FIGURE 8.

Ndfip1 relieves autoinhibition by binding Itch WW domains. A, ubiquitin charging of the HECT domain was tested alone (first lane) or in the presence of WT WW domains (WW1–4, second and third lanes) or mutant WW domains (WW1–4A, fourth and fifth lanes) and in the presence or absence of Ndfip1 (third and fifth lanes). Detection of the ubiquitin-charged HECT domain was by streptavidin. B, GST-tagged Ndfip1 or GST alone was pulled down from cells also expressing the Itch His-MBP-HECT or WW1–4 domains. Enriched proteins were visualized by Coomassie staining, and domain expression was confirmed in bacterial lysates using anti-His antibody. Cells expressing GST-Ndfip1, but no Itch domain, provided a negative control. C. GST-tagged Ndfip1 or GST alone was pulled down from bacteria expressing Itch, Itch^WW2,3A, or Itch^WW1–4A. Proteins were visualized by Coomassie and Itch construct expression was confirmed using anti-Itch. D, the activity of Itch, Itch^WW2,3A, and Itch^WW1–4A was assayed using a homogeneous FRET assay. In all panels, similar results were obtained in at least three independent experiments. HTRF, homogeneous time-resolved FRET; SA, streptavidin.