Abstract
BackgroundIn epithelial cells, tyrosine kinases induce tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which is responsible for the epithelial-mesenchymal transition (EMT). However, the precise mechanisms remain unclear.MethodsProtein antibody microarray analysis and E3 ligase profiling were performed to detect the unique E3 ligase underlying E-cadherin downregulation in lung adenocarcinoma tissues. Gene knockdown was performed using viral shRNA. Immunoblotting, immunofluorescence, immunoprecipitation, and xenograft models in vivo were integratively applied to explore RNF43-induced EMT in lung adenocarcinoma cell lines.ResultsProtein antibody microarray analysis and E3 ligase profiling revealed that the RING finger protein 43 (RNF43) was linked to E-cadherin downregulation within the context of c-Src activation in lung adenocarcinoma tissues. In addition, the c-Src-Caspase-8 interaction markedly increased c-Src activity. Activated c-Src phosphorylated E-cadherin at the tyrosine 797 site to initiate RNF43-mediated E-cadherin ubiquitination at lysine 816 and subsequent degradation, thus allowing the nuclear translocation of β-catenin and upregulation of Vimentin and RNF43 expression in lung adenocarcinoma cells. Decreased E-cadherin expression and increased Vimentin expression induced the EMT phenotype and promoted tumor metastasis. The Frizzled 8 (Frz8)-RNF43-induced ubiquitination of phosphorylated E-cadherin was blocked by a monoclonal antibody against the cysteine-rich domain (CRD) of Frz8 but not by antibodies against the protease domain (PA) of RNF43.ConclusionsOur data suggest that RNF43 participates in the regulation of EMT in the metastasis of lung adenocarcinoma through the ubiquitination and degradation of phosphorylated E-cadherin by activated c-Src.
Highlights
In epithelial cells, tyrosine kinases induce tyrosine phosphorylation and ubiquitination of the Ecadherin complex, which is responsible for the epithelial-mesenchymal transition (EMT)
RING finger protein 43 (RNF43) was associated with E-cadherin downregulation in lung adenocarcinoma in the context of c-Src activation Hakai is characterized as an E-cadherin-binding protein, and E3 ubiquitin ligase induces the ubiquitination of Ecadherin following the c-Src-mediated tyrosine phosphorylation of E-cadherin in Madin Darby canine kidney (MDCK) epithelial cells [13]
To generalize and test these results in human lung adenocarcinoma, protein extracts from 60 pairs of cancerous and paracancerous tissues were incubated with high-profile microarrays containing monoclonal antibodies against 620 E3 ubiquitin ligases and 30 EMT-related molecules (Additional file 2: Table S1, Additional file 3: Table S2, and Fig. 1a)
Summary
Tyrosine kinases induce tyrosine phosphorylation and ubiquitination of the Ecadherin complex, which is responsible for the epithelial-mesenchymal transition (EMT). A previous study revealed that activated c-Src exclusively tagged E-cadherin with phosphotyrosine under growth factor-induced stimulation [12, 13] This event was followed by the ubiquitination and degradation of Ecadherin by an E3 ligase, called Hakai, to facilitate EMT through the nuclear import of β-catenin to form a transcriptional complex with TCF/LEF [12, 13]. RNF43 was shown to ubiquitinate and degrade phosphorylated E-cadherin at the cytoplasmic tyrosine 797 site by c-Src, allowing EMT via the nuclear translocation and subsequent interaction of βcatenin with TCF-4 This process was shown to govern a wide variety of target genes, including Vimentin, and was efficiently abrogated by a monoclonal antibody against RNF43
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