%0 Journal Article
%T An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance
%A Abdolrahman S. Nateri
%A Bradley Spencer-Dene
%A David Kerr
%A David O. Bates
%A Enric Domingo
%A Eugene Tulchinsky
%A Federica Lorenzi
%A Hans Clevers
%A Ian Tomlinson
%A Ningning Li
%A Philip Clarke
%A Robert G. J. Vries
%A Roya Babaei-Jadidi
%A Yihang Pan
%A Yulong He
%J Archive of "Oncogenesis".
%D 2019
%R 10.1038/s41389-019-0125-3
%X a Left, 2DE and MALDI-MS-based identification of novel Fbxw7-associated proteins using crypts (upper panel) isolated from 3-week fbxw7fl/fl and fbxw7¦¤G mice. Yellow circles in the lower panel denote potential Fbxw7-associated proteins. a Right, WB analysis (upper panels), and RT-PCR analysis (lower panels) of fbxw7fl/fl vs. fbxw7¦¤G derived crypts and intestinal proteins and mRNA expression for ZEB2 and ¦Â-actin control. Experiments were performed on at least three independent occasions. b Left, schematic representation of the modified yeast two-hybrid reverse Ras Recruitment Screening (rRRS) system identifying proteins interacting with Fbxw7 in a GSK-3¦Â phosphorylation-dependent manner. GSK-3¦Â under the control of the methionine-regulated MET3 promoter induces phosphorylation of encoded myristoylated proteins through a cDNA library plus positive control expressing FLAG-¦Â-catenin (B¡ªMiddle) which only rescued the growth of cdc25¨C2 mutant yeast by Fbxw7-associated protein(s), if they interact with RasV12-FBXW7¦¤F (i.e. human FBXW7¦Á isoform mutant lacking F-box domain; therefore, interaction with Skp1 is lost and degradation of SCFFbxw7 substrates will not occur in yeast) used as a bait at the restrictive temperature 37£¿¡ãC, in a methionine-dependent manner. In the FBXW7¦¤F mutant, both the N-terminal F-box and Dim-domains are deleted to avoid any interactions with SKP1 and other FBXW7 isoform-associated proteins. Thus, cdc25¨C2 mutant yeasts can grow only at 37£¿¡ãC, when a phosphorylation-dependent interaction between a protein target and RasV12-FBXW7¦¤F takes place. The FBXW7¦¤F(bait)-dependent growth of these clones was further analysed on galactose-containing medium at 37£¿¡ãC (B¡ªRight). Red circles show the GSK-3¦Â-phosphorylation-dependent interactor, including the Zeb2-clone, green circles show the phosphorylation/non-phosphorylation-dependent interactor and blue circles show the revertant clones (B¡ªRight). c Left, subcellular localisation of GFP-fused human ZEB2 in the absence (top; nuclear) and presence (bottom; nuclear spots indicative of protein degradation) of GSK-3¦Â in HCT116 CRC cells. (c¡ªMiddle and c¡ªRight) WB analysis of total ZEB2 protein level following the inhibition of GSK-3¦Â (e.g. WS119 or LiCl treatment, and siRNA against GSK-3¦Â) and of UPS pathways (MG132) in SW620 CRC cells. d Direct binding and ubiquitin-dependent degradation of ZEB2 by FBXW7. Co-immunoprecipitation (IP) of ZEB2 upon pull-down of FBXW7 in HEK-293T cells (Left); co-IP of FBXW7 upon pull-down of ZEB2 using the TNT-coupled reticulocyte lysate (Middle), and ubiquitination assays
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381143/