to phosphorylate Bub3p, as well as itself [42]. Mps1p, a dual-specificity kinase, can phosphorylate Mad1p [44]. Thus, propagation of the checkpoint signal is likely to involve protein phosphorylation. Activation of the checkpoint by over-expression of either MPS1 or a mutant of BUB1 requires that all the other checkpoint genes be fully functional [44,45]. These observations hint at the existence of an interdependent checkpoint complex, where each of the Bub, Mad, and Mps1 proteins is required simultaneously to fully activate the checkpoint response. The intracellular localization of the mitotic checkpoint proteins has shed light on their possible mode of activity. In vertebrates, several of the Mad and Bub proteins (Bub1p, Bub3p, Mad1p, and Mad2p) reside on unattached kinetochores [38]. It is important to note that a significant fraction of these same proteins also appears to be diffusely distributed throughout the mitotic cytoplasm, raising the question of which pool of proteins is the active one. Nonetheless, the kinetochore localization is still significant because it suggests that the checkpoint response may be triggered in response to the state of microtubule±kinetochore binding. Consistent with this possibility, laser ablation of the unattached kinetochore of a mono-oriented chromosome (i.e. only one of the sister chromatids is attached to the spindle) causes mammalian cells to escape from a mitotic delay [46]. Therefore, a signal that inhibits anaphase initiation appears to emanate from kinetochores that are not attached to the spindle apparatus. Indeed, the Bub and Mad proteins disappear from kinetochores by metaphase, when all chromosomes have attained bipolar attachments to spindle microtubules. Although the precise events that trigger the checkpoint response are still unknown, the existing data lead to two models. In one, the mitotic checkpoint is sensitive to the presence or absence of tension at the kinetochore. Tension is exerted on kinetochores that are bound to microtubules in a bipolar fashion, and it arises from the opposing pulling forces exerted by the microtubules. This model was inspired by the observation that the checkpoint arrest in praying mantis spermatocyes can be alleviated by pulling on a mono-oriented chromosome with a microneedle [47]. According to this model, the mitotic checkpoint response is activated by kinetochores that have not established bipolar spindle attachments and thereby do not experience tension. The second model proposes that the mitotic checkpoint is sensitive to the physical binding of microtubules by the kinetochore, regardless of tension. Here, the checkpoint is activated by the presence of unattached kinetochores, and once all kinetochores are bound to microtubules, the checkpoint response is turned off. This model is consistent with the observation that the checkpoint is not induced in cells treated with taxol, where microtubule binding by the kinetochores is established, but without tension [48]. How does the mitotic checkpoint inhibit anaphase initiation? In several organisms, Mad2p interacts with Cdc20p [49±51]. The ternary Mad2p-Cdc20p-cyclosome/APC complex lacks ubiquitin ligase activity, but that activity is restored upon Mad2p dissociation [52]. The Mad2 protein exists in two forms, a tetramer and a monomer [52]. Although both are able to bind Cdc20p, only the Mad2p tetramer can inhibit the cyclosome/ APC in vitro. Therefore, upon checkpoint activation, Mad2p monomers may be induced to form tetramers, which then associate with Cdc20p to inhibit the degradation of the anaphase inhibitor. The molecular roles of the Bub, Mps1, and the other Mad proteins in the checkpoint response are still unclear. SUMMARY The timing of the metaphase to anaphase transition is crucial for proper chromosome segregation. The premature initiation of anaphase at a time when spindle microtubule binding by the sister chromatids is not yet complete would lead to chromosome missegregation, with the possibility of cellular abnormalities or cell death. The cell goes to great lengths to regulate this transition. Anaphase is initiated by the cyclosome/apcdependent degradation of an anaphase inhibitor. The activity of the cyclosome/apc is regulated by several protein kinases and by the mitotic checkpoint, which ensures that anaphase will not start until all paired chromosomes have formed stable bipolar attachments. The spindle assembly checkpoint inhibits the activity of the Cdc20 protein, which, in association with the cyclosome/apc, is required for the degradation of the anaphase inhibitor. The precise function of many of the components of this transition remains to be discovered. However, the evolutionary conservation of most of the participants in this process bears with it the hope that significant discoveries in one organism will quickly be translatable to general principles of mitotic regulation.

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