Mammadova G , Iwasaki T , Tokmakov AA , Fukami Y , Sato K .

	Figure 1. Inhibition of early events of Xenopus egg fertilization by LY294002, a potent PI 3-kinase inhibitor. (A) Xenopus unfertilized eggs were microinjected with DMSO alone (lanes 1 and 2), 10 μM PP2 (lane 3), 10 μM PP3 (lane 4), 10 μM U73122 (lane 5), or 10 μM LY294002 (lane 6), and subjected to no treatment (lane 1, - sperm) or insemination for 5 min (lanes 2-6, + sperm). SDS (0.1%)-solubilized egg membrane fractions (500 μg/lane) abundant in Src and PLCγ were analyzed for tyrosine phosphorylation of Src (top panel, IB: phospho-Src), activation of Src (middle panels, IVKA: phospho-Cdk1; IVKA: protein amounts of the immunoprecipitated Src), and tyrosine phosphorylation of PLCγ (bottom panel, IB: phospho-PLC gamma) as described in "Methods". Asterisks in each panel indicate the positions of the protein bands of interest. (B) Shown are representative traces of sperm-induced Ca2+ release, as monitored by the fluorescent ratio signal, in Xenopus eggs that were co-injected with fura-2 and LY294002 (0 or 10 μM) and inseminated as described in "Methods". (C) Triton X-100-solubilized extracts (100 μg/lane) were prepared from Xenopus eggs that were injected with the indicated inhibitors (lanes 3-5, each 10 μM) and inseminated for 40 min. Extracts from DMSO (0.2%)-injected unfertilized (lane 1) or fertilized eggs (lane 2) were also prepared as controls. Samples were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against phospho-MAPK (top panel), cyclin B2 (middle panel), or Mos (bottom panel) as described in "Methods". (D) The occurrence of first embryonic cleavage was evaluated in fertilized Xenopus embryos that were injected with the indicated concentrations of LY294002 and inseminated for 100 min. Values indicated on each bar are the number of cleaved embryos per number of embryos tested.
	Figure 2. Subcellular localization and tyrosine phosphorylation of an 85-kDa subunit of PI 3-kinase before and after fertilization of Xenopus eggs. (A) Triton X-100-solubi- lized extracts of Xenopus unfertilized eggs (lane 1, 100 μg/ lane) and 293 human embryonic kidney cells (lane 2, 20 μg/ lane) were separated by SDS-PAGE and analyzed by immuno- blotting with the anti-p85 subunit of PI 3-kinase antibody. An asterisk indicates the position of the immunoreactive 85-kDa protein (doublets in the egg extracts). Molecular size mark- ers (in kDa) used are also indicated. (B) Low density, deter- gent-insoluble membrane (LD-DIM) (left panels) and detergent-soluble (right panels) fractions were prepared from Xenopus eggs that had been untreated (lanes 1 and 6) or activated by 106/ml sperm (lanes 2 and 7), 0.5 μM A23187 (lanes 3 and 8), 10 mM H2O2 (lanes 4 and 9), or 5 U/ml cathepsin B (lanes 5 and 10). Protein samples (LD-DIM frac- tions, 5 μg/lane; detergent-soluble fractions, 800 μg/lane) were immunoprecipitated with the anti-p85 subunit of PI 3- kinase antibody and the immunoprecipitates were analyzed by immunoblotting with the same antibody (IB: p85) or with the anti-phosphotyrosine antibody (IB: phospho-p85). Aster- isks indicate the positions of the p85 bands in each panel. (C) LD-DIM fractions were prepared from Xenopus eggs that had been inseminated for the periods indicated (0-40 min). Pro- tein samples (5 μg/lane) were analyzed for the presence of PLC gamma, p85 subunit of PI 3-kinase, or tyrosine-phosphorylated UPIII (pUPIII) by immunoprecipitation and immunoblotting (IB) as described in "Methods". Triton X-100-solubilized extracts of Xenopus unfertilized eggs (100 μg/lane) were also analyzed for the presence of phosphorylated MAPK (pMAPK) by direct immunoblotting as in Figure 1C. Asterisks indicate the positions of the protein bands of interest.
