Inhibitory Phosphorylation of Separase Is Essential for Genome Stability and Viability of Murine Embryonic Germ Cells

Abstract

Activity of separase, a cysteine protease that cleaves sister chromatid cohesin at the onset of anaphase, is tightly regulated to ensure faithful chromosome segregation and genome stability. Two mechanisms negatively regulate separase: inhibition by securin and phosphorylation on serine 1121. To gauge the physiological significance of the inhibitory phosphorylation, we created a mouse strain in which Ser1121 was mutated to Ala (S1121A). Here we report that this S1121A point mutation causes infertility in mice. We show that germ cells in the mutants are depleted during development. We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death. Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

Pttg1/securin is required for the branching morphogenesis of the mammary gland and suppresses mammary tumorigenesis

“…In vertebrates, there are two mechanisms to inhibit separase, phosphorylation and binding by securin (16). We have shown previously that these two mechanisms are redundant in most somatic cells (17), but, in mice, postmigration germ cells rely entirely on phosphorylation to inhibit separase (18). Mammalian securin, PTTG1, has been associated with cancer before it is known as securin.…”

“…Most likely, this is because in vertebrates separase is also inhibited by phosphorylation (16). Only when both inhibitory mechanisms are removed are defects in mitotic separation of sister chromatids seen (17,18). Nonetheless, the function of securin as the inhibitor of separase fueled the speculation that the overexpression of PTTG1 in tumors might be related to chromosome instability.…”

DNA-dependent cohesin cleavage by separase

“…The inhibitory phosphorylation of separase becomes essential during cell division of early embryonic cells and embryonic germ cells when securin levels are low. 21,22 Mutant mice lacking securin and expressing a non-phosphorylatable separase die in embryonic stage. 22 Interestingly, however, mouse embryonic stem cells lacking both these separase regulations can still progress through mitosis in a timely fashion with correct chromosome segregation.…”

“…21,22 Mutant mice lacking securin and expressing a non-phosphorylatable separase die in embryonic stage. 22 Interestingly, however, mouse embryonic stem cells lacking both these separase regulations can still progress through mitosis in a timely fashion with correct chromosome segregation. 23 The mutant cells separate chromosomes prematurely only when challenged with a microtubule poison.…”

The Biochemistry of Mitosis

“…This is because both cyclin B-Cdk and securin inhibit the separase protease (Stemmann et al 2001;Holland and Taylor 2006;Huang et al 2008) that, once active, cleaves the Scc1 subunit of the cohesin complex, which holds the two sister chromatids together (see Hirano 2015). In animal cells, most of the cohesin complex on chromosome arms is removed by the Plk1 kinase (Hauf et al 2005), but cohesin complexes remain at the centromere protected by the Shugoshin protein, which recruits an antagonistic PP2A phosphatase (Kitajima et al 2006;Riedel et al 2006;Tang et al 2006).…”