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t checked whether or not Emi1 co-immunoprecipitates with Mad1 in the immunodepletion experiments (Reimann and Jackson, 2002; Tunquist et al, 2003). Because of the limiting volume of mouse oocytes, such approaches cannot be applied to our system. Nevertheless, our in vivo competition and/or depletion approaches suggest that Emi1 participates in cytostatic arrest also in mouse oocytes.

Our observations suggest that the metaphase-to-anaphase transition in vertebrate eggs is controlled by a relay of phosphorylation events that fine-tune the reciprocal affinity of regulatory components of the APC. In our model, p90Rsk2 is activated by the Mos pathway during oocyte maturation and phosphorylates Emi1, increasing its affinity for Cdc20 and preventing activation of the APC (Figure 9). After metaphase is reached, the Mos pathway becomes dispensable (Tunquist and Maller, 2003), possibly because phosphorylated Emi1 is stable in the oocyte environment. This might be due to the absence of a counteracting phosphatase or to inaccessibility of Emi1 when complexed to Cdc20. In agreement with this second hypothesis, it was noted that Emi1 has a stronger affinity for the APC activator Cdh1 than for Cdc20, and it is rapidly degraded when Cdh1 disappears (Reimann et al, 2001b; Hsu et al, 2002). Since only Cdc20 is present in oocytes (Lorca et al, 1998), it is possible that phosphorylation by p90Rsk2 prolongs Emi1 life in meiosis through the stabilization of the normally loose interaction with Cdc20. At fertilization, activation of CaMKII triggers activation of APCCdc20 and degradation of cyclin B (Lorca et al, 1993). It remains to be established whether CaMKII acts directly through phosphorylation of Emi1 or Cdc20 and whether phosphorylation causes dissociation of the complex. In this regard, our preliminary data suggest that at least Emi1 is not a direct in vitro substrate for CamKII (MP Paronetto and C Sette, unpublished observation).

In conclusion, our results indicate that p90Rsk2 functionally interacts with Emi1 in the establishment of CSF activity in mouse eggs. Our model offers a reconciling view on the connection between two components of CSF that were previously considered independent, leaving puzzling doubts on the relation between the establishment and the maintenance of CSF arrest (Duesbery and Vande Woude, 2002). Moreover, since activation of the MAPK pathway is required also in the response to the spindle- and DNA-damage checkpoints in mitotic cells (Chung and Chen, 2003; Panta et al, 2004), our results may provide a direct link between this pathway and cell cycle arrest through the inhibition of the APC.

 

 
Materials and methods
 

Plasmid construction

Mouse Emi1 was amplified by PCR using a 13 dpc embryo cDNA library and oligonucleotides 5′-AGGAATTCATGAAGTGTTTTAATTGCAACCCT G-3′ (forward) and 5′-GGTCGACTCACAATCTTTGTAAGTTCTTTTTA C-3′ (reverse) and subcloned into the EcoRI and SalI sites of either pCDNA3-myc or pGEX4T1 expression vectors for myc- or GST-tagged Emi1, respectively. Oligonucleotides were derived from the mouse Emi1 homologue deposited in the NCBI database (Fbxo5: NM_025995). Additional oligonucleotides used were as follows: 5-GGTCGACTCATAGGTGCTCCAGGCCCAT-3′ (reverse) for GST-Emi11–181, 5′-AGGAATTCATGCAGCGAGTCATTGAAAGC-3′ (forward) for Emi1236–383, 5′-GGTCGACTCAGGCTTTGAGGCTTTCGTTG-3′ (reverse) for Emi1236–313. Point mutations in Emi1 were introduced by using mutated oligonucleotides and PCR amplification. Pfu polymerase (Stratagene) was used for all amplifications and constructs sequences were verified by direct sequencing. The vector pMT2-HAp90Rsk2 was a generous gift of Dr Mortin Frodin.

Expression and purification of GST fusion proteins Plasmids (pGEX-) containing GST fusion proteins were transformed into the Escherichia coli BL21 strain, and grown at 30°C in LB medium to an OD600=0.6 before induction with 0.5 mM isopropyl-β-thiogalactopyranoside (IPTG, Sigma-Aldrich) for 3 h. GST fusion proteins were purified from bacterial lysates on glutathione-agarose (Sigma-Aldrich) as previously described (Sette et al, 1998) and analysed by SDS–PAGE and Coomassie blue staining to test purity and integrity.

