Cyclin-specific control of ribosomal DNA segregation

Following chromosome duplication in S phase of the cell cycle, the sister chromatids are linked by cohesin. At the onset of anaphase, separase cleaves cohesin and thereby initiates sister chromatid separation. Separase activation results from the destruction of its inhibitor, securin, which is triggered by a ubiquitin ligase called the anaphase-promoting complex (APC). Here, we show in budding yeast that securin destruction and, thus, separase activation are not sufficient for the efficient segregation of the repetitive ribosomal DNA (rDNA). We find that rDNA segregation also requires the APC-mediated destruction of the S-phase cyclin Clb5, an activator of the protein kinase Cdk1. Mutations that prevent Clb5 destruction are lethal and cause defects in rDNA segregation and DNA synthesis. These defects are distinct from the mitotic-exit defects caused by stabilization of the mitotic cyclin Clb2, emphasizing the importance of cyclin specificity in the regulation of late-mitotic events. Efficient rDNA segregation, both in mitosis and meiosis, also requires APC-dependent destruction of Dbf4, an activator of the protein kinase Cdc7. We speculate that the dephosphorylation of Clb5-specific Cdk1 substrates and Dbf4-Cdc7 substrates drives the resolution of rDNA in early anaphase. The coincident destruction of securin, Clb5, and Dbf4 coordinates bulk chromosome segregation with segregation of rDNA.     

A non-proteolytic function of separase links the onset of anaphase to mitotic exit

Separase is a protease that triggers chromosome segregation at anaphase onset by cleaving cohesin, the chromosomal protein complex responsible for sister chromatid cohesion. After anaphase, cells exit from mitosis; that is, they complete downregulation of cyclin-dependent kinase activity, undergo cytokinesis and enter G1 of the next cell cycle. Here we show that separase activation at the onset of anaphase is sufficient to promote release from the nucleolus and activation of the budding yeast phosphatase, Cdc14, a key step in mitotic exit. The ability of separase to activate Cdc14 is independent of its protease function but may involve promoting phosphorylation of the Cdc14 inhibitor Net1. This novel separase function is coregulated with its proteolytic activity by the separase inhibitor securin. This helps to explain the coupling of anaphase and mitotic exit--after securin degradation at anaphase onset, separase cleaves cohesin to trigger chromosome segregation and concurrently uses a non-proteolytic mechanism to initiate mitotic exit.     

Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.     

Mutual inhibition of separase and Cdk1 by two-step complex formation

Stable maintenance of genetic information requires chromosome segregation to occur with high accuracy. Anaphase is triggered when ring-shaped cohesin is cleaved by separase, a protease regulated by association with its inhibitor securin. Dispensability of vertebrate securin strongly suggests additional means of separase regulation. Indeed, sister chromatid separation but not securin degradation is inhibited by constitutively active cyclin-dependent kinase 1 (Cdk1) and can be rescued solely by preventing phosphorylation of separase. We demonstrate that Cdk1-dependent phosphorylation of separase is not sufficient for inhibition. In a second step, Cdk1 stably binds phosphorylated separase via its regulatory cyclin B1 subunit. Complex formation results in inhibition of both protease and kinase, and we show that vertebrate separase is a direct inhibitor of Cdk1. This unanticipated function of separase is negatively regulated by securin but independent of separase's proteolytic activity.     

Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed

Cellular transition to anaphase and mitotic exit has been linked to the loss of cyclin-dependent kinase 1 (Cdk1) kinase activity as a result of anaphase-promoting complex/cyclosome (APC/C)–dependent specific degradation of its cyclin B1 subunit. Cdk1 inhibition by roscovitine is known to induce premature mitotic exit, whereas inhibition of the APC/C-dependent degradation of cyclin B1 by MG132 induces mitotic arrest. In this study, we find that combining both drugs causes prolonged mitotic arrest in the absence of Cdk1 activity. Different Cdk1 and proteasome inhibitors produce similar results, indicating that the effect is not drug specific. We verify mitotic status by the retention of mitosis-specific markers and Cdk1 phosphorylation substrates, although cells can undergo late mitotic furrowing while still in mitosis. Overall, we conclude that continuous Cdk1 activity is not essential to maintain the mitotic state and that phosphatase activity directed at Cdk1 substrates is largely quiescent during mitosis. Furthermore, the degradation of a protein other than cyclin B1 is essential to activate a phosphatase that, in turn, enables mitotic exit.     

The spindle and kinetochore–associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.     

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.     

