Journal: The Journal of Biological Chemistry

Article Title: The SCF–FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage

doi: 10.1016/j.jbc.2022.102703

Figure Lengend Snippet: The regulation of WDR5 by FBXW7 depends in part on GSK3β. A , FLAG-FBXW7 was overexpressed in HEK293T for 24 h, and all cultures were treated with 5 μM MLN4924 for 5 h prior to harvest. FLAG-FBXW7 was immunoprecipitated, and the indicated sample was dephosphorylated with λ-phosphatase at 30 °C for 3 h. B , GFP-WDR5 was overexpressed in HEK293T for 24 h, and cultures were treated with 5 μM CHIR-99021 or DMSO for 5 h prior to cell harvest, as indicated. GFP-WDR5 was immunoprecipitated using GFP-trap beads. ∗∗ p < 0.01, unpaired two-way Student’s t test with Welch’s correction, n = 3. C , U2OS cells were transfected twice with 30 nM against GL2 or GSK3β and harvested 72 h after the first transfection. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA, n = 3. D , GFP-WDR5 and Myc-GSK3β were overexpressed in HEK293T cells for 24 h. GFP-WDR5 was immunoprecipitated using GFP-trap beads. E , the indicated proteins were overexpressed in HEK293T for 24 h. 5 μM CHIR-99021 or 5 μM MLN4924 were added for 9 h and 10 μM MG132 for 5 h prior to harvest. FLAG-WDR5 was immunoprecipitated using α-FLAG M2 beads in the presence of 20 mM N -ethylmaleimide. F , FLAG-FBXW7 or FLAG-FBXW7 ARG (R465H, R479Q, and R505C mutations) were coexpressed with Myc-Cullin1 in HEK293T for 48 h and immobilized on α-FLAG M2 beads. In vitro ubiquitination assays of 200 nM GST-WDR5 or GST-c-Myc were performed with 170 nM Uba1, 250 nM of UbcH5b and CDC34, 30 μM ubiquitin, 200 nM His-GSK3β, 5 mM ATP, and immobilized FLAG-FBXW7 constructs at 37 °C for 90 min. DMSO, dimethyl sulfoxide; GSK3β, glycogen synthase kinase-3β; GST, glutathione- S -transferase; HEK293T, human embryonic kidney 293T cell line; l.e., long exposure; s.e., short exposure.

Article Snippet: Protein expression was induced by addition of 2 μg/ml doxycycline for at least 72 h. GSK3β activity was blocked with 5 μM CHIR-99021 (MedChemExpress; catalog no.: HY-10182) and cullin-RING E3 ubiquitin ligases with 5 μM MLN4924 (Cell Signaling Technology; catalog no.: 85923) for 5 h prior to cell harvest.

Techniques: Immunoprecipitation, Transfection, In Vitro, Ubiquitin Proteomics, Construct