The E3 ubiquitin ligase specificity subunit ASB2beta is a novel regulator of muscle differentiation that targets filamin B to proteasomal degradation

Abstract

Ubiquitin-mediated protein degradation is the main mechanism for controlled proteolysis, which is crucial for muscle development and maintenance. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 gene (ASB2) encodes the specificity subunit of an E3 ubiquitin ligase complex involved in differentiation of hematopoietic cells. Here, we provide the first evidence that a novel ASB2 isoform, ASB2beta, is important for muscle differentiation. ASB2beta is expressed in muscle cells during embryogenesis and in adult tissues. ASB2beta is part of an active E3 ubiquitin ligase complex and targets the actin-binding protein filamin B (FLNb) for proteasomal degradation. Thus, ASB2beta regulates FLNb functions by controlling its degradation. Knockdown of endogenous ASB2beta by shRNAs during induced differentiation of C2C12 cells delayed FLNb degradation as well as myoblast fusion and expression of muscle contractile proteins. Finally, knockdown of FLNb in ASB2beta knockdown cells restores myogenic differentiation. Altogether, our results suggest that ASB2beta is involved in muscle differentiation through the targeting of FLNb to destruction by the proteasome.

Figure 1

Expression of ASB2 mRNA in tissues. (a) Autoradiogram of ASB2 mRNA expression in human primary tissues (upper) and diagram showing the type and position of polyA+ RNAs on the membrane (lower). Autoradiograms of ASB2 mRNAs expression in mouse embryos (b) in mouse adult tissues (c). Northern blot was performed using 2 μg of polyA+ RNA (b) or 5 μg of total RNA (c). To confirm RNA loading and integrity, β-actin was used as a probe (b) or 18S rRNA was methylene blue-stained on membrane after transfer (c).

Figure 2

Expression pattern of ASB2 during chick embryogenesis. ASB2 mRNAs were detected in whole mount preparations (a to d) and sections through the trunk region (e), the intestine (f), the heart (g) or the developing limb skeletal muscle (g) of chick embryos by in situ hybridization. In each panel, the developmental stage is indicated. NT, neural tube; No, notochord; Dm, dermomyotome; My, myotome. Arrows indicate somites in a–c and e, limb skeletal muscle in d and h, and smooth muscle cells of the intestine in f. Arrowhead points to the heart.

Figure 3

Induction of ASB2 mRNA during differentiation of C2C12 myoblasts. C2C12 cells were cultured in growth medium (GM) and shifted to differentiation medium (DM) for 6 days. (a) Morphological changes of C2C12 cells for assessment of alignment, elongation and fusion were observed under a phase-contrast microscope. (b and c) Expression of myogenin, myosin heavy chain (MHC) and troponin T during differentiation of C2C12 cells. 10 μg-aliquots of each whole cell extract were analyzed by western blot using indicated antibodies. (d) Detection of ASB2 mRNA in C2C12 cells cultured in GM or in DM for 1 to 6 days. Northern blot analysis was performed using 5 μg of total RNA. Upper and lower panels are autoradiograms of mRNA of ASB2 and Arbp as assessment of RNA quantities in each lane, respectively.

Figure 4

Characterization of ASB2 isoforms. (a) Schematic representation of the domains of mouse ASB2 isoforms. Positions of the peptide sequences used for production of the 2PNAB1 and 1PLA sera are shown (b) Relative expression of ASB2 mRNAs in human skeletal muscle, heart, smooth muscle and myeloid cells. Quantitative real-time RT-PCR were carried out as described in Materials and Methods. Results are plotted as relative expression for ASB2α and ASB2β. Data corresponding to one out of three independent experiments are shown as mean ± standard deviation. (c) Alignments of the ubiquitin interacting motif (UIM), the BC-box and the Cul5 box of mouse ASB2β with the consensus sequences. Shaded regions represent residues identical (black) or similar (grey) to the domain class consensus sequences. e is a negatively charged residue, Φ represents hydrophobic residue and x is any amino acid. In a and B, amino acid numbering is indicated. (d) Isoform specificity of ASB2-specific antibodies. HeLa cells were transfected with FLAG-tagged ASB2β and FLAG-tagged ASB2α expression vectors or the corresponding empty vector (−). Urea soluble fractions (5 μg) were separated by SDS-PAGE and subjected to immunoblotting with anti-FLAG antibodies and 1PLA or 2PNAB1 sera, as indicated. (e) ASB2β protein is induced during differentiation of C2C12 myoblasts. C2C12 cells were cultured in GM and were shifted to DM for 2 and 4 days. 30 μg-aliquots of each urea soluble fraction were analyzed by western blot using the 2PNAB1 serum. (f) ASB2β associated with Elongins B and C, Cullin 5 and Rbx2. Sf21 cells were infected with baculoviruses expressing the proteins indicated. The lysates were immunoprecipitated (IP) with anti-FLAG antibodies. Crude extracts (input) and immune complexes were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ASB2 isoforms were detected using the 1PLA serum. (g–h) The ASB2β/ElonginBC/Cul5/Rbx2 complex had ubiquitin ligase activity. The cell lysates of (f) were subjected to anti-Flag immunoaffinity purification. The purified ASB2 complexes were incubated with Uba1, UbcH5a, ubiquitin and ATP to assess their ability to stimulate ubiquitylation by UbcH5a by Western blot using anti-polyubiquitin (g), anti-Flag (h, left panel) and anti-HA (h, right panel) antibodies. Arrow indicates the heavy chain of immunoglobulins (Ig(H)).

