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 α oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2α) and a muscle-type (ASB2β) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2α is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2α structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2α, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of β1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2α, together with ankyrin repeats 1 to 10, is necessary for association of ASB2α with filamin A. Importantly, the ASB2α N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and β integrins. Together, these data provide new insights into the molecular mechanisms of ASB2α binding to filamin.
Highlights
Of therapeutic approaches to target leukemia stem cells in vivo, and for engraftment of normal hematopoietic stem cells following transplantation
ASB2␣-induced filamin A (FLNa) Degradation Regulates Adhesion of Hematopoietic Cells to Fibronectin—We previously found that ASB2␣ expression prevented cell spreading and inhibited initiation of migration, and that these effects were recapitulated by knocking down FLNa and FLNb (24, 29)
To assess the role of ASB2␣-induced FLN degradation in hematopoietic cell adhesion, myeloblastic PLB985 cells stably transfected with zinc inducible vectors encoding ASB2␣, E3 ubiquitin ligase defective mutant ASB2␣LA, or the corresponding empty vector control were cultured with or without ZnSO4, labeled with calcein AM, and allowed to adhere on fibronectin-coated wells in the absence or presence of Mn2ϩ to activate integrins
Summary
Cell Lines and Culture Conditions—Myeloblastic PLB985 cells stably transfected with ZnSO4-inducible vectors expressing ASB2␣wt, ASB2␣LA, ASB2⌬N, and ASB2␣Y9F were used as described (24). PLB985 cells stably transfected with zinc-inducible vectors encoding ASB2 proteins or the corresponding empty vector were cultured with or without ZnSO4 for 16 h, loaded with 0.5 M calcein AM in HBSS without Ca2ϩ and Mg2ϩ (HBSSϪ) containing 0.5% BSA, and washed once in HBSSϪ, 1 mM EDTA. The immobilized GFP-ASB2␣ANK(1– 10) protein was further incubated with 10 g of Escherichia coli extracts expressing GST or GST-IgFLNa21 in whole cell extract buffer for 3 h in ice. Beads were washed twice as described above. Adhesion assays were performed as described above (sample size: PLB985 FLNaKD ϭ 18 and PLB985 LucKD ϭ 18; from 6 independent experiments)
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