Molecular Tuning of Filamin A Activities in the Context of Adhesion and Migration.

The dynamic organization of actin cytoskeleton meshworks relies on multiple actin-binding proteins endowed with distinct actin-remodeling activities. Filamin A is a large multi-domain scaffolding protein that cross-links actin filaments with orthogonal orientation in response to various stimuli. As such it plays key roles in the modulation of cell shape, cell motility, and differentiation throughout development and adult life. The essentiality and complexity of Filamin A is highlighted by mutations that lead to a variety of severe human disorders affecting multiple organs. One of the most conserved activity of Filamin A is to bridge the actin cytoskeleton to integrins, thereby maintaining the later in an inactive state. We here review the numerous mechanisms cells have developed to adjust Filamin A content and activity and focus on the function of Filamin A as a gatekeeper to integrin activation and associated adhesion and motility.

Loss of Asb2 Impairs Cardiomyocyte Differentiation and Leads to Congenital Double Outlet Right Ventricle.

Defining the pathways that control cardiac development facilitates understanding the pathogenesis of congenital heart disease. Herein, we identify enrichment of a Cullin5 Ub ligase key subunit, Asb2, in myocardial progenitors and differentiated cardiomyocytes. Using two conditional murine knockouts, Nkx+/Cre.Asb2fl/fl and AHF-Cre.Asb2fl/fl, and tissue clarifying technique, we reveal Asb2 requirement for embryonic survival and complete heart looping. Deletion of Asb2 results in upregulation of its target Filamin A (Flna), and concurrent Flna deletion partially rescues embryonic lethality. Conditional AHF-Cre.Asb2 knockouts harboring one Flna allele have double outlet right ventricle (DORV), which is rescued by biallelic Flna excision. Transcriptomic and immunofluorescence analyses identify Tgfß/Smad as downstream targets of Asb2/Flna. Finally, using CRISPR/Cas9 genome editing, we demonstrate Asb2 requirement for human cardiomyocyte differentiation suggesting a conserved mechanism between mice and humans. Collectively, our study provides deeper mechanistic understanding of the role of the ubiquitin proteasome system in cardiac development and suggests a previously unidentified murine model for DORV.

The E3 Ubiquitin Ligase Asb2α in T Helper 2 Cells Negatively Regulates Antitumor Immunity in Colorectal Cancer.

The escape of cancer cells from host immunosurveillance involves a shift in immune responses, including an imbalance in Th1 and Th2 cells. A Th1-dominated immune response predicts positive outcomes in colorectal cancer. The E3 ubiquitin ligase, Asb2α, is expressed in Th2 cells, but its roles in T-cell maturation and cancer are unclear. We provide evidence that the Th2 master regulator, Gata3, induces Asb2 Loss of Asb2 did not affect Th differentiation ex vivo, but reduced IL4 production from Th2 cells. We found that high ASB2 expression was associated with poor outcome in colorectal cancer. Loss of Asb2 from hematopoietic cells promoted a Th1 response and attenuated colitis-associated tumorigenesis in mice. Diminished Th2 function correlated with increased IFNγ production and an enhanced type 1 antitumor immune response in Asb2-deficient mice. Our work suggests that Asb2α promotes a Th2 phenotype in vivo, which in turn is associated with tumor progression in a mouse model of colitis.

Asb2α-Filamin A Axis Is Essential for Actin Cytoskeleton Remodeling During Heart Development.

RATIONALE: Heart development involves differentiation of cardiac progenitors and assembly of the contractile sarcomere apparatus of cardiomyocytes. However, little is known about the mechanisms that regulate actin cytoskeleton remodeling during cardiac cell differentiation. OBJECTIVE: The Asb2α (Ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) CRL5 (cullin 5 RING E3 ubiquitin ligase) triggers polyubiquitylation and subsequent degradation by the proteasome of FLNs (filamins). Here, we investigate the role of Asb2α in heart development and its mechanisms of action. METHODS AND RESULTS: Using Asb2 knockout embryos, we show that Asb2 is an essential gene, critical to heart morphogenesis and function, although its loss does not interfere with the overall patterning of the embryonic heart tube. We show that the Asb2α E3 ubiquitin ligase controls Flna stability in immature cardiomyocytes. Importantly, Asb2α-mediated degradation of the actin-binding protein Flna marks a previously unrecognized intermediate step in cardiac cell differentiation characterized by cell shape changes and actin cytoskeleton remodeling. We further establish that in the absence of Asb2α, myofibrils are disorganized and that heartbeats are inefficient, leading to embryonic lethality in mice. CONCLUSIONS: These findings identify Asb2α as an unsuspected key regulator of cardiac cell differentiation and shed light on the molecular and cellular mechanisms determining the onset of myocardial cell architecture and its link with early cardiac function. Although Flna is known to play roles in cytoskeleton organization and to be required for heart function, this study now reveals that its degradation mediated by Asb2α ensures essential functions in differentiating cardiac progenitors.

