Fig. 1: SCF^FBXW7 interacts and targets ZEB2 for degradation in a GSK-3β phosphorylation-dependent manner.

a Left, 2DE and MALDI-MS-based identification of novel Fbxw7-associated proteins using crypts (upper panel) isolated from 3-week fbxw7^fl/fl and fbxw7^ΔG 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 fbxw7^fl/fl vs. fbxw7^ΔG derived crypts and intestinal proteins and mRNA expression for ZEB2 and β-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-3β phosphorylation-dependent manner. GSK-3β under the control of the methionine-regulated MET3 promoter induces phosphorylation of encoded myristoylated proteins through a cDNA library plus positive control expressing FLAG-β-catenin (B—Middle) which only rescued the growth of cdc25–2 mutant yeast by Fbxw7-associated protein(s), if they interact with RasV12-FBXW7ΔF (i.e. human FBXW7α isoform mutant lacking F-box ___domain; therefore, interaction with Skp1 is lost and degradation of SCF^Fbxw7 substrates will not occur in yeast) used as a bait at the restrictive temperature 37 °C, in a methionine-dependent manner. In the FBXW7ΔF 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, cdc25–2 mutant yeasts can grow only at 37 °C, when a phosphorylation-dependent interaction between a protein target and RasV12-FBXW7ΔF takes place. The FBXW7ΔF(bait)-dependent growth of these clones was further analysed on galactose-containing medium at 37 °C (B—Right). Red circles show the GSK-3β-phosphorylation-dependent interactor, including the Zeb2-clone, green circles show the phosphorylation/non-phosphorylation-dependent interactor and blue circles show the revertant clones (B—Right). 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-3β in HCT116 CRC cells. (c—Middle and c—Right) WB analysis of total ZEB2 protein level following the inhibition of GSK-3β (e.g. WS119 or LiCl treatment, and siRNA against GSK-3β) 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 with HA-tagged ubiquitin- (HA-Ub) expressing construct in HEK-293T cells (Right). The asterisk indicates a nonspecific band(s). e Co-IP of endogenous ZEB2 upon pull-down of FBXW7 in HCT116 cells with FBXW7 deletion. f ZEB2 pulse-chase stability assays with 15 µg/ml cycloheximide (CHX) in HCT116 cells with or without FBXW7 deletion