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.     

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.     

The dual mechanism of separase regulation by securin

BACKGROUND: Sister chromatid separation and segregation at anaphase onset are triggered by cleavage of the chromosomal cohesin complex by the protease separase. Separase is regulated by its binding partner securin in two ways: securin is required to support separase activity in anaphase; and, at the same time, securin must be destroyed via ubiquitylation before separase becomes active. The molecular mechanisms underlying this dual regulation of separase by securin are unknown.RESULTS: We show that, in budding yeast, securin supports separase localization. Separase enters the nucleus independently of securin, but securin is required and sufficient to cause accumulation of separase in the nucleus, where its known cleavage targets reside. Securin also ensures that separase gains full proteolytic activity in anaphase. We also show that securin, while present, directly inhibits the proteolytic activity of separase. Securin prevents the binding of separase to its substrates. It also hinders the separase N terminus from interacting with and possibly inducing an activating conformational change at the protease active site 150 kDa downstream at the protein's C terminus.CONCLUSIONS: Securin inhibits the proteolytic activity of separase in a 2-fold manner. While inhibiting separase, securin is able to promote nuclear accumulation of separase and help separase to become fully activated after securin's own destruction at anaphase onset.     

Positive and Negative Regulation of Vertebrate Separase by Cdk1-Cyclin B1 May Explain Why Securin Is Dispensable

Sister chromatid cohesion is established during replication by entrapment of both dsDNAs within the cohesin ring complex. It is dissolved in anaphase when separase, a giant cysteine endopeptidase, cleaves the Scc1/Rad21 subunit of cohesin, thereby triggering chromosome segregation. Separase is held inactive by association with securin until this anaphase inhibitor is destroyed at the metaphase-to-anaphase transition by ubiquitin-dependent degradation. The relevant ubiquitin ligase, the anaphase-promoting complex/cyclosome, also targets cyclin B1, thereby causing inactivation of Cdk1 and mitotic exit. Although separase is essential, securin knock-out mice are surprisingly viable and fertile. Capitalizing on our previous finding that Cdk1-cyclin B1 can also bind and inhibit separase, we investigated whether this kinase might be suitable to maintain faithful timing and execution of anaphase in the absence of securin. We found that, similar to securin, Cdk1-cyclin B1 regulates separase in both a positive and negative manner. Although securin associates with nascent separase to co-translationally assist proper folding, Cdk1-cyclin B1 acts on native state separase. Upon entry into mitosis, Cdk1-cyclin B1-dependent phosphorylation of Ser-1126 renders separase prone to inactivation by aggregation/precipitation. Stable association of Cdk1-cyclin B1 with phosphorylated separase counteracts this tendency and stabilizes separase in an inhibited yet activatable state. These opposing effects are suited to prevent premature cleavage of cohesin in early mitosis while ensuring timely activation of separase by anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. Coupling sister chromatid separation with subsequent exit from mitosis by this simplified mode might have been the common scheme of mitotic control prior to the evolution of securin.     

Securin and separase phosphorylation act redundantly to maintain sister chromatid cohesion in mammalian cells

The spindle assembly checkpoint monitors the integrity of the spindle microtubules, which attach to sister chromatids at kinetochores and play a vital role in preserving genome stability by preventing missegregation. A key target of the spindle assembly checkpoint is securin, the separase inhibitor. In budding yeast, loss of securin results in precocious sister chromatid separation when the microtubule spindle is disrupted. However, in contrast to budding yeast, mammalian securin is not required for spindle checkpoint, suggesting that there are redundant mechanisms controlling the dissolution of sister chromatid cohesion in the absence of securin. One candidate mechanism is the inhibitory phosphorylation of separase. We generated a nonphosphorylable point mutant (S1121A) separase allele in securin-/- mouse embryonic stem cells. Securin(-/-)separase(+/S1121A) cells are viable but fail to maintain sister chromatid cohesion in response to the disruption of spindle microtubules, show enhanced sensitivity to nocodazole, and cannot recover from prometaphase arrest.     

