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

Dominik Boos,Christian Kuffer,Rene Lenobel,Roman Körner,Olaf Stemmann

doi:10.1074/jbc.m706748200

Dominik Boos, Christian Kuffer + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m706748200

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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.

Highlights

A sequence homology to the cyclin B1-binding N terminus of S. cerevisiae Cdc6 defined the Cdc6-like domain (CLD) of human separase [12] and spurred the speculation that Cdc6 and separase might bind the regulatory subunit of Cdk1 in the same phosphorylation-dependent manner [4]
For the model to be true the following parallels to Cdc6-cyclin B1 have to be fulfilled: 1) Only phosphorylated CLD should mediate the interaction of separase with Cdk1
Phosphorylations within the Cdc6-like Domain of Separase Are Critical for Cdk1 Binding—Asking whether residues within the CLD might be phosphorylated in vivo, we performed mass spectrometric analysis of endogenous separase immunoprecipitated from mitotically arrested HelaS3 cells

Summary

Introduction

It has been previously shown that Cdk1 binds human separase only when the latter is phosphorylated on serine 1126 and contains an intact, so-called Cdc6-like domain (CLD) [12]. In vitro, arrested 293T cells and probed for association with Cks1/2 and the same CLD-EED mutation renders the binding of Cdk1 to separase.

Results

Conclusion