Hosoda E , Hiraoka D , Hirohashi N , Omi S , Kishimoto T , Chiba K .

	Figure 1. sfSGK protein and phosphorylation of its A-loop are detectable in starfish oocytes. (A) The amino acid sequence of sfSGK was aligned with that of human SGK3. Colored boxes indicate conserved domains: PX domain (blue), A-loop (magenta), HM (purple), and catalytic domain (orange). Magenta and purple asterisks indicate conserved residues phosphorylated by PDK1 and TORC2, respectively (see also Fig. 2). (B) Unstimulated oocytes were incubated with 1-MA for 4 min, followed by immunoblotting with the indicated antibodies. Asterisks, nonspecific bands. (C) Immunoprecipitation (IP) was performed with anti-sfSGK-HM antibody or control (Cont.) IgG using extracts of unstimulated or 1-MA−stimulated oocytes. Input extract (Input), flow-through (FT), and beads (Beads) samples were analyzed by immunoblotting (IB). Asterisk, nonspecific bands. The indicated results in B and C are representative of three and two independent experiments, respectively. Closed and open arrowheads in B and C indicate positions of the upper and lower bands of sfSGK, respectively.
	Figure 2. sfSGK is activated by PDK1 and TORC2 in a PI3K-dependent manner. (A) Unstimulated oocytes were treated with 1-MA and analyzed by immunoblotting with anti-human phospho-SGK (sfSGK-pT312 [A-loop]), anti-sfSGK-HM (sfSGK), anti-sfAkt phospho-Ser477 (Akt-pS477[HM]), anti-sfAkt C-terminal fragment (Akt), anti-sfCdc25 (Cdc25), anti-Cdk1 phospho-Tyr15 (Cdk1-pY15), and anti-PSTAIR (Cdk1) antibodies. GVBD occurred at 17 min. (B) A piece of ovary was separated into oocytes and germinal epithelium, and subjected to immunoblotting. (C) To stimulate ovarian oocytes, 1-MA was injected into the body cavity of female starfish. Pieces of ovaries were then recovered at the indicated times and analyzed via immunoblotting or monitored for GVBD timing. GVBD occurred within 25 min. Spawning started at 30 min. (D) Unstimulated oocytes were treated with 1-MA for 4 min in the presence of indicated inhibitors or DMSO, and then subjected to normal SDS-PAGE, followed by immunoblotting. Closed and open arrowheads in A–D indicate the positions of the upper and lower sfSGK bands, respectively. Asterisks in A–D, nonspecific bands. (E and F) Unstimulated oocytes were treated with 1-MA for 4 min (E) or treated as in D (F), and then subjected to Phos-tag SDS-PAGE, followed by immunoblotting (IB). The results shown are representative of three independent experiments in A, E, and F, and two independent experiments in B–D.
	Figure 3. Activation of sfSGK is required for rapid pHi increase after 1-MA stimulus. (A) Unstimulated oocytes were injected with an anti-sfSGK-HM antibody or control IgG, treated with 1-MA for 4 min, and then subjected to immunoblotting. Asterisk, nonspecific bands. Closed and open arrowheads indicate positions of the upper and lower band of sfSGK, respectively. The result shown is representative of three independent experiments. (B) BCECF-dextran and either anti-sfSGK-HM antibody or control IgG were coinjected into unstimulated oocytes. After a 1-h incubation, 1-MA was added, and the fluorescence intensity ratio was measured before and after 1-MA addition. pHi was calculated from the fluorescence intensity ratio and plotted (means ± standard error [SE] of three independent experiments).
	Figure 4. Activation of sfSGK is required for cyclin B–Cdk1 activation. (A–C) Unstimulated oocytes were injected with anti-sfSGK-HM antibody or control IgG and treated with 1-MA, followed by monitoring GVBD (A; means ± SE of three independent experiments); imaging by DIC microscopy (B); and immunoblotting (C) at the indicated times. An oil drop introduced along with the antibodies as a mark of the injection can be seen on the right of the GV in B. Bar in B, 50 µm. Asterisk in C, nonspecific bands. Closed and open arrowheads in C indicate positions of the upper and lower bands of sfSGK, respectively. (D and E) Unstimulated oocytes were injected with anti-sfSGK-HM antibody, incubated for 1 h, and further injected with mRNA encoding a mutant sfSGK (T479E or K183M/T479E), followed by additional incubation for 22 h. These oocytes were treated with 1-MA, followed by immunoblotting (D) and monitoring GVBD (E; means ± SE of three independent experiments). exo. and endo. in D indicate exogenous and endogenous sfSGK, respectively. Lanes 3 and 12 in D represent oocytes that were collected at the time of GVBD under each condition (∼16 and ∼14 min, respectively). The results shown in B–D are representative of three independent experiments.
