Fig. 3.

Emi2's ₁₉₂RSST motif is critical for CaMKII-induced Plx1 association and is required for Emi2 destruction and CSF release. (A) A schematic of the primary structure of Xenopus Emi2 with candidate CaMKII target/Polo Box-binding sites and equivalent sequences among vertebrate Emi2 orthologs depicted. (B) Both the ₁₉₂RSST and ₃₃₃RLST motifs of Emi2 can bind the PBD in vitro after phosphorylation by CaMKII. Indicated variants of the Emi2 N terminus were phosphorylated by CaMKII and processed as in Fig. 2B to assay Polo Box-binding. (C) The ₁₉₂RSST motif of Emi2 is chiefly responsible for Ca²⁺-induced Plx1 association in CSF extract. Indicated variants of the Emi2 N terminus were incubated in CSF extract for 5 min with or without Ca²⁺ addition. Recombinant protein was purified on amylose resin, electrophoresed, and Coomassie stained. Associated Plx1 was detected by immunoblotting. (D) CaMKII-induced Emi2 destruction requires the ₁₉₂RSST motif of Emi2. Radiolabeled IVT Emi2 was preincubated in CSF extract for 5 min before addition of IVT-active CaMKII fragment or mock IVT. Emi2 stability was monitored by autoradiography. (E) The ₁₉₂RSST motif in Emi2 is required for Ca²⁺-induced Emi2 destruction in CSF extract. Radiolabeled IVT Emi2 variants were incubated in CSF extract with Ca²⁺ addition, and Emi2 stability and gel mobility were monitored by autoradiography. (F) Expression of the Emi2 192–5* mutant in intact oocytes prohibits Ca²⁺-induced CSF release and meiotic exit. Oocytes were injected with in vitro-transcribed myc-Emi2 mRNA, matured with progesterone, and treated with ionophore A23187 at 3 h post-germinal vesicle breakdown to induce Ca²⁺ release and meiotic exit. Anti-Emi2 or anti-myc antibodies were used to detect endogenous or expressed Emi2, respectively, and cyclin B levels and H1 kinase activity were monitored at 5, 15, and 30 min after ionophore treatment.