Two New Competing Pathways Establish the Threshold for Cyclin-B

RESEARCH ARTICLE Two new competing pathways establish the threshold for cyclin-B–Cdk1 activation at the meiotic G2/M transition Daisaku Hiraoka1,*, Ryota Aono2, Shin-ichiro Hanada2, Eiichi Okumura2 and Takeo Kishimoto1,2,*

ABSTRACT G2/M-phase border in somatic cells. Extracellular stimuli through Extracellular ligands control biological phenomena. Cells distinguish maturation-inducing hormones then induce transition from meiotic – physiological stimuli from weak noise stimuli by establishing a ligand- prophase I to metaphase I through activation of cyclin-B Cdk1 concentration threshold. Hormonal control of the meiotic G2/M (equivalent to the G2/M transition in somatic cells) (Kishimoto, transition in oocytes is essential for reproduction. However, the 2003), hallmarked by germinal vesicle breakdown (GVBD). Failure mechanism for threshold establishment is unclear. In starfish to tightly control this transition could result in serious defects in oocytes, maturation-inducing hormones activate the PI3K–Akt reproduction. Previous studies in frog and starfish have focused on signaling induced by supra-threshold doses of hormones (Haccard pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, and Jessus, 2006; Kishimoto, 2003). However, the mechanism by resulting in activation of cyclin-B–Cdk1, which then induces meiotic which subthreshold stimuli are prevented from eliciting transition G2/M transition. Here, we show that cyclin-B–Cdk1 is partially remains unclear. Thus, the nature of threshold-setting for hormone- activated after subthreshold hormonal stimuli, but this triggers dependent regulation of the meiotic G2/M transition is not fully negative feedback, resulting in dephosphorylation of Akt sites on understood. Here, we studied the molecular mechanism of Cdc25 and Myt1, thereby canceling the signal. We also identified threshold-setting using starfish oocytes, a model that has been phosphatase activity towards Akt substrates that exists independent widely used for studying G2/M transition (Kishimoto, 2015). of stimuli. In contrast to these negative regulatory activities, an In starfish oocytes, the maturation-inducing hormone 1- methyladenine (1-MeAde) induces the meiotic G2/M transition atypical Gβγ-dependent pathway enhances PI3K–Akt-dependent phosphorylation. Based on these findings, we propose a model for (Kanatani et al., 1969; Kishimoto, 2011), with no requirement for threshold establishment in which hormonal dose-dependent new protein synthesis (Dorée, 1982). Based on previous studies using competition between these new pathways establishes a threshold; supra-threshold doses of 1-MeAde, the following model of the signaling has been established. In immature oocytes, the kinase Myt1 the atypical Gβγ-pathway becomes predominant over Cdk-dependent – negative feedback when the stimulus exceeds this threshold. Our (UniProt ID Q99640 for the human form) inhibits cyclin-B Cdk1 by findings provide a regulatory connection between cell cycle and phosphorylating residues Thr14 and Tyr15 on Cdk1 (Okumura et al., signal transduction machineries. 2002). 1-MeAde promotes dissociation of the Gβγ complex (comprising the Gβ and Gγ subunits; hereafter referred to as Gβγ)of γ KEY WORDS: G ,Gβ, Oocyte, PI3K, Akt, Threshold, Cyclin B, Cdk1, heterotrimeric G-proteins from Giα (Chiba et al., 1993; Jaffe et al., Meiotic G2/M transition 1993; Shilling et al., 1989) through a putative 1-MeAde receptor located on the oocyte plasma membrane (Kanatani and Hiramoto, INTRODUCTION 1970). The Gβγ complex then binds to and activates PI3K, which Cells must respond to extracellular stimuli, such as hormones and produces phosphatidylinositol 3,4,5-triphosphate (PIP3) at the environmental stresses, in order to adapt to changing environments. plasma membrane (Sadler and Ruderman, 1998; Vanhaesebroeck By establishing a stimulus threshold, cells are able to discriminate et al., 2010). In a PIP3-dependent manner, phosphoinositide- physiologically relevant stimuli from noise stimuli. Supra-threshold dependent kinase 1 (PDK1) and target of rapamycin complex 2 but not subthreshold doses of stimuli elicit cellular responses. Such (TORC2) phosphorylate Akt on its activation loop and C-terminal stimulus-intensity-dependent responses can control progression of hydrophobic motif, respectively, thereby fully activating Akt the cell cycle. An important example is hormonal control of meiosis (Hiraoka et al., 2004, 2011). Akt directly inhibits Myt1 by in oocytes. In most animals, immature oocytes, which have a large phosphorylating residue Ser75 (Okumura et al., 2002). Cdc25, the nucleus, called the germinal vesicle, arrest at prophase of meiosis phosphatase for Myt1 sites on Cdk1, is thought to be I (meiotic prophase I) (Masui, 1985), which is equivalent to the phosphorylated by Akt (Okumura et al., 2002), although the role of this phosphorylation remains unclear. Consequently, Cdc25

1 activity predominates over that of Myt1, resulting in cyclin-B–Cdk1 Science and Education Center, Ochanomizu University, Tokyo 112-8610, Japan. 2Laboratory of Cell and Developmental Biology, Graduate School of Bioscience, activation through dephosphorylation of Cdk1 (Okumura et al., Tokyo Institute of Technology, Yokohama 226-8501, Japan. 1996, 2002). Cyclin-B–Cdk1-dependent positive feedback further

*Authors for correspondence ([email protected]; activates Cdc25 and inactivates Myt1 through phosphorylation of [email protected]) residues other than Akt sites (Hara et al., 2012; Kishimoto, 2015; Okumura et al., 2014). Finally, fully activated cyclin-B–Cdk1 D.H., 0000-0002-3875-2684 causes an irreversible meiotic G2/M-phase transition. This is an Open Access article distributed under the terms of the Creative Commons Attribution The PI3K–Akt pathway is also essential in various signal License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. transduction events induced by ligands such as insulin, growth factors and neurotransmitters in somatic cells (Alessi et al., 1997a,b;

Received 19 October 2015; Accepted 1 July 2016 Hoeffer and Klann, 2010; Manning and Cantley, 2007; Sarbassov Journal of Cell Science

