PP 2A delays APC /C‐dependent degradation of separase‐associated but not free securin

Abstract

The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase-associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase-associated securin is destabilized by introduction of phosphorylation-mimetic aspartates or extinction of separase-associated PP2A activity. G2-or prometaphase-arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate-mutant securin. Thus, PP2A-dependent stabilization of separase-associated securin prevents precocious activation of separase during checkpointmediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase.

Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs

“…In support, inhibition of phosphatases with okadaic acid prevented mitotic exit and dephosphorylation, indicating that the PP1/2A/ 4/6 families of phosphatases are responsible for dephosphorylating substrates during EME. This is supported by previous studies which have shown that both PP1 (23,59,60) and PP2A (45,57,61) are responsible for dephosphorylating critical substrates during EME in mammals. However, this still does not answer the question of how these phosphatases specifically dephosphorylate only a discrete subset of phosphorylation sites during EME.…”

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

“…Cells were grown for at least 24 h before synchronization procedures were applied. Analysis of DNA content by flow cytometry was performed as described (9).…”

“…Phosphorylation turns vertebrate securin into a better anaphase-promoting complex/cyclosome substrate. This is counteracted by separase, which mediates the PP2A (protein phosphatase 2A)-dependent dephosphorylation and hence stabilization of associated securin (9). Although this provides an explanation for the positive effect of separase on securin, data are lacking that address how vertebrate securin positively affects separase.…”

Cyclin B3 promotes APC/C activation and anaphase I onset in oocyte meiosis

“…It is noteworthy in this context that free securin is degraded earlier than separase-bound securin, which is stabilized by maintenance in a non-phosphorylated state through association with phosphatase PP2A [50]. Securin phosphorylation enhances its affinity for the APC/C [50,51]. Regardless of the source of the distinction, our data clearly demonstrate that APC/C becomes partially active on time, but not sufficiently active to robustly provoke degradation of all of its intended targets and to trigger anaphase I. .…”

“…It is also possible that the fluorescent protein tags interfere slightly with this binding. It is noteworthy in this context that free securin is degraded earlier than separase-bound securin, which is stabilized by maintenance in a non-phosphorylated state through association with phosphatase PP2A [50]. Securin phosphorylation enhances its affinity for the APC/C [50,51].…”