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	Fig. 1. Signaling by subthreshold levels of 1-MeAde is canceled through the characteristic dephosphorylation of Akt substrates, including Cdc25. (A) 1-MeAde dose-response curve in starfish oocytes. Thirty immature oocytes were treated with various concentrations of 1-MeAde. After 90 min, the proportion of oocytes that had undergone GVBD, a marker of the meiotic G2/M-phase transition that occurs at approximately 18 min after 1-MeAde addition, were counted. Data represent mean values±s.d. from three independent experiments. (B) Immature oocytes were treated with sub- (30 nM) or supra-threshold (500 nM) concentrations of 1-MeAde, collected at the indicated times and subjected to immunoblot with antibodies against phospho-Akt (at Ser477; pS477), Akt (Akt), phospho-Cdc25 (pS188), Cdc25 (Cdc25), pan phospho-Akt substrates (PAS), phospho-Cdk1 (at Tyr15; pY15) or the PSTAIR epitope (Cdk1). (C) The signal intensity of each band in B was quantified and normalized against a standard sample [an aliquot of a batch sample of 1-MeAde-treated (500 nM, 3 min) oocytes was loaded on an extra lane (not shown) as a standard in every independent experiment]. Phosphorylation levels (pS477, pS188 and pY15) were further normalized against total amounts of each protein (Akt, Cdc25 and Cdk1, respectively). Data represent mean values±s.d. from three independent experiments. (D,E) Immature oocytes were treated with sub- (40 nM) or supra-threshold (500 nM) concentrations of 1-MeAde, followed by analysis with the H1 kinase assay [autoradiography (D; H1K) and liquid scintillation counting (E)] and immunoblot. The data shows a representative of two independent experiments. (F) Immature oocytes were injected with the Myt1-S75 peptide (S75 pep.) followed by treatment with subthreshold (7 nM) levels of 1-MeAde (i), or the Myt1-S75 peptide was phosphorylated by human Akt1 in vitro and then injected into immature oocytes (ii). After incubation, the oocytes were collected at the indicated times and subjected to immunoblot analysis to detect phosphorylation and total protein levels. (G) Phosphorylation levels of the Myt1-S75-peptide were quantified from the immunoblot images shown in F and normalized against the total amount of each protein. The phosphorylation levels, relative to those at 4 min in ‘i’, are indicated as the mean value±s.d. from three independent experiments. Black lines indicate the linear approximation to show the dephosphorylation rate. inj., injection.
	Fig. 2. The characteristic dephosphorylation of Akt substrates after treatment with subthreshold concentrations of 1-MeAde depends on cyclin-B–Cdk1 activity. (A) Hypothesis tested in B and C. (B) Immature oocytes were treated with subthreshold levels of 1-MeAde (30 nM) in the presence of 30 μM roscovitine or 0.15% DMSO (as a negative control) and collected at the indicated times, followed by immunoblotting to detect phosphorylation and total protein levels. (C) Phosphorylation levels were quantified from the images in B, as described in Fig. 1C. Data represent mean values±s.d. from three independent experiments. (D) Immature oocytes were injected with mRNA encoding starfish Akt (sfAkt), incubated for 3 h, treated with a subthreshold concentration (30 nM) of 1-MeAde, and collected at the indicated times, followed by immunoblotting for phosphorylation and total proteins. (E) Phosphorylation levels were quantified from the images in D and normalized against the total amounts of each protein. Phosphorylation levels relative to those in uninjected oocytes at 2 min are shown. Data represent mean values±s.d. from three independent experiments. cyc, cyclin; exp., exposure; PPase, phosphatase.