3153 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3153-3166 doi:10.1242/jcs.182170 et al., 2005). Akt phosphorylates diverse substrates and thereby stimuli. Fig. 1A shows a typical dose–response curve. The highest regulates cellular functions, including cell survival, glucose concentration at which GVBD failed to occur in any oocytes was metabolism and cell cycle control (Manning and Cantley, 2007). used as the subthreshold 1-MeAde dose (Fig. 1A; 30 nM). A supra- In these cases, pathways downstream of Akt further affect PI3K threshold dose of 1-MeAde (500 nM) induced phosphorylation of activity, forming feedback loops. For example, Akt-dependent Akt and Cdc25 within 2 min [Fig. 1B,C; phosphorylated residue activation of mammalian target of rapamycin complex 1 (mTORC1) Ser477 (pS477) and phosphorylated residue Ser188 (pS188)], leads to suppression of transcription of PDGF receptor (Zhang et al., followed by full activation of cyclin-B–Cdk1, as indicated by 2007). In insulin signaling, the adaptor protein insulin receptor complete dephosphorylation of Cdk1 [Fig. 1B,C; phosphorylation substrate 1 (IRS1) mediates PI3K activation (Esposito et al., 2001; of residue Y15 (pY15)], resulting in GVBD at approximately Sun et al., 1991). Akt indirectly activates p70S6K, which then 18 min. Subthreshold 1-MeAde induced Akt and Cdc25 phosphorylates IRS1, thereby suppressing PI3K activation phosphorylation within 2 min (Fig. 1B,C; pS477, pS188). At (Harrington et al., 2004). To date, no such regulatory signaling 8 min, we observed a decrease in Cdk1 phosphorylation on Tyr15, networks have been reported in 1-MeAde signaling in starfish indicating initiation of cyclin-B–Cdk1 activation. Simultaneously, oocytes. however, residue Ser188 of Cdc25 was rapidly dephosphorylated to In this study, our initial analysis focused on the signaling dynamics basal levels, suggesting that signaling had been disrupted. Consistent induced by subthreshold levels of 1-MeAde. We found that with this, Cdk1 phosphorylation on Tyr15 returned to a level similar such stimuli initiate activation of cyclin-B–Cdk1 but that the to that in immature oocytes, indicating that cyclin-B–Cdk1 was cyclin-B–Cdk1-dependent negative-feedback pathway causes rapid inactivated without reaching maximum activity. This partial and dephosphorylation of Akt substrates, including Cdc25 and Myt1, transient activation of cyclin-B–Cdk1 was confirmed by performing thereby canceling signaling by subthreshold stimuli. This implied the a direct kinase assay using histone H1 as a substrate (Fig. 1D,E). At existence of an overriding mechanism that counteracts this negative 14 min, Cdc25 was again weakly phosphorylated and then gradually feedback and is thereby able to fully activate cyclin-B–Cdk1 in dephosphorylated to basal levels (Fig. 1B,C; Fig. S2A,B; pS188). response to supra-threshold levels of 1-MeAde. Consistently, we Next, in order to examine whether a characteristic identified a novel ‘atypical Gβγ-pathway’ in which Gβγ enhances dephosphorylation after subthreshold 1-MeAde stimulus is specific PI3K–Akt-dependent phosphorylation of Cdc25 and Myt1. to Ser188 of Cdc25, we used glutathione S-transferase (GST)- Furthermore, we found that, in addition to acting as an overriding conjugated peptide substrates of Akt, which are the Myt1-S75-peptide mechanism, this atypical pathway is also required for the PI3K–Akt and another Akt substrate peptide (AS-peptide) derived from Cdc25 pathway to induce phosphorylation of Akt substrates even in the (residues Ala181–Gly193) in which the Akt consensus region was absence of the negative feedback. Based on these findings, we propose replaced with another efficient Akt substrate sequence RPRAATF a model for full activation of cyclin-B–Cdk1 at the meiotic G2/M (Alessi et al., 1996). Immature oocytes were injected with these transition in which competition between newly identified mutually peptides, then treated with a subthreshold concentration of 1-MeAde. opposing pathways establishes the 1-MeAde-dose threshold. As a result, a characteristic phosphorylation and subsequent dephosphorylation of the peptides were observed, as well as of RESULTS Ser188 of Cdc25 (Fig. S2C–F). We also used a pan antibody that Ser188 on Cdc25 is phosphorylated by Akt recognizes phospho-Akt substrates (PASs) with phosphorylated Ser To monitor signaling dynamics, we performed immunoblotting with and Thr residues in the Akt consensus site. Following subthreshold phosphorylation (phospho)-specific antibodies against residue stimulus, this antibody detected multiple bands at 2 min, many (but Ser477 (the TORC2 phosphorylation site) on Akt (Hiraoka et al., not all) of which grew weaker or were lost in a manner concomitant 2011) and residue Tyr15 on Cdk1 to monitor the activity of cyclin-B– with the characteristic Cdc25 dephosphorylation (Fig. 1B; PAS). Cdk1. In addition, to monitor Akt substrate phosphorylation, we Thus, we concluded that the characteristic dephosphorylation occurs generated phospho-specific antibodies against Myt1 and Cdc25. The on a wide range of Akt substrates, including Cdc25 and Myt1. Taken anti-phospho-Myt1 (residue Ser75) antibody detected together, these findings indicate that although subthreshold stimuli can phosphorylation of an exogenously introduced Myt1-derived cause Akt activation and subsequent partial cyclin-B–Cdk1 activation, peptide containing Ser75 (Myt1-S75-peptide) in starfish oocytes rapid Akt substrate dephosphorylation disrupts signaling. (Fig. S1A) but was not suitable for detecting endogenous Myt1 Remarkably, the dynamics of Akt Ser477 phosphorylation were owing to high background and reactivity with non-specific bands distinct from those of Cdc25 phosphorylation, peaking at 4 min and (Fig. S1B). With respect to Cdc25, Akt phosphorylated and activated then decreasing gradually to near-basal levels (Fig. 1B,C; Fig. S2A,B; recombinant starfish Cdc25 in vitro (Fig. S1C). Cdc25 contains five pS477). In addition, when we monitored phosphorylation of the Akt consensus sites (RXRXXS/T, where X is any amino acid), and PDK1-target site on Akt using exogenously introduced human Akt1 we successfully generated an antibody able to detect endogenous as we have reported previously (Hiraoka et al., 2011), we found that Cdc25 phosphorylation on residue Ser188 following 1-MeAde the dynamics of phosphorylation were similar to those of the stimulation (Fig. S1B). Inhibition of Akt activation by an anti-TOR phosphorylation of the TORC2-target site [Fig. S2G,H; neutralizing antibody (recognizing starfish TOR, which is a catalytic phosphorylation of residue Thr308 (pT308), PDK1-target site; subunit of TOR complexes) (Hiraoka et al., 2011) disrupted this phosphorylation of residue Ser473 (pS473), TORC2-target site]. phosphorylation, suggesting that Cdc25 is phosphorylated at Ser188 Thus, the characteristic rapid dephosphorylation of Akt substrates by Akt in starfish oocytes (Fig. S1D). seems to occur while Akt remained active (at least at a level that can induce Cdc25 phosphorylation, Fig. 1B,C; compare pS477 at 2 min Subthreshold levels of 1-MeAde trigger cyclin-B–Cdk1 with 10 min). activation, but a subsequent dephosphorylation of Akt A possible mechanism for the characteristic dephosphorylation of substrates cancels the signaling Akt substrates could be activation of phosphatases. We examined a To elucidate the threshold-setting mechanism, we first compared phosphatase activity for Akt substrates in starfish oocytes. When the

1-MeAde signaling dynamics between supra- and subthreshold Myt1-S75-peptide or AS-peptide was phosphorylated by human Journal of Cell Science

3154 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3153-3166 doi:10.1242/jcs.182170

Fig. 1. See next page for legend.

