Fig. 6.
FGFR4-Shp2 are required for Y67-FXR phosphorylation by Src. a PMH were transfected with siRNA as indicated, and 48 h later, cells were treated with FGF19 for 10 min, and p-Y416-Src, p-Y527-Src, Src, Shp2 and insulin receptor (INSR) levels were detected by IB. b PMH were transfected with siRNA for Src as indicated and treated with FGF19 for 10 min, and p-Y-Shp2 levels were detected by IP/IB. Protein levels in input samples were detected by IB. c PMH were transfected with siRNA for Src as indicated and treated with FGF19 for 10 min, and FRS2 or Shp2 in anti-Shp2 or anti-FRS2 immunoprecipitates, respectively, were detected by IB. Protein levels in input samples were detected by IB. d PMH were transfected with siRNA as indicated and infected with Ad-flag-FXR, and then, 48 h later, the cells were treated with FGF19 for 10 min. p-Y-FXR and p-Y67-FXR levels were detected by IP/IB. Protein levels in input samples were detected by IB, and relative intensities of the bands, determined using Image J, are plotted below the blots. e CoIP: PMH were treated with FGF19 for the indicated times, and FRS2, Shp2, Src, FXR, and ERK in anti-FGFR4 immunoprecipitates were detected by IB. f CoIP: hepatocytes were treated with FGF19 for 10 or 30 min, and Shp2, FXR, and ERK were detected by IB in anti-Src immunoprecipitates. Protein levels in input samples are shown below. g PMH were transfected with siRNA for ERK and treated with FGF19 for 10 min, and levels of indicated proteins were detected by IB. h PMH were transfected with siRNA for Src and treated with FGF19 for 10 min, and levels of Src, p-ERK, and ERK were detected by IB. i Model: an FGF19-FGFR4-Shp2 signaling cascade leads to activation of Src and Src-mediated phosphorylation of FXR at Y67, which is important for nuclear localization of FXR and transcriptional regulation of BA levels by FXR. FGF19-activated Src also activates ERK independently of the MAPK/ERK signaling pathway, which leads to transcriptional repression of Cyp7a138,39