Phosphorylation-dependent Binding of Cyclin B1 to a Cdc6-like Domain of Human Separase

Abstract

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.

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

“…4C). The above loss-offunction phenotypes do not contradict the observation that overexpression of ⌬CLD causes premature separation of sister chromatids in prometaphase-arrested cells (8,16). ⌬CLD is a hypermorph in that it can no longer be bound and inhibited by Cdk1-cyclin B1, but at the same time, it is a hypomorph because FIGURE 4.…”

“…Although necessary, these phosphorylations are not sufficient for inhibition of separase, which additionally requires Cdk1-cyclin B1 to stably associate, via its regulatory cyclin B1 subunit, with the phosphorylated CLD of separase (17). Within the Cdk1-cyclin B1-separase complex, Ser-1126 is probably not in direct contact with the kinase because this residue is dispensable for binding of Cdk1-cyclin B1 to separase fragments (16). However, Ser-1126 phosphorylation is absolutely required for Cdk1-cyclin B1 to associate with full-length separase.…”

“…This securin-independent inhibition of human separase requires that Ser-1126 and several residues within a Cdc6-like domain (CLD) 2 centered around position 1370 are phosphorylated by Cdk1-cyclin B1 (and possibly other mitotic kinases) (15,16). Although necessary, these phosphorylations are not sufficient for inhibition of separase, which additionally requires Cdk1-cyclin B1 to stably associate, via its regulatory cyclin B1 subunit, with the phosphorylated CLD of separase (17).…”

A conditional mutation in Arabidopsis thaliana separase induces chromosome non-disjunction, aberrant morphogenesis and cyclin B1;1 stability

“…In animals, one way that separase is regulated is through binding cyclin B1, as part of the CDK complex (Gorr et al, 2005;Holland and Taylor, 2006;Boos et al, 2008). This interaction has been viewed in terms of cyclin B negatively regulating separase.…”

The sister bonding of duplicated chromosomes

“…The biochemical mechanism of this inhibition remains to be worked out. The current model is that the CDK1/cyclin B complex catalyzes the phosphorylation, binds to separase with higher affinity, and inhibits separase activity [89, 90]. This was nicely demonstrated in vitro.…”