Akt1 in vitro and then injected into immature oocytes, these was once phosphorylated in the oocytes and then subjected peptides were dephosphorylated, indicating existence of a to dephosphorylation) by performing sampling every 1 min phosphatase activity even in unstimulated oocytes (Fig. 1F,G; ii, (Fig. 1F,G; i). The rate of dephosphorylation was faster after Myt1-S75-peptide; Fig. S2I for AS-peptide). Then we compared the subthreshold 1-MeAde stimulus than in unstimulated oocytes rate of this dephosphorylation with that observed after subthreshold (Fig. 1G; i versus ii, black lines), suggesting that a phosphatase

1-MeAde stimulus (in this situation, unphosphorylated peptide was activated during the characteristic dephosphorylation event. Journal of Cell Science

3155 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3153-3166 doi:10.1242/jcs.182170

Fig. 1. Signaling by subthreshold levels of 1-MeAde is canceled through supra-threshold stimuli, oocytes must override this pathway. The the characteristic dephosphorylation of Akt substrates, including Cdc25. 1-MeAde dose-dependent increase in Akt phosphorylation on (A) 1-MeAde dose-response curve in starfish oocytes. Thirty immature oocytes Ser477 (see Fig. 1C; pS477 increases less than twofold) is less likely were treated with various concentrations of 1-MeAde. After 90 min, the proportion of oocytes that had undergone GVBD, a marker of the meiotic to be sufficient to override the negative feedback mediated by G2/M-phase transition that occurs at approximately 18 min after 1-MeAde cyclin-B–Cdk1 because even a fivefold increase in the amount of addition, were counted. Data represent mean values±s.d. from three Akt as a result of exogenous Akt expression failed to prevent Cdc25 independent experiments. (B) Immature oocytes were treated with sub- dephosphorylation (see Fig. 2D,E). Therefore, another mechanism (30 nM) or supra-threshold (500 nM) concentrations of 1-MeAde, collected at could exist. Because microinjection of Gβγ protein induces GVBD the indicated times and subjected to immunoblot with antibodies against in starfish oocytes (Chiba et al., 1993; Jaffe et al., 1993), we phospho-Akt (at Ser477; pS477), Akt (Akt), phospho-Cdc25 (pS188), Cdc25 – (Cdc25), pan phospho-Akt substrates (PAS), phospho-Cdk1 (at Tyr15; pY15) hypothesized that the pathway necessary to override the cyclin-B or the PSTAIR epitope (Cdk1). (C) The signal intensity of each band in B was Cdk1-dependent negative feedback is activated by Gβγ or its quantified and normalized against a standard sample [an aliquot of a batch downstream factors, such as PI3K (Fig. 3A). sample of 1-MeAde-treated (500 nM, 3 min) oocytes was loaded on an extra First, we tested whether activation of PI3K is sufficient to induce lane (not shown) as a standard in every independent experiment]. meiotic G2/M transition. We constructed a constitutively active Phosphorylation levels (pS477, pS188 and pY15) were further normalized form of PI3K (CA-PI3K). We confirmed PIP3 production using a against total amounts of each protein (Akt, Cdc25 and Cdk1, respectively). GFP fusion of the pleckstrin homology (PH) domain of starfish Akt 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) (PH GFP), which binds to PIP3 (Hirohashi et al., 2008). A greater concentrations of 1-MeAde, followed by analysis with the H1 kinase assay increase in the ratio of GFP fluorescence in the plasma membrane to [autoradiography (D; H1K) and liquid scintillation counting (E)] and that in the cytoplasm was detected after CA-PI3K expression, immunoblot. The data shows a representative of two independent through mRNA injection, than after 1-MeAde treatment (4.6-fold experiments. (F) Immature oocytes were injected with the Myt1-S75 peptide and 1.9-fold, respectively), indicating that more PIP3 was produced (S75 pep.) followed by treatment with subthreshold (7 nM) levels of 1-MeAde at the plasma membrane through CA-PI3K expression (Fig. 3B–D). (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 Immunoblot analysis showed that CA-PI3K induced Akt collected at the indicated times and subjected to immunoblot analysis to detect phosphorylation on Ser477 at a level comparable to that induced phosphorylation and total protein levels. (G) Phosphorylation levels of the by supra-threshold levels of 1-MeAde (Fig. 3E). In addition, when Myt1-S75-peptide were quantified from the immunoblot images shown in F and monitored on human Akt1, phosphorylation of the PDK1-target site normalized against the total amount of each protein. The phosphorylation was also induced (Fig. S4A, pT308). Nevertheless, no or less ‘ ’ levels, relative to those at 4 min in i , are indicated as the mean value±s.d. from phosphorylation of Akt substrates was observed in CA-PI3K- three independent experiments. Black lines indicate the linear approximation to show the dephosphorylation rate. inj., injection. expressing oocytes (Fig. 3E; pS188). Furthermore, CA-PI3K failed to induce GVBD (Fig. 3F). In contrast, we have previously reported that constitutively active Akt (CA-Akt) induces GVBD (Hiraoka Dephosphorylation of Akt substrates after subthreshold et al., 2011; Okumura et al., 2002). However, based on our re- stimuli depends on partially activated cyclin-B–Cdk1 evaluation, extremely high activity (40-fold higher than that of Because the peak of partial cyclin-B–Cdk1 activation preceded endogenous Akt) is required for induction of Akt substrate Cdc25 dephosphorylation, we hypothesized that cyclin-B–Cdk1- phosphorylation and GVBD by CA-Akt alone (Fig. 3G,H). These dependent negative feedback was occurring. To test this idea, we results suggest that activation of the PI3K–Akt pathway alone is not used the Cdk-inhibitor roscovitine (Fig. 2A). After subthreshold sufficient for full phosphorylation of Akt substrates and meiotic G2/ 1-MeAde treatment in the presence of roscovitine, phosphorylation M transition. of Ser188 on Cdc25 and Akt substrates, detected by using the antibody against PASs, was maintained (Fig. 2B,C; pS188 and Gβγ activates an atypical pathway that enhances PI3K–Akt- PAS). A similar effect was observed when yeast Suc1 protein was dependent Akt substrate phosphorylation to induce meiotic used to inhibit cyclin-B–Cdk1 (Fig. S3A). These results suggest that G2/M transition cyclin-B–Cdk1 causes Akt substrate dephosphorylation in order to Next, we investigated signaling from Gβγ (Fig. 4A). Expression of prevent signaling after subthreshold stimuli. Because Ser477 exogenous Gβγ complex, but neither the Gβ nor Gγ subunits alone, phosphorylation on Akt was slightly elevated through inhibition induced GVBD (Fig. 4B). The amount of exogenously expressed of cyclin-B–Cdk1 (Fig. 2B,C; Fig. S3A), we examined whether this Gβγ was physiologically relevant – mRNA injection of the starfish increase in Ser477 phosphorylation was sufficient to maintain Gβ- and Gγ-encoding genes resulted in an approximately twofold Akt substrate phosphorylation. Following injection of starfish Akt increase in Gβγ protein, implying that exogenous Gβγ was expressed mRNA, oocytes were treated with a subthreshold dose of 1-MeAde at a level similar to that of the endogenous proteins (Fig. 4C). (Fig. 2D,E). The total protein and phosphorylation levels of Akt Immunoblot analysis of the oocytes immediately after GVBD were increased approximately fivefold. However, the characteristic showed that phosphorylation of Akt and its substrates was similar to dephosphorylation of Akt substrates occurred as efficiently as in that induced by supra-threshold levels of 1-MeAde (Fig. 4C; pS477, uninjected oocytes (Fig. 2D,E, pS188; see also Fig. S3B for PAS), pS188 and PAS). Such similarity between 1-MeAde stimulation suggesting that loss of the characteristic dephosphorylation event by and Gβγ expression was also observed when cell cycle progression roscovitine could not be attributed solely to increased was inhibited with roscovitine (Fig. 4B,C). Furthermore, the PI3K phosphorylation of Ser477 on Akt. inhibitor wortmannin abolished all phosphorylation events (Fig. S4B). These observations suggest that signaling from Gβγ is PI3K–Akt activity alone is not sufficient for phosphorylation sufficient to mimic 1-MeAde signaling, leading to full activation of of Akt substrates and meiotic G2/M transition cyclin-B–Cdk1 that is dependent on the PI3K–Akt pathway. Cyclin-B–Cdk1-dependent negative feedback should be present Considering that CA-PI3K expression was not sufficient for even when oocytes receive supra-threshold doses of 1-MeAde. phosphorylation of Akt substrates or meiotic G2/M transition, it is

