Low nuclear body formation and tax SUMOylation do not prevent NF-kappaB promoter activation

Abstract

BackgroundThe Tax protein encoded by Human T-lymphotropic virus type 1 (HTLV-1) is a powerful activator of the NF-κB pathway, a property critical for HTLV-1-induced immortalization of CD4+ T lymphocytes. Tax permanently stimulates this pathway at a cytoplasmic level by activating the IκB kinase (IKK) complex and at a nuclear level by enhancing the binding of the NF-κB factor RelA to its cognate promoters and by forming nuclear bodies, believed to represent transcriptionally active structures. In previous studies, we reported that Tax ubiquitination and SUMOylation play a critical role in Tax localization and NF-κB activation. Indeed, analysis of lysine Tax mutants fused or not to ubiquitin or SUMO led us to propose a two-step model in which Tax ubiquitination first intervenes to activate IKK while Tax SUMOylation is subsequently required for promoter activation within Tax nuclear bodies. However, recent studies showing that ubiquitin or SUMO can modulate Tax activities in either the nucleus or the cytoplasm and that SUMOylated Tax can serve as substrate for ubiquitination suggested that Tax ubiquitination and SUMOylation may mediate redundant rather than successive functions.ResultsIn this study, we analyzed the properties of a new Tax mutant that is properly ubiquitinated, but defective for both nuclear body formation and SUMOylation. We report that reducing Tax SUMOylation and nuclear body formation do not alter the ability of Tax to activate IKK, induce RelA nuclear translocation, and trigger gene expression from a NF-κB promoter. Importantly, potent NF-κB promoter activation by Tax despite low SUMOylation and nuclear body formation is also observed in T cells, including CD4+ primary T lymphocytes. Moreover, we show that Tax nuclear bodies are hardly observed in HTLV-1-infected T cells. Finally, we provide direct evidence that the degree of NF-κB activation by Tax correlates with the level of Tax ubiquitination, but not SUMOylation.ConclusionsThese data reveal that the formation of Tax nuclear bodies, previously associated to transcriptional activities in Tax-transfected cells, is dispensable for NF-κB promoter activation, notably in CD4+ T cells. They also provide the first evidence that Tax SUMOylation is not a key determinant for Tax-induced NF-κB activation.

The Major Histocompatibility Complex Class II Transactivator CIITA Inhibits the Persistent Activation of NF-κB by the Human T Cell Lymphotropic Virus Type 1 Tax-1 Oncoprotein

“…In the presence of CIITA253-1130, both Tax-1 and RelA migrated to the nucleus without accumulating in NB and maintained their functional activities. This result is in line with the work of Bonnet et al (46), showing that the formation of Tax-1 NB is dispensable for Tax-1-induced RelA nuclear translocation and the triggering of NF-B-responsive gene expression. In contrast, CIITA⌬955-959 inhibits the activation of NF-B by Tax-1 and, similarly to full-length CIITA, interacts with Tax-1 and retains both Tax-1 and RelA in the cytoplasm, where the three factors colocalize.…”

“…In this study, we have not addressed this point. However, recent papers have challenged the strategic importance of Tax-1 sumoylation, because Tax-1 sumoylation and the formation of Tax-1 NB do not seem to be strictly required for NF-B-promoted gene expression (46,47). Moreover, in contrast to the findings of Turci et al (48), Tax-2-mediated activation of NF-B has been reported to be independent of sumoylation and ubiquitination 293T cells were transfected with the empty pCDNA3 vector (pC) or with Tax-1 in the presence or absence of FLAG-tagged CIITA.…”

“…For instance, it has been shown that ubiquitin-conjugated Tax-1 activates IKK␥ in the cytoplasm, whereas sumoylation of Tax-1 is required for its assembly in NB and activation of NF-Bresponsive genes (26). However, more recent papers have reported that the formation of Tax-1 NB and Tax-1 sumoylation are not strictly required for NF-B-driven gene induction (46,47). Similarly, the requirement of Tax-2 ubiquitination and sumoylation for the activation of NF-B is still debated (28,48,49).…”

Human T Cell Leukemia Virus Type 2 Tax-Mediated NF-κB Activation Involves a Mechanism Independent of Tax Conjugation to Ubiquitin and SUMO

“…Tax SUMOylation was initially proposed to regulate the nuclear steps of the pathway by facilitating NF-B promoter activation within Tax nuclear speckles (23,52). However, this conclusion was challenged by the recent finding that a Tax1 mutant that supports only low levels of SUMOylation still activates an NF-B promoter (56), further highlighting the key role played by Tax1 ubiquitination.…”

“…Our results show that Tax2-mediated activation of NF-B involves a mechanism independent of the lysine residues of Tax2 and therefore independent of Tax2 conjugation to ubiquitin and SUMO. Of note, we recently demonstrated that Tax1 SUMOylation, initially believed to be required for Tax1-mediated NF-B activation, is indeed dispensable for this process (56). In this respect, Tax2 is similar to Tax1.…”