Abstract
In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr152 and Thr176) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
Highlights
Arrests further progression of the cell cycle until fertilization
It was reported that Thr[195], which is located in 192RSST195 motifs of Xenopus, is phosphorylated by CaMKII and recognized by Plk1-PBD10
We examined the structure of the Plk1-Polo-box domain (PBD)·Emi2146–177 complex containing various complexes of Plk1-PBD with cdc[25] originated peptide or chemical 4j(Fig. S1), and we found that Emi2′ s peptide-binding characteristics in relation to Plk1-PBD are similar to those previously reported for phosphopeptides[24]
Summary
The high protein levels of MPF are decreased via degradation of cyclin B by ubiquitin-mediated proteolysis, which is promoted by ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). “Cytostatic factor” (CSF) is a collective name of biochemical activities responsible for the process that prevents degradation of cyclin B; CSF serves to maintain the arrest of the cell cycle. One of CSFs, Emi[2] ( known as F-box only protein 43) inhibits APC/C activity by binding to APC/C-cdc[20]; Emi[2] blocks the ubiquitin-mediated proteolysis of MPF8–10. The activated APC/C can initiate degradation of cyclin B and downregulation of MPF; cell cycle progression can be resumed and meiosis II can be completed, as illustrated as Fig. 1A. We tested whether each peptidomimetic can act as an antagonist of oocyte maturation or fertilization
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