Positive and negative regulation of vertebrate separase by Cdk1-cyclin B1 may explain why securin is dispensable

Abstract

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.

Keywords: Cdk1-Cyclin B1; Centriole Disengagement; Chromosomes; Cyclin-dependent Kinase (CDK); Cysteine Protease; Mitosis; Phosphorylation; Securin; Separase; Sister Chromatid Separation.

FIGURE 1.

Studying the positive effect of securin on separase. A, securin does not influence the mRNA level of separase. The relative amounts of securin and separase mRNAs in securin^−/− and parental HCT116 cells were quantified on an electrochemical biochip. B, endogenous separase (Sep.) is prone to precipitation in the absence of securin (Sec.). Lysates (input) of mitotically arrested securin^−/− (SEC^−/−) and parental HCT116 cells were centrifuged to assess the solubility of separase by Western analysis of the resulting supernatant (Supernat.) and pellet fractions. α-Tubulin (αTub.) and Ser-10-phosphorylated histone H3 (H3 pS10) served as loading controls. C, association with securin prevents precipitation of transiently overexpressed separase. Following its overexpression in HEK293T cells with or without co-overexpression of securin, the solubility of separase was determined by centrifugation. D, co-translational association of securin with nascent separase. mRNAs lacking or containing a stop codon and coding for amino acids 1–425, 1–627, 1–873, or 1–1105 of human separase or amino acids 1–942 of human exportin-1 (Crm1) were translated in vitro in the presence of [³⁵S]methionine and ³⁵S-labeled securin. Input (I) samples were fractionated by ultracentrifugation into supernatant (S) and RNC-containing pellet (P) fractions prior to SDS-PAGE and autoradiography. Crm1 was chosen as a negative control because, similar to the N-terminal half of separase, it contains a superhelical structure.

FIGURE 2.

Mitosis-specific phosphorylation of Ser-1126 decreases the solubility of separase. A, aggravated aggregation tendency of separase (Sep.) in mitosis. Synchronized interphase (Int., Interph.) or prometaphase (Mit.) transgenic HEK293 cells induced to express WT separase were stained with propidium iodide and analyzed by flow cytometry or lysed and subjected to ultracentrifugation and Western analysis. Signals for pelleted (Pel.) versus soluble (Sup.) separase were quantified by densitometry and are depicted as quotients (gray bars). αTub., α-tubulin; H3 pS10, Ser-10-phosphorylated histone H3; Supernat., supernatant; AU, arbitrary units. B, preventing phosphorylation of Ser-1126 stabilizes securin-less separase. HEK293T cells transiently overexpressing securin (Sec.) and/or Myc-tagged WT separase or S1126A were arrested in prometaphase before the solubility of separase was determined by pelleting assay and quantified as described for A.

FIGURE 3.

Ser-1126 phosphorylation and binding of Cdk1-cyclin B1 have opposing effects on separase solubility. A, mutational inactivation of its cyclin B1-binding site decreases the solubility of separase (Sep.). Transgenic HEK293 cells transfected with securin (siSECURIN) or GL2 (siGL2) siRNA and expressing WT separase or ΔCLD were arrested in prometaphase before the solubility of separase was determined by pelleting assay and quantified as described for Fig. 2A. I, input; S, supernatant; P, pellet; αTub., α-tubulin; Sec., securin; H3 pS10, Ser-10-phosphorylated histone H3; AU, arbitrary units; Pel., pelleted separase; Sup., soluble separase. B, precipitation of separase in mitosis is alleviated by the S1126A mutation but aggravated by CLD deletion. Lysates of transgenic, prometaphase-arrested HEK293 cells expressing the indicated separase variants were immunodepleted of securin before the solubility of securin-free separase was determined by quantitative pelleting assay as described for Fig. 2A. IP, immunoprecipitation.

FIGURE 4.

Positive and negative effects of Cdk1-cyclin B1 define the minimal requirements of separase regulation in mitosis. A, WT separase and mutants S1126A and ΔCLD exhibit some, high, or no cohesin cleavage activity upon isolation from prometaphase-arrested cells, respectively. Taxol-treated HEK293T cells transiently overexpressing securin (Sec.) and/or Myc₆-TEV₂-tagged WT separase (Sep.), S1126A, or ΔCLD were lysed and subjected to anti-Myc immunoprecipitation without prior centrifugation to avoid the removal of insoluble separase fractions. Where indicated, separase-associated Cdk1-cyclin B1 (Cyc.B1) was washed away with a high salt buffer. TEV protease-eluted separases were analyzed by immunoblotting and ³⁵S-labeled Scc1 cleavage assay/autoradiography. B and C, in ΔCLD-expressing cells, the short half-life of proteolytic activity correlates with reduced centriole disengagement. Transgenic HEK293 cells transfected with separase or GL2 siRNA were thymidine-arrested and, where indicated, induced to express WT separase or ΔCLD. Cells were harvested 16 h after release into fresh medium and analyzed by immunoblotting, propidium iodide flow cytometry (B), and immunofluorescence microscopy of purified centrosomes (C). Fixed centrosomes were co-stained for centrin-2 and C-Nap1. Depending on a centrin-2 to C-Nap1 signal ratio of 2:1 or 2:2, centrioles were classified as engaged or disengaged, respectively. In three independent experiments (dots), the engagement status of at least 200 centrosomes per cell line and condition was analyzed and blotted as mean values (bars). Scale bar = 1 μm. αTub., α-tubulin; Dox., doxycycline.

FIGURE 5.

Model of how phosphorylation and mutually exclusive association with securin or Cdk1-cyclin B1 determine the fate of separase across the cell cycle. The minimal module of separase control (shown in boldface) relies only on the positive and negative effects of Cdk1-cyclin B1 (Cyc.B1) on separase to maintain faithful chromosome segregation in the absence of securin. pSeparase, phosphorylated separase; APC/C, anaphase-promoting complex/cyclosome.