Thus, to achieve meiotic G2/M-phase transition in response to likely that Gβγ activates not only the PI3K–Akt pathway but also Journal of Cell Science

3156 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3153-3166 doi:10.1242/jcs.182170

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. Journal of Cell Science

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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.

another pathway (here referred to as an ‘atypical Gβγ pathway’) that (Ford et al., 1998; Li et al., 1998), and we found that the D246S mutant enhances the PI3K–Akt-dependent phosphorylation of Akt produced the desired phenotype. Expression of FLAG-tagged Gβ- substrates to induce the meiotic G2/M transition. D246S with Myc-tagged Gγ (referred to as the mutant Gβγ) failed to induce Akt phosphorylation, Akt substrate phosphorylation and Expression of mutant Gβγ lowers the 1-MeAde threshold GVBD (Fig. 5A,B). Nevertheless, co-expression of the mutant Gβγ To test whether the atypical Gβγ-pathway can act as an overriding with CA-PI3K resulted in Akt substrate phosphorylation and GVBD mechanism in establishment of the threshold for 1-MeAde to induce (Fig. 5A,B). Then, we treated the mutant-Gβγ-expressing oocytes with the meiotic G2/M transition, first we attempted to construct a Gβγ subthreshold levels of 1-MeAde. As a result, these oocytes underwent mutant that activatesthe atypical pathway without activating the PI3K– GVBD, suggesting that an increase in activity of the atypical Gβγ- Akt pathway. To this end, we introduced point mutations into Gβ at pathway is likely to override cyclin-B–Cdk1-dependent negative residues involved in interactions between Gβγ and its target proteins feedback (Fig. 5C,D). Journal of Cell Science

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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.

The atypical Gβγ pathway is required for PI3K–Akt-dependent Akt at a similar levels to that induced by 1-MeAde-treatment (Fig. 6E– phosphorylation of Cdc25 and Myt1, even in the absence of H; pS477). Nonetheless, CA-PI3K induced significantly less Akt cyclin-B–Cdk1-dependent negative feedback substrate phosphorylation than Gβγ (Fig. 6E,F for pS188 and PAS, Considering Gβγ-dependent signaling is first activated before Fig. 6G,H for pS75; see Fig. S4C for DMSO or kinase-dead control of cyclin-B–Cdk1 activation, the atypical Gβγ pathway might not CA-PI3K). Taken together, these results suggest that even in the absence only act as overriding machinery against cyclin-B–Cdk1-dependent of activation of cyclin-B–Cdk1-dependent negative feedback, activation negative feedback but could also play a positive role in of the PI3K–Akt pathway alone is not sufficient for efficient phosphorylation of Akt substrates, even in the absence of this phosphorylation of Akt sites on Cdc25 and Myt1, and that the negative-feedback pathway. atypical Gβγ pathway enhances these phosphorylation events. To test this idea, CA-PI3K or Gβγ were expressed, through injection of mRNA, in immature oocytes in the presence of roscovitine, which The phosphatase activity towards Akt substrates is not prevents activation of cyclin-B–Cdk1-dependent negative feedback altered by the atypical Gβγ pathway (Fig.6A).WhenPIP3 production was monitored by using PH-GFP, both In order to upregulate phosphorylation levels of Akt substrates, the CA-PI3K and Gβγ increased the membrane-to-cytoplasm fluorescence atypical Gβγ pathway might reduce phosphatase activity towards ratio (5.4- and 1.8-fold, respectively; Fig. 6B–D), indicating PIP3 Akt substrates. Thus, we examined whether the atypical Gβγ production at the plasma membrane at higher (or at least comparable) pathway alters the phosphatase activity. levels than that induced by supra-threshold amounts of 1-MeAde (see To this end, the phosphatase activity for Akt substrates in

Fig. 3C,D). Furthermore, both constructs induced phosphorylation of immature oocytes was compared with that after 1-MeAde treatment Journal of Cell Science

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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.