Cell culture and transfections Hek293 cells were maintained in Dulbecco's medium supplemented with 10% fetal bovine serum (FBS) (Gibco BRL) in 90 mm dishes. Subconfluent monolayers were processed for CaPO4 transfection with 1–10 μg of the appropriate plasmids or by Fugene (Stratagene) with 0.2–2 μg of the appropriate plasmids as previously described (Sette et al, 2002). At 24–48 h after transfection, cells were harvested in lysis buffer (50 mM Hepes, pH 7.5, 75 mM NaCl, 10 mM β-glycerophosphate, 2 mM EGTA, 15 mM MgCl2, 0.1 mM sodium orthovanadate, 1 mM DTT, 0.5% Triton X-100, protease inhibitor cocktail (Sigma-Aldrich)) and incubated for 10 min on ice. Lysates were centrifuged for 10 min at 10 000 g at 4°C and used for further analysis. Protein concentration was determined using a protein assay kit (Bio-Rad) following the manufacturer's instructions.

FACS sorting Transfected cells were separated based on size (forward scatter) and green fluorescence (GFP-positive) using a FACSVantage cell sorter (Beckton and Dickinson). Purity of GFP-positive and -negative populations was >98%. Sorted cells were used for Western blot analysis as described below.

Pull-down assays Cell extracts (500 μg of total proteins) were added to 2 μg of GST fusion protein adsorbed on glutathione-agarose (Sigma-Aldrich) in 250 μl (final volume) of lysis buffer supplemented with 0.05% bovine serum albumin (BSA). After incubation for 90 min at 4°C under constant shaking, beads were washed three times with lysis buffer without Triton X-100, and absorbed proteins were eluted in SDS sample buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% (wt/vol) SDS, 0.7 M 2-mercaptoethanol and 0.0025% (wt/vol) bromophenol blue) and resolved on a 10% SDS–PAGE for subsequent Western blot analysis.

Immunoprecipitation assay Cell extracts (500 μg of total proteins) were incubated with 1 μg of anti-myc antibody for 2 h at 4°C under constant shaking. Protein A–Sepharose or protein G–Sepharose (Sigma-Aldrich) was preadsorbed with 0.05% BSA before incubation with the immunocomplexes for an additional hour. Hence, beads were washed three times with lysis buffer and absorbed proteins were eluted in SDS sample buffer for Western blot analysis.

Kinase assays For p90Rsk2 assays, 1 μg of each GST-Emi1 fusion protein was incubated at 30°C for 20 min with the purified active form of the kinase (5 U, Upstate Biotechnology) in reaction buffer: 50 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 10 mM β-glycerophosphate, 0.5 mM NaVO4, 50 μM ATP and 5 μCi of 32P-γ-ATP. In some experiments, GST fusion proteins were phosphorylated while still bound to the GSH-agarose beads and at the end of the incubation the kinases were washed by rinsing three times with an excess of kinase buffer (without label) before using the proteins for pull-down assays. H1 kinase assays were performed on cell extracts of GFP-positive Hek293 cells as previously described (Bhatt and Ferrell, 1999).

Western blot analysis Cell extracts or immunoprecipitated proteins were diluted in SDS sample buffer as described above and boiled for 5 min. For oocyte extracts, 300 metaphase II oocytes/sample were collected and immediately frozen in sample buffer. After thawing, oocytes were sonicated and boiled before loading. Proteins were separated on 10% SDS–PAGE gels and transferred to polyvinylidene fluoride Immobilon-P membranes (Millipore) using a semidry blotting apparatus (Bio-Rad). The membranes were saturated with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature, and incubated with the following primary antibodies (1:1000 dilution) overnight at 4°C: mouse anti-HA (for HA-p90Rsk2, from BabCO Berkeley antibody company); rabbit anti-actin (Sigma-Aldrich); mouse anti-Myc (for myc-Emi1); rabbit anti-Emi1 (Gentaur); goat anti-p90Rsk2, rabbit anti-Cdc20, rabbit anti-cyclin A2, mouse anti-cyclin-B1. Primary antibodies were all from SantaCruz Biotechnology, unless specified otherwise. Secondary anti-mouse or anti-rabbit IgGs conjugated to horseradish peroxidase (Amersham) were incubated with the membranes for 1 h at room temperature at a 1:10 000 dilution in PBS containing 0.1% Tween 20. Immunostained bands were detected by chemiluminescent method (SantaCruz Biotechnology).