Cullin 5-RING E3 ubiquitin ligases, new therapeutic targets?

Ubiquitylation is a reversible post-translational modification of proteins that controls a myriad of functions and cellular processes. It occurs through the sequential action of three distinct enzymes. E3 ubiquitin ligases (E3s) play the role of conductors of the ubiquitylation pathway making them attractive therapeutic targets. This review is dedicated to the largest family of multimeric E3s, the Cullin-RING E3 (CRL) family and more specifically to cullin 5 based CRLs that remains poorly characterized.

Substrates of the ASB2α E3 ubiquitin ligase in dendritic cells.

Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functions that are mediators of innate and adaptive immune responses. Transcriptomic and proteomic approaches have been used so far to identify transcripts and proteins that are differentially expressed in these subsets to understand the respective functions of cDCs subsets. Here, we showed that the Cullin 5-RING E3 ubiquitin ligase (E3) ASB2α, by driving degradation of filamin A (FLNa) and filamin B (FLNb), is responsible for the difference in FLNa and FLNb abundance in the different spleen cDC subsets. Importantly, the ability of these cDC subsets to migrate correlates with the level of FLNa. Furthermore, our results strongly point to CD4 positive and double negative cDCs as distinct populations. Finally, we develop quantitative global proteomic approaches to identify ASB2α substrates in DCs using ASB2 conditional knockout mice. As component of the ubiquitin-proteasome system (UPS) are amenable to pharmacological manipulation, these approaches aimed to the identification of E3 substrates in physiological relevant settings could potentially lead to novel targets for therapeutic strategies.

Phosphorylation of serine 323 of ASB2α is pivotal for the targeting of filamin A to degradation.

ASB proteins are the specificity subunits of cullin5-RING E3 ubiquitin ligases (CRL5) that play roles in ubiquitin-mediated protein degradation. However, how their activity is regulated remains poorly understood. Here, we unravel a novel mechanism of regulation of a CRL5 through phosphorylation of its specificity subunit ASB2α. Indeed, using mass spectrometry, we showed for the first time that ASB2α is phosphorylated and that phosphorylation of serine-323 (Ser-323) of ASB2α is crucial for the targeting of the actin-binding protein filamin A (FLNa) to degradation. Mutation of ASB2α Ser-323 to Ala had no effect on intrinsic E3 ubiquitin ligase activity of ASB2α but abolished the ability of ASB2α to induce degradation of FLNa. In contrast, the ASB2α Ser-323 to Asp phosphomimetic mutant induced acute degradation of FLNa. Moreover, inhibition of the extracellular signal-regulated kinases 1 and 2 (Erk1/2) activity reduced ASB2α-mediated FLNa degradation. We further showed that the subcellular localization of ASB2α to actin-rich structures is dependent on ASB2α Ser-323 phosphorylation and propose that the interaction with FLNa depends on the electrostatic potential redistribution induced by the Ser-323 phosphate group. Taken together, these data unravel an important mechanism by which ASB2α-mediated FLNa degradation can be regulated.

ASB2α regulates migration of immature dendritic cells.

The actin-binding protein filamins (FLNs) are major organizers of the actin cytoskeleton. They control the elasticity and stiffness of the actin network and provide connections with the extracellular microenvironment by anchoring transmembrane receptors to the actin filaments. Although numerous studies have revealed the importance of FLN levels, relatively little is known about the regulation of its stability in physiological relevant settings. Here, we show that the ASB2α cullin 5-ring E3 ubiquitin ligase is highly expressed in immature dendritic cells (DCs) and is down-regulated after DC maturation. We further demonstrate that FLNs are substrates of ASB2α in immature DCs and therefore are not stably expressed in these cells, whereas they exhibit high levels of expression in mature DCs. Using ASB2 conditional knockout mice, we show that ASB2α is a critical regulator of cell spreading and podosome rosette formation in immature DCs. Furthermore, we show that ASB2(-/-) immature DCs exhibit reduced matrix-degrading function leading to defective migration. Altogether, our results point to ASB2α and FLNs as newcomers in DC biology.

Quantitative proteomic analysis to decipher the differential apoptotic response of bortezomib-treated APL cells before and after retinoic acid differentiation reveals involvement of protein toxicity mechanisms.