in the presence of wortmannin (in this situation, it is assumed that feedback pathway, which induces dephosphorylation of Akt 1-MeAde treatment activates the atypical Gβγ pathway while the substrates and thereby prevents subthreshold stimulus-induced PI3K-Akt-pathway is inhibited) (Fig. 7A). In order to evaluate the noise signaling, and an atypical Gβγ-pathway, which is distinct phosphatase activity in these oocytes, pre-phosphorylated AS- from the PI3K–Akt pathway and enhances Akt-catalyzed peptide was injected. Then dephosphorylation of the injected phosphorylation. We also found that full activation of the PI3K– phospho-peptide in the oocytes was analyzed by immunoblotting. Akt pathway alone was not sufficient for efficient phosphorylation The AS-peptide was dephosphorylated to similar extents in both of Akt sites on Cdc25 and Myt1 regardless of activation of cyclin- situations (Fig. 7B,C). This suggests that the atypical Gβγ pathway B–Cdk1-dependent negative feedback. Cooperation of the atypical does not alter phosphatase activity towards Akt substrates, at least in Gβγ pathway with the PI3K–Akt pathway first initiates activation of the absence of cyclin-B–Cdk1-dependent negative feedback, and cyclin-B–Cdk1 and then overrides the negative feedback that arises that the atypical Gβγ pathway can enhance Akt-catalyzed as a result; thereby, inducing full activation of cyclin-B–Cdk1 and phosphorylation without affecting phosphatase activity. the meiotic G2/M transition. In the context of threshold establishment, it is plausible to assume DISCUSSION that the activity of the atypical Gβγ-pathway depends on the dose of In this study, we identified two new pathways that regulate PI3K– 1-MeAde – higher concentrations of the hormone induce Akt-pathway-dependent phosphorylation of Akt substrates in an dissociation of a larger number of Gβγ subunits, which in turn opposing manner – the cyclin-B–Cdk1-dependent negative- enhances activation of the atypical Gβγ pathway. The point at which Journal of Cell Science

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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) Hypothesistested 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 −8 −3 oocytes for immature and Gβγ, six oocytes for CA-PI3K). P-values (one-tailed t-test): *1, P<10 ;*2,P<10 . (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, −5 −4 P>0.05 (n.s.); *2, *3, P<0.01; *4, P<10 . 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 . Journal of Cell Science

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scenarios that can be applicable to our results, we provide a simple one by introducing some speculation – first, the phosphatase activity towards Akt substrates detected in immature oocytes and in cyclin- B–Cdk1-dependent negative feedback events are attributed to the same molecule (an unknown phosphatase, referred to as PPase X); second, the atypical Gβγ pathway only enhances phosphorylation by Akt; third, the negative feedback resulting from cyclin-B–Cdk1 activation only activates PPase X. In unstimulated immature oocytes (Fig. 8A), all pathways are inactive, but an unknown phosphatase for Akt substrates, including Myt1 and Cdc25 (PPase X in Fig. 8), is active. Subthreshold stimuli (Fig. 8B) activate the PI3K–Akt pathway (Fig. 8; green) and the atypical Gβγ pathway (Fig. 8; cyan). Under these conditions, the atypical Gβγ pathway is activated to a level sufficient to override the PPase X basal activity but not to override the activity of PPase X that is enhanced by cyclin-B–Cdk1- dependent negative feedback. Thus, subthreshold signaling can initiate cyclin-B–Cdk1 activation, but this is reversed through the negative-feedback effects. The cyclin-B–Cdk1-dependent positive- feedback pathway (Fig. 8; black arrow from cyclin-B–Cdk1) (Kishimoto, 2015; Hara et al., 2012; Okumura et al., 2014) might be inactivated as a result of inactivation of cyclin-B–Cdk1, and thereby fail to form a strong positive-feedback loop. The activity of the atypical Gβγ pathway appears to depend on the dose of 1-MeAde. Supra-threshold doses of 1-MeAde sufficiently activate the atypical Gβγ pathway to override the cyclin-B–Cdk1-dependent negative feedback, thus maintaining phosphorylation of Akt sites on Cdc25 and Myt1 (Fig. 8C). Cyclin-B–Cdk1 then becomes maximally activated through a fully functional positive-feedback loop.

Phosphatase for Akt substrates in starfish oocytes We revealed an important role of phosphatase(s) in the regulation of phosphorylation levels of Akt substrates in starfish oocytes, although the enzyme responsible remains unclear. Protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) are reported to dephosphorylate Akt sites on Bad and GSK3β, respectively (Ayllón et al., 2000; Sutherland et al., 1993). In addition, microinjection of okadaic acid, which inhibits PP1, PP2A, protein phosphatase 4 and protein phosphatase 6, induces Fig. 7. The atypical Gβγ pathway does not alter phosphatase activity for – the meiotic G2/M-phase transition in starfish oocytes (Picard et al., Akt substrates in the absence of cyclin-B Cdk1-dependent negative 1989). Thus, PP1 and PP2A could be the presumptive candidates 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 for the Akt substrate phosphatases in starfish oocytes. Cyclin-B treated with a supra-threshold concentration of 1-MeAde (500 nM) for 4 min Cdk1-dependent negative feedback seems to activate a after pre-incubation with wortmannin were injected with the phosphorylated phosphatase because the rate of dephosphorylation of the Myt1- AS-peptide (AS-pep). Oocytes were collected at 45 s after injection, and S75-peptide was faster in the cyclin-B–Cdk1 negative-feedback analyzed by immunoblotting for phosphorylated and total protein (B). pathway than that in immature oocytes. Generally, cyclin-B–Cdk1 Phosphorylation of the AS-peptide (PAS) was quantified from images in shown is known to indirectly downregulate PP2A that contains the B55 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 regulatory subunit (PP2A-B55) through the inhibitory proteins experiments (C). P-values (one-tailed t-test): *1, *2, *3, P>0.05 (n.s.). Ensa or Arpp19 in M-phase (Castilho et al., 2009; Gharbi-Ayachi et al., 2010; Mochida et al., 2009, 2010; Vigneron et al., 2009). Thus, if PP2A is responsible for Akt substrate dephosphorylation activity of the atypical Gβγ pathway becomes predominant to that in starfish oocytes, its regulatory mechanism would be distinct of cyclin-B–Cdk1-dependent negative feedback could be the from that of the PP2A-B55 and Ensa (or Arpp19) system, rather threshold. Although we cannot yet directly test contribution of the using other regulatory subunits and regulatory proteins. atypical Gβγ pathway to threshold establishment with loss of function experiments, so far, our result that expression of the mutant Possible mechanisms of the cyclin-B–Cdk1-dependent Gβγ-D246S lowers the threshold for 1-MeAde to induce the meiotic negative feedback G2/M transition supports this idea. The finding of negative feedback through cyclin-B–Cdk1 is surprising because all known cyclin-B–Cdk1-dependent feedback A model for threshold-setting for the meiotic G2/M transition pathways are positive (Kishimoto, 2015; Lindqvist et al., 2009). The Based on present findings, we propose a model, shown in Fig. 8, for initial activation of cyclin-B–Cdk1 has been thought to lead to threshold-setting for the meiotic G2/M transition. In multiple formation of a positive-feedback loop that accelerates activation of Journal of Cell Science

