Cdc20 Is Critical for Meiosis I and Fertility of Female Mice

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.     

The APC/C recruits cyclin B1–Cdk1–Cks in prometaphase before D box recognition to control mitotic exit

The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated at prometaphase by mitotic phosphorylation and binding of its activator, Cdc20. This initiates cyclin A degradation, whereas cyclin B1 is stabilized by the spindle checkpoint. Upon checkpoint release, the RXXL destruction box (D box) was proposed to direct cyclin B1 to core APC/C or Cdc20. In this study, we report that endogenous cyclin B1–Cdk1 is recruited to checkpoint-inhibited, phosphorylated APC/C in prometaphase independently of Cdc20 or the cyclin B1 D box. Like cyclin A, cyclin B1 binds the APC/C by the Cdk cofactor Cks and the APC3 subunit. Prior binding to APC/C^Cdc20 makes cyclin B1 a better APC/C substrate in metaphase, driving mitotic exit and cytokinesis. We conclude that in prometaphase, the phosphorylated APC/C can recruit both cyclin A and cyclin B1 in a Cks-dependent manner. This suggests that the spindle checkpoint blocks D box recognition of APC/C-bound cyclin B1, whereas distinctive complexes between the N terminus of cyclin A and Cdc20 evade checkpoint control.     

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

Loss of Cdc20 causes a securin-dependent metaphase arrest in two-cell mouse embryos

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.     

Cdc20 proteolysis requires p38 MAPK signaling and Cdh1‐independent APC/C ubiquitination during spindle assembly checkpoint activation by cadmium

Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, initiates the destruction of key mitotic regulators to facilitate mitosis, while it is negatively regulated by the spindle assembly checkpoint (SAC) to prevent premature anaphase entry. Activation of the p38 mitogen‐activated protein kinase could contribute to mitotic arrest, but the underlying mechanism is unknown. Here we report a novel pathway in which the p38 signaling triggers Cdc20 destruction under SAC elicited by cadmium, a human carcinogen. We found that the cadmium‐induced prometaphase arrest was linked to decreased Cdc20 and accumulated cyclin A protein levels in human cells, whereas the activity of cyclin B1–Cdk1 was unaffected. The Cdc20 half‐life was markedly shortened along with its ubiquitination and degradation via 26S proteasome in cadmium‐treated asynchronous or G₂‐enriched cells. Depletion of APC3 markedly suppressed the cadmium‐induced Cdc20 ubiquitination and proteolysis, while depletion of Cdh1, another activator of APC/C, did not. Intriguingly, blockage of p38 activity restored the Cdc20 levels for continuing mitosis under cadmium, while inhibition of JNK activity had no effect. The cadmium‐induced Cdc20 proteolysis was also suppressed during transient depletion of p38α or stable expression a dominant negative form of p38. Inhibition of p38 abolished the induction of Mad2–Cdc20–APC3 complex by cadmium. Moreover, forced expression of MKK6–p38 signaling could promote Cdc20 degradation in a Cdh1‐independent APC/C pathway. In summary, accelerated ubiquitination and proteolysis of Cdc20 is essential for prometaphase arrest that is mediated via the p38 signaling during SAC activation. J. Cell. Physiol. 223: 327–334, 2010. © 2010 Wiley‐Liss, Inc.     

Mitotic regulation of the human anaphase-promoting complex by phosphorylation

The anaphase-promoting complex (APC) or cyclosome is a ubiquitin ligase that initiates anaphase and mitotic exit. APC activation is thought to depend on APC phosphorylation and Cdc20 binding. We have identified 43 phospho-sites on APC of which at least 34 are mitosis specific. Of these, 32 sites are clustered in parts of Apc1 and the tetratricopeptide repeat (TPR) subunits Cdc27, Cdc16, Cdc23 and Apc7. In vitro, at least 15 of the mitotic phospho-sites can be generated by cyclin-dependent kinase 1 (Cdk1), and 3 by Polo-like kinase 1 (Plk1). APC phosphorylation by Cdk1, but not by Plk1, is sufficient for increased Cdc20 binding and APC activation. Immunofluorescence microscopy using phospho-antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of cyclin B1. In prometaphase phospho-APC accumulates on centrosomes where cyclin B ubiquitination is initiated, appears throughout the cytosol and disappears during mitotic exit. Plk1 depletion neither prevents APC phosphorylation nor cyclin A destruction in vivo. These observations imply that APC activation is initiated by Cdk1 already in the nuclei of late prophase cells.     