The ubiquitin-proteasome system allows the targeted degradation of proteins and plays a critical role in the regulation of many cellular processes. Proteasome inhibition is a recent antitumor therapeutic strategy and bortezomib was the first proteasome inhibitor approved for clinical use. In this study, we used the NB4 cell line to investigate the effects of bortezomib toward acute promyelocytic leukemia cells before and after retinoic acid-induced differentiation. We showed that apoptosis level after bortezomib treatment is higher in NB4 cells than in differentiated NB4 cells. To compare early protein variations upon bortezomib treatment in both NB4 cell populations, we performed a quantitative proteomic analysis based on iTRAQ peptide labeling followed by data analysis with in-house developed scripts. This strategy revealed the regulation of 14 proteins principally involved in protein stress response and apoptosis in NB4 cells after proteasome inhibition. Altogether, our results suggest that the differential level of apoptosis induced by bortezomib treatment in both NB4 cell populations could result from distinct protein toxicity level.

Filamins but not Janus kinases are substrates of the ASB2α cullin-ring E3 ubiquitin ligase in hematopoietic cells.

The ASB2α protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2α substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2α biological effects is crucial for the understanding of ASB2α functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2α induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates.

Macrophage mesenchymal migration requires podosome stabilization by filamin A.

Filamin A (FLNa) is a cross-linker of actin filaments and serves as a scaffold protein mostly involved in the regulation of actin polymerization. It is distributed ubiquitously, and null mutations have strong consequences on embryonic development in humans, with organ defects which suggest deficiencies in cell migration. We have reported previously that macrophages, the archetypal migratory cells, use the protease- and podosome-dependent mesenchymal migration mode in dense three-dimensional environments, whereas they use the protease- and podosome-independent amoeboid mode in more porous matrices. Because FLNa has been shown to localize to podosomes, we hypothesized that the defects seen in patients carrying FLNa mutations could be related to the capacity of certain cell types to form podosomes. Using strategies based on FLNa knock-out, knockdown, and rescue, we show that FLNa (i) is involved in podosome stability and their organization as rosettes and three-dimensional podosomes, (ii) regulates the proteolysis of the matrix mediated by podosomes in macrophages, (iii) is required for podosome rosette formation triggered by Hck, and (iv) is necessary for mesenchymal migration but dispensable for amoeboid migration. These new functions assigned to FLNa, particularly its role in mesenchymal migration, could be directly related to the defects in cell migration described during the embryonic development in FLNa-defective patients.

Functional and structural insights into ASB2alpha, a novel regulator of integrin-dependent adhesion of hematopoietic cells.

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 (ASB2ß) 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 ß1 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 ß integrins. Together, these data provide new insights into the molecular mechanisms of ASB2α binding to filamin.

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

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 subunit ASBα 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 ASB2α triggers filamin degradation and recapitulates early events crucial for cell differentiation. ASB2α is thought to link substrates to the ubiquitin transferase machinery; however, the mechanism by which ASB2α interacts with filamin to induce degradation remained unknown. Here, we use cell-based and biochemical assays to show that the subcellular localization of ASB2α to actin-rich structures is dependent on filamin and that the actin-binding domain (ABD) of filamin mediates the interaction with ASB2α. Furthermore, we show that the ABD is necessary and sufficient for ASB2α-mediated filamin degradation. We propose that ASB2α exerts its effect by binding the ABD and mediating its polyubiquitylation, so targeting filamins for degradation. These studies provide the molecular basis for ASB2α-mediated filamin degradation and unravel an important mechanism by which filamin levels can be acutely regulated.

Filamins regulate cell spreading and initiation of cell migration.

Mammalian filamins (FLNs) are a family of three large actin-binding proteins. FLNa, the founding member of the family, was implicated in migration by cell biological analyses and the identification of FLNA mutations in the neuronal migration disorder periventricular heterotopia. However, recent knockout studies have questioned the relevance of FLNa to cell migration. Here we have used shRNA-mediated knockdown of FLNa, FLNb or FLNa and FLNb, or, alternatively, acute proteasomal degradation of all three FLNs, to generate FLN-deficient cells and assess their ability to migrate. We report that loss of FLNa or FLNb has little effect on migration but that knockdown of FLNa and FLNb, or proteolysis of all three FLNs, impairs migration. The observed defect is primarily a deficiency in initiation of motility rather than a problem with maintenance of locomotion speed. FLN-deficient cells are also impaired in spreading. Re-expression of full length FLNa, but not re-expression of a mutated FLNa lacking immunoglobulin domains 19 to 21, reverts both the spreading and the inhibition of initiation of migration.Our results establish a role for FLNs in cell migration and spreading and suggest that compensation by other FLNs may mask phenotypes in single knockout or knockdown cells. We propose that interactions between FLNs and transmembrane or signalling proteins, mediated at least in part by immunoglobulin domains 19 to 21 are important for both cell spreading and initiation of migration.