Figure 5

ASB2β induced ubiquitin-mediated FLNb degradation. (a) Expression of FLNa, FLNb and FLNc in C2C12 cells induced to differentiate. C2C12 cells were cultured in growth medium (GM) and shifted to differentiation medium (DM) for 6 days. Expression of FLNa, FLNb, FLNc and Erk2 was analyzed by Western blot using 20 μg-aliquots of whole cell extracts. (b) Quantification of FLNb-turnover following CHX-treatment. C2C12 cells were cultured in GM or shifted to DM for 18 hours and subsequently treated with cycloheximide (CHX) to block protein synthesis for various time (1.5, 3 and 6 hours). (c) FLNb degradation in differentiating C2C12 cells is dependent on the proteasome. C2C12 cells were cultured in DM for 16 hours and subsequently treated with MG132 or DMSO for 8 hours. In b and c, 10-μg aliquots of each whole cell extract was immunoblotted with antibodies to FLNb and Erk2 and quantification of FLNb level relative to Erk2 by densitometric scanning of three independent experiments is shown. (d) ASB2β induces polyubiquitylation of FLNb. Recombinant ASB2/elongin BC/cullin5/Rbx2 complexes were purified as in Figure 3f. All samples contained purified Uba1, UbcH5a, ubiquitin, APP-BP1/Uba3, Ubc12 and NEDD8. Purified FLNb-GFP was also provided as indicated and subjected to ubiquitylation. Aliquots of the reaction mixture were analyzed by western blotting using anti-GFP (upper panel) and antibodies to polyubiquitylated proteins (lower panel). (e) ASB2β-induced FLNb degradation is dependent on ASB2 ubiquitin ligase activity and the proteasome. NIH3T3 cells were mock-transfected or transfected for 24 h with FLNb-GFP together with GFP (−), or GFP-ASB2αwt, GFP-ASB2αLA, GFP-ASB2βwt, GFP-ASB2βLA expression vectors in the absence or presence of 1 μM MG132 for 18 h, as indicated. (f) ASB2β does not induce FLNa degradation. NIH3T3 cells were mock-transfected or transfected for 24 h with FLNa-GFP together with GFP (−), or GFP-ASB2 expression vectors, as indicated. In (e) and (f), 20 μg aliquots of whole cell extracts were immunoblotted with antibodies to GFP. (g) ASB2β induced degradation of endogenous FLNb. C2C12 myoblasts were transfected with GFP-ASB2βwt or GFP-ASB2βLA expression vectors, plated on fibronectin-coated coverslips 5 h after transfection, fixed 15 h later and analyzed using an antibody directed against FLNb. Scale bar, 10 μm.

Figure 6

Down-regulation of FLNb in primary myoblasts induced to differentiate correlates with ASB2β induction. Human myoblasts were cultured in growth medium (GM) and shifted to differentiation medium (DM) for 5 days. (a) Morphological changes of primary cells for assessment of alignment, elongation and fusion were observed under a phase-contrast microscope. Scale bar, 500 μm. (b) Expression of Myogenin, MHC, Troponin T and Erk2 during differentiation of primary cells. (c) Expression of ASB2β, FLNb and Erk2 during differentiation of primary cells. The numbers under the blot represent the percentages of FLNb relatively to day 0, calculated after normalization to Erk2. In b and c, 5 μg-aliquots of each whole cell extract were analyzed by western blot using indicated antibodies.

Figure 7

ASB2β knockdown delayed differentiation of C2C12 myoblasts. (a to f) Stable C2C12 cell populations expressing shRNAs directed against ASB2β (ASB2β KD1 and ASB2β KD2) or transfected with the empty vector (ctrl) were shifted to DM for 5 days. (a) 20 μg-aliquots of urea soluble fractions were analyzed by western blot using 2PNAB1 serum. 5 μg-aliquots of whole cell extracts were analyzed by western blot using anti-FLNb (N-16) and anti-Erk2 antibodies. (b) Expression of FLNb relative to Erk2 based on densitometric scanning. Results are mean ± standard deviation of three independent experiments. (c) Images of differentiating C2C12 cell populations (1, 3 and 5 days after initiation of differentiation) observed with a phase-contrast microscope are shown for each stable cell populations. Scale bar, 100 μm. (d) Fluorescence images of C2C12 cell populations, 5 days after induction of differentiation. Troponin T (green) and nuclei (blue) were stained to identify troponin T positive cells and facilitate myotube identification. Scale bar, 20 μm. (e) Histograms represent the fusion index calculated for C2C12 cell populations at day 3 and 5. Results are mean ± standard deviation from 2 independent experiments, where at least 200 nuclei/experiment were counted. (f) Expression of myosin heavy chain (MHC) and troponin T during differentiation of C2C12 cell populations. Ten μg-aliquots of each whole cell extract were analyzed by western blot, using anti-MHC and anti-troponin T antibodies. (g) FLNb knockdown in ASB2β knockdown C2C12 cells. ASB2β KD2 cells were stably transfected with constructs that generate shRNAs targeting ASB2β (ASB2β/FLNb KD) or luciferase (ASB2β/Luc KD). Ten μg-aliquots of each whole cell extract were immunoblotted with antibodies to FLNb, FLNc and Erk2. (h) Expression of myosin heavy chain (MHC) and troponin T during differentiation of ASB2β and FLNb double knockdown C2C12 cells. ASB2β/FLNb KD and ASB2β/Luc KD cells were shifted to DM for 6 days. Ten μg-aliquots of each whole cell extract were analyzed by western blotting using indicated antibodies.