Securin and separase modulate membrane traffic by affecting endosomal acidification

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans-Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V-) ATPase complexes, mimicking the effect of the specific V-ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V-ATPase assembly and function.     

Dual inhibition of sister chromatid separation at metaphase.

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.     

PP2A delays APC/C‐dependent degradation of separase‐associated but not free securin

The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase‐associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase‐associated securin is destabilized by introduction of phosphorylation‐mimetic aspartates or extinction of separase‐associated PP2A activity. G2‐ or prometaphase‐arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate‐mutant securin. Thus, PP2A‐dependent stabilization of separase‐associated securin prevents precocious activation of separase during checkpoint‐mediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase.     

Securin and not CDK1/cyclin B1 regulates sister chromatid disjunction during meiosis II in mouse eggs

Mammalian eggs remain arrested at metaphase of the second meiotic division (metII) for an indeterminate time before fertilization. During this period, which can last several hours, the continued attachment of sister chromatids is thought to be achieved by inhibition of the protease separase. Separase is known to be inhibited by binding either securin or Maturation (M-Phase)-Promoting Factor, a heterodimer of CDK1/cyclin B1. However, the relative contribution of securin and CDK/cyclin B1 to sister chromatid attachment during metII arrest has not been assessed. Although there are conditions in which either CDK1/cyclinB1 activity or securin can prevent sister chromatid disjunction, principally by overexpression of non-degradable cyclin B1 or securin, we find here that separase activity is primarily regulated by securin and not CDK1/cyclin B1. Thus the CDK1 inhibitor roscovitine and an antibody we designed to block the interaction of CDK1/cyclin B1 with separase, both failed to induce sister disjunction. In contrast, securin morpholino knockdown specifically induced loss of sister attachment, that could be restored by securin cRNA rescue. During metII arrest separase appears primarily regulated by securin binding, not CDK1/cyclin B1.     

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.     

Overexpression of the Pituitary Tumor Transforming Gene Induces p53-dependent Senescence through Activating DNA Damage Response Pathway in Normal Human Fibroblasts

Pituitary tumor transforming gene (PTTG1, securin) is involved in cell-cycle control through inhibition of sister-chromatid separation. Elevated levels of PTTG1 were found to be associated with many different tumor types that might be involved in late stage tumor progression. However, the role of PTTG1 in early stage of tumorigenesis is unclear. Here we utilized the adenovirus expression system to deliver PTTG1 into normal human fibroblasts to evaluate the role of PTTG1 in tumorigenesis. Expressing PTTG1 in normal human fibroblasts inhibited cell proliferation. Several senescence-associated (SA) phenotypes including increased SA-β-galactosidase activities, decreased bromodeoxyuridine incorporation, and increased SA-heterochromatin foci formation were also observed in PTTG1-expressing cells, indicating that PTTG1 overexpression induced a senescent phenotype in normal cells. Significantly, the PTTG1-induced senescence is p53-dependent and telomerase-independent, which is distinctively different from that of replicative senescence. The mechanism of PTTG1-induced senescence was also analyzed. Consistent with its role in regulating sister-chromatid separation, overexpression of PTTG1 inhibited the activation of separase. Consequently, the numbers of cells with abnormal nuclei morphologies and chromosome separations were increased, which resulted in activation of the DNA damage response. Thus, we concluded that PTTG1 overexpression in normal human fibroblasts caused chromosome instability, which subsequently induced p53-dependent senescence through activation of DNA-damage response pathway.     

PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

Human securin, also known as human pituitary tumor-transforming gene 1 (pttg1), plays a key role in cell-cycle regulation. Two homologous genes, pttg2 and pttg3, have been identified although very little is known about their physiological function. In this study, we aimed at the characterization of these two pttg1 homologs. Real-time PCR analysis using specific probes demonstrated that Pttg2 is expressed at very low levels in various cell lines and tissues whereas Pttg3 was largely undetectable. We focused on the study of Pttg2 and found that, unlike PTTG1, PTTG2 lacks transactivation activity and does not bind to separase, making improbable a role in the control of sister chromatids separation. To further investigate the biological role of pttg2, we used short hairpin RNA inhibition of Pttg2 and found that cells with reduced Pttg2 levels assumed a rounded morphology compatible with a defect in cell adhesion and died by apoptosis in a p53- and p21-dependent manner. Using microarray technology, we generated a gene expression profile of Pttg2-depleted cells versus wild-type cells and found that knockdown of PTTG2 results in concomitant downregulation of E-cadherin and elevated vimentin levels, consistent with EMT induction. The observation of aberrant cellular behaviors in Pttg2-silenced cells reveals functions for pttg2 in cell adhesion and provides insights into a potential role in cell invasion.     

Separase sensor reveals dual roles for separase coordinating cohesin cleavage and cdk1 inhibition

Complete dissociation of sister chromatid cohesion and subsequent induction of poleward movement of disjoined sisters are two essential events underlying chromosome segregation; however, how cells coordinate these two processes is not well understood. Here, we developed a fluorescence-based sensor for the protease separase that mediates cohesin cleavage. We found that separase undergoes an abrupt activation shortly before anaphase onset in the vicinity of chromosomes. This activation profile of separase depends on the abilities of two of its binding proteins, securin and cyclin B1, to inhibit its protease activity and target it to chromosomes. Subsequent to its proteolytic activation, separase then binds to and inhibits a subset of cyclin B1-cdk1, which antagonizes cdk1-mediated phosphorylation on chromosomes and facilitates poleward movement of sisters in anaphase. Therefore, by consecutively acting as a protease and a cdk1 inhibitor, separase coordinates two key processes to achieve simultaneous and abrupt separation of sister chromatids.     

Fission Yeast MAP Kinase Is Required for the Increased Securin-Separase Interaction That Rescues Separase Mutants Under Stress

Sister chromatid separation requires two steps of proteolysis. Securin, the chaperon and inhibitor of separase, is destructed in anaphase after polyubiquitination, and resulting activated separase cleaves the cohesin subunit Scc1/Rad21. Fission yeast securin/Cut2 and separase/Cut1 that form the complex are essential for viability and a number of temperature-sensitive (ts) mutants have been isolated. We here report that the stresses such as 1.2 M sorbitol, 0.6 M KCl and 0.1 M CaCl2 in the medium suppress the ts phenotypes of all the cut1 mutants and two of the three cut2 mutants examined. This unexpected finding led us to study how the ts phenotypes of cut1 and cut2 mutants were rescued by the increased stresses. The stresses caused a temporal arrest in the cell number increase, and this arrest was dependent on Spc1/Sty1 but not Rad3 and Mad2. During the 2-3 hr arrested period that occurred prior to the re-start of division cycle, the level of securin dramatically increased, apparently accompanying the increased complex formation with mutant separase protein. Securin bound to separase was hyperphosphorylated. The stresses could not rescue the indestructible Cut2 and Rad21 mutants. We postulate that the stresses produce the hyperchaperonic form of Cut2 that can rescue separase mutations.     

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.     

Elucidation of novel budding yeast separase mutants

The mitotic separase cleaves Scc1 in cohesin to allow sister chromatids to separate from each other upon anaphase onset. Separase is also required for DNA damage repair. Here, we isolated and characterized 10 temperature-sensitive (ts) mutants of separase ESP1 in the budding yeast Saccharomyces cerevisiae. All mutants were defective in sister chromatid separation at the restricted temperature. Some esp1-ts mutants were hypersensitive to the microtubule poison benomyl and/or the DNA-damaging agent bleomycin. Overexpression of securin alleviated the growth defect in some esp1-ts mutants, whereas it rather exacerbated it in others. The Drosophila Pumilio homolog MPT5 was isolated as a high-dosage suppressor of esp1-ts cells. We discuss various features of separase based on these findings.     

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.     

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.