A label-free quantitative proteomics strategy to identify E3 ubiquitin ligase substrates targeted to proteasome degradation.

The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded.

JAM-L-mediated leukocyte adhesion to endothelial cells is regulated in cis by alpha4beta1 integrin activation.

Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L-VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.

ASB2 targets filamins A and B to proteasomal degradation.

The ordered series of proliferation and differentiation from hematopoietic progenitor cells is disrupted in leukemia, resulting in arrest of differentiation at immature proliferative stages. Characterizing the molecular basis of hematopoietic differentiation is therefore important for understanding and treating disease. Retinoic acid induces expression of ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) in acute promyelocytic leukemia cells, and ASB2 expression inhibits growth and promotes commitment, recapitulating an early step critical for differentiation. ASB2 is the specificity subunit of an E3 ubiquitin ligase complex and is proposed to exert its effects by regulating the turnover of specific proteins; however, no ASB2 substrates had been identified. Here, we report that ASB2 targets the actin-binding proteins filamin A and B for proteasomal degradation. Knockdown of endogenous ASB2 in leukemia cells delays retinoic acid-induced differentiation and filamin degradation; conversely, ASB2 expression in leukemia cells induces filamin degradation. ASB2 expression inhibits cell spreading, and this effect is recapitulated by knocking down both filamin A and filamin B. Thus, we suggest that ASB2 may regulate hematopoietic cell differentiation by modulating cell spreading and actin remodeling through targeting of filamins for degradation.

Ubiquitin-mediated proteasomal degradation in normal and malignant hematopoiesis.

Understanding the molecular mechanisms controlling normal hematopoietic differentiation is critical to develop new treatments for blood diseases and to manipulate stem cells. Despite the identification of many players in hematopoiesis, the molecular mechanisms controlling hematopoietic differentiation remain poorly understood. Due to a number of recent findings, the targeting of regulators of hematopoiesis to proteasomal degradation might be an important step in control of this developmental program.

PRAM-1 potentiates arsenic trioxide-induced JNK activation.

The promyelocytic leukemia RARalpha target gene encoding an adaptor molecule-1 (PRAM-1) is involved in a signaling pathway induced by retinoic acid in acute promyelocytic leukemia (APL) cells. To better understand the function of PRAM-1, we have undertaken the identification of its partners through a yeast two-hybrid screen. Here, we show that the proline-rich domain of PRAM-1 interacted with the Src homology 3 (SH3) domain of hematopoietic progenitor kinase 1 (HPK-1)-interacting protein of 55 kDa (HIP-55, also called SH3P7 and Abp1) known to stimulate the activity of HPK-1 and c-Jun N-terminal kinase (JNK). Overexpression of PRAM-1 in the NB4 APL cell line increased arsenic trioxide-induced JNK activation through a caspase 3-like-dependent activity. Dissociation of the SH3 domain from the rest of the HIP-55 protein was observed in the NB4 APL cell line treated with arsenic trioxide due to specific cleavage by caspase 3-like enzymes. The cleavage of HIP-55 correlated with the induction of PRAM-1 mRNA and protein expression. Taken together, our results suggest that the caspase 3-cleaved SH3 domain of HIP-55 is likely involved in PRAM-1-mediated JNK activation upon arsenic trioxide-induced differentiation of NB4 cells.

ASB2 is an Elongin BC-interacting protein that can assemble with Cullin 5 and Rbx1 to reconstitute an E3 ubiquitin ligase complex.

The ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB2) gene was identified as a retinoic acid-response gene and a target of the promyelocytic leukemia-retinoic acid receptor-alpha oncogenic protein characteristic of acute promyelocytic leukemia. Expression of ASB2 in myeloid leukemia cells inhibits growth and promotes commitment, recapitulating an early step known to be critical for differentiation. Here we show that ASB2, by interacting with the Elongin BC complex, can assemble with Cullin5.Rbx1 to form an E3 ubiquitin ligase complex that stimulates polyubiquitination by the E2 ubiquitin-conjugating enzyme Ubc5. This is a first indication that a member of the ASB protein family, ASB2, is a subunit of an ECS (Elongin C-Cullin-SOCS box)-type E3 ubiquitin ligase complex. Altogether, our results strongly suggest that ASB2 targets specific proteins to destruction by the proteasome in leukemia cells that have been induced to differentiate.