ASB2-induced degradation of FLNa is dependent on ASB2 ubiquitin ligase activity and proteasome in myeloid leukemia cells. (A) ASB2wt was induced in PLB985/MT-ASB2 treated with 65 μM ZnSO4 for the indicated times (lanes 1-4). ASB2wt was induced in PLB985/MT-ASB2 cells treated with 10 μM ZnSO4 (lane 5). ASB2LA (lane 6) and ASB2-Δ (lane 7) were induced in PLB985/MT-ASB2LA and PLB985/MT-ASB2-Δ cells treated with 100 μM ZnSO4. PLB985/MT-Flag cells were used as controls and treated with 100 μM ZnSO4 (lane 8). Protein extracts (20 μg) were separated by SDS-PAGE and immunoblotted for ASB2, FLNa (N-ter), and FLNb. * indicate a nonspecific band. PLB985 cells were nucleofected with the GFP, GFP-ASB2wt, and GFP-ASB2LA expression vectors for 8 hours and treated without (B) or with 0.5 μM MG132 (C). Expression of FLNa was analyzed by immunocytochemistry using an antibody directed against the N-ter of FLNa. Colocalization of ASB2 and FLNa is indicated in the merged image (yellow). Scale bar represents 20 μm. (D-F) Whereas FLNa expression is not influenced by GFP or GFP-ASB2-Δ, FLNa expression is dramatically reduced in GFP-ASB2wt–expressing leukemia cells. GFP, GFP-ASB2wt, and GFP-ASB2-Δ were induced in PLB985/MT-GFP, PLB985/MT-GFP-ASB2wt, and PLB985/MT-GFP-ASB2-Δ cells with 100 μM ZnSO4 for 48 hours, respectively. After permeabilization and fixation, cells were stained with anti-FLNa or anti-IgG1 antibodies and Alexa Fluor 647–conjugated anti-mouse antibodies. A total of 10 000 cells were analyzed by flow cytometry. (D) Dot plots of a representative experiment showing FLNa expression before and after ZnSO4 treatment. (E) Expression of FLNa in GFP–ASB2wt- and GFP–ASB2-Δ–expressing cells. (F) Expression of GFP-ASB2wt and GFP–ASB2-Δ after ZnSO4 treatment. Results are mean plus or minus SEM from 3 independent experiments.
