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Science 338 (6106): 520-524

Copyright © 2012 by the American Association for the Advancement of Science

The APC/C Inhibitor XErp1/Emi2 Is Essential for Xenopus Early Embryonic Divisions

Thomas Tischer*, Eva Hörmanseder*, and Thomas U. Mayer{dagger}

Department of Biology and Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstr. 10, 78457 Konstanz, Germany.


Figure 1
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  		Fig. 1. XErp1 is essential for early embryonic divisions. (A) Immunoblot analyses of calf intestinal phosphatase (CIP)–treated samples harvested at the indicated time points. (B and C) One-cell embryos were injected with XErp1-MO or control MO and H2O or mRNA encoding WT, Fbox– mutant, or ZBR– mutant XErp1; 24-hpf phenotypes were quantified (B) or images were taken at the indicated times (C). (D) Embryos used in (B) were lysed at 6 hpf and immunoblotted. Asterisk marks nonspecific band. Cyc, cyclin.

 

Figure 2
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  		Fig. 2. XErp1 is required for timely destruction of APC/C substrates. (A) One-cell embryos were injected with control MO (Ctrl) or XErp1-MO, lysed at the indicated time points, and immunoblotted. (B) Embryo extract depleted of XErp1 or control immunoglobulin G extract was supplemented with 35S-labeled securin and at the indicated time points, samples were analyzed by immunoblot and autoradiography analysis. (C) XErp1-depleted extract was supplemented with buffer or IVT myc-XErp1WT and samples were analyzed as in (B). (D) Extract from (C) was treated with CIP and immunoblotted for XErp1. Asterisk marks nonspecific band. (E) At 4 hpf, embryos were lysed, and XErp1 and control immunoprecipitates were immunoblotted for XErp1 and Cdc27. (F) Non–CIP-treated embryo lysates were immunoblotted. Gwl, greatwall kinase.

 

Figure 3
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  		Fig. 3. XErp1 is regulated by phosphorylation. (A and B) One-cell embryos were injected with XErp1-MO and H2O or mRNA encoding WT, Cdk1 phosphomutant ST5 -> 6A (6A), or Cdk1 phosphomutant XErp1 defective in APC/C-inhibition (6A,ZBR–). At 6 hpf, images were taken (A) and phenotypes quantified (B). (C) Embryo extract was supplemented with nondegradable cyclin B (CycB{Delta}90) and analyzed by immunoblot. Time course started with CycB{Delta}90 addition. (D) Cdc27 was immunoprecipitated from extracts treated as in (C) and supplemented with MG-262. Samples were CIP-treated before immunoblotting for Cdc27 and XErp1. (E) Embryo extract was supplemented with 1 μM OA or dimethyl sulfoxide (DMSO) as solvent control, and samples were CIP-treated as indicated and analyzed as in (C). (F) From extracts treated as in (E) and supplemented with MG-262, Cdc27 was immunoprecipitated. Samples were CIP-treated and immunoblotted for XErp1 and Cdc27. (G) One-cell embryos were injected with 50 nM CycB{Delta}90 or incubated in 2 μM OA for 30 min. Cdc27 immunoprecipitates were CIP-treated and analyzed by immunoblot. (H) mRNA encoding FLAG-tagged PP2A B subunits or H2O (control) was incubated in embryo extract and immunoprecipitated using antibodies to FLAG. Precipitates were analyzed for XErp1 and FLAG epitope. (I) Extracts were incubated with B'56 mRNAs and GST-tagged XErp1301-400 WT or PKA phosphomutant (4A) XErp1. After reisolation of GST-tagged proteins, interacting B'56 subunits were analyzed by immunoblot. Inputs [(H) and (I)] are shown in fig. S3, F and G. CBB, Coomassie Brilliant Blue.

 

Figure 4
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  		Fig. 4. PKA phosphorylates XErp1 at sites critical for PP2A B'56-recruitment. (A) In vitro PKA phosphorylation assay using [{gamma}-32P]ATP and MBP-tagged full-length WT or PKA phosphomutant (4A) XErp1. {gamma}-32P incorporation was analyzed by autoradiography. (B) Embryo lysate depleted of PKA ({Delta}PKA) or treated with control antibodies ({Delta}Ctrl) were immunoblotted for PKA and tubulin. (C) Embryo lysates from (B) were supplemented with [{gamma}-32P]ATP and phosphorylation of MBP-XErp1BD was analyzed by autoradiography. Recombinant PKA was added to {Delta}PKA lysate. (D) MBP-XErp1BD was phosphorylated by PKA in vitro and incubated for 20 min in embryo extract. After repurification of MBP-XErp1BD, the associated PP2A catalytic subunit (PP2A-C) was analyzed by immunoblot. (E) One-cell embryos were injected with XErp1-MO or control MO and H2O or mRNA encoding WT or PKA phosphomutant (4A) XErp1; at 24 hpf, the indicated phenotypes were quantified. Images and immunoblots are shown in fig. S4, B and C. (F) One-cell embryos were incubated with H89 or injected with PKI to inhibit PKA; at 24 hpf, the indicated phenotypes were quantified. Images are shown in fig. S4H. (G) Embryos were treated as in (F); samples were taken at 6 hpf, CIP-treated as indicated, and immunoblotted for XErp1 and the phosphorylated PKA consensus motif (RXXpS/pT, where X stands for any amino acid and pS/pT for phosphorylated serine or threonine) to monitor PKA activity. (H) Model of APC/C regulation in early mitotic divisions.

 

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