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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.

cyclin-B–Cdk1 itself. Our results show for the first time that cyclin- Possible mechanisms for the atypical Gβγ pathway B–Cdk1 can put the brakes on its activation by itself. A remarkable observation in present study is that activation of In addition to upregulation of a phosphatase as discussed above, PI3K–Akt alone is not enough to cause Akt substrate cyclin-B–Cdk1-dependent negative feedback might downregulate phosphorylation, even in the absence of activation of the cyclin- the kinase activity of Akt. As for Cdk-dependent regulation of Akt B–Cdk1-dependent negative-feedback pathway, suggesting that activity, Liu et al. have reported that cyclin-A–Cdk2 promotes Akt Akt requires the input of additional mechanisms in order to activation through direct phosphorylation of Akt proteins in phosphorylate its substrates in intracellular environments. When the mammalian cells (Liu et al., 2014). However, the phosphorylation PI3K–Akt pathway is activated by Gβγ through G-protein-coupled sites are not conserved in starfish Akt, and the effect of the receptors, such as endothelin or serotonin receptors (Liu et al., 2003; cyclin-B–Cdk1-dependent negative feedback on Akt is, if any, Liu and Fanburg, 2006), the atypical Gβγ pathway would be rather negative. Because phosphorylation of Akt by TORC2 is activated and required for Akt to execute its cellular function. The independent of phosphorylation by PDK1 (Hiraoka et al., 2011), molecular identity of the supporting pathway might be different occurrence of TORC2-mediated phosphorylation does not always depending on cell and ligand type. ensure phosphorylation by PDK1. In addition, we can not directly Although the molecular details of the atypical Gβγ pathway are evaluate the phosphorylation of the PDK1 site on endogenous Akt unclear, our results indicate that this pathway can work so far. Therefore, we cannot completely exclude the possibility that independently of cyclin-B–Cdk1-dependent negative feedback phosphorylation of Akt by PDK1 is negatively regulated by and not alter the phosphatase activity towards Akt substrates. One cyclin-B–Cdk1-dependent negative feedback. However, this possibility would be the upregulation of the kinase activity of Akt mechanism is a less likely possibility because, in the present through Akt-binding regulators (upregulation of activators or study, the dynamics of PDK1-mediated phosphorylation of downregulation of inhibitors). exogenous human Akt1 were similar to those of TORC2- So far, we cannot completely exclude another possibility that the mediated phosphorylation in starfish oocytes. atypical Gβγ pathway upregulates another kinase (not Akt) that Alternatively, Akt-binding proteins could be involved. For could phosphorylate Akt substrates. Akt belongs to the AGC kinase example, c-Jun NH2-terminal kinase (JNK)-interacting protein 1 family (Pearce et al., 2010). Some kinases in the family have (JIP1) binds to and inhibits Akt independently of Akt redundant substrate specificity with Akt. Particularly, serum and phosphorylation in human prostate adenocarcinoma cells (Song glucocorticoid-regulated kinase (SGK) is reported to be activated by and Lee, 2005). This type of Akt regulator could function in PDK1 and TORC2, depending on PI3K (García-Martínez and cyclin-B–Cdk1-dependent negative feedback. Furthermore, Alessi, 2008; Kobayashi and Cohen, 1999). Although a regulatory another possibility that the cyclin-B–Cdk1-dependent negative mechanism of SGK activity that fits with the atypical Gβγ pathway feedback might downregulate the atypical Gβγ pathway should be has not been reported so far, investigation of the involvement of this considered. kinase could be interesting. Journal of Cell Science

