ASB2-induced FLNa degradation is dependent on ASB2 ubiquitin ligase activity and the proteasome. (A) Schematic representation of ASB2, ASB2LA, a BC box mutant, and ASB2-Δ, a deletion mutant lacking the SOCS box. The BC box mutation is indicated in bold. (B) ASB2-Δ and ASB2LA do not activate formation of polyubiquitin chains by the E2 enzyme. Elongin B, elongin C, cullin 5, and Rbx2 together with ASB2wt, ASB2LA, or ASB2-Δ were expressed in Sf21 cells. ASB2 complexes were immunoprecipitated (IP) with anti-flag or -ASB2 antibodies as indicated, and incubated with Uba1, Ubc5a, ubiquitin, and adenosine triphosphate. Their ability to stimulate polyubiquitination was assessed by Western blotting with antibodies to polyubiquitinylated proteins. ASB2 immunoprecipitation was assessed by blotting with anti-ASB2 antibodies. (C) ASB2 ubiquitin ligase activity is required for FLNa degradation. HeLa cells were transfected with GFP, GFP-ASB2wt, GFP-ASB2LA, or GFP-ASB2-Δ expression vectors and analyzed 24 hours after transfection using an antibody directed against the N-ter of FLNa. Scale bar represents 20 μm. (D) ASB2-induced FLNa degradation is dependent on proteasome activity. Ten hours after transfection with GFP-ASB2wt, HeLa cells were left untreated (−) or treated with 1 μM MG132 for 14 hours. Percentages of FLNa-negative GFP-ASB2-positive cells were counted among a randomly selected pool of at least 100 cells expressing GFP-ASB2. Results are mean plus or minus SEM from 3 independent experiments. (E) FLNa-GFP degradation by ASB2 is dependent on proteasome activity. NIH3T3 cells were transfected with FLNa-GFP, Myc-ubiquitin, and DsRed (−), DsRed-ASB2wt (wt), or DsRed-ASB2LA (LA) expression vectors. Seven hours after transfection, cells were left untreated or incubated with 1 μM MG132 or 20 μM LLnL for 17 hours, as indicated. A total of 20-μg aliquots of whole cell extracts were immunoblotted with antibodies directed against the N-ter of human FLNa and ASB2. Antibodies to human FLNa do not recognize endogenous mouse FLNa.

ASB2-induced FLNb degradation is dependent on ASB2 ubiquitin ligase activity and the proteasome. (A) ASB2-induced FLNb degradation depends on ASB2 ubiquitin ligase activity. NIH3T3 cells were transfected with GFP-ASB2wt or GFP-ASB2LA expression vectors, replated on coverslips coated with 5 μg/mL fibronectin 5 hours after transfection, fixed 15 hours after replating, and analyzed using an antibody directed against FLNb. Scale bar represents 50 μm. (B) FLNb-GFP degradation by ASB2 is dependent on proteasome activity. NIH3T3 cells were transfected for 24 hours with FLNb-GFP together with the GFP (−), GFP-ASB2wt (wt), or GFP-ASB2LA (LA) expression vectors in the absence or presence of 1 μM MG132 for 18 hours, as indicated. (C) HEK293T cells were transfected for 24 hours with FLNb-GFP (left) or GFP-talin (right) together with the GFP (−), GFP-ASB2wt (wt), or GFP-ASB2LA (LA) expression vectors, as indicated. In panels B and C, 20-μg aliquots of whole cell extracts were immunoblotted with antibodies to GFP.

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.

