Figure 2: Nedd4 induces IRS-2 monoubiquitination.
From: Nedd4-induced monoubiquitination of IRS-2 enhances IGF signalling and mitogenic activity
(a) Cell-free ubiquitination assay results. HEK293T cells overexpressing FLAG-IRS-2 or FLAG were serum starved and lysed. Immunoprecipitates adsorbed on anti-FLAG antibody-conjugated beads were subjected to cell-free ubiquitination assay using GST-Nedd4 or GST. (b–e) IRS-2 ubiquitination by Nedd4 overexpression. HEK293 cells (b) or HEK293T cells expressing the indicated proteins (c–e) were serum starved. A portion of the cell lysate was mixed with 2% SDS, boiled for 5 min and diluted. These denatured samples (indicated lanes in b and all immunoprecipitates in c–e) and native samples were subjected to immunoprecipitation and immunoblotting as indicated. In c, purified monoubiquitin was subjected to immunoblotting, to confirm that FK1 antibody does not recognize monoubiquitin (upper panel). UbK0 is an ubiquitin mutant in which all Lys residues were substituted to Arg. Nedd4Y592A/F694A is a mutant lacking ubiquitin chain-elongation activity. (f) Location of Lys residues in IRS-2. IPK, intermediate region flanking the PTB domain and the KRLB region; KRLB, kinase regulatory loop-binding region; C1-3, C-terminal region 1–3. (g,h) Effects of the indicated Lys-Arg substitution in IRS-2 on its ubiquitination by Need4. HEK293T cells overexpressing indicated proteins were serum starved. Cell lysates were denatured and analysed by immunoprecipitation and immunoblotting as indicated (g). Densitometric analyses were performed. Ubiquitination levels of IRS-2 were normalized to its protein levels, and the means±s.d. of four independent experiments were shown in h. *Significant difference (P<0.05, one-way analysis of variance followed by Tukey–Kramer test). (i) Quantitative MS analysis results. IRS-2 immunoprecipitates derived from HEK293T cells expressing IRS-2 (-Nedd4) or IRS-2 and Nedd4 (+Nedd4) were treated with trypsin and Asp-N. Peptides with ubiquitin remnant motif were enriched and subjected to LC-MS/MS analysis. The graph shows means±s.d. of three independent experiments. *Significant difference from control (P<0.05, Student’s t-test).
