Fig. 2: GSK-3-mediated phosphorylation-dependent degradation of ZEB2 by FBXW7α. | Oncogenesis

Fig. 2: GSK-3-mediated phosphorylation-dependent degradation of ZEB2 by FBXW7α.

From: An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance

Fig. 2

a Schematic mapping and identifying FBXW7 phosphodegrons on ZEB2 protein. Constructs of D1–D9 represent the structure of the GFP-fused ZEB2 deletions. Serine and threonine residues within the potential GSK-3β phosphorylation sites (i.e. degron sequences) are shown in red and green, and proline residues are in blue in wild-type ZEB2-D9; whilst the small letter “a” indicates S/T residues replaced by alanine in the mutant ZEB2-D9 (AA1 to AA7, where AA1 + AA2 + AA3 = AA6, and AA4 + AA5 + AA6 = AA7). b ZEB2-D8 directly binds to FBXW7 for ubiquitin and GSK-3β-mediated degradation. HEK-293T cells transfected with the indicated constructs (D6–D8) together with FLAG-GSK-3β plasmid and HA-tagged ubiquitin (HA-Ub) followed by IP and IB. The red arrowhead (fourth panel) denotes the co-IP of the ZEB2-D8 mutant in the FBXW7 precipitates. Co-IP and IB experiments were performed in triplicate. c FBXW7 controls the degradation of ZEB2-D9, the shorter version of ZEB2-D8. HCT116 cells ±FBXW7 were transfected with the ZEB2-D9 construct, treated with cycloheximide (CHX) for 1 h and whole-cell lysates were subjected to IB. d Phosphorylation of ZEB2-D9 may be a prerequisite for its degradation. Lambda protein phosphatase (λPPase) treatment leads to faster motility due to the release of phosphate groups from phosphodegrons. e ZEB2-D9 protein stability depends on phosphorylation and proteasome. HEK-293T cells were transfected with the ZEB2-D9 construct, treated with Okadaic acid (inhibitor of PP1 and PP2A phosphatases; lane2), LiCl (GSK-3β inhibitor; lane3) or MG132 proteasome inhibitor I (Prot Inhib; lane4) for 8 h and whole-cell lysates were subjected to IB. f Phosphodegrons within ZEB2-D9 are collectively essential to its stability. HEK-293T cells were transfected with wild-type (WT) ZEB2-D9 and D9-phosphorylation-defective mutants (AA1–AA7 constructs) together with FLAG-GSK-3β plasmid and whole-cell lysates subjected to IB. g HCT116FBXW7(−/−) cells were transfected with GFP-ZEB2-D9 wild-type (WT) and mutant (AA7) and the activated FLAG-GSK3β. FLAG-GSK3β was immunoprecipitated with anti-FLAG and then detected with the phospho-S/T antibody. GFP-ZEB2-D9 phosphorylation status was examined by immunoblot analysis after immunoprecipitation using an anti-phospho-(Ser/Thr) antibody that efficiently detected phospho wild-type GFP-ZEB2-D9-WT

Back to article page