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Threshold-setting in Xenopus oocytes δ, encoded by PIK3CA, PIK3CB, PIK3CG and PIK3CD, respectively), Xenopus oocytes are a well-established model for studying meiotic p110β and p110γ can be activated by Gβγ (Vanhaesebroeck et al., 2010). G2/M transition (Haccard and Jessus, 2006). Cyclin-B–Cdk1 Because p110γ exists only in mammals, we isolated a cDNA encoding the β activity has been shown to be bistable due to positive feedback by starfish homolog of p110 from a total mRNA library of immature oocytes using Xenopus egg extract (Novak and Tyson, 1993; Pomerening using the SMART RACE cDNA Amplification kit (Clontech). A partial cDNA fragment was isolated by performing reverse-transcriptase (RT)-PCR et al., 2003). In addition, mitogen-activated protein kinase (MAPK) ′ ′ Xenopus with degenerate primers. The 5 and 3 ends were identified by performing activity, which is involved in meiotic resumption in but not rapid amplification of cDNA ends (RACE) with specific primers. The full- in starfish oocytes, has been reported to exhibit an all-or-nothing length open reading frame was amplified by using RT-PCR and cloned into response to hormonal stimuli in Xenopus oocytes (Ferrell and the pGEM-T Easy vector (Promega). All accession numbers and primer Machleder, 1998). Furthermore, PKA-dependent phosphorylation sequences are shown in Tables S2 and S3, respectively. All primers were of Arpp19 at residue Ser109 has been reported to prevent the synthesized by Eurofins Genomics (Tokyo, Japan). meiotic G2/M transition. Dephosphorylation of this residue is required for the transition, and the extent of dephosphorylation is DNA constructs hormone-dose dependent (Dupré et al., 2014). These properties are DNA constructs used in this study are shown in Table S4. important to ensure an appropriate response to maturation-inducing hormones. However, to our knowledge, signaling dynamics in Protein preparation To prepare the recombinant GST-fused Akt peptide substrates, Escherichia response to subthreshold stimuli have never been investigated in the coli Xenopus strain BL21-CodonPlus-RIL (Agilent Technologies) was transformed system. Our study emphasizes the importance of with the pGEX-4T-1 constructs. Expression of recombinant proteins was examining these dynamics in the Xenopus model to more fully induced through a 2-h incubation with 1 mM isopropylthiogalactoside elucidate the network structure responsible for regulating the (IPTG), and proteins were purified using glutathione–Sepharose-4B (GE meiotic G2/M transition. However, the increased signaling Healthcare). For in vitro phosphorylation, the peptides were dialyzed against complexity in Xenopus oocytes, which includes de novo synthesis Tris-buffered saline (TBS) using EasySep membrane (TOMY; Tokyo, of proteins – such as Mos – and multiple phosphorylation events, Japan). Then, 240 μg of peptides were incubated in a 240-μl reaction might present a challenge (Haccard and Jessus, 2006; Schmitt and mixture containing active human Akt1 for 3 h at 30°C. The reaction was stopped by addition of EDTA. The phosphorylated peptide was purified Nebreda, 2002). – In summary, we demonstrate cyclin-B–Cdk1-dependent negative using glutathione Sepharose-4B to remove human Akt1. For – microinjection, sample concentration and buffer exchange (to PBS) were feedback, insufficiency of the PI3K Akt pathway for inducing Akt performed using a 10-k cutoff Vivaspin column (GE Healthcare). NP-40 substrate phosphorylation, and enhancement of Akt substrate was added at a final concentration of 0.1%. For His6–Cdc25, Gβ–His6 and βγ phosphorylation through an atypical G pathway. These findings p110β–His6, E. coli strain BL21 (DE3) (Invitrogen) was transformed with provide a model for establishing a hormonal dose threshold for each pET21a construct. His6–Cdc25 and Gβ–His6 were purified from cyclin-B–Cdk1 activation at the meiotic G2/M transition through inclusion bodies under denaturing conditions (6 M urea) using Probond- – regulating the phosphorylation of Akt sites on Cdc25 and Myt1. The Resin (Invitrogen). Purified His6 Cdc25 was refolded by performing features of these two new pathways refine our knowledge of the field stepwise reduction of the urea concentration with dialysis using EasySep for of cell cycle control and signal transduction. 2 h against TBS containing 4 M urea, for 2 h against TBS containing 2 M urea and then for 2 h against TBS without urea; the protein was then used for the in vitro phosphatase assay. Purified Gβ–His6 was dialyzed against PBS MATERIALS AND METHODS and used as the antigen. Starfish p110β–His6 was contained almost entirely Oocyte preparation in inclusion bodies. For antigen preparation, the protein was separated by Starfish Asterina pectinifera (renamed Patiria pectinifera in the 2007 SDS-PAGE, and the gels were stained with Coomassie brilliant blue R-250 β– National Center for Biotechnology Information Taxonomy Browser) were (CBB). The band corresponding with p110 His6 was excised from the gel collected during the breeding season and kept in laboratory aquaria that were and extracted using an Electro-Eluter instrument (Bio-Rad); it was then supplied with circulating seawater at 14°C. Fully grown immature oocytes dialyzed against PBS. For GST-tagged starfish p110β, the E. coli BL21 without follicle cells were released from isolated ovaries after treatment with strain was transformed with the pGEX-4T-1 construct. The expressed Ca2+-free artificial seawater (Ookata et al., 1992). All treatments with protein was almost entirely contained in inclusion bodies, thus separated by 1-MeAde or other chemicals, as well as microinjections, were performed in SDS-PAGE, transferred onto a PVDF membrane and stained with Ponceau artificial seawater at 23°C; 500 nM 1-MeAde was used as the supra- S. The band representing GST–p110β was excised, destained with TBS and threshold stimulus. The subthreshold concentration of 1-MeAde (the highest used for antibody purification. Recombinant fission yeast Suc1 protein concentration at which oocytes did not undergo GVBD) was determined for was bacterially produced and purified as described previously (Kusubata each animal and is indicated in the figure legends. et al., 1992), and then dissolved in 0.05% NP-40 at a concentration of 12.35 mg/ml for microinjection. Chemicals Roscovitine (Calbiochem) and wortmannin (LC Laboratories; Woburn, Antibody generation MA) were dissolved in DMSO at 20 mM as a stock solution, and used at a All antibodies in Table S5 were generated by immunizing rabbits, then final concentration of 30 μM and 40 μM, respectively. purified using antigen. Antigens for immunization and antibody purification are indicated in Table S5. Control IgG for microinjection was purified from Buffers rabbit pre-immune serum using protein-A–Sepharose-4B (Sigma-Aldrich). Composition of buffers and reaction mixtures are shown in Table S1. For microinjection, concentration and buffer exchange (to PBS) were performed using a 50-k cutoff Vivaspin column. NP-40 was added at a final cDNA cloning concentration of 0.1%. cDNA clones of starfish Gβ and Gγ were identified by performing homology searches in a cDNA library of starfish expressed sequence tags (ESTs) using Microinjection GNB1 GNGT1 human Gβ1 (encoded by ) and human Gγ1 (encoded by ), Microinjection was performed as described previously (Kishimoto, 1986). respectively, as queries, and verified by sequencing. PI3K is a heterodimer mRNAs for exogenous protein expression in starfish oocytes were consisting of a catalytic subunit (p110) and an adaptor subunit transcribed from pSP64-S constructs using the mMESSAGE

(Vanhaesebroeck et al., 2010). In four reported isoforms of p110 (α, β, γ, mMACHINE kit (Ambion), dissolved in water and injected into immature Journal of Cell Science

