Figure 4

Activation of Xenopus unfertilized eggs by bp(V), a potent inhibitor of PTEN. (A) Shown are representative photographs 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 control. Src activity in the absence of phospholipids was taken as 100%.