Figure 5: Nedd4 enhances IGF-I signalling in thyrocytes.

In the experiments described below, FRTL-5 cells were treated with or without 1 mM dibutyryl cAMP for 24 h. In samples indicated ‘+IGF-I’, cells were then stimulated with 100 ng ml⁻¹ IGF-I for 1 min (a,d,e) or 24 h (g) after cAMP treatment. *Significant difference (P<0.05). (a) Effects of cAMP treatment on the Nedd4 and IRS-2 complex formation. Cell lysates were subjected to immunoprecipitation and immunoblotting as indicated. (b) Effects of cAMP treatment on the Nedd4 and IRS-2 binding in cell-free binding assay. Cell lysates were subjected to pull-down analysis using indicated GST-fused proteins. (c) Effects of cAMP treatment on IRS-2 ubiquitination. Native samples and denatured samples were subjected to immunoprecipitation and immunoblotting as indicated. (d) Effects of cAMP and/or IGF-I treatments on IRS-2 subcellular localization. Cell lysates were fractionated into membrane/cytosol fractions, followed by immunoblotting. Right panel shows the densitometric analysis results. Mean±s.d. of three independent experiments. *Significant difference (P<0.05, one-way analysis of variance (ANOVA) followed by Tukey–Kramer test). (e–g) Effect of Nedd4 knockdown on IGF-I signalling and IGF-I-induced DNA synthesis. Cells transfected with Nedd4 siRNAs were treated with dibutyryl cAMP followed by IGF-I stimulation. Lysates were subjected to immunoprecipitation and immunoblotting (e). Densitometric analyses were performed. Tyrosine phosphorylation levels of IGF-IRβ and IRS-2 were normalized to their protein levels in immunoprecipitates and p85 PI3K bound to IRS-2 was normalized to IRS-2 levels in immunoprecipitates. Means±s.d. of seven independent experiments are shown in f. [³H]thymidine incorporation into DNA during 20–24 h of IGF-I treatment time was measured. The values were normalized and the means±s.d. of three independent experiments are shown in g. *Significant difference (P<0.05, one-way ANOVA followed by Tukey–Kramer test).