Fig. 3.

PID autophosphorylation is induced by PDK1 phosphorylation. (A) His:PDK1-containing bacterial lysate was incubated with GST, GST:PID, or GST:MPID bound to glutathione-agarose beads. Half of the sample was used to confirm PDK1 binding by Western blot analysis by using anti-polyhistidine antibody. (B) The remaining sample was used in a kinase assay in the presence of [γ-³²P]ATP and subjected to PAGE. Incorporation of radiolabeled phosphate was determined by autoradiography. Binding and phosphorylation analyses were confirmed by three independent experiments. (C) An activation loop serine is required for PDK1 activation of PID. Wild-type and ALS mutant protein (see Fig. 1A) phosphorylation was analyzed in the absence or presence of PDK1 protein. Results shown were representative of two independent experiments. Autoradiographs were exposed for 1 h in B and C.