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	Figure 1. SGK is activated after 1-MeAde stimulation in a PDK1- and TORC2-dependent manner. (A) The region of sfAkt used to raise the anti-phospho-A-loop antibody (Lys304–Pro320 of sfAkt) is aligned with the corresponding region of sfSGK (Lys301–Pro317; see also Fig. S1 A). The arrowhead indicates the residue phosphorylated by PDK1. Dots indicate identical residues. (B) A-loop phosphorylation of endogenous sfAkt is undetectable in whole oocyte extracts. Oocytes injected with or without sfAkt-mRNA were treated with 1-MeAde and analyzed by immunoblotting with the anti-phospho-A-loop (pA-loop) and anti-sfAkt-C (Akt) antibodies. Closed arrowheads indicate A-loop phosphorylation of Akt. Open arrowheads indicate the positions of active SGK (see E and F). exp., exposure. (C and D) A-loop phosphorylation of endogenous Akt is detectable in immunoprecipitates. Oocytes were incubated with 1-MeAde in the presence or absence of a PDK1 inhibitor, BX795, and subjected to immunoprecipitation with control IgG or the anti-sfAkt-C antibody. Input and flow-through (FT) extracts (C) and precipitates (D) were analyzed by immunoblotting (Akt-pHM represents phosphorylation of Ser477 in the HM of Akt). Closed arrowheads in D indicate A-loop phosphorylation of Akt. A-loop phosphorylation of nonspecifically included SGK was detected in both control and Akt immunoprecipitates (D, open arrowheads in pA-loop). IP, immunoprecipitation. (E) Unstimulated and 1-MeAde–treated oocytes were subjected to immunoblotting (IB) with anti-phospho-A-loop (left), anti-sfSGK-HM (middle), and anti-sfAkt-C (right) antibodies. Closed and open arrowheads indicate positions of Akt and active SGK, respectively. (F) The anti-phospho-A-loop antibody against sfAkt cross-reacts with A-loop-phosphorylated endogenous SGK. Oocytes were treated with 1-MeAde. Immunoprecipitation was performed with control IgG or the anti-sfSGK-HM antibody. (G) SGK is activated simultaneously with Akt activation. Oocytes were treated with 1-MeAde for indicated durations and analyzed by immunoblotting. GVBD occurred at ∼20 min. (H) SGK activation depends on TOR. Oocytes were injected with control IgG or the anti-TOR antibody, treated with 1-MeAde, and analyzed by immunoblotting. Open arrowheads in B–H indicate the position of active SGK. The data are representative of two independent experiments in B, E, F, and H and three independent experiments in C, D, and G. Full blots for B–H are presented in Fig. S5.
	Figure 2. SGK, but not Akt, is required for the meiotic G2/M transition. (A) Immunoprecipitation (IP) with control IgG or the anti-sfAkt-C antibody was performed using extracts of unstimulated oocytes, followed by immunoblotting (IB) with the anti-sfAkt-C antibody. FT, flow-through. (B–E) Inhibition of activation of Akt and SGK by specific antibodies. Oocytes were injected with the anti-sfAkt-C antibody (B and C), the anti-sfSGK-HM antibody (D and E), or control IgG (B–E) and then treated with 1-MeAde for 4 min. Immunoprecipitation with the anti-sfAkt-C antibody was performed. Input extracts (B and D) and precipitates (C and E) were analyzed by immunoblotting. Open arrowheads indicate the position of active SGK. Asterisks indicate nonspecific bands. The data are representative of two independent experiments in A–C and three independent experiments in D and E. (F and G) SGK, but not Akt, is required for the meiotic G2/M transition. Oocytes were injected with the anti-sfAkt-C antibody (F), the anti-sfSGK-HM antibody (G), or control IgG (F and G) and then treated with 1-MeAde. GVBD was monitored as a marker of the meiotic G2/M transition. The graphs show the percentage of oocytes that had undergone GVBD by the indicated time points (mean ± SD from three independent experiments). Full blots for A–E are presented in Fig. S6.
	Figure 3. SGK directly phosphorylates Cdc25 and Myt1. (A) SGK, but not Akt, is required for Cdk-independent phosphorylation of Cdc25 and Myt1 upon 1-MeAde stimulation. Oocytes were injected with the indicated antibodies, treated with 1-MeAde in the presence of roscovitine, and then analyzed by immunoblotting using a normal or Phos-tag–containing SDS-PAGE gel. Open arrowheads indicate position of active SGK. (B) His-tagged recombinant WT sfCdc25 or the S188A mutant was incubated with or without GST-tagged active hSGK3 in the presence of ATP and MgCl2. Thereafter, immunoblotting was performed. (C) GST-tagged recombinant kinase-dead (KD) or the S75A mutant of sfMyt1 was phosphorylated by hSGK3 as described in B. Asterisks indicate minor fragments of recombinant Myt1. The data in A–C are representative of two independent experiments. Full blots for A–C are presented in Fig. S7.
