Figure 4.

Instability of Emi2 in MI results from high Cdc2 kinase activity. (A) Oocytes were treated with progesterone, and MI and MII lysates were prepared. Recombinant GST-Emi2 protein (aa 319-375), conjugated to glutathione Sepharose beads, was (left) incubated in either MI or MII extract for 1 h at 4°C. Beads were washed five times with PBS (supplemented with 300 mM NaCl and 0.1% Triton). The amount of bound PP2A was detected by immunoblotting. Right, Rsk kinase was immunoprecipitated from both MI and MII extracts, washed five times with PBS (supplemented with 300 mM NaCl and 0.1% Triton), and its activity was measured by in vitro kinase assay using recombinant GST-Emi2 (aa 489-651) wild-type or T545/551A as substrate. (B) Oocytes were treated with progesterone, and lysates were made at the indicated times. Cdc2 kinase activity was measured by in vitro HHI kinase assay followed by autoradiography (top). Results were quantified by phosphorimager (bottom, left). Quantification of the average Cdc2 kinase activity in MII relative to MI is shown on the bottom right; error bar, SD of three independent experiments. (C) Left, CSF extract supplemented with recombinant GST-Emi2 (aa 489-651) T545/551A mutant protein was treated with or without recombinant nondegradable cyclin B (40 nM). ³⁵S-labeled Emi2 protein was added, and samples were taken at the indicated times and analyzed by SDS-PAGE and autoradiography. Right, Cdc2 kinase activity was measured by in vitro HHI kinase assay. Quantification of kinase activity was shown; error bar, SD of three independent experiments.