Figure 3: Need4 enhances IGF-I signalling by regulating IRS-2 ubiquitination.

(a–c) Effects of Nedd4 overexpression on IGF-I signalling. HEK293 cells overexpressing Nedd4 or Nedd4^C854S (a ubiquitin ligase inactive mutant) together with IRS-2 were serum starved, followed by IGF-I stimulation (100 ng ml⁻¹) for 1 min (a) or the indicated durations (c). Lysates were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. The results of a were quantified by densitometric analysis. IGF-I-induced 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. The means±s.d. of three independent experiments are shown in b. *Significant difference from control (P<0.05, one-way analysis of variance (ANOVA) followed by Tukey–Kramer test). (d,e) Effects of the expression of IRS-2 ubiquitination site mutants (d) and an IRS-2 -ubiquitin chimeric protein (e) on IRS-2 tyrosine phosphorylation. HEK293 cells overexpressing indicated proteins were subjected to experiments similar to a. Ub^I44A, ubiquitin unable to interact with ubiquitin-binding domains. IGF-I-induced tyrosine phosphorylation levels of IRS-2 and PI3K bound to IRS-2 were normalized to IRS-2 protein levels in immunoprecipitates. Means±s.d. of six (d) or three (e) independent experiments are shown. *Significant difference from control (P<0.05, one-way ANOVA followed by Tukey–Kramer test); NS, not significant.