The spindle checkpoint,APC/CCdc20, and APC/CCdh1 play distinct roles in connecting mitosis to S phase

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/C^Cdc20. Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/C^Cdh1 leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/C^Cdc20, and APC/C^Cdh1 act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.     

How APC/C-Cdc20 changes its substrate specificity in mitosis

Progress through mitosis requires that the right protein be degraded at the right time. One ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) targets most of the crucial mitotic regulators by changing its substrate specificity throughout mitosis. The spindle assembly checkpoint (SAC) acts on the APC/C co-activator, Cdc20 (cell division cycle 20), to block the degradation of metaphase substrates (for example, cyclin B1 and securin), but not others (for example, cyclin A). How this is achieved is unclear. Here we show that Cdc20 binds to different sites on the APC/C depending on the SAC. Cdc20 requires APC3 and APC8 to bind and activate the APC/C when the SAC is satisfied, but requires only APC8 to bind the APC/C when the SAC is active. Moreover, APC10 is crucial for the destruction of cyclin B1 and securin, but not cyclin A. We conclude that the SAC causes Cdc20 to bind to different sites on the APC/C and this alters APC/C substrate specificity.     

Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A

Successful mitosis requires the right protein be degraded at the right time. Central to this is the spindle checkpoint that prevents the destruction of securin and cyclin B1 when there are improperly attached chromosomes. The principal target of the checkpoint is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C). A Drosophila Cdc20/fizzy mutant arrests in mitosis with high levels of cyclins A and B, but paradoxically the spindle checkpoint does not stabilize cyclin A. Here, we investigated this paradox and found that Cdc20 is rate limiting for cyclin A destruction. Indeed, Cdc20 binds efficiently to cyclin A before and in mitosis, and this complex has little associated Mad2. Furthermore, the cyclin A complex must bind to a Cks protein to be degraded independently of the checkpoint. Thus, we identify a crucial role for the Cks proteins in mitosis and one mechanism by which the APC/C can target substrates independently of the spindle checkpoint.     

The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time

In Drosophila cells cyclin B is normally degraded in two phases: (a) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator; and (b) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. We show that the APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box-mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]-GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM-GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM-GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint.     

DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint

BACKGROUND: DNA damage during mitosis triggers an ATM kinase-mediated cell cycle checkpoint pathway in yeast and fly embryos that delays progression through division. Recent data suggest that this is also true for mammals. Here we used laser microsurgery and inhibitors of topoisomerase IIalpha to break DNA in various mammalian cells after they became committed to mitosis. We then followed the fate of these cells and emphasized the timing of mitotic progression, spindle structure, and chromosome behavior. RESULTS: We find that DNA breaks generated during late prophase do not impede entry into prometaphase. If the damage is minor, cells complete mitosis on time. However, more significant damage substantially delays exit from mitosis in many cell types. In human (HeLa, CFPAC-1, and hTERT-RPE) cells, this delay occurs during metaphase, after the formation of a bipolar spindle and the destruction of cyclin A, and it is not dependent on a functional p53 pathway. Pretreating cells with ATM kinase inhibitors does not abrogate the metaphase delay due to chromosome damage. Immunofluorescence studies reveal that cells blocked in metaphase by chromosome damage contain one or more Mad2-positive kinetochores, and the block is rapidly overridden when the cells are microinjected with a dominant-negative construct of Mad2 (Mad2deltaC). CONCLUSIONS: We conclude that the delay in mitosis induced by DNA damage is not due to an ATM-mediated DNA damage checkpoint pathway. Rather, the damage leads to defects in kinetochore attachment and function that, in turn, maintain the intrinsic Mad-2-based spindle assembly checkpoint.     