	Figure 5. Reduced pHi delays GVBD and blocks cytoplasmic granule invasion into the GV region. (A–C) To clamp the pHi at ∼6.7, 7.0, and 7.2, unstimulated oocytes were incubated with modified ASW for 20 min (see also Fig. S1 B). As a control, unstimulated oocytes were incubated in ASW for 20 min, in which the basal pHi is ∼7.0 but increases to ∼7.2 after 1-MA stimulation (Moriwaki et al., 2013; see also Fig. S1 B). These oocytes were stimulated with 1-MA, followed by immunoblotting (A); monitoring GVBD (B; means ± SE of three independent experiments); and time-lapse DIC imaging (C; images captured every 10 s; selected images after GVBD are shown; see Videos 1–4 for complete image sequences). Asterisk in A, nonspecific bands. Closed and open arrowheads in A indicate the positions of the upper and lower sfSGK bands, respectively. Bar in C, 50 µm. GVBD morphology is shown in Fig. S3. Z-stacks acquired after the time-lapse imaging are shown in Fig. S4. The results in A and C are representative of two independent experiments.
	Figure 6. F-actin shell, meshwork, and patches were formed at all clamped pHi values. (A and B) Oocytes stimulated with 1-MA in ASW or in modified ASW to clamp the pHi at ∼7.2, 7.0, or 6.7 were fixed upon GVBD or 5 min after GVBD and then triple-stained with phalloidin for F-actin (gray), anti-α-tubulin antibody for microtubules (green), and DAPI for chromosomes (magenta). Typical images of GV region (max projections and single slices) from two independent experiments are shown in A. Single slices including the actin patches (white boxes with enlarged images) observed 5 min after GVBD are shown in B. Bars in A and B, (main images) 10 µm, (insets) 5 µm.
	Figure 7. Reduced pHi perturbed chromosome transport and microtubule organization for spindle assembly. (A) Oocytes were treated and stained as in Fig. 6 at the indicated time points after GVBD. Typical images of GV region or spindles (max projection and/or single slices) from two independent experiments are shown. GVBD occurred around 17 min after 1-MA stimulation in ASW and around 13, 10, and 18 min at clamped pHi values of 7.2, 7.0, and 6.7, respectively. Bars, 10 µm. The stained samples were also used in B–D. (B) Oocytes with spindles were counted at the indicated times after GVBD. n.d., not determined. (C) Spindles assembled at clamped pHi values of 7.0 (45−50 min after GVBD) and 7.2 (20−30 min after GVBD) were checked for chromosome alignment (aligned or unaligned; typical images are shown). Arrowheads indicate unaligned chromosomes. Bars, 10 µm. (D) At the indicated time points after GVBD, oocytes at a clamped pHi of 6.7 were classified according to their chromosome distribution (typical images are shown): (i, cyan) scattered within the entire GV region; (ii, green) somewhat gathered toward the animal pole, but not in a compact group; (iii, magenta) gathered in a compact group near the animal pole. Dotted lines, boundary of the GV region. Bars, 10 µm. In B–D, results from two females (starfish 1 and 2) are shown. In B–D, 20 and 25 oocytes were observed at each time point in starfish 1 and 2, respectively.
	Figure 8. Model for sfSGK-dependent meiotic resumption in ovarian oocytes. A model for 1-MA–induced meiotic resumption in ovarian oocytes was prepared on the basis of previous and current findings. 1-MA stimulus induces Gβγ-dependent PI3K activation. In a manner that depends on PI3K, PDK1 and TORC2 activate sfSGK. Activation of sfSGK is essential for cyclin B–Cdk1 activation and sfNHE3-dependent pHi increase. These SGK-dependent pathways prefer lower pHi (magenta box). Processes leading to GVBD after cyclin B–Cdk1 activation prefer higher pHi (green box). After GVBD, actin-dependent chromosome gathering and microtubule organization for spindle assembly as well as cytoplasmic granule invasion into the GV region require a pHi of ∼7.0 or higher (cyan box) although spindles contains unaligned chromosomes at pHi ∼7.0. Because the pHi of ovarian oocytes before 1-MA stimulation in the body cavity is ∼6.7, the sfSGK-dependent pHi increase accelerates GVBD and is required for the processes leading to meiotic spindle assembly in ovarian oocytes. By contrast, the pHi of isolated oocytes before 1-MA stimulation in ASW is ∼7.0, which is already permissive for the processes toward spindle assembly.

