Isoda M , Kanemori Y , Nakajo N , Uchida S , Yamashita K , Ueno H , Sagata N .

	Figure 1. Induction of SCFβ-TrCP-dependent degradation of Xe-Cdc25A by the ERK pathway. (A) Artificially activated eggs (or eggs after 40 min of calcium ionophore treatment) were injected or not with 9 ng of JNK mRNA, reinjected 2.5 h later (time 0) with 9 ng of either MEK-CA, MKK6-CA, or MKK7-CA mRNAs, and analyzed at 20-min intervals by immunoblotting (IB) with anti-Xe-Cdc25A, anti-phospho-JNK, anti-phospho-ERK, and anti-phospho-p38 antibodies. (B) Activated eggs were injected with 9 ng of GST mRNA (Control) or MEK-CA mRNA. Egg extracts were then treated (+λ) or not (−λ) with λ phosphatase and analyzed for Xe-Cdc25A by immunoblotting. (C) Activated eggs were injected with 18 ng of GST mRNA (Control) or dominant-negative β-TrCPΔF mRNA, reinjected 2.5 h later with 9 ng of MEK-CA mRNA, and then analyzed for Xe-Cdc25A by immunoblotting. (D) Activated eggs were injected or not with 9 ng of MKP3 mRNA, reinjected 40 min later with 2 ng of mRNA encoding Myc-tagged WT or D231A Xe-Cdc25A, further injected 2.5 h later with 9 ng of MEK-CA mRNA, and then analyzed for Myc-Xe-Cdc25A constructs and phospho-ERK by immunoblotting. Five, three, four, and four independent experiments were performed for A, B, C, and D, respectively, and, for each, a typical result is shown.
	Figure 2. p90rsk phosphorylation, and its requirement for the ERK pathway-induced degradation, of Xe-Cdc25A. (A) Activated eggs were injected with 2 ng of mRNA encoding the indicated Myc-Xe-Cdc25A constructs, reinjected 2.5 h later with 9 ng of MEK-CA mRNA, and then analyzed for Myc-Xe-Cdc25A constructs by immunoblotting. (B) GST-fused Xe-Cdc25A peptides (GST-S120, GST-A120, etc., each named after the relevant Ser or substituted Ala residue numbers) were incubated with [γ-32P]ATP and p90rsk protein, subjected to SDS-polyacrylamide gel electrophoresis, stained with Coomassie Brilliant Blue (CBB), and then autoradiographed (32P). (C) Activated eggs were injected with 2 ng of mRNA encoding the indicated Myc-Xe-Cdc25A constructs, reinjected 2.5 h later with either 9 ng of MEK-CA mRNA or 36 ng of p90rsk-CA mRNA, and analyzed for Myc-Xe-Cdc25A constructs and phospho-ERK by immunoblotting. Four, three, and four independent experiments were performed for A, B, and C, respectively, and, for each, a typical result is shown.
	Figure 3. Phosphorylation of Xe-Cdc25A by ERK. (A) Conservation of consensus ERK phosphorylation motifs (Ser-Pro) in Xenopus and human Cdc25A proteins. (B) The indicated GST-fused Xe-Cdc25A peptides were incubated with [γ-32P]ATP and ERK protein and analyzed as described in Figure 2B. (C) GST-tagged full-length Xe-Cdc25A proteins (WT or S85A) were synthesized in wheat germ extracts, purified by GST-pull-down, incubated with either buffer (Cont.) or ERK protein, and analyzed by immunoblotting with anti-GST and anti-phospho-S85 antibodies. (D) Activated eggs were injected with 2 ng of mRNA encoding Myc-Xe-Cdc25A (WT or S85A), reinjected or not 2.5 h later with 9 ng of MEK-CA mRNA, and cultured for 50 min. Egg extracts were treated or not with λ-phosphatase and analyzed for Myc-Xe-Cdc25A and phospho-S85 by immunoblotting. (E) Activated eggs were injected with either buffer (Control), 18 ng of p21Cip1 mRNA, or 9 ng of MKP3 mRNA, reinjected 40 min later with 2 ng of Myc-Xe-Cdc25A mRNA, further injected 2.5 h later with 9 ng of MEK-CA mRNA, collected at the indicated times, and analyzed by immunoblotting as described in D. Four, three, four, and five independent experiments were performed for B, C, D, and E, respectively, and, for each, a typical result is shown.
