FIGURE 7.

ASK1 phosphorylates PDK1. A, for the in vitro kinase assay, ∼5 μg of recombinant PDK1(KD) proteins were mixed with 20 μm ATP, 0.3 μCi of [γ-³²P]ATP, and 20 mm MgCl₂ in 30 μl of kinase buffer and incubated with the HA immunoprecipitates for HA-ASK1 at 30 °C for 30 min with frequent gentle mixing. B, identification of ASK1 phosphorylation sites on PDK1. The recombinant ASK1 protein was analyzed for its kinase activity by an in vitro kinase assay using recombinant PDK1-N, PDK1-C, substitution mutants (S258A/S262A, S394A/S398A, or T518A/T522A) of PDK1-C, and the positive control MKK6(K82A) as substrates. C, effect of ASK1 on PI3K/PDK1-mediated signaling. HEK293 cells, transfected with the expression vectors indicated, were lysed, and the GST precipitates were then analyzed for PDK1 activity by an in vitro kinase assay using serum/glucocorticoid-regulated kinase as a substrate and by immunoblot analysis with an anti-phospho-PDK1(Ser²⁴¹) antibody (left). The Akt and Bad immunoprecipitates were also analyzed for Akt and Bad activation by immunoblotting with anti-phospho-Akt(Thr³⁰⁸) and anti-phospho-Bad(Ser¹³⁶) antibodies (middle and right). The circled letters P indicate phosphorylation. re., recombinant. WB, Western blot.