Baltz, Intracellular pH regulation in the early embryo. 1993, Pubmed, Echinobase

Baltz, Intracellular pH regulation in the early embryo. 1993, Pubmed , Echinobase
Biondi, The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. 2001, Pubmed
Bun, A disassembly-driven mechanism explains F-actin-mediated chromosome transport in starfish oocytes. 2018, Pubmed , Echinobase
Burdyniuk, F-Actin nucleated on chromosomes coordinates their capture by microtubules in oocyte meiosis. 2018, Pubmed , Echinobase
Chiba, Evolution of the acquisition of fertilization competence and polyspermy blocks during meiotic maturation. 2011, 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
Chiba, The primary structure of the alpha subunit of a starfish guanosine-nucleotide-binding regulatory protein involved in 1-methyladenine-induced oocyte maturation. 1992, Pubmed , Echinobase
Dubé, Intracellular pH increase driven by an Na+/H+ exchanger upon activation of surf clam oocytes. 1997, Pubmed
Eltschinger, TOR Complexes and the Maintenance of Cellular Homeostasis. 2016, Pubmed
Flament, Xenopus oocyte maturation: cytoplasm alkalization is involved in germinal vesicle migration. 1996, Pubmed
García-Martínez, mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). 2008, Pubmed
Gasser, SGK3 mediates INPP4B-dependent PI3K signaling in breast cancer. 2014, Pubmed
Grandin, Cycling of intracellular pH during cell division of Xenopus embryos is a cytoplasmic activity depending on protein synthesis and phosphorylation. 1990, Pubmed
Grillo-Hill, Increased H⁺ efflux is sufficient to induce dysplasia and necessary for viability with oncogene expression. 2015, Pubmed
Hamaguchi, Regulation of intracellular pH in sea urchin eggs by medium containing both weak acid and base. 1997, Pubmed , Echinobase
Harada, Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH. 2003, Pubmed , Echinobase
Harada, Regulation of intracellular pH by p90Rsk-dependent activation of an Na(+)/H(+) exchanger in starfish oocytes. 2010, Pubmed , Echinobase
Harris, Conditions for assembly of tubulin-based structures in unfertilized sea urchin eggs. Spirals, monasters and cytasters. 1992, Pubmed , Echinobase
Hayashi, BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase. 2001, Pubmed
He, Serum- and glucocorticoid-induced kinase 3 in recycling endosomes mediates acute activation of Na+/H+ exchanger NHE3 by glucocorticoids. 2011, Pubmed
Hijikata, Revisiting gap locations in amino acid sequence alignments and a proposal for a method to improve them by introducing solvent accessibility. 2011, Pubmed
Hiraoka, Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition. 2016, Pubmed , Echinobase
Hiraoka, SGK phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation at the meiotic G2/M transition. 2019, Pubmed , Echinobase
Hiraoka, Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt. 2011, Pubmed , Echinobase
Hiraoka, PDK1 is required for the hormonal signaling pathway leading to meiotic resumption in starfish oocytes. 2004, Pubmed , Echinobase
Hotulainen, Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells. 2005, Pubmed
Hunt, Maturation promoting factor, cyclin and the control of M-phase. 1989, Pubmed
Kanatani, Isolation and indentification on meiosis inducing substance in starfish Asterias amurensis. 1969, Pubmed , Echinobase
Kanellos, Cellular functions of the ADF/cofilin family at a glance. 2016, Pubmed
Kinoshita, Phosphate-binding tag, a new tool to visualize phosphorylated proteins. 2006, Pubmed
Kishimoto, MPF-based meiotic cell cycle control: Half a century of lessons from starfish oocytes. 2018, Pubmed , Echinobase
Kishimoto, Entry into mitosis: a solution to the decades-long enigma of MPF. 2015, Pubmed
Kishimoto, Microinjection and cytoplasmic transfer in starfish oocytes. 1986, 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
Kobayashi, Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. 1999, Pubmed
Lee, pH changes associated with meiotic maturation in oocytes of Xenopus laevis. 1981, Pubmed
Lénárt, Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes. 2003, Pubmed , Echinobase
Lénárt, A contractile nuclear actin network drives chromosome congression in oocytes. 2005, Pubmed , Echinobase
