The transcription factor NF-B is critical for the induction of cancer, including adult T-cell leukemia, which is linked to infection by human T-cell leukemia virus type 1 and the expression of its regulatory protein Tax. Although activation of the NF-B pathway by Tax involves its interaction with the regulatory subunit of the IB kinase (IKK) complex, NEMO/IKK␥, the mechanism by which Tax activates specific cellular genes in the nucleus remains unknown. Here, we demonstrate that the attachment of SUMO-1 to Tax regulates its localization in nuclear bodies and the recruitment of both the RelA subunit of NF-B and free IKK␥ in these nuclear structures. However, this sumoylation step is not sufficient for the activation of the NF-B pathway by Tax. This activity requires the prior ubiquitination and colocalization of ubiquitinated Tax with IKK complexes in the cytoplasm and the subsequent migration of the RelA subunit of NF-B to the nucleus. Thus, the ubiquitination and sumoylation of Tax function in concert to result in the migration of RelA to the nucleus and its accumulation with IKK␥ in nuclear bodies for activation of gene expression. These modifications may result in targets for the treatment of adult T-cell leukemia.The human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia and a virus-associated neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (31,36,47). These two diseases have been linked to the expression of the HTLV-1 regulatory protein Tax (10,13,29,41). Tax is a potent transcriptional activator of viral genes as well as specific cellular genes and has a clear oncogenic potential since Tax is able to transform T lymphocytes and fibroblasts and induces tumors in transgenic mice (19). A substantial part of the oncogenic properties of Tax is associated with its ability to activate the expression of cellular genes that control T-cell proliferation and differentiation by inducing constitutive activation of the NF-B pathway (2, 38).In nonstimulated cells, inactive NF-B complexes composed of p50 and RelA heterodimers are retained in the cytoplasm by NF-B inhibitors of the IB family (15,23,40). Tax activation of the NF-B pathway involves its interaction with the regulatory subunit of the IB kinase (IKK) complex, NEMO/IKK␥, leading to the activation of the two catalytic subunits, IKK␣ and IKK␤ (8,14). Activation of the IB kinase complex by Tax determines the phosphorylation of the IB proteins, leading to their ubiquitination and degradation by the proteasome and to the migration of the NF-B complexes to the nucleus (18,45,46). It also determines the phosphorylation of the RelA subunit of NF-B, a prerequisite for activation of RelA and p50 heterodimers in the nucleus (30). However, Tax also colocalizes in discrete nuclear bodies (NB) with the two subunits of NF-B, p50 and RelA, in addition to RNA polymerase II, and the assembly of these nuclear structures correlates with Tax transcriptional activity (4-6, 37). Thus, Tax-mediated activation o...

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.

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 identi-

Human T-cell leukemia virus type 1 (HTLV-1) is the retrovirus responsible for adult T-cell leukemia and HTLV-1-associated myelopathy. Adult T-cell leukemia development is mainly due to the ability of the viral oncoprotein Tax to promote T-cell proliferation, whereas the appearance of HTLV-1-associated myelopathy involves the antigenic properties of Tax. Understanding the events regulating the intracellular level of Tax is therefore an important issue. How Tax is degraded has not been determined, but it is known that Tax binds to proteasomes, the major sites for degradation of intracellular proteins, generally tagged through polyubiquitin conjugation. In this study, we investigated the relationship between Tax, ubiquitin, and proteasomes. We report that mono-and polyubiquitinated Tax proteins can be recovered from both transfected 293T cells and T lymphocytes. We also show that lysine residues located in the carboxy-terminal domain of Tax are the principal targets of this process. Remarkably, we further demonstrate that mutation of lysine residues in the C-terminal part of Tax, which massively reduces Tax ubiquitination, impairs proteasome binding, and conversely, that a Tax mutant that binds poorly to this particle (M22) is faintly ubiquitinated, suggesting that Tax ubiquitination is required for association with cellular proteasomes. Finally, we document that comparable amounts of ubiquitinated species were found whether proteasome activities were inhibited or not, providing evidence that they are not directly addressed to proteasomes for degradation. These findings indicate that although it is ubiquitinated and binds to proteasomes, Tax is not massively degraded via the ubiquitinproteasome pathway and therefore reveal that Tax conjugation to ubiquitin mediates a nonproteolytic function.Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia, a malignant monoclonal proliferation of CD4 ϩ T lymphocytes and of a chronic myelopathy called HTLV-1-associated myelopathy/tropical spastic paraparesis (36). Although these two diseases are definitely divergent in term of pathogenic mechanisms, the HTLV-1 Tax regulatory protein can be considered a key actor in both cases. First, via its ability to activate the viral promoter (31, 34), chronic Tax production is required to sustain viral replication. Second, HTLV-1-mediated immortalization of T lymphocytes, a fundamental event for subsequent cell transformation, results mainly from the ability of Tax to trigger T-cell proliferation through various mechanisms, including transcriptional transactivation of cellular genes (reviewed in reference 21) and promotion of cell cycle and deregulation of apoptosis (reviewed in reference 13).HTLV-1-associated myelopathy/tropical spastic paraparesis is not related to T-cell transformation and is considered as an immune-mediated pathology (reviewed in reference 15). Complex mechanisms are involved, among which exacerbation of the antiviral cytotoxic T-cell response (7, 23) and cross recognition of cell...

