Stable Ubiquitination of Human T-Cell Leukemia Virus Type 1 Tax Is Required for Proteasome Binding

Abstract

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...

Ubiquitylated Tax targets and binds the IKK signalosome at the centrosome

“…However, ubiquitylation of some transcription factors (reviewed in Conaway et al, 2002) increases their transcriptional activity in a degradation-independent manner. We and others have shown that Tax is ubiquitylated (Chiari et al, 2004;Peloponese et al, 2004). We also showed that Tax ubiquitylation on C-terminal lysines is critical for both Tax binding to IKK and for the nuclear translocation of RelA (Nasr et al, 2006).…”

“…To investigate whether Tax is modified by distinct poly-ubiquitylation, we used ubiquitin mutants harboring lysine to arginine mutation on all lysine residues of ubiquitin (Ub-K0), or on all lysine residues except lysine 48 (Ub-K48) or lysine 63 (Ub-K63). HeLa cells were co-transfected with a Tax-6 histidine (6His) construct and wild type or mutated HA-ubiquitin plasmids, and proteins were purified by Ni-NTA using highly stringent conditions as described (Chiari et al, 2004). It should be pointed out that the two ubiquitin mutants conserve the C-terminal glycine that mediates conjugation of the initial ubiquitin monomer to target lysines.…”

“…Ni-NTA pull-down Ni-NTA pull-down was performed as described (Chiari et al, 2004). Briefly, 36 h post-transfection, cells were lysed in reducing and highly denaturing conditions and incubated with Ni-NTA beads.…”

Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

“…The critical lysine residues within Tax for ubiquitination are lysines 263, 280, and 284 (31), and mutation of these lysines to arginine residues results in a defect in NF-B activation (17). It was of interest to determine the role of these lysines in Tax-mediated relocalization of IKK.…”

Proapoptotic regimes for HTLV-I-transformed cells: targeting Tax and the NF-κB pathway

“…77,82 A recent report indicated that Tax is ubiquitylated in both transfected cells and T-lymphocytes, and that Tax ubiquitylation is needed for proteasome binding. 92 The proteasome inhibitor PS-341 has demonstrated clinical effect in both multiple myeloma and non-Hodgkin's lymphoma. Several reports investigated its effects on ATL-derived cells in both in vitro and in vivo models.…”