Fig. 3. Met phosphorylates β-catenin at Y142. (A) In vitro kinase assay with recombinant Met kinase and WT or mutant GST–β-catenin. Anti-phospho-Tyr (anti-Tyr-P) western blot shows phosphorylated Met kinase upon addition of ATP and its basal activation (–ATP condition). Anti-Y142-P-β-catenin western blot (top) indicates that WT and Y654F GST–β-catenin are phosphorylated by Met kinase at Y142, whereas phosphorylation of Y142F GST–β-catenin is as low as in basal condition (–ATP). (B) Control (His) and β-catenin immunoprecipitation from untreated hippocampal neurons or those treated with 50 ng/ml HGF for 10 minutes with or without pervanadate. The level of Y142-P β-catenin increases with HGF treatments, whereas phosphorylation at Y654 is not stimulated by HGF signalling. Met and -catenin co-immunoprecipitate with β-catenin, and the recovery of both proteins decreases upon phosphorylation of β-catenin at Y142. (C) β-catenin immunoprecipitation from untreated neurons or those treated with 50 ng/ml HGF for the indicated times. The level of Y142-P β-catenin peaks at 10 minutes, is maintained at similar levels up to 1 hour and starts decaying slightly after overnight (o/n) stimulation. Quantifications in B and C correspond to % of the increase in the intensity of the phosphospecific β-catenin band relative to the control normalized to total β-catenin.