ASB2 induces FLNa degradation. GFP-tagged ASB2 colocalizes with F-actin (A) and FLNa (B) in stress fibers (top), lamellae (middle), and filopodiae (bottom). HeLa cells were imaged 10 hours after transfection with GFP-ASB2 expression vector. Actin was detected with Alexa 568-phalloidin and FLNa with an antibody against its N terminus. Higher magnification views of the lower panels are also shown (bottom). (C) HeLa cells were transfected with GFP or GFP-ASB2 expression vectors as indicated; 24 hours later, FLNa was localized by staining the cells with antibodies directed against the N terminus (N-ter) or C terminus (C-ter) of the protein. Scale bars represent 20 μm. (D) FLNa-negative, GFP-ASB2-positive HeLa cells were counted 10 and 24 hours after transfection among a randomly selected pool of at least 100 cells expressing GFP-ASB2. Results are mean plus or minus SEM from 3 independent experiments. (E) NIH3T3 cells were mock-transfected or transfected with FLNa-GFP and GFP-ASB2 expression vectors for 24 hours, as indicated; 20-μg aliquots of whole cell extracts were immunoblotted with antibodies directed against the N-ter of human FLNa and GFP. Antibodies to human FLNa do not recognize endogenous mouse FLNa. (F) Triple staining showing F-actin organization and FLNa expression in HeLa cells expressing GFP or GFP-ASB2.

ASB2, but not ASB2LA, induces polyubiquitylation of FLNa. Recombinant ASB2/elongin BC/cullin5/Rbx2 complexes were purified as in Figure 2B. All samples contained purified Uba1, UbcH5a, and ubiquitin. The 3 components of the NEDD8 pathway were added as indicated. Purified FLNa was also provided as indicated and subjected to ubiquitylation. Aliquots of the reaction mixture were analyzed by Western blotting using anti-FLNa (upper panel) and anti-ASB2 antibodies (lower panel).

ASB2 induces degradation of FLNa and FLNb in differentiating myeloid leukemia cells. (A-C) Down-regulation of FLNa and FLNb in myeloid leukemia cells induced to differentiate correlates with ASB2 induction. NB4 and PLB985 cells were treated with 10⁻⁶ M of all-trans RA. Differentiation was assessed by cell morphology on May-Grünwald-Giemsa-stained cytospin slides (A). Expression of ASB2, FLNa, FLNb, and non-muscle myosin IIA (NM myosin IIA) was analyzed by Western blot using 20-μg aliquots of whole cell extracts (B) and by immunofluorescence (C). (D) ASB2 knockdown delayed RA-induced FLNa and FLNb degradation. Stable PLB985 cell populations expressing shRNAs directed against ASB2 (no. 1 and no. 2) or transfected with the empty vector (−) were treated with 10⁻⁶ M RA for different times as indicated; 10-μg aliquots of the protein extracts were analyzed by Western blotting. (E) PLB985 cell populations expressing shRNA no. 1 or transfected with the empty vector (−) were untreated and treated with RA. (Top panel) Representative experiment of 3 showing numbers of viable cells. (Bottom panel) Percentages of CD11b-positive cells. Error bars represent SDs from the results of 3 independent experiments.

ASB2-induced degradation of FLNs inhibits cell spreading on fibronectin. (A,B) NIH3T3 cells were transfected with GFP, GFP-ASB2wt, GFP-ASB2LA, or GFP–ASB2-Δ expression vectors for 24 hours, trypsinized, and serum arrested for 1 hour in suspension. Cells were plated on fibronectin-coated coverslips and fixed after 45 minutes. (A) Cells were stained with anti-FLNa antibodies as indicated. Scale bar represents 20 μm. (B) Percentages of nonspread (▩) and spread (■) cells are plotted as mean plus or minus SEM from 3 independent experiments. (C) FLNa and FLNb double knockdown in HT1080. (D) Spreading quantification. HT1080 WT or HT1080 FLNabKD cells were replated on fibronectin-coated coverslips and fixed after 40 minutes. Cell areas of at least 100 cells were measured and plotted as mean plus or minus SEM. (E) HT1080 WT were transfected with GFP-ASB2wt and 24 hours later replated on fibronectin-coated coverslips, fixed after 40 minutes, and stained for FLNa. Cell areas were measured and cells expressing GFP-ASB2wt (outlined in the image) were compared with untransfected cells on the same coverslip. Scale bar represents 20 μm. The plot shows the mean area plus or minus SEM.