Protein phosphatase 2A and separase form a complex regulated by separase autocleavage

The onset of anaphase is triggered by the activation of a site-specific protease called separase. Separase cleaves the chromosomal cohesins holding the duplicated sister chromatids together, allowing sisters to simultaneously separate and segregate to opposite ends of the cell before division. Activated separase cleaves not only cohesin, but also itself; however, the biological significance of separase self-cleavage has remained elusive. Before anaphase, separase is inhibited by at least two mechanisms. The first involves the binding of securin, whereas the second requires the phosphorylation-dependent binding of cyclin-dependent kinase 1 (Cdk1)/cyclin B1. Because securin and Cdk1/cyclin B1 interact with separase in a mutually exclusive manner, the degradation of both these inhibitors plays an important role in activating separase at anaphase. Here we identify a new separase interacting partner, a specific subtype of the heterotrimeric protein phosphatase 2A (PP2A). PP2A associates with separase through the B' (B56) regulatory subunit and does so independently of securin and cyclin B1 binding. The association of PP2A with separase requires a 55-amino acid domain closely juxtaposed to separase autocleavage sites. Strikingly, mutation of these cleavage sites increases PP2A binding, suggesting that separase cleavage disrupts the interaction of PP2A with separase. Furthermore, expression of a non-cleavable separase, but not a non-cleavable mutant that cannot bind PP2A, causes a premature loss of centromeric cohesion. Together these observations provide a new mechanistic insight into a physiological function for separase self-cleavage.     

Phosphorylation-dependent binding of cyclin B1 to a Cdc6-like domain of human separase

Sister chromatids are held together by the ring-shaped cohesin complex, which likely entraps both DNA-double strands in its middle. This tie is resolved in anaphase when separase, a giant protease, becomes active and cleaves the kleisin subunit of cohesin. Premature activation of separase and, hence, chromosome missegregation are prevented by at least two inhibitory mechanisms. Although securin has long been appreciated as a direct inhibitor of separase, surprisingly its loss has basically no phenotype in mammals. Phosphorylation-dependent binding of Cdk1 constitutes an alternative way to inhibit vertebrate separase. Its importance is illustrated by the premature loss of cohesion when Cdk1-resistant separase is expressed in mammalian cells without or with limiting amounts of securin. Here, we demonstrate that crucial inhibitory phosphorylations occur within a region of human separase that is also shown to make direct contact with the cyclin B1 subunit of Cdk1. This region exhibits a weak homology to Saccharomyces cerevisiae Cdc6 of similar Cdk1 binding behavior, thereby establishing phosphoserine/threonine-mediated binding of partners as a conserved characteristic of B-type cyclins. In contrast to the Cdc6-like domain, the previously identified serine 1126 phosphorylation is fully dispensable for Cdk1 binding to separase fragments. This suggests that despite its in vivo relevance, it promotes complex formation indirectly, possibly by inducing a conformational change in full-length separase.     

Role for cyclin-dependent kinase 2 in mitosis exit

Mitosis requires cyclin-dependent kinase (cdk) 1-cyclin B activity [1]. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B [2]. Cdk2 is also active during mitosis [3, 4]. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable [5]. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2 [6]. Here, we show that cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. Our findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit.     

Securin regulates entry into M-phase by modulating the stability of cyclin B

Timely progression into mitosis is necessary for normal cell division. This transition is sensitive to the levels of cyclin B, the regulatory subunit of the master mitotic kinase, Cdk1. Cyclin B accumulates during G2 and prophase when its rate of destruction by the anaphase promoting complex (APC) is low. Securin is also an APC substrate and is known for its role in inactivating the cohesin-cleaving enzyme, separase, until the metaphase to anaphase transition. Here we show that securin has an additional role in cell-cycle regulation, that of modulating the timing of entry into M-phase. In mouse oocytes, excess securin caused stabilization of cyclin B and precocious entry into M-phase. Depletion of securin increased cyclin B degradation, resulting in delayed progression into M-phase. This effect required APC activity and was reversed by expression of wild-type securin. These data reveal a role for securin at the G2-M transition and suggest a more general mechanism whereby physiological levels of co-competing APC substrates function in modulating the timing of cell-cycle transitions.     

Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint

The spindle checkpoint prevents activation of the anaphase-promoting complex (APC/C) until all chromosomes are correctly attached to the mitotic spindle. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C by binding the APC/C activating protein CDC20 until the chromosomes are properly aligned and attached to the mitotic spindle, at which point MCC disassembly releases CDC20 to activate the APC/C. Once the APC/C is activated, it targets cyclin B and securin for degradation, and the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. We sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined the effect on the MCC and APC/C activation. Using this approach we found that 2 phosphatase inhibitors, calyculin A and okadaic acid (1 μM), caused MCC dissociation and APC/C activation leading to cyclin A and B degradation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.     

Nuclear Exclusion of Separase Prevents Cohesin Cleavage in Interphase Cells

During mitosis, equal transmission of the duplicated chromosomes demands a strict regulation of separase, which cleaves cohesin and triggers sister chromatid separation in anaphase. Vertebrate separase is inhibited by securin and the inhibitory phosphorylation of separase. However, knockout experiments indicate that securin is dispensable and the inhibitory phosphorylation was observed only in M phase cells. This begs the question how cohesin cleavage by separase is prevented in the absence these two mechanisms. Here we show that separase is excluded from cohesin by the nuclear envelope, which forms in telophase and disassembles in mitosis. The exclusion is achieved passively by its large physical mass and may be backed up by the CRM1-dependent nuclear export. A functional NES motif is identified in separase. We demonstrated that the nuclear envelope is sufficient to prevent active separase from cleaving nuclear cohesin. We propose that the nuclear exclusion is important to prevent cohesin cleavage during interphase in the absence of securin and the phosphorylation inhibition.     

