FIG. 3.

Inactivation of the PDK1-Akt signaling pathway during VP-16-induced 293T-cell apoptosis. (A and B) 293T cells were treated with the indicated concentrations of VP-16 for 36 h. (A) Endogenous Akt was immunoprecipitated from each cell lysate and subjected to Akt kinase activity assay as described in Materials and Methods. Each vertical bar represents the mean ± the standard deviation of three independent experiments. Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis with an anti-phospho-Akt (Ser⁴⁷³) antibody or an anti-Akt antibody (bottom). The results shown are representative of at least two independent experiments. (B) Cell lysates were incubated with DEVD-AMC for 1 h at 37°C. The increase in caspase 3-like protease (DEVDase) activity in the cell lysates was determined as described in Materials and Methods. Each vertical bar represents the mean ± the standard deviation of three independent experiments. (C) 293T cells were incubated with 10 μg of VP-16 per ml. At the indicated time points, cells were harvested and subjected to Western blot analysis with an anti-phospho-Akt (Ser⁴⁷³) antibody, an anti-Akt antibody, an anti-phospho-MAPK antibody, or an anti-MAPK antibody. (D) 293T cells were incubated with medium alone, 10 μg of VP-16 per ml, or 10 μg of VP-16 per ml plus 100 μg of Z-Asp per ml for 36 h. The increase in caspase 3-like protease (DEVDase) activity in the cell lysates was determined as described in Materials and Methods. Each vertical bar represents the mean ± the standard deviation of three independent experiments. Cell lysates were subjected to Western blot analysis with an anti-phospho-Akt (Ser⁴⁷³) antibody or an anti-Akt antibody (bottom). (E and F) 293T cells were incubated with the indicated concentration of VP-16 for 36 h. (E) Phosphotyrosine-containing proteins were immunoprecipitated with an antiphosphotyrosine antibody (PY20) and then incubated with phosphatidylinositol in the presence of 50 μM ATP containing 20 μCi of [γ-³²P]ATP. PI3K kinase activity was assessed by resolving the lipid fractions on a thin-layer chromatography plate following autoradiography. The amount of immunoprecipitated PI3K was confirmed by Western blot analysis with an anti-p85α subunit of PI3K (bottom). (F) PDK1 kinase activity was evaluated as described in Materials and Methods. The PDK1 kinase activity in untreated 293T cell lysates was normalized as 100%. Each vertical bar represents the mean ± the standard deviation of three independent experiments. The amount of immunoprecipitated PDK1 was confirmed by Western blot analysis with an anti-PDK1 antibody (bottom).