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 ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of filamins. This led to the proposal that ASB2α 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 ASB2α-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 ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of filamins.
From the Departments of Cell Biology and.
Ziba
Z
Razinia
Massimiliano
M
Baldassarre
Gaia
G
Cantelli
David A
DA
Calderwood
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D064448:Filamins / Q000235:genetics / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
C437987:ASB2 protein, human; C578663:FLNA protein, human; D064448:Filamins; D050826:Suppressor of Cytokine Signaling Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex
Actin; Calponin Homology Domain; Cell Biology; Cytoskeleton; E3 Ubiquitin Ligase; Filamin; Stress Fiber