Okumura E , Morita A , Wakai M , Mochida S , Hara M , Kishimoto T .

	Figure 1. Arpp19 is required for cyclin B–Cdk1 activation regardless of the presence or absence of nucleus. (A) PP2A-B55 activity is possibly suppressed after 1-MeAde addition in enucleated as well as nucleated oocytes. Fizzy-pSer50 was injected into nucleated or enucleated oocytes at G2 phase (1-MeAde untreated, −) or M phase (1-MeAde treated, +; at a time equivalent to GVBD). 15 min later, phosphorylation states of Fizzy-Ser50 were analyzed by Western blot. Asterisk, nonspecific band. (B) Anti-Arpp19 antibody inhibits cyclin B–Cdk1 activation in nucleated oocytes. Immature oocytes were uninjected (none) or injected with either anti-Arpp19 antibody or control IgG, and then treated with 1-MeAde. After collection of oocytes at the indicated times, immunoblots were performed with the indicated antibodies. Numbers on the right indicate molecular mass. (C) Arpp19 is largely cytoplasmic. Protein amounts in immature oocytes were compared with immunoblots. Gwl is a nuclear marker and cyclin B is a cytoplasmic marker. (D) Anti-Arpp19 antibody inhibits cyclin B–Cdk1 activation also in enucleated oocytes. After enucleation (which removes Gwl), oocytes were injected and treated as in B. Note that even after 1-MeAde addition, phospho-Tyr15 in Cdk1 (pTyr15) remained detectable and Cdc25 remained inactive in anti-Arpp19 antibody-injected nucleated (B) and enucleated (D) oocytes. Brightness, contrast, and gamma settings were adjusted in the image presentation.
	Figure 2. Arpp19 is phosphorylated on Ser69 by cyclin B–Cdk1 regardless of the presence or absence of Gwl. (A and B) Arpp19 becomes phosphorylated even in enucleated oocytes after 1-MeAde addition. Nucleated or enucleated oocytes were treated with 1-MeAde, and 40 min later (equivalent to metaphase of meiosis I) conventional (A) and phos-tag (B) SDS-PAGE followed by anti-Arpp19 immunoblots were performed. Entry into M phase was confirmed by loss of pTyr15 in Cdk1. (C) Gwl restores enucleation effects on the phosphorylation pattern of Arpp19. Enucleated immature oocytes were uninjected or injected with recombinant Gwl (recGwl; inactive, WT; control kinase-dead, KD) and then treated with 1-MeAde. Phos-tag analysis as in B was performed in C–F. (D) Inhibition of Cdk1 prevents Arpp19 phosphorylation in enucleated oocytes. Enucleated oocytes were treated with 1-MeAde in the presence of the Cdk inhibitor roscovitine (20 µM; Ros) or control DMSO. (E) Arpp19 becomes phosphorylated not only on the Gwl site, Ser106, but also on a possible Cdk1 site, Ser69, after entry into M phase in nucleated oocytes. Nucleated immature oocytes were injected with wild type (WT) or each mutant of Arpp19 proteins (4A [T42A, S69A, S106A, and T155A], 2A [S69A and S106A], and 3A [T42A, S106A, and T155A]), and then treated with 1-MeAde. The 3A mutant, which retains Ser69, showed the band upshift. (F) Cyclin B–Cdk1 directly phosphorylates Arpp19 on Ser69 in vitro. Each of wild-type Arpp19 and the mutant proteins described in E was mixed with purified cyclin B–Cdk1. Wild type and the 3A mutant, both of which retain Ser69, showed the band upshift. (G and H) Arpp19 is phosphorylated on Ser69 in vivo after entry into M phase both in nucleated and enucleated oocytes. Nucleated (G) or enucleated (H) oocytes were treated with 1-MeAde, and oocyte extracts were prepared at the indicated times. Anti-Arpp19 immunoprecipitates were analyzed with anti-pSer69 antibody. Entry into M phase was confirmed by loss of pTyr15 in Cdk1 and by histone H1 kinase (H1K) activation. Enucleation was confirmed by loss of MCM2, a nuclear protein marker (H). Brightness, contrast, and gamma settings were adjusted in the image presentation.