Lien, PI3K signaling in cancer: beyond AKT. 2017, Pubmed
Liu, H+- and Na+- elicited rapid changes of the microtubule cytoskeleton in the biflagellated green alga Chlamydomonas. 2017, Pubmed
Lu, mTOR complex-2 activates ENaC by phosphorylating SGK1. 2010, Pubmed
Malik, Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks. 2018, Pubmed
Masui, From oocyte maturation to the in vitro cell cycle: the history of discoveries of Maturation-Promoting Factor (MPF) and Cytostatic Factor (CSF). 2001, Pubmed
Miyazaki, Fast polyspermy block and activation potential. Correlated changes during oocyte maturation of a starfish. 1979, Pubmed , Echinobase
Miyazaki, Potassium rectifications of the starfish oocyte membrane and their changes during oocyte maturation. 1975, Pubmed , Echinobase
Mori, An Arp2/3 nucleated F-actin shell fragments nuclear membranes at nuclear envelope breakdown in starfish oocytes. 2014, Pubmed , Echinobase
Mori, Intracellular transport by an anchored homogeneously contracting F-actin meshwork. 2011, Pubmed , Echinobase
Moriwaki, Arrest at metaphase of meiosis I in starfish oocytes in the ovary is maintained by high CO2 and low O2 concentrations in extracellular fluid. 2013, Pubmed , Echinobase
Nurse, Universal control mechanism regulating onset of M-phase. 1990, Pubmed
Ochi, Block of CDK1-dependent polyadenosine elongation of Cyclin B mRNA in metaphase-i-arrested starfish oocytes is released by intracellular pH elevation upon spawning. 2016, Pubmed , Echinobase
Oita, Degradation of polyubiquitinated cyclin B is blocked by the MAPK pathway at the metaphase I arrest in starfish oocytes. 2004, Pubmed , Echinobase
Okano-Uchida, In vivo regulation of cyclin A/Cdc2 and cyclin B/Cdc2 through meiotic and early cleavage cycles in starfish. 1998, Pubmed , Echinobase
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
Orlowski, Diversity of the mammalian sodium/proton exchanger SLC9 gene family. 2004, Pubmed
Pearce, The nuts and bolts of AGC protein kinases. 2010, Pubmed
Regula, Microtubule assembly and disassembly at alkaline pH. 1981, Pubmed
Rezai, Protooncogene product, c-mos kinase, is involved in upregulating Na+/H+ antiporter in Xenopus oocytes. 1994, Pubmed
Sadler, Components of the signaling pathway linking the 1-methyladenine receptor to MPF activation and maturation in starfish oocytes. 1998, Pubmed , Echinobase
Schatten, Intracellular pH shift initiates microtubule-mediated motility during sea urchin fertilization. 1986, Pubmed , Echinobase
Sellier, Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes. 2006, Pubmed
Shilling, Pertussis toxin inhibits 1-methyladenine-induced maturation in starfish oocytes. 1989, Pubmed , Echinobase
Stith, Increased intracellular pH is not necessary for ribosomal protein s6 phosphorylation, increased protein synthesis, or germinal vesicle breakdown in Xenopus oocytes. 1985, Pubmed
Strickland, Light microscopy of echinoderm embryos. 2004, Pubmed , Echinobase
Tachibana, c-Mos forces the mitotic cell cycle to undergo meiosis II to produce haploid gametes. 2000, Pubmed , Echinobase
Tachibana, MAP kinase links the fertilization signal transduction pathway to the G1/S-phase transition in starfish eggs. 1997, Pubmed , Echinobase
Takahashi, p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1. 1999, Pubmed
Tessier, Serum and glucocorticoid-regulated protein kinases: variations on a theme. 2006, Pubmed
Tessier, Role of the Phox homology domain and phosphorylation in activation of serum and glucocorticoid-regulated kinase-3. 2006, Pubmed
Usui, Involvement of mitogen-activating protein kinase and intracellular pH in the duration of the metaphase I (MI) pause of starfish oocytes after spawning. 2008, Pubmed , Echinobase
Vanhaesebroeck, The emerging mechanisms of isoform-specific PI3K signalling. 2010, Pubmed
Wang, Activation of NHE3 by dexamethasone requires phosphorylation of NHE3 at Ser663 by SGK1. 2005, Pubmed
Watanabe, Effects of intracellular pH on the mitotic apparatus and mitotic stage in the sand dollar egg. 1997, Pubmed , Echinobase
Yeoh, Determining the differences in actin binding by human ADF and cofilin. 2002, Pubmed
Yun, Concerted roles of SGK1 and the Na+/H+ exchanger regulatory factor 2 (NHERF2) in regulation of NHE3. 2003, Pubmed