Year-end Sale % OFF 
Abstract
BackgroundTransformation by oncogene Ras overcomes TGF-β mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism of how these two pathways interact with each other is controversial.MethodsMolecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-β receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-β signalling activities is also determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion.ResultsRas interacts with the SPSB1’s SPRY domain to enhance TGF-β signaling. Ras interacts and colocalizes with the TGF-β type II receptor’s (TβRII) negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TβRII, in turn the increase of receptor levels significantly enhance Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-β signaling and its mediated migration and invasion.ConclusionRas positively cooperates with TGF-β signaling by reducing the cellular protein levels of TβRII negative regualtor SPSB1.
Highlights
Transformation by oncogene Ras overcomes TGF-β mediated growth inhibition in epithelial cells
This study describes a new mechanism of how Ras up-regulates the TGF-β signaling: Ras interacts with the newly identified TβRII negative regulator SPSB1 and causes its degradation via ubiquitination
Ras reduces SPSB1 expression levels We have previously generated a mesenchymal cellular model 21D1 by transforming Madin Darby Canine Kidney (MDCK) cells using v-HaRas [34, 38]. While activation of both Ras and TGF-β signalings are required for the maintenance of 21D1 cells mesenchymal phenotype, it is interesting that Smad2 phosphorylation levels are increased in 21D1 cells in comparision to the partental epithelial MDCK cells (Fig. 1a)
Summary
Molecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-β receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-β signalling activities is determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