	Figure 3. Phosphorylation and translocation of a serine/threonine-specific protein kinase Akt in fertilized Xenopus eggs. (A) Triton X-100-solubilized extracts of Xenopus unfertilized eggs (lane 1, 100 μg/lane) and 293 human embryonic kidney cells (lane 2, 20 μg/lane) were separated by SDS-PAGE and analyzed by immunoblotting with the anti-mammalian Akt antibody. Molecular size markers (in kDa) used are also indicated. (B) Xenopus unfertilized eggs were microinjected with DMSO alone, 10 μM PP2, or 10 μM LY294002, and subjected to no treatment (0 min) or insemination for the periods indicated (1-20 min). Triton X-100-solubilized extracts (100 μg/lane) were prepared and analyzed by immunoblotting with the anti-Akt antibody (IB: Akt), anti-phosphorylated serine-473-specific antibody (IB: pSer473-Akt), or anti-phosphorylated threonine-308-specific anti- body (IB: pThr308-Akt). (C) Effect of microinjection of PP2 (lane 3), LY294002 (lane 4), or PP3 (lane 5) on sperm-induced phosphorylation of Akt was evaluated using unfertilized (UF, lane 1) and 5-min inseminated egg samples (F5, lanes 2-5) by immunoblotting as in panel B. (D) Xenopus unfertilized eggs (UF) and 5-min inseminated eggs (F5) were subjected to subcellular fractionation by discontinuous sucrose density gradient ultracentrifugation as described in "Methods". The twelve fractions obtained were analyzed by immunoblotting with anti-Akt antibody (IB: Akt). The positions of LD-DIM fractions (fractions 4-6, 10-time concentrated) and detergent-soluble, non-microdomain fractions (fractions 10-12) are indicated. (E) LD-DIM fractions and soluble, non-microdomain fractions were prepared from unfertilized eggs (UF) and fertilized eggs (1-20 min after insemina- tion, F1-F20). Protein samples of the same egg-equivalent amounts (about 10 eggs per lane) were analyzed for the presence of Akt by immunoprecipitation and immunoblotting with the anti-Akt antibody (IP: Akt, IB: Akt). (F) Detergent-soluble, non-microdomain fractions (20 eggs per lane) and LD-DIM fractions (2 eggs per lane) that had been prepared from unfertilized eggs (UF) and 5-min inseminated eggs (F5) were analyzed by immunoblotting with either the anti-Akt antibody (IB: Akt) or anti- phosphorylated threonine-308-specific antibody (IB: pThr308-Akt). (G) Effect of LY or PP2 on the sperm-induced translocation of the p85 subunit of PI 3-kinase (upper panel) and Akt (lower panel) was evaluated by immunoblotting of LD-DIM fractions that had been prepared from unfertilized eggs (UF) or 5-min fertilized eggs (F5). In all panels, asterisks indicate the positions of Akt or p85.
	Figure 4. Activation of Xenopus unfertilized eggs by bp(V), a potent inhibitor of PTEN. (A) Shown are representative photo- graphs of Xenopus eggs before and after 20-min treatment with 200 μM bp(V). (B) The occurrence of cortical contraction as a function of time was monitored in Xenopus eggs that had been treated with sperm alone (sperm, open squares), 200 μM bp(V) alone (bp(V), open circles), sperm plus pre-injected 10 μM LY294002 (sperm + LY, closed squares), or 200 μM bp(V) plus pre- injected 10 μM LY294002 (bp(V) + LY, closed circles). (C) Xenopus eggs that had been treated with or without 200 μM bp(V) and 10 μM LY294002 for 30 min were subjected to extraction of the membrane fractions. The SDS-solubilized membrane extracts (500 μg per lane) were analyzed for tyrosine phosphorylation of Src as in Figure 1A. (D) Purified Src was pretreated with several phospholipids at the indicated concentrations (0-100 μM) and subjected to in vitro kinase assays as described in "Methods". Autophosphorylation of Src was analyzed by immunoblotting of the reaction mixtures with the phosphorylated tyrosine-419-specific antibody. Shown on the left are representative immunoblotting data. Results shown in the right are the mean s.e.m. of four independent experiments (at 100 μM of each phospholipids). *P < 0.01 compared with levels in the con- trol. Src activity in the absence of phospholipids was taken as 100%.

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