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 (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.     

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.The ubiquitin-proteasome system (UPS)¹ plays an essential role in the regulation of protein stability in eukaryotic cells. Degradation of a protein by the UPS entails two successive steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate and its degradation by the 26 S proteasome (1, 2). Ubiquitylation of protein substrates occurs through the sequential action of distinct enzymes: a ubiquitin-activating enzyme, E1; a ubiquitin-conjugating enzyme, E2; and a ubiquitin ligase, E3, responsible for the specific recognition of substrates. Increasing attention has been recently given to the UPS leading to the identification of hundreds of E3 ubiquitin ligases (E3s). Two major classes of E3s have been described: (i) E3s of the HECT (homologous to the E6-associated protein carboxyl terminus) domain family that function as ubiquitin carriers (3, 4) and (ii) E3s of the RING (really interesting new gene) or of the U box families that have no inherent catalytic activity but recruit an E2 enzyme toward substrates (5–7).Classical approaches to identify substrates of E3s are based on the identification of interacting proteins. Although these have successfully led to the identification of a number of substrates of monomeric E3s, identification of substrates of multimeric E3s is very challenging because of the weak affinity of substrates for their requisite specificity subunit and because of the labile nature of the substrate complexed with the specificity subunit (8).Acute promyelocytic leukemia (APL) is associated with six reciprocal translocations always involving the retinoic acid receptor α (RARα) gene (9–11). The RARα protein is a member of the nuclear receptor superfamily that stimulates myeloid differentiation in the presence of its ligand, all-trans-retinoic acid (RA). In more than 95% of APL, the t(15;17) translocation between the promyelocytic leukemia (PML) gene on chromosome 15 and the RARα gene on chromosome 17 produces the PML-RARα fusion protein (12). The PML-RARα protein enhances the repression of RARα target genes by increasing associations with corepressors (13–15) and by recruiting DNA methyltransferases (16). These complexes dissociate from the PML-RARα fusion protein in the presence of pharmacological concentrations of RA perhaps explaining why APL cells are sensitive to RA treatment. Indeed at pharmacological concentrations, RA induces complete remission in a high percentage of APL patients (17–19). By studying RA-induced differentiation of APL cells we have attempted to identify some of the genes that may be up-regulated during this process to further understand the control of growth and differentiation in leukemia (20). One gene identified in this manner, ASB2 (ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) is an RA-response gene involved in induced differentiation of myeloid leukemia cells (21–23).The ASB2 protein is a subunit of a multimeric E3 ubiquitin ligase of the cullin-RING ligase family (24, 25). The ASB2 suppressor of cytokine signaling box can be divided into a BC box that defines a binding site for the Elongin BC complex and a Cul5 box that determines the binding specificity for Cullin5 (24, 26). Indeed the ASB2 protein, by interacting with the Elongin BC complex, can assemble with a Cullin5/Rbx1 or -2 module to reconstitute an active E3 ubiquitin ligase complex (23–25). Within this complex, the ASB2 protein is the specificity subunit involved in the recruitment of specific substrate(s). Furthermore endogenous ASB2 protein was copurified with ubiquitin ligase activity in RA-treated APL cells suggesting that, during induced differentiation of leukemia cells, the ASB2 protein may target proteins involved in blocking differentiation to destruction by the proteasome machinery (24). We recently identified actin-binding proteins filamin A (FLNa) and filamin B (FLNb) as ASB2 targets and showed that ASB2 triggers ubiquitylation and drives proteasome-mediated degradation of these proteins during RA-induced differentiation of myeloid leukemia cells (23).With the aim to develop a strategy to identify E3 substrates that are degraded by the proteasome, we used an MS approach to identify ASB2 substrates in physiologically relevant settings. Indeed we used label-free quantitative proteomics to identify proteins that are absent or less abundant in cells that express wild-type ASB2 but that accumulate in cells expressing an ASB2 E3 ligase-defective mutant. Application of label-free MS methods that have the advantage to be simple, fast, and cheap enabled the identification of FLNa and FLNb as ASB2 substrates. This study provides a new strategy for the identification of E3 substrates that have to be degraded.     

