Figure 4. Mutation of both PPXY domains disrupts Itch and LITAF interaction.

(A) Extracts from HEK-293T cells transfected with GFP-LITAF WT or GFP-LITAF Y23,61A were incubated with either GST alone, GST-Itch WT, GST-Itch PRD or GST-Itch WW pre-coupled to glutathione-Sepharose. Aliquot from total cell lysate (CL) and proteins specifically bound to the beads were processed by immunoblot with a polyclonal antibody against GFP. (B) HEK-293T cells were transiently transfected with either GFP-LITAF WT, GFP-LITAF Y61A, GFP-LITAF Y23A or GFP-LITAF Y23,61A. Aliquots of CL were processed by immunoblot with GFP antibody to show protein expression. The rest of the extracts were incubated with either GST or GST-Itch WW fusion proteins pre-coupled to gluthatione-Sepharose. Proteins specifically bound to the beads were immunoblotted with GFP antibody to reveal protein interactions. The bands representing the GST-fusions in the Ponceau staining are marked by a red asterisk. Additional staining in the GST-Itch-WT lane likely represents degradation products of the fusion protein. (C) 293T cells were co-transfected with constant amount of rLuc-Itch and various amounts of either GFP-LITAF Y61A or GFP-LITAF Y23,61A. The graph is a representative example of the saturation studies performed to provide evidence for a specific interaction between the proteins. BRET ratios were plotted as a function of the excited GFP activity to total rLuc activity ratio, allowing comparison of BRET ratios between GFP-LITAF Y61A and GFP-LITAF Y23,61A when expressed at the same level. (D) Quantification of the interaction between Itch WW domains and the different LITAF constructs. The densitometry of GFP signal in the fraction bound to GST-Itch-WW beads relative to the densitometry of the GFP signal in 1/10 volume of protein extract is represented as described in materials and methods. Data are mean ± s.e.m. from n = 4 experiments. * p<0.05 compared to binding of GFP-LITAF WT (ANOVA post-hoc Tukey).