	Figure 3. Ser69 phosphorylation of Arpp19 by cyclin B–Cdk1 converts Arpp19 into an active inhibitor of PP2A-B55. (A) Anti-Arpp19 antibody prevents in vitro phosphorylation on Ser69 of Arpp19 by cyclin B–Cdk1, but not on Ser106 by Gwl. Wild-type Arpp19 was preincubated with anti-Arpp19, control IgG, or PBS, and then phosphorylated with cyclin B–Cdk1 (left) or Gwl (right). Arpp19 phosphorylation was monitored by immunoblot with anti-pSer69 (left) or anti-pSer106 (right). (B) Estimation of the endogenous concentrations of B55 and Arpp19 proteins in starfish oocytes. Indicated amounts of recombinant GST-B55 (recB55; left) or recombinant Arpp19 (recArpp19; right) and/or five starfish oocytes were loaded and analyzed by Western blot. The concentrations of endogenous B55 (left) and Arpp19 (right) were calculated to be 0.3 and 1.3 µM, respectively, from protein amounts of 46 and 72 pg per 3 nl of oocyte volume. Asterisk, nonspecific bands. (C) Arpp19 phosphorylated in vitro on Ser69 by cyclin B–Cdk1 suppresses PP2A-B55 activity. Wild-type Arpp19 or the Ser69Ala (S69A) mutant protein was thiophosphorylated by cyclin B–Cdk1, Gwl, or both kinases, and then mixed with recombinant PP2A-B55 heterotrimers. Final concentrations of PP2A-B55 and Arpp19 in the mixture were adjusted to be 50 and 200 nM, respectively. After removal of kinases, phosphatase activity of PP2A-B55 was measured using Fizzy-pSer50 as a substrate. Each error bar indicates mean value ± SD from three independent experiments.
	Figure 4. Chromosome segregation in meiosis I is abortive in Gwl-inhibited oocytes. (A and B) Immature starfish oocytes were first injected with HiLyte 488–labeled tubulin (green) and Alexa 568–labeled histone H1 (magenta), and then injected with neutralizing anti-Gwl antibody (anti-Gwl) or control IgG (cont. IgG) along with ZZ-IBB (that can deliver cytoplasmically injected IgG into the nucleus) or uninjected (None). After 1-MeAde addition, live-cell images were obtained using a confocal microscope to monitor formation of the meiosis I spindle. Gwl-inhibited oocytes failed to properly segregate homologous chromosomes (A). This phenotype may be classified into three types, although they overlapped in some cases: lagging (#1, magenta), congressed (#2, yellow), or scattered (#3, blue) chromosomes along with frequently multipolar spindles (asterisks). However, further coinjection into immature oocytes with Arpp19 that had been in vitro thiophosphorylated by Gwl (pS106-Arpp19), but not with wild-type Arpp19 (wt-Arpp19), restored homologous chromosome segregation (B). Time after 1-MeAde addition is indicated in each frame. Arrow, chromosomes; arrowhead, the first polar body. Bars, 5 µm. (C) Quantification of chromosome segregation failure displayed in A and B. Each color corresponds to #1, #2, and #3 in A and B, respectively. Numbers of independent experiments (more than three females) are indicated at each bar.
	Figure 5. Model for the two-step phosphorylation of Arpp19 and the distinct roles of the two phosphorylations in governing M phase. First, phosphorylation on Ser69 by cyclin B–Cdk1 (red line) is involved in the autoregulatory activation of cyclin B–Cdk1. Second, further phosphorylation on Ser106 by Gwl (blue line) is needed for proper chromosome segregation, although it is not essential for the autoactivation. Initial activation corresponds to a trigger that reverses the balance between Cdc25 and Myt1/Wee1 before the first activation of cyclin B–Cdk1. Although molecular identity of the trigger remains elusive in most systems, it is clearly identified as Akt/PKB in the starfish oocyte (Okumura et al., 2002; Kishimoto, 2011): 1-MeAde causes activation of Akt/PKB, which in turn directly phosphorylates and inhibits Myt1 and phosphorylates and activates Cdc25, thus tipping the balance toward the initial activation of cyclin B–Cdk1.

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