Fig. 6
c-Src underlay RNF43-mediated E-cadherin ubiquitination through E-cadherin phosphorylation at tyrosine 797. a, H522 cells were stably transfected with wild-type c-Src (Src WT) and a constitutively active c-Src, Src Y527F. p-Src, c-Src, E-cadherin (E-cad), Vimentin, RNF43, total β-catenin, and β-actin via adenovirus and detected by western blot analysis of total cell lysates (TCLs) (Upper blot). β-catenin in the nuclear extract was evaluated using an antibody against β-catenin (lower blot). b, TCLs were subjected to immunoprecipitation (IP) using an anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected with an anti-ubiquitin antibody. p-Src, RNF43, nonspecific phosphorylated tyrosine of E-cadherin (p-Tyr-100), and E-cadherin in the immunocomplex were detected by western blot. c, EMT morphological changes and metastatic incidences in H522 cells transfected with Src WT and Src Y527F (χ2 test, *, P < 0.05). Scale bars, 50 μm. d, Tyrosine 797 of E-cadherin was conserved across various species (upper panel). MALDI-mass spectrometry analysis of CNBr-cleaved endogenous E-cadherin expression in A549 cells with or without endogenous c-Src (lower panel). e, E-cadherin-knockdown A549 cells transfected with unmutated E-cadherin (E-cad WT) or E-cadherin mutated at Y755-757F (E-cad Y755-757F) or Y797F (E-cad Y797F) were lysed to obtain TCLs. E-cadherin (anti-HA) was immunoprecipitated from TCLs and probed for ubiquitin-E-cadherin, p-Src, nonspecific phosphorylated tyrosine of E-cadherin, RNF43, and E-cadherin (anti-HA) by western blot. f, E-cadherin-knockdown A549 cells transfected with HA-tagged E-cad WT, E-cad Y755-757F, or E-cad Y797F were lysed to obtain TCLs. p-Src, c-Src, Vimentin, E-cadherin (anti-HA), RNF43, total β-catenin, and β-actin were detected by western blot analysis of the TCLs (upper blot). Nuclear extracts were subjected to western blot analysis of β-catenin (lower blot). g, EMT morphological changes and metastatic incidences in E-cadherin-knockdown cells transfected with HA-tagged E-cad WT, E-cad Y755-757F, or E-cad Y797F (χ2 test, *, P < 0.05). Scale bars, 50 μm