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

Ziba Razinia 1 2 Massimiliano Baldassarre 1 Mohamed Bouaouina 1 Isabelle Lamsoul 0 3 Pierre G. Lutz 0 3 David A. Calderwood () 1 0 Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale , 31077 Toulouse , France 1 Deparment of Pharmacology and Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine , New Haven, CT 06520-8066 , USA 2 Department of Cell Biology, Yale University School of Medicine , New Haven, CT 06520-8066 , USA 3 Universite de Toulouse , Universite Paul Sabatier , Institut de Pharmacologie et de Biologie Structurale , 31077 Toulouse , France Summary Filamins 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 ce subunit ASB and that acute degradation of filamins through this ubiquitin-proteasome pathway correlates with cell differentiation. en Specifically, in myeloid leukemia cells retinoic-acid-induced expression of ASB2 triggers filamin degradation and recapitulates early ic events crucial for cell differentiation. ASB2 is thought to link substrates to the ubiquitin transferase machinery; however, the lleS amsescahyasntiosmshboywwthhaicththAeSsBu2bceilnl utelararcltoscwaliitzhaftiiloanmoinf AtoSiBnd2uc etodeagctriand-aritciohnsrterumcatuinreeds iusndkenpoewnnd.eHnteroen, wfielaumsiencaenlld-btahsaetdthanedacbtiionc-hbeinmdiicnagl C domain (ABD) of filamin mediates the interaction with ASB2. Furthermore, we show that the ABD is necessary and sufficient for fo ASB2-mediated filamin degradation. We propose that ASB2 exerts its effect by binding the ABD and mediating its polyubiquitylation, l so targeting filamins for degradation. These studies provide the molecular basis for ASB2-mediated filamin degradation and unravel a rn an important mechanism by which filamin levels can be acutely regulated. u Jo Key words: Filamin, ASB2, Calponin homology domain, Ubiquitylation - Introduction Filamins (FLNs) are essential, evolutionarily conserved, actinbinding proteins that crosslink and bundle actin filaments and link transmembrane receptors to intracellular cytoskeletal and signaling networks. Humans have three highly homologous FLN isoforms (FLNa, FLNb and FLNc). FLNa and FLNb show ubiquitous cellular and tissue expression patterns, whereas FLNc is thought to be largely restricted to skeletal and cardiac muscles (Thompson et al., 2000; van der Flier and Sonnenberg, 2001). FLN expression is essential to mammalian development (Zhou et al., 2007), and mutations in the human FLN genes result in diverse congenital anomalies including defects in the brain, bone, cardiovascular system and many other organs (Krakow et al., 2004; Robertson et al., 2003; Sheen et al., 2001). Although the mechanisms underlying the genetic mutations that disrupt development are unknown, the wide range of diseases suggests involvement of FLNs in a diverse variety of interactions. FLNs are cytoplasmic homodimers composed of two ~250 kDa subunits. Each FLN subunit consists of an N-terminal actin-binding domain (ABD), composed of two calponin homology (CH) domains, followed by 24 tandem immunoglobulin-like domains (IgFLN1IgFLN24) of ~96 amino acids interrupted by two hinge regions (Gorlin et al., 1990; Pudas et al., 2005; van der Flier and Sonnenberg, 2001). Dimerization of FLN subunits through their Cterminal IgFLN domain allows formation of a 160-nm V-shaped flexible structure that tethers actin filaments (Gorlin et al., 1990; Hartwig et al., 1980). In addition to F-actin, FLNs bind to more than 70 diverse proteins, including transmembrane receptors, signaling and adaptor proteins, and act as scaffolds for a wide range of signaling complexes (Feng and Walsh, 2004; Stossel et al., 2001; Zhou et al., 2010; Zhou et al., 2007). Through these interactions, mainly mediated by IgFLN16IgFLN24, FLNs link matrix and cytoskeletal signaling pathways and regulate reorganization of the actin cytoskeleton, cell shape, cell adhesion and migration (Stossel et al., 2001; Zhou et al., 2010). The importance of FLNs in a range of cellular processes and the numerous FLN-binding proteins suggests that FLN activity and interactions will be tightly regulated. A number of regulatory mechanisms have been identified, including dynamic phosphorylation and dephosphorylation of FLN or FLN-binding proteins (Jay et al., 2000; Kiema et al., 2006; Vadlamudi et al., 2002; Woo et al., 2004), intramolecular autoinhibition (Lad et al., 2007), mechanical force (Chen et al., 2009; Glogauer et al., 1998; Pentikainen and Ylanne, 2009) and competition between FLNbinding partners (Ithychanda et al., 2009; Lad et al., 2008). In addition, FLNs can be regulated by calpain and caspase proteolysis and, in turn, affect upon many processes, including apoptosis and motility (Browne et al., 2000; OConnell et al., 2009; Umeda et al., 2001). Finally, we recently found that FLN levels can be acutely controlled by the ubiquitinproteasome pathway, and that transient loss of FLNs is important during cell differentiation (Bello et al., 2009; Heuze et al., 2008). This is achieved through the action of ASB2 [for ankyrin repeat containing protein with a suppressor of cytokine signaling (SOCS) box 2] proteins, the specificity subunits of E3 ubiquitin ligase complexes (Bello et al., 2009; Heuze et al., 2008). ASB2, initially identified as a retinoic-acid-response gene in myeloid leukemia cells (Guibal et al., 2002), encodes two isoforms, ASB2 and ASB2, which are expressed in hematopoietic and muscle cells, respectively (Bello et al., 2009). ASB2 contains an N-terminal region followed by 15 predicted ankyrin repeats and a C-terminal SOCS box. The SOCS box mediates interaction with a cullin family member (cullin 5) and RING finger proteins (Rbx1 or Rbx2) by interacting with elongin BC to form an E3 ubiquitin ligase complex (Heuze et al., 2005; Kohroki et al., 2005). It has been proposed that ASB2 mediates polyubiquitylation and proteasomal degradation of bound proteins and regulates myeloid cell proliferation and differentiation by targeting regulators of hematopoiesis for degradation (Heuze et al., 2005). Other members of the ASB family have also been proposed to exert their effects by targeting specific regulatory proteins for degradation (Chung et al., 2005; Debrincat et al., 2007; Wilcox et al., 2004) and have been implicated in many biological processes (Boengler et al., 2003; Diks et al., 2006; Kile et al., 2001; Kohroki et al., 2001; ce McDaneld et al., 2004; McDaneld et al., 2006). en We recently identified FLNa, FLNb and FLNc as ASB2 ic substrates and showed that ASB2 induces proteasome-mediated lleS tdheegrSaOdaCtiSonboofx,FrLeNn (...truncated)