Positive feedback sharpens the anaphase switch

Abstract

At the onset of anaphase, sister-chromatid cohesion is dissolved abruptly and irreversibly, ensuring that all chromosome pairs disjoin almost simultaneously. The regulatory mechanisms that generate this switch-like behavior are unclear. Anaphase is initiated when a ubiquitin ligase, the Anaphase-Promoting Complex (APC), triggers the destruction of securin, thereby allowing the protease, separase, to disrupt sister-chromatid cohesion 1-4. Here we demonstrate that Cdk1-dependent phosphorylation of securin near its destruction-box motif inhibits securin ubiquitination by the APC. The phosphatase Cdc14 reverses securin phosphorylation, thereby increasing the rate of securin ubiquitination. Because separase is known to activate Cdc14 5 , 6, our results support the existence of a positive feedback loop that increases the abruptness of anaphase. Consistent with this model, we show that mutations that disrupt securin phosphoregulation decrease the synchrony of chromosome segregation. Our results also suggest that coupling securin degradation with changes in Cdk1 and Cdc14 activities helps coordinate the initiation of sister-chromatid separation with changes in spindle dynamics.Securin is known to integrate multiple signaling pathways to delay anaphase in response to perturbations such as DNA damage 7,8 . We hypothesized that securin might also receive regulatory inputs that make separase activation more switch-like. To address this possibility, we used mass spectrometry to analyze the phosphorylation state of securin from mitotic budding yeast cells (Fig. 1a) 9 . We identified six phosphorylation sites, including four of the five sites corresponding to the consensus sequence of the cyclin-dependent-kinase, Cdk1 (S/ T*-P) (see also Suppl. Fig. 1). Three Cdk1 sites were found in securin's C-terminal domain, which is known to interact with separase 10 ; mutations at these sites are known to modulate securin's ability to bind separase and import it into the nucleus 11 . The fourth Cdk1 phosphopeptide, near the N terminus of securin, has not been characterized.The N-terminal Cdk1 site is near the destruction-box of securin, a motif important for recognition of substrates by the APC 2-4, 12 . To assess whether phosphorylation of this site affects securin ubiquitination by the APC, we tested the ability of purified APC to ubiquitinate purified securin before and after phosphorylation in vitro by Cdk1. We found that if securin was phosphorylated by Cdk1, the rate of ubiquitination by APC Cdc20 (the APC-activator complex that controls anaphase onset) was reduced 10-fold (Fig. 1b, c) and the rate of ubiquitination by APC Cdh1 was reduced 5-fold (Suppl. Fig. 2).This regulatory mechanism may be conserved -Drosophila and human homologues of securin have S/T-P motifs near their destruction-boxes, and both were ubiquitinated less efficiently by the APC after phosphorylation by Cdk1 (Suppl. Fig. 3 Cdc14 is a phosphatase that removes phosphates from Cdk1 substrates during anaphase and late mitosis 4,[13][14][15][16] . We found t...

Cyclin B3 controls anaphase onset independent of spindle assembly checkpoint in meiotic oocytes

“…There are reports in buddying yeast that Cdk1 phosphorylates securin and protects it from being degraded by APC/C. [32][33][34] However, in human cells Cdk1 is less important in the metaphase-anaphase transition, and the main mechanism is that securin and cyclin B1 inhibit separase activity to protect cohesion so that chromosomes are not able to separate. 34,35 Considering others' findings and our results we conclude that securin is the main molecule that protects cohesion.…”

Multisite phosphoregulation of Cdc25 activity refines the mitotic entrance and exit switches

“…Cdc14 dephosphorylates Securin to target it for ubiquitylation and degradation. Degradation of Securin activates Separase, which also activates Cdc14 (13). Our laboratory and other groups found that Clp1, the Schizosaccharomyces pombe Cdc14 ortholog, dephosphorylates Cdc25 on Cdk1 phosphosites, and this dephosphorylation correlates with Cdc25 inactivation and degradation (17,18).…”

“…Mitotic exit and the spindle assembly checkpoint may also be modulated by a bistable switch (12)(13)(14). In Saccharomyces cerevisiae, Cdc14, a phosphatase that dephosphorylates Cdk1 substrates (15,16), adds abruptness to the metaphase-anaphase switch by interacting with Securin, a protein that protects sister chromatid separation until anaphase onset in an ultrasensitive positive feedback loop.…”

CDC23 regulates cancer cell phenotype and is overexpressed in papillary thyroid cancer

“…The phosphorylation of securin by Cdk1 is critical for its binding with separase and also reduces the ubiquitination of itself by APC (Holt et al 2008). In Homo sapiens, pituitary tumortransforming 1 (PTTG1) is a homolog of yeast securin protein for which the function is unknown.…”