	Figure 4. SGK is the major kinase that phosphorylates the Akt/SGK consensus motif in starfish oocytes. (A and B) Kinase activity for the AS peptide is detectable in an extract of 1-MeAde–treated oocytes. Extracts were prepared from unstimulated or 1-MeAde–treated (3 min) oocytes and incubated with the GST-AS peptide for the indicated durations. Phosphorylation of the peptide was detected by immunoblotting with an anti-pan-phospho-Akt/SGK substrate antibody (PAS), which recognizes phosphorylated Ser or Thr in the Akt/SGK consensus motif (A). Phosphorylation of the peptide was quantitated from the blots in A and normalized against the total amount of the peptide. The graph shows the levels of phosphorylation relative to that at 60 min (B; mean ± SD from three independent experiments). (C–H) SGK, but not Akt, is responsible for the detectable AS-peptide kinase activity in the extract. An extract was prepared from 1-MeAde–treated oocytes (Crude). Immunodepletion was performed with control IgG (mock), the anti-sfAkt-C antibody (ΔAkt), or the anti-sfSGK-HM antibody (ΔSGK). Immunoblot of the extracts (C and F) and GST-AS peptide kinase assay using the extracts (D and G) are shown. Phosphorylation was quantitated as described in A and B. Graphs show the levels of phosphorylation relative to those at 15 min in the crude extract of 1-MeAde–treated oocytes (E from the blot in D, and H from the blot in G; mean ± SD from three independent experiments). Open arrowheads in F indicate the position of active SGK. Full blots for A, C, D, F, and G are presented in Fig. S7.
	Figure 5. SGK is sufficient for the meiotic G2/M transition. (A–C) CA-SGK expression induces the meiotic G2/M transition. Unstimulated oocytes were injected with mRNA encoding CA-SGK. GVBD was monitored every 15 min during incubation. The graph shows percentage of oocytes that had undergone GVBD by the indicated time points (A; mean ± SD from three independent experiments). CA-SGK–expressing oocytes at GVBD, unstimulated oocytes, and 1-MeAde–treated oocytes were analyzed by immunoblotting (B). Phosphorylation levels of the A-loop of endogenous SGK in 1-MeAde–treated oocytes and CA-SGK at GVBD were quantitated from the immunoblots in B. The graph shows relative phosphorylation levels (C; mean ± SD from three independent experiments). NS, P = 0.06 (one-tailed t test). (D) CA-SGK expression induces regulatory phosphorylation of Cdc25 and Myt1 to trigger cyclin B–Cdk1 activation. Unstimulated oocytes were treated with 1-MeAde for 4 min in the presence of roscovitine. For expression of CA-SGK, unstimulated oocytes were injected with the mRNA, incubated with roscovitine, collected at the time point at which 50% of CA-SGK–expressing oocytes exhibited GVBD in the absence of roscovitine (5 h 45 min in this sampling), and analyzed by immunoblotting. Note that in the immunoblot of phospho-A-loop after a long exposure (pA-loop, long exp.), weak nonspecific bands were detected at almost the same size as CA-SGK (asterisk). The data are representative of two independent experiments. Full blots for B and D are presented in Fig. S8.
	Figure 6. SGK is activated by cooperation of the Gβγ-PI3K and atypical Gβγ pathways. (A and B) Exogenous expression of Gβγ induces the meiotic G2/M transition in a SGK-dependent manner. Unstimulated oocytes were injected with the indicated antibodies and subsequently injected with mRNA encoding Gβγ. Thereafter, GVBD was monitored (A; mean ± SD from three independent experiments). Oocytes were collected when GVBD occurred. Anti-sfSGK-HM antibody–injected oocytes, which did not undergo GVBD, were collected 5 h after mRNA injection. For 1-MeAde treatment, unstimulated oocytes were treated with 1-MeAde for 4 or 25 min (GVBD occurred at ∼21 min) and then analyzed by immunoblotting (B). (C) Exogenous Gβγ expression induces SGK activation. Unstimulated oocytes were treated with 1-MeAde for 4 min in the presence of roscovitine or injected with mRNA encoding Gβγ and then incubated with roscovitine for 6 h. Thereafter, extracts were prepared from these oocytes and analyzed by immunoblotting. (D) Gβγ-induced SGK activation requires PI3K. Unstimulated oocytes were treated with 1-MeAde or injected with mRNA encoding Gβγ, incubated in the presence of roscovitine with or without wortmannin, and analyzed by immunoblotting. (E) PI3K is not sufficient for activation of SGK. Unstimulated oocytes were treated with 1-MeAde for 4 min or injected with mRNA encoding CA-PI3K and incubated for 3 h. Extracts of CA-PI3K–expressing oocytes were immunodepleted using control IgG (Mock) or the anti-sfAkt-C antibody (ΔAkt) and analyzed by immunoblotting. A closed arrowhead in the blot of pA-loop indicates the position of Akt. (F) Expression of exogenous Gβγ fails to induce A-loop phosphorylation of Akt. Endogenous Akt was immunoprecipitated (IP) from the extracts in C and analyzed by immunoblotting. Open arrowheads in B–F indicate the position of active SGK. The data are representative of two independent experiments in B, D, and E and three independent experiments in C and F. Full blots for B–F are presented in Fig. S8.
	Figure 7. SGK triggers cyclin B–Cdk1 activation in 1-MeAde signaling. Downstream of 1-MeAde stimulation, Gβγ activates the PI3K pathway (green arrow) and the atypical Gβγ pathway (cyan arrow). SGK is activated by cooperation of these pathways and subsequently activates and inactivates Cdc25 and Myt1, respectively, via direct phosphorylation. Consequently, reversal of the balance of Cdc25 and Myt1 activities triggers cyclin B–Cdk1 activation. Akt is weakly activated after 1-MeAde stimulation and may phosphorylate Cdc25 and Myt1 at undetectable levels. However, Akt is dispensable for cyclin B–Cdk1 activation and may have other functions in oocytes.

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