The E3 ubiquitin ligase specificity subunit ASB2α targets filamins for proteasomal degradation by interacting with the filamin actin-binding domain

Abstract

SummaryFilamins are an important family of actin-binding and crosslinking proteins that mediate remodeling of the actin cytoskeleton and maintain extracellular matrix connections by anchoring transmembrane proteins to actin filaments and linking them to intracellular signaling cascades. We recently found that filamins are targeted for proteasomal degradation by the E3 ubiquitin ligase specificity subunit ASB and that acute degradation of filamins through this ubiquitin-proteasome pathway correlates with cell differentiation. Specifically, in myeloid leukemia cells retinoic-acid-induced expression of ASB2 triggers filamin degradation and recapitulates early events crucial for cell differentiation. ASB2 is thought to link substrates to the ubiquitin transferase machinery; however, the mechanism by which ASB2 interacts with filamin to induce degradation remained unknown. Here, we use cell-based and biochemical assays to show that the subcellular localization of ASB2 to actin-rich structures is dependent on filamin and that the actin-binding domain (ABD) of filamin mediates the interaction with ASB2. Furthermore, we show that the ABD is necessary and sufficient for ASB2-mediated filamin degradation. We propose that ASB2 exerts its effect by binding the ABD and mediating its polyubiquitylation, so targeting filamins for degradation. These studies provide the molecular basis for ASB2-mediated filamin degradation and unravel an important mechanism by which filamin levels can be acutely regulated.

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

“…A definitive test of filamins' roles in ASB2␣ function requires a better understanding of ASB2␣-mediated degradation and identification of functional ASB2␣-resistant filamin mutants. In our previous studies, we provided insight into the mechanism by which ASB2␣ targets filamins for degradation, and we identified the ABD of filamin as sufficient and necessary for ASB2␣-mediated degradation (7). We now map the ASB2␣ target region to the calponin homology domain 1 (CH1) in the FLNa actin-binding domain and identify a specific cluster of lysine residues in CH1 that are required for FLNa proteasomal degradation.…”

“…1 mg/ml fibronectin solution and MG132 were purchased from Sigma. Full-length FLNa-GFP, FLNaABD (aa 1-275)-GFP, FLNa⌬ABD (aa 276 -2647)-GFP, and ␣-actinin1 ABD (aa 1-253)-GFP have been described previously (7). FLNaCH1 (aa 1-154) and FLNaCH2 (aa 156 -275) were generated using polymerase chain reaction and subcloned into GFP pCDNA3, a modified version of pCDNA3 expression vector (Invitrogen).…”

“…GST-FLNaABD K42R/K43R/K135R was generated by QuikChange site-directed mutagenesis. dsRed-ASB2␣ and dsRed-ASB2␣⌬S expression constructs were described previously (5,7,31).…”

“…Flow Cytometry Assays of ASB2␣ Activity-Flow cytometry assays were described previously (7). Briefly, for the serial transfection, CHO cells were transfected with the indicated GFPtagged constructs, and 24 h after transfection, cells were re-transfected with either dsRed-ASB2␣ or dsRed-ASB2␣⌬S.…”

“…We have shown that ASB2␣ expression triggers rapid proteasomal degradation of filamins in various cell types (5)(6)(7). Furthermore, retinoic acid-induced expression of ASB2␣ correlates with filamin down-regulation in myeloid leukemia cells induced to differentiate (5), and knock down of endogenous ASB2␣ delays filamin degradation and differentiation (5).…”