Structural basis for recognition of Emi2 by Polo-like kinase 1 and development of peptidomimetics blocking oocyte maturation and fertilization

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.

Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions

“…The overall binding mode for the phosphopeptide of HEF1 to Plk1 is quite similar to that reported previously ( i.e. the interactions with the β-sheet of Polo box 1 and the interactions around the phosphothreonine are almost the same as in the previous report), whereas the interactions seen for the terminal ends of the peptide seem to vary somewhat, depending on the sequence ( 25 , 26 ). Therefore, the structural analysis clearly suggests that the phosphothreonine at position 804 of HEF1 (pThr-804) plays an important role in specific interaction and recognition by Plk1 PBD.…”

“…3 A ) was expressed as a recombinant protein containing an N-terminal His 6 tag in the pET28a vector and a tobacco etch virus protease cleavage site (sequences ENLYFQS, where the amino acid residue between Q and S is cleaved) was engineered between the affinity tag and Plk1 PBD. Recombinant protein was expressed and purified following the procedure described earlier ( 25 ). Briefly, the protein was expressed in E. coli BL21 (DE3) CodonPlus RIL (Stratagene) at 18 °C; the expression was induced by 0.5 m m IPTG, and the cells were cultured for 16 h. The cells were harvested and lysed in 25 m m HEPES (pH 7.5), 300 m m NaCl, 5 m m β-mercaptoethanol, and 0.1 m m PMSF by sonication and centrifugation.…”

Maturation promoting factor destabilization mediates human chorionic gonadotropin induced meiotic resumption in rat oocytes

“…; Jia et al . ). Further, CSFs prevent degradation of cyclin B1 and maintains cell cycle arrest (Madgwick & Jones ; Kubiak et al .…”

“…; Jia et al . ). MAD2 is another CSF that inhibits APC/C activity by binding with cdc20 (Homer et al .…”

Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop

“…The EMBO Journal 40: e107516 | 2021 (Jia et al, 2015). Also, peptide-bound Plk1-PBD can form a dimer through a hydrophobic patch in PBD (Zhu et al, 2016).…”