Fig 7.

Insulin-dependent formation of an active Pak1-SKIP complex. (A) Pak1 constructs used in this study (left) and binding of SKIP to the Pak1 kinase domain (right). (B) Competitive binding for the Pak1 kinase domain between SKIP and the Pak1 inhibitory switch region (Pak1 aa 67 to 150). GST-Pak1 kinase domain (1 μg) immobilized to glutathione-Sepharose 4B beads was incubated with SKIP (1 μg) and various amounts of Pak1 aa 67 to 150. *, P < 0.05; **, P < 0.01 (t test). (C) Binding of SKIP to the constitutively active form of Pak1 (Pak1 L107F) in insulin-stimulated C2C12 cells. C2C12 cells transfected with 3× FLAG-tagged Pak1 L107F or Pak1 T422A mutant were stimulated with insulin for the indicated times. Lysates were immunoprecipitated (I.P.) with anti-FLAG antibody.