	Fig. 3. PI3K–Akt activation alone is not sufficient to cause phosphorylation of Ser188 on Cdc25 and meiotic G2/M transition. (A) A working hypothesis of the overriding pathways (cyan). (B–D) Immature oocytes were injected with mRNA encoding PH–GFP. After 1.5 h, a group of these oocytes were further injected with mRNA encoding FLAG-tagged CA-PI3K, then incubated for 5 h. Another group of oocytes injected with mRNA encoding PH–GFP were incubated for 6.5 h and then treated with a supra-threshold concentration of 1-MeAde (500 nM) for 4 min. These oocytes were analyzed by immunoblotting (B). Fluorescence images of oocytes treated under the same conditions as in B were obtained with confocal laser microscopy analysis (C). Typical fluorescence images of the oocytes are shown. The focal plane was set on the equatorial plane. Note that in all cases, oocytes had intact germinal vesicles in which some PH–GFP had accumulated. Scale bar: 50 μm. The plasma-membrane-to-cytoplasm fluorescence density ratio was calculated for each oocyte from the fluorescence images, as shown in C. Mean values of the ratio are indicated in D. n indicates the number of oocytes observed. Error bars represent the s.d. P-values (one-tailed t-test): *1, P<10−3; *2, P<10−4. (E,F) After the addition of a supra-threshold concentration of 1-MeAde or injection of mRNA encoding FLAG–CA-PI3K, immunoblotting was performed (E) or the GVBD ratio was determined (F). n indicates the number of oocytes observed. (G) Immature oocytes were injected with mRNA encoding CA-Akt and then collected immediately after GVBD (approximately 3 h after injection), or were treated with a supra-threshold (500 nM) concentration of 1-MeAde, followed by immunoblotting. (H) To compare expression levels of CA-Akt with those of endogenous Akt, the sample of CA-Akt-expressing oocytes immediately after GVBD were diluted 20- or 40-fold (1/20 or 1/40) with the sample of 1-MeAde-treated (500 nM, 4 min) oocytes and were then analyzed by immunoblotting. Endo, endogenous; exo, exogenous.
	Fig. 4. Exogenous Gβγ expression mimics 1-MeAde-stimulated signaling. (A) Hypothesis tested in B and C. (B) Immature oocytes were injected with mRNA encoding either untagged Gβ or Gγ, or an equimolar mixture of both mRNAs followed by incubation with 30 μM roscovitine (Ros) or 0.15% DMSO as a control. The proportion of oocytes that had undergone GVBD was determined following incubation for the indicated times. n indicates the number of oocytes observed. (C) Gβ, Gγ or Gβγ was expressed with roscovitine, as described in B. The oocytes were collected at the indicated times after mRNA injection. Gβγ-expressing oocytes that had been incubated with DMSO were collected immediately after GVBD (asterisk; approximately 3–4 h). For 1-MeAde-treated oocyte samples, immature starfish oocytes were incubated with roscovitine or DMSO for 6 h and then treated with a supra-threshold (500 nM) concentration of 1-MeAde for the indicated times. In the absence of roscovitine, GVBD occurred approximately 22 min after 1-MeAde addition. Samples were analyzed by immunoblotting to detect phosphorylation and total protein.
	Fig. 5. Gβγ activates a pathway that is distinct from, but works cooperatively with, the PI3K–Akt pathway to induce the meiotic G2/M-phase transition by enhancing Akt substrate phosphorylation. (A) Equimolar mixtures of mRNAs encoding starfish FLAG–Gγ and either Myc-tagged wild-type Gβ (Gβγ-WT) or the D246S mutant Gβ (Gβγ-D246S) were injected into immature oocytes. mRNA encoding CA-PI3K was injected or co-injected with the Gβγ-D246S mRNA mixture. The proportion of oocytes that had undergone GVBD was determined following incubation for the indicated times. (B) The oocytes described in A were collected 7 h after injection or immediately after GVBD. For the 1-MeAde-treated samples, immature oocytes were treated with a supra-threshold concentration (500 nM) of 1-MeAde for the indicated times. GVBD occurred approximately 20 min after 1-MeAde addition. The oocytes were then analyzed by immunoblotting. (C) mRNA encoding either FLAG–Gγ or Myc–Gβ-D246S, or an mRNA mixture of them (Gβγ-D246S) was injected into immature oocytes. After 8 h, a subthreshold concentration (3 nM; low) of 1-MeAde was added. Then, the number of oocytes displaying GVBD was counted at the indicated times. (D) mRNA-injected oocytes (8 h) in C and oocytes treated with a supra-threshold concentration of 1-MeAde (4 min) were analyzed by immunoblotting. n indicates the number of oocytes observed (A,D). Data are representative of two independent experiments.