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

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.     

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.     

Ubiquitination of APOBEC3G by an HIV-1 Vif-Cullin5-Elongin B-Elongin C complex is essential for Vif function

The human immunodeficiency virus type 1 (HIV-1) virion infectivity factor (Vif) overcomes the antiviral activity of APOBEC3G to protect HIV-1 DNA from G-to-A hypermutation. Vif targets APOBEC3G for ubiquitination and proteasomal degradation by forming an SCF-like E3 ubiquitin ligase complex composed of Cullin5, Elongin B, and Elongin C (Vif-BC-Cul5) through a novel SOCS-box motif. In this paper, we have established an in vitro ubiquitin conjugation assay with purified Vif-BC-Cul5 complex and reported that the Vif-BC-Cul5 complex could function as an E3 ligase for APOBEC3G in vitro. A Vif-BC-Cul5 complex promotes the in vitro ubiquitination of the wild type, APOBEC3G but not that of D128K mutant, which does not interact with Vif. We have also investigated several loss-of-function Vif mutants. One mutant, SLQ144/146AAA, lost its activity on APOBEC3G because it could not form a complex due to mutations in SOCS-box motif. Other mutants, C114S and C133S, also lost their activity because of loss of the E3 ligase activity of a Vif-BC-Cul5 complex, although these mutants retained the ability to bind to APOBEC3G as well as Cul5 complex. These findings suggest that the E3 ubiquitin ligase activity of the Vif-BC-Cul5 complex is essential for Vif function against APOBEC3G.     

Regulation of DNA topoisomerase IIα stability by the ECV ubiquitin ligase complex

In this study, we attempted to elucidate the E3 ubiquitin ligase for topo IIα. When cullins and VHL were ectopically expressed in HT1080 and HEK293T cells, topo IIα was degraded most prominently in cullin 2- and VHL-expressing cells. Cullin 2 and the β domain (aa 114-123) of VHL, a subunit of the ECV (Elongin B/C-cullin 2-VHL protein) complex, specifically interact with the ATPase domain of topo IIα. We identified that topo IIα associated with endogenous Elongin C. In HT1080 cells co-transfected with deletion mutants of topo IIα GRDD (glucose-regulated destruction domain) and VHL, topo IIα was degraded by VHL expression. These results demonstrate that ECV acts as E3 ubiquitin ligase targeting GRDD-independent topo IIα to the ubiquitin-proteasome pathway.     

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.     

Ankyrin repeat and SOCS box 3 (ASB3) mediates ubiquitination and degradation of tumor necrosis factor receptor II

Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-alpha by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.     

Identification of Elongin C and Skp1 sequences that determine Cullin selection

The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) complexes belong to families of structurally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit, which is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that activates ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzyme Ubc5. In SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits, which are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that activates ubiquitylation of target proteins, in most cases by the E2 Cdc34. In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases. Our findings provide evidence that E2 selection by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate recognition subunit but by yet to be elucidated features of these macromolecular complexes.     

Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

The ankyrin and SOCS (suppressor of cytokine signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting 18 members in humans, the identity of the physiological targets of the Asb proteins remains largely unexplored. To increase our understanding of the function of ASB proteins, we conducted a family-wide SILAC (stable isotope labeling by amino acids in cell culture)-based protein/protein interaction analysis. This investigation led to the identification of novel as well as known ASB-associated proteins like Cullin 5 and Elongins B/C. We observed that several proteins can be bound by more than one Asb protein. The additional exploration of this phenomenon demonstrated that ASB-Cullin 5 complexes can oligomerize and provides evidence that Cullin 5 forms heterodimeric complexes with the Cullin 4a-DDB1 complex. We also demonstrated that ASB11 is a novel endoplasmic reticulum-associated ubiquitin ligase with the ability to interact and promote the ubiquitination of Ribophorin 1, an integral protein of the oligosaccharyltransferase (OST) glycosylation complex. Moreover, expression of ASB11 can increase Ribophorin 1 protein turnover in vivo. In summary, we provide a comprehensive protein/protein interaction data resource that can aid the biological and functional characterization of ASB ubiquitin ligases.     

