Journal: PLoS ONE

Article Title: Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

doi: 10.1371/journal.pone.0035826

Figure Lengend Snippet: (A) RCS cells were transfected with wt FGFR3 or activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M, K650E), and analyzed for the indicated molecules by WB 48 hours later. The levels of ERK phosphorylation vary among the tested mutants, reflecting the different strength of FGFR3 activation by each particular mutation . K508M - kinase inactive FGFR3 mutant. GFP and empty vectors serve as transfection controls. (B) LRP6 phosphorylation at Thr1572 caused by highly activating FGFR3 mutants R248C and K650E. (C) Cells were transfected with the indicated FGFR3 vectors together with Topflash reporter vectors, treated with WNT3a and analyzed for luciferase activity. Data represent an average from three transfections (each measured twice), with the indicated standard deviations. A logarithmic scale of the y -axis is necessary to express the massive Topflash activation in WNT3a-treated cells expressing activating FGFR3 mutants (* p <0.001; Student’s t -test; compared to wt FGFR3). Results are representative of four experiments. (D) Cells were transfected with wt FGFR2 or activating FGFR2 mutants (S252W, P253R, C342R, C342Y, Y375C), and analyzed for the indicated molecules by WB. Note the significant ERK and LRP6 phosphorylation caused by C342R, C342Y and Y375C mutants, which correlates with increased basal (E; upper graph) and WNT3a-induced (E; lower graph) β-catenin activity, evidenced by Topflash experiment. Results are representative for three experiments (* p <0.001; Student’s t -test; compared to wt FGFR2).

Article Snippet: For the recombinant RTK kinase assays, the reactions were carried-out with 400 ng of recombinant FGFR2, FGFR3, TRKA or EGFR (SignalChem, Richmond, Canada) and 400 ng of recombinant β-catenin (Abcam, Cambridge, MA, USA) in 50 μl of kinase buffer in the presence of 50 μM ATP for 60 minutes at 30°C.

Techniques: Transfection, Phospho-proteomics, Activation Assay, Mutagenesis, Luciferase, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

doi: 10.1371/journal.pone.0035826

Figure Lengend Snippet: (A) RCS cells were treated for indicated times with FGF2 (10 ng/ml) in the presence of heparin (1 µg/ml), and analyzed for β-catenin phosphorylation at Tyr142 by WB (arrow). (B) HEK293 cells were transfected with wt FGFR2 or its activating mutant Y375C, and analyzed for indicated molecules 48 hours later. Note the increased β-catenin phosphorylation at Tyr142 (arrow). (C) Active recombinant FGFR3, FGFR2, TRKA and EGFR were subjected to a cell-free kinase assay with recombinant β-catenin as a substrate. Samples with ATP or kinase omitted serve as controls for kinase reaction.

Article Snippet: For the recombinant RTK kinase assays, the reactions were carried-out with 400 ng of recombinant FGFR2, FGFR3, TRKA or EGFR (SignalChem, Richmond, Canada) and 400 ng of recombinant β-catenin (Abcam, Cambridge, MA, USA) in 50 μl of kinase buffer in the presence of 50 μM ATP for 60 minutes at 30°C.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Recombinant, Kinase Assay