Schwesterchromatidtrennung in Vertebraten: Charakterisierung von Separase und seinen Substraten
Final Report Abstract
The final catalytic step in separating sister chromatids during cell divisions is carried out by separase. This giant protease cleaves the Scc1 subunit of the cohesin complex, which connects the replicated DNA strands. Hyper- or hypoactivity of separase results in premature chromatid separation (PCS) or chromatid nondisjunction, respectively. Separase functions not only at chromosomes but also at centrosomes: By triggering centriole disengagement it lisences later centrosome duplication. Given these critical functions, separase must be tightly regulated to ensure stable inheritance of genomic information and to prevent aneuploidies resulting in cell death or malignant transformation. Prior to anaphase, separase is held inactive by association with securin. Both proteins are co-regulated and positively influence each other by as-yet unknown mechanisms. Securin is frequently overexpressed in tumors but, surprisingly, its loss leaves mice and cultured human cells largely unaffected. Previously, we have identified the essential kinase MPF (mitosis promoting factor = Cdk1-cyclin B1) as a crucial securin-independent inhibitor of separase. Within the separase-MPF complex both enzymes, the protease and the kinase are inhibited. While MPF-dependent inhibition of separase becomes essential during early mitosis in the absence of securin, separase-dependent inhibition of residual MPF is important for late mitotic/meiotic events and cytokinesis. Here, we have: • used biochemical experiments to reveal the sequential, multiple steps of separase-MPF complex formation and to identify an evolutionary conserved cyclin B1 binding site on separase, • unraveled unanticipated parallels between the chromosome and the centrosome cycles by demonstrating, amongst others, that separase cleaves centrosomal cohesin to bring about centriole disengagement, • discovered that a key cellular function of the newly discovered separase-PP2A interaction is to stabilize by de-phosphorylation separase associated- over free securin, and • established the efficient generation of doubly transgenic, stable cell lines by two consecutive rounds of site-specific recombination using Flp and Cre.
Publications
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(2007) Protein phosphatase 2A and separase form a complex regulated by separase autocleavage. J Biol Chem, 282, 24623-24632
Holland, A.J., Böttger, F., Stemmann, O. & Taylor, S.S.
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(2008) Phosphorylation-dependent binding of cyclin B1 to a Cdc6-like domain of human separase. J Biol Chem, 283, 816-823
Boos, D., Kuffer, C., Lenobel, R., Körner, R. & Stemmann, O.