3164 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 3153-3166 doi:10.1242/jcs.182170 oocytes. Incubation time for protein expression (indicated in each legend) intensity and the area of each region were quantified. To avoid variation in was determined on the basis of the time to induce GVBD or to induce total intensity in the cytoplasmic region resulting from the presence or comparable Akt phosphorylation to that by 1-MeAde. The amounts of absence of the germinal vesicle on the focal plane, we used the fluorescence injected mRNAs or proteins per oocyte are shown in Table S6. intensity per area (fluorescence density) to calculate the ratio. In addition, the average autofluorescence density of intact immature oocytes, which do Immunoblotting not express PH–GFP, was defined as the background and subtracted from Oocytes were lysed by vortexing in Laemmli sample buffer, and then boiled the density of PH–GFP-expressing oocytes. Finally, the fluorescence for 5 min. The proteins were separated by performing SDS-PAGE density in the plasma membrane region was divided by that in the (Laemmli, 1970) and transferred onto a PVDF membrane (Millipore) cytoplasmic region to calculate the plasma-membrane-to-cytoplasmic ratio. (Towbin et al., 1979). Membranes were blocked in a blocking buffer for Translocation of PH–GFP was estimated by using the change in this ratio. detection of starfish phospho-Myt1 (at Ser75), starfish Myt1 and GST, but not for other proteins. The primary antibodies are indicated in the Table S7. Statistical analysis Alkaline-phosphatase- or horseradish peroxidase (HRP)-conjugated Statistical significance was calculated using unpaired one-tailed Student’s secondary antibodies were used. Proteins reacting with the antibodies t-test in Microsoft Excel with StatPlus (AnalystSoft; Walnut, CA). Statistical were detected as follows: for HRP, visualization with ECL prime (GE significance was defined as P<0.05. Healthcare), followed by acquisition of digital images on a LAS4000 imager (Fujifilm); for alkaline phosphatase, visualization with the BCIP/NBT Acknowledgements phosphatase substrate system (Kirkegaard and Perry Laboratories, We thank K. Tachibana for helpful discussion and suggestions, M. Terasaki Gaithersburg, MD), followed by acquisition of digital images with an (University of Connecticut Health Center, Farmington, CT) for the gift of the pSP64-S MP630 scanner (Canon; Tokyo, Japan). Quantification was performed vector and K. Chiba for generous support. We also thank Y. Mori and M. Katsuki for using Multi Gauge software version 3.0 (Fujifilm). Graph drawing and previous work. linear approximation were performed using Microsoft Excel. Brightness and contrast were adjusted equally across whole images using Photoshop Competing interests (Adobe). The authors declare no competing or financial interests. Author contributions In vitro Cdc25 phosphatase assay D.H. designed and performed all experiments other than the following. R.A. The phosphatase activity of Cdc25 was measured indirectly. For performed experiments with the Gβ-D246S mutant, S.H. and E.O. generated the phosphorylation of Cdc25 by Akt, recombinant His6-tagged starfish anti-phospho-Cdc25 Ser188 antibody and performed in vitro Cdc25 phosphatase Cdc25 was incubated with active human Akt1 in 6 μl of reaction mixture assays, D.H. and T.K. wrote the manuscript, and T.K. obtained funding and for 30 min at 30°C. Next, 1 μl of inactive cyclin-B–Cdk1 [100 ng; purified supervised the study. from immature starfish oocytes as described previously (Okumura et al., 1996)] was added. After additional incubation for 10 min at 30°C to allow Funding activation of cyclin-B–Cdk1 by Cdc25, 4 μl of histone H1 mixture was D.H. was supported by a postdoctoral fellowship from the Global Centers of Excellence program of the Japan Society for the Promotion of Science. This work added and incubated for an additional 20 min at 30°C to allow – μ was supported by grants-in-aid to T.K. from the Ministry of Education, Culture, phosphorylation of histone H1 by activated cyclin-B Cdk1. Finally, 11 l Sports, Science, and Technology [grant numbers 25291043, 15K14504]; and the of 2× Laemmli sample buffer was added, and the sample was boiled for Uehara Memorial Foundation. Deposited in PMC for immediate release. 3 min. The samples were run on a 12% SDS-PAGE gel and stained with CBB. The gels were autoradiographed using an imaging plate on a Supplementary information BAS2000 scanner (Fujifilm). Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.182170.supplemental H1 kinase assay References 7.5 μl of lysis buffer was added to 30 oocytes in 5 μl of artificial seawater. – Alessi, D. R., Caudwell, F. B., Andjelkovic, M., Hemmings, B. A. and Cohen, P. After one freeze thaw cycle, oocytes were lysed by vortexing, followed (1996). Molecular basis for the substrate specificity of protein kinase B; by centrifugation (16,000 g, for 15 min). 0.5 μl of supernatant was used for comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett. 399, 333-338. the H1 kinase assay. The residual supernatant was analyzed by Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R. J., immunoblotting. For the H1 kinase assay, the supernatant was mixed with Reese, C. B. and Cohen, P. (1997a). Characterization of a 3-phosphoinositide- 9.5 μl of reaction mixture containing histone H1 and incubated at 25°C for dependent protein kinase which phosphorylates and activates protein kinase 15 min. The reaction was stopped by adding Laemmli sample buffer, Balpha. Curr. Biol. 7, 261-269. Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., Norman, followed by boiling. After SDS-PAGE analysis on a 12.5% acrylamide gel, D. G., Gaffney, P., Reese, C. B., MacDougall, C. N., Harbison, D. et al. (1997b). phosphorylation of histone H1 was detected by autoradiography and 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional quantified by performing liquid scintillation counting. homology with the Drosophila DSTPK61 kinase. Curr. Biol. 7, 776-789. Ayllón, V., Martınez-A,́ C., Garcıa,́ A., Cayla, X. and Rebollo, A. (2000). Protein phosphatase 1α is a Ras-activated Bad phosphatase that regulates interleukin-2 Fluorescence imaging deprivation-induced apoptosis. EMBO J. 19, 2237-2246. Fluorescence images were obtained with the Fluoview FV1000 confocal Castilho, P. V., Williams, B. C., Mochida, S., Zhao, Y. and Goldberg, M. L. (2009). microscope (Olympus) using a 10× objective lens (UPLSAPO 10×; The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a Olympus). Living oocytes expressing PH–GFP in artificial seawater were phosphatase directed against CDK phosphosites. Mol. Biol. Cell 20, 4777-4789. set under the microscope. Fluoview software was used to acquire digital Chiba, K., Kontani, K., Tadenuma, H., Katada, T. and Hoshi, M. (1993). Induction of starfish oocyte maturation by the βγ subunit of starfish G protein and possible images from the photon detector of the FV1000 system. ImageJ software existence of the subsequent effector in cytoplasm. Mol. Biol. Cell 4, 1027-1034. (National Institutes of Health) was used for quantification. Translocation of Dorée, M. (1982). Protein synthesis is not involved in initiation or amplification of the PH–GFP was quantitatively estimated as follows. First, a region of interest maturation-promoting factor (MPF) in starfish oocytes. Exp. Cell Res. 139, (ROI) was defined along the cell periphery (ROI-out). Next, another ROI 127-133. was defined by reducing the size of the first ROI by 7 μm inward (ROI-in). Dupré, A., Daldello, E. M., Nairn, A. C., Jessus, C. and Haccard, O. (2014). We defined the area between ROI-out and ROI-in as the plasma membrane Phosphorylation of ARPP19 by protein kinase A prevents meiosis resumption in region and the area inside ROI-in as the cytoplasmic region. When the Xenopus oocytes. Nat. Commun. 5, 3318. Esposito, D. L., Li, Y., Cama, A. and Quon, M. J. (2001). Tyr(612) and Tyr(632) in germinal vesicle was included in the focal plane, an ROI outlining the human insulin receptor substrate-1 are important for full activation of insulin- germinal vesicle was defined, and the area outside of this ROI and inside stimulated phosphatidylinositol 3-kinase activity and translocation of GLUT4 in

ROI-in was defined as the cytoplasmic region. Next, the fluorescence adipose cells. Endocrinology 142, 2833-2840. Journal of Cell Science

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