- Eukaryotic chromosome segregation: New aspects of separase regulation by securin, Cdk1, PP2A and auto-cleavage (2011)
- The universal triggering event of eukaryotic chromosome segregation is the proteolytic cleavage of chromosomal cohesin by separase. The activity of this essential but potentially also very dangerous protease must be tightly controlled. Prior to the onset of anaphase separase is kept inactive by association with either securin or cyclin-dependent kinase 1 (Cdk1) in conjunction with cyclin B1. Only when all chromosomes interact properly with the mitotic spindle apparatus does the anaphase promoting complex or cyclosome (APC/C), a multisubunit E3 ligase, mediate the ubiquitylation of securin and cyclin B1. Their subsequent proteasomal degradation then releases active separase. Murine embryonic stem cells, which lack securin and express a Cdk1-resistant phosphorylation site mutant separase are viable. Thus, additional regulations of sister chromatid separation by separase must exist. It was reported that human separase cleaves not only cohesin but also itself and, furthermore, that it interacts with protein phosphatase 2A (PP2A). However, the functions of separase's auto-cleavage and PP2A-interaction remain enigmatic. Moreover, securin was reported to also interact with PP2A but, strangely, with a different isoform of the phosphatase. Thus, the question needs clarification of whether separase or securin or both interact with which isoform of PP2A. In this study, further insights into the relationship between separase auto-cleavage and PP2A binding are presented. Phosphorylation of a serine residue in close proximity to the major cleavage site of separase was found to stimulate auto-cleavage of separase. Interestingly, a quantitative mass-spectrometric approach (SILAC) identified this serine residue as a substrate of separase-bound PP2A. Furthermore, a point mutation within separase was identified, which totally abolishes PP2A binding and which maps to the immediate vicinity of the auto-cleavage sites. Thus, PP2A prevents the auto-cleavage of separase both catalytically and sterically. It could further be shown that non-cleavable separase exhibits increased association with PP2A and that forced cleavage of separase displaces PP2A. Taken together, these results demonstrate that auto-cleavage and PP2A binding constitute two antagonistic mechanisms of separase regulation. Evidence is provided that the interaction of PP2A with securin is indirect and bridged by separase, and that it is the B56- and not the B55-isoform of PP2A which associates with the separase-securin complex. Moreover, free securin is shown to be degraded in early mitosis in a phosphorylation- and APC/C-dependent manner, while separase-associated securin is kept dephosphorylated and, thus, protected by PP2A. Securin levels are frequently increased in tumors. In normal cells, the early removal of excessive securin might later ensure swift separase activation and anaphase onset, thereby contributing to faithful chromosome segregation.