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 antibody (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 insemination, 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.