Immunofluorescence analysis Oocytes were processed for immunofluorescence analysis using anti-tubulin antibody (1:100, Sigma-Aldrich) or anti-Emi1 antibody (1:200, Gentaur) or anti-p90Rsk2 antibody (1:200, SantaCruz Biotechnology) as previously documented (Sette et al, 2002).

 

 
 
Oocyte collection, microinjection and in vitro culture

Two-cell embryos and GV oocytes were collected from hormonally primed 6- to 7-week-old CD1 female mice (Charles River Italia) and cultured in M16 medium under mineral oil as previously described (Hogan et al, 1994). Oocytes were allowed to undergo GVBD by incubation in the absence of an exogenous cAMP source and used for microinjection either immediately (for dsRNAi) or after GVBD (approximately 2 h after collection). Before injection, oocytes and embryos were washed in M2 medium and then transferred to 50 μl drops of the same medium under mineral oil. Microinjection manipulations were performed as previously described (Sette et al, 1998). Briefly, into the cytoplasm of one blastomere of a two-cell embryo we injected 2–5 pl of a purified p90Rsk2 (1–5 U, Upstate Biotechnology) together with either GST or GST-Emi1 diluted to a protein concentration of 1 mg/ml in injection buffer (20 mM Hepes, pH 7.4, 120 mM KCl, 100 μM EGTA, 10 mM β-glycerophosphate, 1 mM DTT, 10 μg/ml leupeptin, 10 μg/ml pepstatin). Microinjections were performed using an Olympus invertoscope (Olympus) equipped with Hoffman modulation contrast optics (Modulation optics Inc., Greenvale, NY) and two Leitz mechanical micromanipulators (Leica AG, Heerbrugg, Switzerland). After microinjections, embryos or oocytes were returned to M16 medium drops and cultured at 37°C under a humidified atmosphere of 5% CO2 in air. At 12–14 h after injection, cells were scored for mitotic or meiotic divisions or processed for immunofluorescence analysis.

 

dsRNA preparation

To generate templates for dsRNA synthesis, we employed forward and reverse oligonucleotides containing at the 5′ end a T7 promoter sequence. For Emi1 amplification, the following primers were designed: forward 5′-GTAATACGACTCACTACTATAGGGCATGCAGC GAGTCA-3′; reverse 5′-GTAATACGACTCACTACTATAGGGCTCACAAT CTTTGT. These oligonucleotides amplify a region of 447 bp from base 945 to 1392 at the 3′ end of mouse Emi1 (AK011820). For GFP amplification, the following primers were designed: forward 5′-GTAATACGACTCACTACTATAGGGCATGCATA AAGGAG-3′; reverse 5′-GTAATACGACTCACTACTATAGGGCTCAATGC ATTAGTTC-3′. These oligonucleotides amplify a region of 600 bp of the GFP sequence. pCDNA3-mycEmi1 and pCMV5-GFP expression vectors were used as templates for PCR amplification. Amplified bands were gel-purified and used as templates (1 μg) for in vitro RNA transcription in order to obtain sense and antisense RNA sequences as previously described (Svoboda et al, 2000). RNA was extracted and precipitated by standard procedures and dissolved in RNAse-free H2O. Equimolar amounts of sense and antisense RNA were annealed in DEPC-water supplemented with 1 U/μl of RNasin (Invitrogen) and 4 μg of RNA was boiled for 1 min and allowed to cool down at room temperature before phenol/chloroform extraction and ethanol precipitation. dsRNA was resuspended in H2O, assayed by agarose electrophoresis and stored at −80°C prior to use.

 

 
Acknowledgments
 

We thank Drs Francesco Cenci and Federica Capolunghi for their help with FACS analysis, Drs Manuela Pellegrini and Susanna Dolci for critically reading the manuscript and Dr Mortin Frodin for the gift of the pMT2-HA-p90Rsk2 vector. This work was supported by MIUR Cofin 2002 and 2003.

 

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