Figure 4: Epsin1 mediates the Nedd4 effect on IGF-I signalling.

(a) Effects of Nedd4 overexpression on the IRS-2 and Epsin1 interaction. HEK293T cells overexpressing indicated proteins were serum starved. Lysates were subjected to immunoprecipitation and immunoblotting as indicated. (b) Effects of IRS-2 ubiquitination site mutation or deletion of Epsin1 UIM motif. Epsin1 structure is shown in the upper panel. ENTH, Epsin N-terminal homology; UIM, ubiquitin-interacting motif; DPW, Asp-Pro-Trp; AP-2, adaptor protein-2; NPF, Asn-Pro-Phe; EH, Eps15 homology. HEK293T cells overexpressing indicated proteins were serum starved. Lysates were subjected to immunoprecipitation and immunoblotting as indicated. (c) Role of Epsin1 in IGF-I-induced IRS-2 tyrosine phosphorylation. HEK293 cells transfected with indicated plasmids and/or siRNAs were serum starved and then treated with 100 ng ml⁻¹ IGF-I for 1 min. Cell lysates were subjected to immunoprecipitation and immunoblotting. Densitometric analyses of cells with IGF-I stimulation were performed and tyrosine phosphorylation levels of IRS-2 were normalized to IRS-2 protein levels in immunoprecipitates. The means±s.d. of three independent experiments are shown in the graph. *Significant difference (P<0.05, one-way analysis of variance (ANOVA) followed by Tukey–Kramer test). (d) Regulation of IRS-2 subcellular localization by Nedd4. HEK293 cells overexpressing Nedd4 and IRS-2 were serum starved, followed by IGF-I stimulation (100 ng ml⁻¹, 1 min). Cell lysates were fractionated into membrane/cytosol fractions, followed by immunoblotting using the indicated antibodies. Densitometric analyses were performed and the ratio of IRS-2 in the membrane fraction to IRS-2 in the cytosol fraction was calculated. The graph shows the mean±s.d. of the densitometric analysis of six independent experiments. *Significant difference (P<0.05, one-way ANOVA followed by Tukey–Kramer test).