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability ¹. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease ^2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle¹. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/C^Cdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths¹.Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects ⁵.To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B ⁶.To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety ⁶ (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) ^7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium ⁷ (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation ⁶ (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable ^9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium ⁶.     

Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti‐apoptotic protein Mcl‐1 is regulated during the cell cycle and peaks at mitosis. Mcl‐1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin‐dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl‐1 in cells arrested in mitosis by microtubule poisons. Mcl‐1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase‐promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl‐1 during mitotic arrest by mutation of either Thr92 or a D‐box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl‐1 by CDK1–cyclin B1 and its APC/C^Cdc20‐mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.     

Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes.

Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.     

Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity

Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.     

Differential mRNA translation and meiotic progression require Cdc2-mediated CPEB destruction

Translational activation of several dormant mRNAs in vertebrate oocytes is mediated by cytoplasmic polyadenylation, a process controlled by the cytoplasmic polyadenylation element (CPE) and its binding protein CPEB. The translation of CPE-containing mRNAs does not occur en masse at any one time, but instead is temporally regulated. We show here that in Xenopus, partial destruction of CPEB controls the temporal translation of CPE-containing mRNAs. While some mRNAs, such as the one encoding Mos, are polyadenylated at prophase I, the polyadenylation of cyclin B1 mRNA requires the partial destruction of CPEB that occurs at metaphase I. CPEB destruction is mediated by a PEST box and Cdc2-catalyzed phosphorylation, and is essential for meiotic progression to metaphase II. CPEB destruction is also necessary for mitosis in the early embryo. These data indicate that a change in the CPEB:CPE ratio is necessary to activate mRNAs at metaphase I and drive the cells' entry into metaphase II.     

Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.     

Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex

We have discovered an early mitotic inhibitor, Emi1, which regulates mitosis by inhibiting the anaphase promoting complex/cyclosome (APC). Emi1 is a conserved F box protein containing a zinc binding region essential for APC inhibition. Emi1 accumulates before mitosis and is ubiquitylated and destroyed in mitosis, independent of the APC. Emi1 immunodepletion from cycling Xenopus extracts strongly delays cyclin B accumulation and mitotic entry, whereas nondestructible Emi1 stabilizes APC substrates and causes a mitotic block. Emi1 binds the APC activator Cdc20, and Cdc20 can rescue an Emi1-induced block to cyclin B destruction. Our results suggest that Emi1 regulates progression through early mitosis by preventing premature APC activation, and may help explain the well-known delay between cyclin B/Cdc2 activation and cyclin B destruction.     

Cell cycle-regulated proteolysis of mitotic target proteins

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.     

The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila

Background: Exit from mitosis is a tightly regulated event. This process has been studied in greatest detail in budding yeast, where several activities have been identified that cooperate to downregulate activity of the cyclin-dependent kinase (CDK) Cdc28 and force an exit from mitosis. Cdc28 is inactivated through proteolysis of B-type cyclins by the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) and inhibition by the cyclin-dependent kinase inhibitor (CKI) Sic1. In contrast, the only mechanism known to be essential for CDK inactivation during mitosis in higher eukaryotes is cyclin destruction.Results: We now present evidence that the Drosophila CKI Roughex (Rux) contributes to exit from mitosis. Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos. In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase. Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable cyclin A. Rux has numerous functional similarities with Sic1. While these proteins share no sequence similarity, we show that Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo.Conclusions: Rux inhibits Cdk1-cyclin A kinase activity during metaphase, thereby contributing to exit from mitosis. To our knowledge, this is the first mitotic function ascribed to a CKI in a multicellular organism and indicates the existence of a novel regulatory mechanism for the metaphase to anaphase transition during development.