Hiraoka D , Aono R , Hanada S , Okumura E , Kishimoto T .

	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.

Alessi, 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. 1997, Pubmed

Alessi, 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. 1997, Pubmed
Alessi, Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. 1997, Pubmed
Alessi, Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. 1996, Pubmed
Ayllón, Protein phosphatase 1alpha is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis. 2000, Pubmed
Castilho, The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites. 2009, Pubmed
Chiba, Induction of starfish oocyte maturation by the beta gamma subunit of starfish G protein and possible existence of the subsequent effector in cytoplasm. 1993, Pubmed , Echinobase
Dorée, Protein synthesis is not involved in initiation or amplification of the maturation-promoting factor (MPF) in starfish oocytes. 1982, Pubmed , Echinobase
Dupré, Phosphorylation of ARPP19 by protein kinase A prevents meiosis resumption in Xenopus oocytes. 2014, Pubmed
Esposito, Tyr(612) and Tyr(632) in human insulin receptor substrate-1 are important for full activation of insulin-stimulated phosphatidylinositol 3-kinase activity and translocation of GLUT4 in adipose cells. 2001, Pubmed
Ferrell, The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. 1998, Pubmed
Ford, Molecular basis for interactions of G protein betagamma subunits with effectors. 1998, Pubmed
García-Martínez, mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). 2008, Pubmed
Gharbi-Ayachi, The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A. 2010, Pubmed
Haccard, Oocyte maturation, Mos and cyclins--a matter of synthesis: two functionally redundant ways to induce meiotic maturation. 2006, Pubmed
Hara, Greatwall kinase and cyclin B-Cdk1 are both critical constituents of M-phase-promoting factor. 2012, Pubmed , Echinobase
Harrington, The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. 2004, Pubmed
Hiraoka, PDK1 is required for the hormonal signaling pathway leading to meiotic resumption in starfish oocytes. 2004, Pubmed , Echinobase
Hiraoka, Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt. 2011, Pubmed , Echinobase
Hirohashi, Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish. 2008, Pubmed , Echinobase
Hoeffer, mTOR signaling: at the crossroads of plasticity, memory and disease. 2010, Pubmed
Jaffe, Oocyte maturation in starfish is mediated by the beta gamma-subunit complex of a G-protein. 1993, Pubmed , Echinobase
Kanatani, Site of action of 1-methyladenine in inducing oocyte maturation in starfish. 1970, Pubmed , Echinobase
Kanatani, Isolation and indentification on meiosis inducing substance in starfish Asterias amurensis. 1969, Pubmed , Echinobase
Kishimoto, Cell-cycle control during meiotic maturation. 2003, Pubmed
Kishimoto, Microinjection and cytoplasmic transfer in starfish oocytes. 1986, Pubmed , Echinobase
Kishimoto, Entry into mitosis: a solution to the decades-long enigma of MPF. 2015, Pubmed
Kishimoto, A primer on meiotic resumption in starfish oocytes: the proposed signaling pathway triggered by maturation-inducing hormone. 2011, Pubmed , Echinobase
Kobayashi, Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. 1999, Pubmed
Kusubata, p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro. 1992, Pubmed
Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. 1970, Pubmed
Li, Sites for Galpha binding on the G protein beta subunit overlap with sites for regulation of phospholipase Cbeta and adenylyl cyclase. 1998, Pubmed
Lindqvist, The decision to enter mitosis: feedback and redundancy in the mitotic entry network. 2009, Pubmed
Liu, Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt. 2003, Pubmed
Liu, Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus. 2014, Pubmed
Liu, Serotonin-induced growth of pulmonary artery smooth muscle requires activation of phosphatidylinositol 3-kinase/serine-threonine protein kinase B/mammalian target of rapamycin/p70 ribosomal S6 kinase 1. 2006, Pubmed
Manning, AKT/PKB signaling: navigating downstream. 2007, Pubmed
Mochida, Regulated activity of PP2A-B55 delta is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts. 2009, Pubmed
Mochida, Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis. 2010, Pubmed
Novak, Numerical analysis of a comprehensive model of M-phase control in Xenopus oocyte extracts and intact embryos. 1993, Pubmed
Okumura, Initial triggering of M-phase in starfish oocytes: a possible novel component of maturation-promoting factor besides cdc2 kinase. 1996, Pubmed , Echinobase
Okumura, Akt inhibits Myt1 in the signalling pathway that leads to meiotic G2/M-phase transition. 2002, Pubmed , Echinobase
Ookata, Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes. 1992, Pubmed , Echinobase
Pearce, The nuts and bolts of AGC protein kinases. 2010, Pubmed
Picard, Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish. 1989, Pubmed , Echinobase
Pomerening, Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2. 2003, Pubmed
Sadler, Components of the signaling pathway linking the 1-methyladenine receptor to MPF activation and maturation in starfish oocytes. 1998, Pubmed , Echinobase
Sarbassov, Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. 2005, Pubmed
Schmitt, Signalling pathways in oocyte meiotic maturation. 2002, Pubmed
Shilling, Pertussis toxin inhibits 1-methyladenine-induced maturation in starfish oocytes. 1989, Pubmed , Echinobase
Song, Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop. 2005, Pubmed
Sun, Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. 1991, Pubmed
Sutherland, Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling. 1993, Pubmed
Towbin, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979, Pubmed
Vanhaesebroeck, The emerging mechanisms of isoform-specific PI3K signalling. 2010, Pubmed
Vigneron, Greatwall maintains mitosis through regulation of PP2A. 2009, Pubmed
Zhang, PDGFRs are critical for PI3K/Akt activation and negatively regulated by mTOR. 2007, Pubmed