The Caspase-like Cell Cycle Protease Separase : Upstream Regulations and Downstream Functions

Separase universally triggers eukaryotic anaphases by cleavage of sister chromatid cohesion mediating cohesin. Until then, this essential protease is kept inactive by association with securin. Separase can alternatively be inhibited by association with Cdk1-cyclin B1 but the corresponding complex is scarce in early mitosis and cannot explain why vertebrate securin is dispensable. Protein phosphatase 2A (PP2A) also binds separase but the physiological role of this interaction remains enigmatic. Silencing of the spindle assembly checkpoint (SAC) in metaphase enables the ubiquitin ligase APC/C to mediate the proteasomal destruction of securin (and cyclin B1), thereby activating separase. Despite being structurally related to caspases, separase has not been previously linked to apoptosis. Instead, two studies suggested a role of yeast separase in DNA damage repair but left unanswered whether this non-canonical interphase function of separase is conserved in mammals. Studying upstream regulations and downstream functions of vertebrate separase, I discovered the following: 1) Securin associates co-translationally with separase and prevents its aggregation. This suggests that it might assist the proper folding of this giant protease and offers a first mechanistic explanation for the genetic evidence that securin is not only an inhibitor but also an activator of separase. 2) APC/C prefers phosphorylated securin over un-phosphorylated securin as a substrate. While free securin is phosphorylated in mitosis, separase-bound securin is kept in an unphosphorylated state by associated PP2A. This effecuates supernumerous securin to be degraded first and largely gone by the time separase-associated securin is targeted for proteolysis. Thereby, premature activation of separase is prevented and the metaphase-to-anaphase transition sharpened. 3) Once liberated, separase is subject to conformational change by the peptidyl-prolyl isomerase Pin1 and thereby rendered resistant against residual securin. At the same time, this limits separase's proteolytic half-life and allows cohesin to be reloaded onto telophase chromatin without being cleaved. 4) Pin1 catalyzed trans-to-cis isomerization of separase is essential for Cdk1-cyclin B1-dependent inhibition and explains why the kinase and securin bind separase in a mutually exclusive manner. 5) Formation of the Cdk1-cyclin B1-separase complex is counter-acted by phosphorylation of cyclin B1 in early mitosis. Dephosphorylation results in a second peak of Cdk1-cyclin B1-separase complex formation in late mitosis when most cyclin B1 has already been degraded. 6) Upon destruction of this last cyclin B1 in early G1 phase, separase is released from the late Cdk1-cyclin B1-separase complex and triggers centriole disengagement, thereby licensing later centrosome duplication. 7) Human shugoshin 2 (Sgo2), a protector of meiotic cohesin with hitherto unknown function in somatic cells, represents a crucial second branch of anaphase regulation. It is enabled by SAC-activated Mad2 to bind and…