	Figure 4. Requirement of ERK phosphorylation for Xe-Cdc25A degradation. (A–C) Activated eggs were injected with 2 ng of mRNA encoding the indicated Myc-Xe-Cdc25A constructs, reinjected 2.5 h later with 9 ng of MEK-CA mRNA, and analyzed for Myc-Xe-Cdc25A constructs by immunoblotting. In B, 3A/S36, etc., are reversion mutants of SP:4A (see text). Four independent experiments were performed for A–C, and, for each, a typical result is shown.
	Figure 5. ERK pathway-dependent degradation of Xe-Cdc25A during oocyte maturation. (A) Immature oocytes (IMO) were treated with progesterone (PG) to induce maturation, whereas ovulated eggs were fertilized in vitro. Maturing oocytes and fertilized eggs were collected at the indicated times, and analyzed for endogenous Xe-Cdc25A and phospho-ERK by immunoblotting. GVBD denotes germinal vesicle breakdown. (B) Immature oocytes were coinjected with 2 ng of Myc-Xe-Cdc25A mRNA and 20 ng of either GST mRNA (Control) or β-TrCPΔF mRNA, cultured overnight, treated with progesterone, and analyzed for Myc-Xe-Cdc25A by immunoblotting. (C) Immature oocytes were injected with 2 ng of Myc-Xe-Cdc25A mRNA, cultured overnight, pretreated with dimethyl sulfoxide (Control) or 100 μM U0126 for 1 h, treated with progesterone, and analyzed for Myc-Xe-Cdc25A and phospho-ERK by immunoblotting. (D) Immature oocytes were injected with 2 ng of mRNA encoding the indicated Myc-Xe-Cdc25A constructs, cultured overnight, treated with progesterone, and analyzed for Myc-Xe-Cdc25A constructs by immunoblotting. In B–D, all the Myc-Xe-Cdc25A constructs were, in fact, phosphatase-dead C428S forms to avoid premature maturation of the oocytes. Four, three, four, and five independent experiments were performed for A, B, C, and D, respectively, and, for each, a typical result is shown.
	Figure 6. Cell cycle arrest in early embryos by ERK-induced Xe-Cdc25A degradation. (A) One-cell embryos 25 min after fertilization were injected with either water (Control) or 200 ng of Erp1 antisense morpholino oligos (Erp1-MO) and cultured for 1.5 h; embryo extracts were treated with λ-phosphatase and analyzed for endogenous Erp1 by immunoblotting. (B) One-cell embryos injected with Erp1 morpholino oligos as above were cultured for 35 min, injected with either water (Control), 5 ng of MEK-CA mRNA, or both 5 ng of MEK-CA mRNA and 200 pg of Myc-Xe-Cdc25A (WT or 8A) mRNA, cultured for the indicated times, and analyzed for Xe-Cdc25A (endogenous plus exogenous), phospho-ERK, and phospho-T14/Y15 by immunoblotting. (C) The embryos treated as in B were photographed 3 h after injection of MEK-CA mRNA (together with or without Myc-Xe-Cdc25A mRNA). Three and four independent experiments were performed for A and B, respectively, and, for each, a typical result is shown.
	Figure 7. Model for the mechanism of Xe-Cdc25A degradation and cell cycle arrest induced by the ERK pathway. On strong activation of the ERK pathway, ERK phosphorylates Xe-Cdc25A on S85 (which normally is phosphorylated by Cdk) and other Ser residues (omitted), whereas the downstream kinase p90rsk phosphorylates Xe-Cdc25A on other multiple Ser residues (which overlap with Chk1 phosphorylation sites). These phosphorylations facilitate ubiquitination of Xe-Cdc25A by SCFβ-TrCP, thereby targeting the phosphatase for degradation and causing cell cycle arrest at interphase. For details, see text. RD, regulatory domain; DDG, DDG motif; CD, catalytic domain.

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