The role of cyclin B3's phosphate-binding pocket in female meiosis I of Xenopus laevis

Abstract

Cyclins are the main driver of the cell cycle and are indispensable for correct cell division. Cyclins prevent aneuploidy, which is a hallmark of cancer in the case of mitosis and a risk factor for embryonic mortality or the development of trisomy disorders in the case of meiosis. The different cyclin classes are involved in different cell cycle phases with B-type cyclins mainly responsible for mitosis and meiosis. Cyclin B3 (cycB3) is an understudied member of B-type cyclins with sequence similarity to other B-type cyclins as well as to A-type cyclins. This leads to a unique mix type cyclin with the function of preventing premature metaphase I arrest in meiosis. This metaphase I arrest is circumvented by phosphorylation of the anaphase-promoting complex/cyclosome (APC/C) inhibitor XErp1 by cycB3 in complex with the cyclin-dependent kinase 1 (Cdk1) which leads to further polo-like kinase 1 (Plx1) phosphorylation and results in XErp1 degradation. For the first time, a cycB3-specific phosphate-binding pocket mediating the main function of cycB3 in meiosis is described in this thesis. By comparison of a phosphate-pocket deficient cycB3 version, cycB3TEM, with wild-type cycB3, cycB3WT, the role of the phosphate-binding pocket in XErp1 degradation was investigated. The phosphate-binding pocket in cycB3 directly interacted with threonine 97 residue in XErp1, which was pre-phosphorylated by cycB3/Cdk1. The binding of cycB3/Cdk1 to XErp1 mediated by the phosphate-binding pocket then led to multisite phosphorylation of XErp1 by cycB3/Cdk1 and consequently to binding of Plx1. The number of phosphorylation sites, sufficient for resembling wild-type XErp1 degradation pattern, could be narrowed down to four phosphorylation sites in the M-cluster and several phosphorylation sites in the N-cluster. Therefore, the order of events occurring on XErp1 in metaphase I—consisting of preceding cycB3/Cdk1 phosphorylation of T97, then multisite phosphorylation mediated by phosphate-binding pocket dependent cycB3 binding, followed by Plx1 phosphorylation—was characterised. Furthermore, the comparison of different B-type cyclins revealed a difference in substrate binding which might depend on the phosphate-binding pocket and further structural difference of cycB3 and cycB1. Finally, further possible phosphate-binding pocket dependent substrates of cycB3/Cdk1 were identified. Thus, this thesis contributes to a better understanding of cycB3 function in meiosis and broadens our knowledge about cyclin-mediated Cdk phosphorylation by identifying a phosphate-binding pocket in cycB3.