Fig. 2. Trk phosphorylates β-catenin at Y654. (A) In vitro kinase assay using recombinant TrkA kinase and WT or mutant GST-β-catenin. Anti-phospho-Tyr (anti-Tyr-P) western blot shows phosphorylated TrkA kinase and GST–β-catenin. WT GST–β-catenin is phosphorylated by TrkA kinase upon addition of ATP, whereas K252a inhibits TrkA-induced β-catenin phosphorylation. Phosphorylation of mutant Y654F and Y142F GST–β-catenin by TrkA kinase is 45% and 15% lower than that of WT GST–β-catenin, respectively. The panel below shows the anti-β-catenin western blot for the GST–β-catenin input in the kinase assay (0.8 µg). (B) In vitro kinase assay using TrkA immunoprecipitated from PC12 6/15 cells, and recombinant WT and Y654F β-catenin. Anti-Tyr-P western blot shows TrkA (140-kDa and 110-kDa bands) and the tyrosine phosphorylation of β-catenin. Phosphorylation of WT β-catenin is decreased by preincubation with K252a (100 nM), whereas phosphorylation of Y654F β-catenin is lower than that of WT β-catenin and is not inhibited by K252a. In the first lane, no recombinant β-catenin was added to control for phosphorylation of any endogenous β-catenin that might have been immunoprecipitated with TrkA. Quantification corresponds to % of the increase in the intensity of Tyr-P β-catenin relative to –ATP divided by total β-catenin. (C) Control (Myc) or β-catenin immunoprecipitation from PC12 6/15 cells untreated or treated with NGF (100 ng/ml) and pervanadate for the last 10 minutes of stimulation. Phosphorylation of β-catenin Y654 increases after NGF treatment in parallel with TrkA stimulation as detected by anti-Tyr-P western blot. (D) Control (His) or β-catenin immunoprecipitation from untreated hippocampal neurons or those treated for 10 minutes with 50 ng/ml BDNF or NT-3 in the presence of pervanadate. Upon NT stimulation, phosphorylation of β-catenin at Y654 increases parallel with phosphorylation of TrkB and/or TrkC detected by anti-phospho-Tyr western blot. (E) Control or β-catenin immunoprecipitation from hippocampal neurons untreated or treated for 10 minutes with 50 ng/ml BDNF with or without pervanadate. Level of Y654-P β-catenin increases with BDNF treatment both with and without pervanadate, whereas phosphorylation at Y142 is maintained at the basal level, increasing only slightly with pervanadate. TrkB and N-cadherin co-immunoprecipitate with β-catenin, and N-cadherin association with β-catenin shows a small decrease after pervanadate treatment. (F) β-catenin immunoprecipitation from untreated neurons or those treated with 50 ng/ml BDNF for the indicated times in the presence of pervanadate for the last 10 minutes of stimulation. The level of Y654-P β-catenin peaks at 1 hour and is reduced after overnight (o/n) stimulation. Quantifications in C-F correspond to % of the increase in the intensity of the phosphospecific β-catenin (Y654-P or Y142-P) band relative to the control normalized to total β-catenin.