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.     

The ASB2β Ubiquitin-interacting motif is involved in its monoubiquitination

ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2α and ASB2β. ASB2β has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2α does not. Here, we provide the first evidence that ASB2β but not ASB2α is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2β and hemagglutinin (HA)-tagged Ub were co-expressed in HEK293 cells using the pCMV expression vector. Immunoprecipitation with an anti-Myc antibody followed by immunoblotting with anti-Myc and anti-HA antibodies showed an additional ASB2β protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2β, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2β is produced in cells. ASB2β with mutations in the UIM motif; either Glu·Asp·Glu27-29Ala·Ala·Ala mutations (ASB2β M1) or a Ser38Ala mutation, (ASB2β M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2β monoubiquitination. Furthermore, an ASB2β mutant that lacked a SOCS box (ASB2β ΔC) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2β. In contrast, an ASB2β mutant that lacked the UIM-containing domain (ASB2β ΔN) was not monoubiquitinated. These results suggest that ASB2β but not ASB2α might be monoubiquitinated and that the ASB2β UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination.     

ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism

The molecular mechanisms of endothelial differentiation into a functional vascular network are incompletely understood. To identify novel factors in endothelial development, we used a microarray screen with differentiating embryonic stem (ES) cells that identified the gene for ankyrin repeat and SOCS box protein 4 (ASB4) as the most highly differentially expressed gene in the vascular lineage during early differentiation. Like other SOCS box-containing proteins, ASB4 is the substrate recognition molecule of an elongin B/elongin C/cullin/Roc ubiquitin ligase complex that mediates the ubiquitination and degradation of substrate protein(s). High levels of ASB4 expression in the embryonic vasculature coincide with drastic increases in oxygen tension as placental blood flow is initiated. However, as vessels mature and oxygen levels stabilize, ASB4 expression is quickly downregulated, suggesting that ASB4 may function to modulate an endothelium-specific response to increasing oxygen tension. Consistent with the hypothesis that ASB4 function is regulated by oxygen concentration, ASB4 interacts with the factor inhibiting HIF1alpha (FIH) and is a substrate for FIH-mediated hydroxylation via an oxygen-dependent mechanism. Additionally, overexpression of ASB4 in ES cells promotes differentiation into the vascular lineage in an oxygen-dependent manner. We postulate that hydroxylation of ASB4 in normoxia promotes binding to and degradation of substrate protein(s) to modulate vascular differentiation.     

Differential regulation of PML-RARα stability by the ubiquitin ligases SIAH1/SIAH2 and TRIAD1

The ubiquitin proteasome system plays an important role in normal and malignant hematopoiesis and relies on the concerted action of three enzyme families. The E2 ubiquitin conjugase UBCH8 (ubiquitin conjugating enzyme [human] 8) cooperates with the E3 ubiquitin ligases SIAH1 and SIAH2 (seven in absentia homolog 1/2) to mediate the proteasomal degradation of oncoproteins. One such protein is the leukemia fusion protein PML-RARα (promyelocytic leukemia-retinoic acid receptorα) that is associated with acute promyelocytic leukemia. A limited number of UBCH8 interaction partners that participate in the UBCH8-dependent depletion of cancer-relevant proteins are known. We report here that TRIAD1 (two RING fingers and DRIL [double RING finger linked] 1), an E3 ubiquitin ligase relevant for the clonogenic growth of myloid progenitors, binds UBCH8 as well as PML-RARα. Moreover, there is concurrent induction of TRIAD1 and UBCH8 upon combinatorial treatment of acute promyelocytic leukemia cells with the pro-apoptotic epigenetic modulator valproic acid and the differentiation inducing agent all-trans retinoic acid. However, in sharp contrast to SIAH1/SIAH2 and UBCH8, TRIAD1 binding to PML-RARα has no effect on its turnover. In summary, our data exclude TRIAD1 as crucial regulator of the leukemic determinant PML-RARα, but highlight the prominence of the UBCH8/SIAH axis in PML-RARα degradation.