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.     

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.     

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.     

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.     

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.     

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.     

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.     

Arsenic-induced SUMO-dependent recruitment of RNF4 into PML nuclear bodies

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation

In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.     

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.