FIGURE 8.

ASK1 negatively regulates PDK1-dependent apoptosis and cell growth. A, effect of ASK1 on PDK1-mediated apoptosis. 293T cells were transiently transfected with the expression vectors indicated. After 24 h, cells were treated with TNF-α (20 ng/ml) and cycloheximide (10 μg/ml) for 14 h to induce apoptosis. Apoptotic cell death was determined by the GFP system and TUNEL staining. B, effect of ASK1 on PDK1-mediated cell cycle progression. HaCaT cells (2 × 10⁵/dish), transfected with the indicated combinations of plasmid vectors, were synchronized in G₀/G₁ by hydroxyurea treatment (30). Cells were collected after 10% serum treatment for 24 h and evaluated for determination of cell numbers in G₀/G₁, S, and G₂/M phases by flow cytometry analysis. PDK1(S394A/S398A), the wild-type PDK1 mutant S394A/S398A. C, ASK1-intact MEFs or ASK1-deficient MEFs were incubated for 30 min with or without 100 nm wortmannin and then treated with 100 nm insulin for 20 min. The cell lysates were subjected to immunoblot analysis with the antibodies indicated. β-Actin was used as a loading control. D, a model for the regulation of PDK1-ASK1 complex formation in response to signals that act on PDK1 or ASK1. The dotted and solid lines represent ASK1 and PDK1 signals, respectively. Details of this model are given under “Discussion.” WB, Western blot.