ASB2α, an E3 Ubiquitin Ligase Specificity Subunit, Regulates Cell Spreading and Triggers Proteasomal Degradation of Filamins by Targeting the Filamin Calponin Homology 1 Domain

Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2 alpha, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of filamins. This led to the proposal that ASB2 alpha regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of filamins. Here, we show that the calponin homology domain 1 (CH1), within the filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2 alpha-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2 alpha-mediated degradation without altering ASB2 alpha binding. These lysines lie within previously predicted actin-binding sites, and the ASB2 alpha-resistant filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of alpha-actinin1, which is resistant to ASB2 alpha-mediated degradation, we generated an ASB2 alpha-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2 alpha expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2 alpha-mediated effects on cell spreading are due to loss of filamins.