	Fig. 6. The PI3K–Akt pathway is not sufficient for phosphorylation of Akt substrates, including Cdc25 and Myt1, but the atypical Gβγ pathway enhances phosphorylation even in the absence of cyclin-B–Cdk1-dependent negative feedback. (A) Hypothesis tested in B–H. (B–D) PH–GFP was expressed in immature oocytes, followed by expression of either FLAG–CA-PI3K or Gβγ through incubation with roscovitine for 5 h after mRNA injection. For treatment with 1-MeAde, immature oocytes were pre-incubated with roscovitine, then treated with a supra-threshold concentration of 1-MeAde for 4 min. The oocytes were analyzed by immunoblotting (B) or confocal laser microscopy (C). In all cases, each oocyte had an intact germinal vesicle, within which some PH–GFP had accumulated, although in some cases it was out of the focal plane. Scale bar: 50 μm. The plasma-membrane-to-cytoplasm ratio of fluorescence density is shown in D. Data represent mean values±s.d. (five oocytes for immature and Gβγ, six oocytes for CA-PI3K). P-values (one-tailed t-test): *1, P<10−8; *2, P<10−3. (E,F) After treatment with a supra-threshold concentration of 1-MeAde (500 nM), or FLAG–CA-PI3K or Gβγ expression with roscovitine, oocytes were analyzed by immunoblotting (E). Phosphorylation levels relative to those after treatment with 1-MeAde were quantified. Data represent mean values±s.d. from three independent experiments (F). P-values (one-tailed t-test) for Akt: *1, *5, *6, P>0.05 (not significant, n.s.); *4, P<10−3; *2, *3, P<10−4. For Cdc25, *3, P<0.05; *1, P<0.01; *5, P<10−3; *2, *4, *6, P<10−4. (G,H) Immature oocytes were injected with the GST-Myt1-S75-peptide, followed by expression of either CA-PI3K or Gβγ by mRNA-injection, then incubated with roscovitine for 5 h. For 1-MeAde treatment, oocytes were treated with a supra-threshold (500 nM) concentration of 1-MeAde for 4 min after pre-incubation with roscovitine for 5 h. The oocytes were analyzed by immunoblotting (G). Phosphorylation levels relative to those after treatment with 1-MeAde were quantified. Data represent mean values±s.d. from three independent experiments (H). In F and H, the numbers showing comparisons (*1 to *6) apply to both the upper and the lower panels. P-values (one-tailed t-test) for Akt: *1, *5, *6, P>0.05 (n.s.); *2, *3, P<0.01; *4, P<10−5. For the Myt1-S75-peptide (S75-pep): *1, P>0.05 (n.s.); *5, *6, P<0.05; *2, *3, P<0.01; *4, P<10−4.
	Fig. 7. The atypical Gβγ pathway does not alter phosphatase activity for Akt substrates in the absence of cyclin-B–Cdk1-dependent negative feedback. (A) Hypothesis tested in B and C. (B,C) Immature oocytes after pre-incubation with 40 μM wortmannin (WM) or 0.2% DMSO (DM), or oocytes treated with a supra-threshold concentration of 1-MeAde (500 nM) for 4 min after pre-incubation with wortmannin were injected with the phosphorylated AS-peptide (AS-pep). Oocytes were collected at 45 s after injection, and analyzed by immunoblotting for phosphorylated and total protein (B). Phosphorylation of the AS-peptide (PAS) was quantified from images in shown in B and normalized against total protein (GST). Phosphorylation levels relative to those in the input are given as mean values±s.d. from three independent experiments (C). P-values (one-tailed t-test): *1, *2, *3, P>0.05 (n.s.).
	Fig. 8. A model for threshold-setting by noise canceling and overriding pathways in 1-MeAde signaling. (A) In unstimulated immature oocytes, an unknown phosphatase for Akt substrates is active (PPase X), and all pathways shown in B and C with colors are inactive. (B) When oocytes receive a subthreshold 1-MeAde stimulus, the atypical Gβγ pathway (cyan) works cooperatively with the PI3K–Akt pathway (green) to phosphorylate Myt1 and Cdc25, thereby initiating activation of cyclin-B–Cdk1. However, cyclin-B–Cdk1 activates cyclin-B–Cdk1-dependent negative feedback (magenta), resulting in activation of an unknown phosphatase (note that here we drew this phosphatase as the PPase X, although it remains unclear whether they are the same molecule or not), thereby dephosphorylating Cdc25 and Myt1. Finally, cyclin-B–Cdk1 is inactivated by suppressive phosphorylation without reaching maximum activity. Although the cyclin-B–Cdk1-dependent positive-feedback pathway (black arrow from cyclin-B–Cdk1) might be partially activated, it is inactivated along with cyclin-B–Cdk1 before formation of an autonomously sustainable positive-feedback loop. (C) When oocytes receive supra-threshold 1-MeAde stimulus, the atypical Gβγ pathway and PI3K–Akt pathway activate cyclin-B–Cdk1. Although cyclin-B–Cdk1-dependent negative feedback might be active under these conditions, the atypical Gβγ pathway would be strongly activated enough to override this negative-feedback mechanism to maintain phosphorylation of Akt substrates. Thus, cyclin-B–Cdk1 reaches maximum activity through a fully functional positive-feedback loop, and oocytes irreversibly undergo the meiotic G2/M transition.

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