Regulation of human separase by securin binding and autocleavage

BACKGROUND: Sister chromatid separation is initiated by separase, a protease that cleaves cohesin and thereby dissolves sister chromatid cohesion. Separase is activated by the degradation of its inhibitor securin and by the removal of inhibitory phosphates. In human cells, separase activation also coincides with the cleavage of separase, but it is not known if this reaction activates separase, which protease cleaves separase, and how separase cleavage is regulated.RESULTS: Inhibition of separase expression in human cells by RNA interference causes the formation of polyploid cells with large lobed nuclei. In mitosis, many of these cells contain abnormal chromosome plates with unseparated sister chromatids. Inhibitor binding experiments in vitro reveal that securin prevents the access of substrate analogs to the active site of separase. Upon securin degradation, the active site of full-length separase becomes accessible, allowing rapid autocatalytic cleavage of separase at one of three sites. The resulting N- and C-terminal fragments remain associated and can be reinhibited by securin. A noncleavable separase mutant retains its ability to cleave cohesin in vitro.CONCLUSIONS: Our results suggest that separase is required for sister chromatid separation during mitosis in human cells. Our data further indicate that securin inhibits separase by blocking the access of substrates to the active site of separase. Securin proteolysis allows autocatalytic processing of separase into a cleaved form, but separase cleavage is not essential for separase activation.     

Mnd2, an essential antagonist of the anaphase-promoting complex during meiotic prophase

Meiotic cohesin serves in sister chromatid linkage and DNA repair until its subunit Rec8 is cleaved by separase. Separase is activated when its inhibitor, securin, is polyubiquitinated by the Cdc20 regulated anaphase-promoting complex (APC(Cdc20)) and consequently degraded. Differently regulated APCs (APC(Cdh1), APC(Ama1)) have not been implicated in securin degradation at meiosis I. We show that Mnd2, a factor known to associate with APC components, prevents premature securin degradation in meiosis by APC(Ama1). mnd2Delta cells lack linear chromosome axes and exhibit precocious sister chromatid separation, but deletion of AMA1 suppresses these defects. Besides securin, Sgo1, a protein essential for protection of centromeric cohesion during anaphase I, is also destabilized in mnd2delta cells. Mnd2's disappearance prior to anaphase II may activate APC(Ama1). Human oocytes may spend many years in meiotic prophase before maturation. Inhibitors of meiotic APC variants could prevent loss of chiasmata also in these cells, thereby guarding against aberrant chromosome segregation.     

Subunits and substrates of the anaphase-promoting complex

The initiation of anaphase and exit from mitosis depend on a ubiquitination complex called the anaphase-promoting complex (APC) or cyclosome. The APC is composed of more than 10 constitutive subunits and associates with additional regulatory factors in mitosis and during the G1 phase of the cell cycle. At the metaphase-anaphase transition the APC ubiquitinates proteins such as Pds1 in budding yeast and Cut2 in fission yeast whose subsequent degradation by the 26S proteasome is essential for the initiation of sister chromatid separation. Later in anaphase and telophase the APC promotes the inactivation of the mitotic cyclin-dependent protein kinase 1 by ubiquitinating its activating subunit cyclin B. The APC also mediates the ubiquitin-dependent proteolysis of several other mitotic regulators, including other protein kinases, APC activators, spindle-associated proteins, and inhibitors of DNA replication.     

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.     

Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae

Sister chromatid separation and mitotic exit are triggered by the anaphase-promoting complex (APC/C) which is a multi-subunit ubiquitin ligase required for proteolytic degradation of various target proteins. Cdc20 and Cdh1 are substrate-specific activators of the APC/C. It was previously proposed that Cdh1 is essential for proteolysis of the yeast mitotic cyclin Clb2. We show that Clb2 proteolysis is triggered by two different modes during mitosis. A fraction of Clb2 is degraded during anaphase in the absence of Cdh1. However, a second fraction of Clb2 remains stable during anaphase and is degraded in a Cdh1-dependent manner as cells exit from mitosis. Most of cyclin Clb3 is degraded independently of Cdh1. Our data imply that degradation of mitotic cyclins is initiated by a Cdh1-independent mechanism.     

Inactivating Cdc25, Mitotic Style

Mitotic entry and exit require activation and inactivation of the Cdk1-cyclin B kinase complex, respectively. The Cdc25 protein phosphatase family activates Cdk1-cyclin B at the G2/M transition by removing inhibitory phosphate groups. Cdc25 family members, held inactive during interphase, are activated during mitotic progression in an amplification loop involving Cdk1-cyclin B. While Cdc25 activation at the G2/M transition is required for the timely initiation of mitosis, recent evidence suggests that the inactivation of Cdc25 in late mitosis may play a role in supporting Cdk1-cyclin B inactivation. Here, we discuss the mechanisms of Cdc25 regulation and how they pertain to both mitotic entry and exit.     

‘… The End of the Beginning’: Cdk1 Thresholds and Exit from Mitosis

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