No abstract

Ubiquitin-mediated protein degradation is the main mechanism for controlled proteolysis, which is crucial for muscle development and maintenance. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 gene (ASB2) encodes the specificity subunit of an E3 ubiquitin ligase complex involved in differentiation of hematopoietic cells. Here, we provide the first evidence that a novel ASB2 isoform, ASB2b, is important for muscle differentiation. ASB2b is expressed in muscle cells during embryogenesis and in adult tissues. ASB2b is part of an active E3 ubiquitin ligase complex and targets the actin-binding protein filamin B ( The ubiquitin-proteasome system (UPS) is one of the major mechanisms for controlled proteolysis, which is a crucial determinant of many cellular events in eukaryotes. Degradation of a protein by the ubiquitin-proteasome pathway entails two successive events: the covalent attachment of ubiquitin chains to lysine residues in a substrate protein leading to its recognition and ATP-dependent proteolysis by the proteasome. 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 through dedicated interaction domains. 1 ASB2 is one of 18 members of the ankyrin repeat-containing suppressor of cytokine signaling (SOCS) box protein family (ASB) that are characterized by variable numbers of N-terminal ankyrin repeats. 2 The ASB2 gene was originally identified as an retinoic acid-inducible gene involved in induced differentiation of myeloid leukemia cells. 3,4 We have previously demonstrated that, by interacting with the elongin BC complex, ASB2 can assemble with a Cullin5/Rbx module to form an E3 ubiquitin ligase complex that stimulates polyubiquitylation by the E2 ubiquitin-conjugating enzyme UbcH5a. 5,6 This strongly suggests that ASB2 targets specific proteins to destruction by the proteasome during differentiation of hematopoietic cells. We have recently shown that ASB2 ubiquitin ligase activity drives proteasome-mediated degradation of the ubiquitously expressed actin-binding protein filamins (FLNs), FLNa and b, and can regulate integrin-mediated cell spreading. 6 During muscle development, dramatic changes in protein expression and cell morphology rely on the turnover of regulatory and structural components. Indeed, myogenic transcription factors such as MyoD and its E2A partner or negative Id regulator as well as myofibrillar proteins were shown to be degraded by the UPS. 7-11 Although some E3 ubiquitin ligases active during myogenesis have been identified, 12-23 a precise understanding of the function of ubiquitylation in muscle development and the identities of specific ubiquitin ligases and their potential substrates is lacking.Here we show that ASB2 expression is not restricted to hematopoietic cells but is also expressed and regulated in muscle cells during mouse and chick embryogenesis.

The oncogenic potential of the HTLV-1 Tax protein involves activation of the NF-κB pathway, which depends on Tax phosphorylation, ubiquitination and sumoylation. We demonstrate that the nuclei of Tax-expressing cells, including HTLV-1 transformed T-lymphocytes, contain a pool of Tax molecules acetylated on lysine residue at amino acid position 346 by the transcriptional coactivator p300. Phosphorylation of Tax on serine residues 300/301 was a prerequisite for Tax localization in the nucleus and correlated with its subsequent acetylation by p300, whereas sumoylation, resulting in the formation of Tax nuclear bodies in which p300 was recruited, favored Tax acetylation. Overexpression of p300 markedly increased Tax acetylation and the ability of a wild type HTLV-1 provirus, –but not of a mutant provirus carrying an acetylation deficient Tax gene–, to activate gene expression from an integrated NF-κB-controlled promoter. Thus, Tax acetylation favors NF-κB activation and might play an important role in HTLV-1-induced cell transformation.

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. 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 ubiquitinactivating 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 ␣ (RAR␣) gene (9 -11). The RAR␣ protein is a member of the nuclear receptor superfamily that stimulates myeloid differentiation in the presence of its ligand, all-transretinoic acid (RA). In more than 95% of APL, the t(15;17) translocation between the promyelocytic leukemia (PML) gene on chromosome 15 and the RAR␣ gene on chromosome 17 produces the PML-RAR␣ fusion protein (12). The PML-RAR␣ protein enhances the rep...