CaMKII and polo-like kinase 1 sequentially phosphorylate the cytostatic factor Emi2/XErp1 to trigger its destruction and meiotic exit

Abstract

In vertebrate meiosis, unfertilized eggs are arrested in metaphase II by cytostatic factor (CSF), which is required to maintain mitotic cyclin-dependent kinase activity. Fertilization triggers a transient increase in cytosolic free Ca(2+), which leads to CSF inactivation and ubiquitin-dependent cyclin destruction through the anaphase promoting complex or cyclosome (APC/C). The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the Polo-like kinase Plx1 are essential factors for Ca(2+)-induced meiotic exit, but the critical targets of these kinases were unknown. The APC/C inhibitor Emi2 or XErp1 has recently been characterized as a pivotal CSF component, required to maintain metaphase II arrest and rapidly destroyed in response to Ca(2+) signaling through phosphorylation by Plx1 and ubiquitination by the SCF(betaTrCP) complex. An important question is how the increase in free Ca(2+) targets Plx1 activity toward Emi2. Here, we demonstrate that CaMKII is required for Ca(2+)-induced Emi2 destruction, and that CaMKII functions as a "priming kinase," directly phosphorylating Emi2 at a specific motif to induce a strong interaction with the Polo Box domain of Plx1. We show that the strict requirement for CaMKII to phosphorylate Emi2 is a specific feature of CSF arrest, and we also use phosphatase inhibitors to demonstrate an additional mode of Emi2 inactivation independent of its destruction. We firmly establish the CSF component Emi2 as the first-known critical and direct target of CaMKII in CSF release, providing a detailed molecular mechanism explaining how CaMKII and Plx1 coordinately direct APC/C activation and meiotic exit upon fertilization.

Fig. 1.

Destruction of Emi2 in CSF extract requires CaMKII. (A) Ca²⁺-induced destruction of Emi2 during CSF release requires CaMKII activation. Radiolabeled IVT Emi2 was incubated in CSF extract with or without Ca²⁺ addition and with or without 281–309 peptide. Activation of CaMKII and destruction of cyclin B and Emi2 were monitored by immunoblot or autoradiography. (B) Addition of active CaMKII to CSF extract is sufficient to drive destruction of Emi2 and cyclin B2 without Ca²⁺ addition. A constitutively active fragment of CaMKII or mock IVT was added to CSF extract. Destruction of Emi2 and cyclin B2 was monitored by immunoblot.

Fig. 2.

CaMKII directly phosphorylates Emi2 as a priming kinase to induce association of Plx1 with Emi2. (A) Ca²⁺ addition to CSF extract induces Plx1/Emi2 association. An MBP fusion of the Emi2 N terminus (or MBP alone) was incubated in CSF extract for five min with or without Ca²⁺ addition and with or without 281–309 peptide. Recombinant protein was purified on amylose resin, and associated Plx1 was detected by immunoblotting. (B) Emi2 binds the PBD of Plk1 after phosphorylation by CaMKII. Indicated MBP proteins were phosphorylated in vitro with CaMKII, Cdc2, or mock treatment, incubated with a GST fusion of the Plk1 PBD, and purified on amylose resin. Captured proteins were detected by Coomassie stain.

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.

Fig. 4.

Multiple activities contribute to Emi2 stability and function in CSF extract. (A) Emi2 is destroyed in Δ90 mitotic extract in a CaMKII-independent manner, and Ca²⁺ addition stimulates its rate of destruction. Radiolabeled IVT Emi2 variants were incubated in Δ90 extract with or without Ca²⁺ addition and with or without 281–309 peptide. Emi2 stability and gel mobility were monitored by autoradiography, and CaMKII activation was detected by immunoblotting. The phospho-CaMKII blot is representative of all ±Ca²⁺ pairs except those involving the 281–309 peptide, for which no signal for phospho-CaMKII was detected. (B) Phosphatase inhibition in CSF extract causes cyclin B destruction independent of CaMKII activation and Emi2 destruction. CSF extracts were stimulated with either Ca²⁺ or okadaic acid with or without 281–309 peptide. The stability and gel mobility of radiolabeled IVT Emi2 were monitored by autoradiography. Destruction of cyclin B and activation of CaMKII were monitored by immunoblotting. (C) Emi2 destruction can be induced in okadaic acid-treated CSF extracts, which requires CaMKII and the ₁₉₂RSST and ₃₂DSGYSDS motifs of Emi2. CSF extracts previously stimulated to destroy cyclin by phosphatase inhibition with or without 281–309 peptide were further stimulated by Ca²⁺ addition. Stability and gel mobility of radiolabeled IVT Emi2 variants were monitored by autoradiography.