Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A - PubMed (original) (raw)

Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A

Judy Qiju Wu et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Mos requires Emi2 to induce M phase arrest. (A) Cycling extracts were incubated at room temperature for 45 min and were then supplemented with MBP or MBP-Mos. Aliquots were withdrawn at indicated times and immunoblotted for phosphoMAPK (pMAPK), cyclin B2, cyclin A, and phosphoCdc2 (pCdc2). (B) Two sets of cycling extracts in the presence of control or Emi2 antisense morpholinos were incubated at room temperature for 45 min. Mos protein was added to one set of the extracts. Samples were taken at indicated times. Samples taken from extracts without Mos were used to immunoblot for Emi2, and samples taken from extracts with Mos were used to immunoblot for pMAPK and cyclin B2. The asterisk marks a nonspecific band. (C) Cycling extracts were incubated at room temperature in the presence of Emi2 antisense morpholinos. After 45 min, Mos protein, Emi2 protein, or both were added. Samples were taken at indicated times, and pMAPK and cyclin B2 were examined by immunoblotting.

Fig. 2.

Mos regulates Emi2 T545/551 dephosphorylation. (A) CSF extracts were incubated at room temperature in the presence of DMSO or 200 μM U0126. Samples were taken at the indicated times and were immunoblotted for pMAPK and Emi2. Arrows indicate Emi2 mobility shift. (B) CSF extracts were incubated at room temperature in the presence of DMSO or 250 μM U0126. At the indicated times, samples were withdrawn to examine pMAPK and cyclin B2. Samples were also taken to examine endogenous Emi2 after λ-phosphatase treatment and to measure Cdc2/cyclin B kinase activity. (C) (Upper) CSF extracts were supplemented with 40 nM nondegradable cyclin B1. After 90 min, IVT 35S-labeled Emi2 was added in the presence or absence of Rsk inhibitor, SL0101. After 30 min of incubation, Cdc27 was immunoprecipitated and washed, and bound Emi2 was examined by SDS/PAGE and autoradiography. (Lower) The amount of 35S-labeled Emi2 bound to Cdc27 was quantified, normalized, and plotted. Error bars represent the standard deviation of three replicates. (D) Cycling extracts were incubated at room temperature in the presence of MBP, MBP-Emi2 (489–651) WT, or MBP-Emi2 (489–651) T545/551A proteins. Samples were taken at indicated times, and levels of cyclins A and B2 were examined by immunoblotting. (E) (Left) GST-Emi2 (326–651) bound to glutathione Sepharose was phosphorylated in vitro with Cdc2/cyclin B and [γ-32P]ATP and then incubated in interphase extracts pretreated with MBP or MBP-Mos. At indicated times, beads were washed, and the remaining phospho-Emi2 was examined by SDS/PAGE and autoradiography. (Right) The amount of 32P-labeled Emi2 remaining was quantified, normalized, and plotted. Each column represents the average of three replicates, and error bars represent the standard deviation.

Fig. 3.

A group of Cdc2 sites controlling slow Emi2 degradation are dephosphorylated in response to Mos/MAPK signaling. (A) A series of Emi2 deletion mutants was made to examine the stability of radiolabeled Emi2 fragments in CSF extracts. Constructs listed as stable in CSF extracts were as stable as full-length Emi2. The 1–321 and 1–279 proteins were unstable (

SI Fig. 6_A_

). The sequence of Xenopus Emi2 (amino acids 322–350) was aligned with four other species, and the conserved residues are highlighted. (B) Indicated radiolabeled IVT Emi2 proteins were added to CSF extracts and incubated at room temperature. Samples were taken at indicated times and examined by SDS/PAGE and autoradiography. (C) Indicated Emi2 proteins were processed as in B. (D) Indicated Emi2 proteins were processed as in B. (E) Radiolabeled IVT Emi2 (WT, 4AP, or ND) proteins were added into CSF extract pretreated for 30 min at room temperature with DMSO or 250 μM U0126. Samples were taken at indicated times after IVT protein addition, and 35S-labeled Emi2 was detected by SDS/PAGE and autoradiography. (F) GST-Emi2 (164–651, T545/551A) protein was processed as in Fig. 2_E_ except that each column represents the average of four replicates.

Fig. 4.

The Mos/MAPK/Rsk pathway promotes interaction of Emi2 with PP2A. (A) CSF extracts were preincubated for 5 min with 0.5 mg/ml GST or GST-Emi2 (amino acids 319–375, SD), after which IVT 35S-labeled Emi2 was added to the extract. Samples were taken at indicated times to detect cyclin B2 and added GST by immunoblotting, Cdc2 kinase activity, and full-length Emi2 tracer by autoradiography. In addition, samples were taken to examine Hoechst-stained nuclei supplemented into the extract. (Scale bars: 25 μm.) (B) MBP or MBP-Emi2N (amino acids 1–350) prebound to amylose resin was added to CSF extracts in the presence of DMSO or 250 μM U0126. After 30 min of incubation at room temperature, the beads were washed, and bound PP2A was examined by immunoblotting. (C) Indicated GST-SD proteins prebound to glutathione Sepharose were incubated in interphase extracts pretreated with MBP or Mos protein. After incubation, beads were retrieved and washed, and bound PP2A was examined by immunoblotting. (D) Indicated GST-SD prebound to glutathione Sepharose was phosphorylated in vitro by using recombinant Rsk and [γ-32P]ATP. After washing, proteins were separated by SDS/PAGE. Proteins were Coomassie blue stained, and incorporated 32P was detected by autoradiography. (E) Indicated GST-SD proteins were incubated in CSF extracts and handled as in B. (F) Indicated radiolabeled IVT Emi2 was added to CSF extracts and incubated at room temperature. Samples were taken at indicated times, and levels of 35S-labeled Emi2 were examined by SDS/PAGE and autoradiography. (G) Indicated radiolabeled IVT Emi2 were mixed into CSF extracts. After 2 h of incubation at 4°C, Cdc27 was immunoprecipitated and washed, and bound IVT Emi2 was examined by SDS/PAGE and autoradiography. (H) Mos-activated Rsk phosphorylates Emi2 at S335 and T336. This phosphorylation promotes Emi2–PP2A association, enhancing Emi2 dephosphorylation at both N- and C-terminal Cdc2 phosphorylation sites. Dephosphorylation of the N-terminal sites promotes Emi2 stability. Dephosphorylation of the C-terminal sites enhances APC-inhibitory binding of Emi2.

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Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A - PubMed (original) (raw)