Functional and Structural Insights into ASB2α, a Novel Regulator of Integrin-dependent Adhesion of Hematopoietic Cells

Abstract

By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor ␣ oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2␣) and a muscle-type (ASB2␤) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2␣ is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2␣ structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2␣, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of ␤1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2␣, together with ankyrin repeats 1 to 10, is necessary for association of ASB2␣ with filamin A. Importantly, the ASB2␣ N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and ␤ integrins. Together, these data provide new insights into the molecular mechanisms of ASB2␣ binding to filamin.

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

“…Western blotting, immunofluorescence, and flow cytometry assays, we previously showed that ASB2␣ expression triggers polyubiquitination and proteasomal degradation of filamins (5)(6)(7)31). In keeping with our earlier studies (7), flow cytometric degradation assays show that the FLNa ABD is both necessary and sufficient for ASB2␣-mediated degradation as the isolated ABD is efficiently degraded following ASB2␣ expression, although FLNa lacking the ABD is resistant to degradation (Fig.…”

“…We have established that ASB2␣ targets filamins for proteasomal degradation (5,6,31), have shown that the N-terminal region of ASB2␣ along with the first 10 ankyrin repeats supports ASB2␣ binding to filamins (31), and have revealed that the ABD of filamin mediates interaction with ASB2␣ and is sufficient for ASB2␣-mediated degradation (7). Here, we 155-165).…”

“…ASB2␣ is thought to regulate hematopoietic cell differentiation by modulating cell spreading, actin remodeling, and cell adhesion (5,31), and the importance of ASB2␣ is supported by a recent knock-out mouse study showing that ASB2␣ is a critical regulator of spreading and migration in immature dendritic cells (26). We have established that ASB2␣ targets filamins for proteasomal degradation (5,6,31), have shown that the N-terminal region of ASB2␣ along with the first 10 ankyrin repeats supports ASB2␣ binding to filamins (31), and have revealed that the ABD of filamin mediates interaction with ASB2␣ and is sufficient for ASB2␣-mediated degradation (7).…”

“…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).…”

“…As cell knockdown for both FLNa and FLNb is also impaired in spreading (5, 6), it was proposed that ASB2␣ regulates hematopoietic cell spreading by targeting filamins for degradation (5,26,31). To test whether filamin degradation is the only mechanism by which ASB2␣ impacts cell spreading, we first sought to generate ASB2␣-resistant filamin constructs for use in reconstitution studies.…”