The HTLV-1 Tax oncoprotein is a potent intracellular activator of NF-κB by promotion of the constitutive activation of IKK. The stable ubiquitination of Tax has emerged as an important regulator of Tax-mediated NF-κB activation (39
). However, previous studies have relied on Tax point mutants to demonstrate the importance of ubiquitination in NF-κB activation. In this study, we have determined that Tax polyubiquitin chains are primarily K63 linked and that the E2 ubiquitin-conjugating enzyme Ubc13 is essential for Tax ubiquitination. Tax interacts with Ubc13 both in transfected cells and in HTLV-1-transformed cell lines. Moreover, Tax requires Ubc13 for the activation of NF-κB and the induction of NF-κB-dependent target genes, including those for IL-2 and its high-affinity receptor IL-2Rα in T cells. Finally, in the absence of Ubc13, Tax is impaired in NEMO binding, suggesting that the role of Tax ubiquitination is to mediate protein-protein interactions.
The findings from this study suggest that Ubc13 is essential for the activation of NF-κB by Tax, but not by IL-1 or TNF-α stimulation. Ubc13 has previously been shown to be dispensable for IL-1- and TNF-α-mediated NF-κB activation in MEFs (56
). Furthermore, the processing of p100 to p52 was normal in Ubc13-deficient B cells stimulated with α-CD40 or B-cell-activating factor of the TNF family (56
). Thus, the mechanisms used by Tax to activate NF-κB are clearly different from the IL-1R and TNF receptor signaling pathways, although NEMO is required for NF-κB activation by Tax and cytokine stimulation (20
). In Tax-mediated NF-κB activation, the role of Ubc13 is to facilitate Tax K63-linked polyubiquitination (Fig. and ), which in turn mediates the interaction with NEMO (Fig. ). The exact mechanism by which Tax stimulates IKK activity is poorly understood but may involve upstream kinases such as transforming growth factor β-activated kinase 1 (52
). Interestingly, the Tax M22 mutant defective for NF-κB activation is unable to interact with either NEMO (23
) or Ubc13 (Fig. ), suggesting that an association of Tax with NEMO and/or Ubc13 is critical for NF-κB activation. Since the fusion of Tax M22 with NEMO restores NF-κB activation (54
), it will be interesting to determine if the fusion of Ubc13 to Tax M22 will similarly activate NF-κB.
Our results are not in agreement with those of a recent study demonstrating that Tax activation of NF-κB is independent of Ubc13 (15
). However, the conclusions by Gohda et al. were derived from a single experiment, an NF-κB luciferase assay with siRNA-mediated knockdown of Ubc13 in 293T cells (15
). It is unclear to us why our results are inconsistent with the findings by Gohda et al. Our findings in four different cell types, 293T, Ubc13−/−
MEFs, Jurkat, and C8166 cells, using a variety of different assays, consistently support a role for Ubc13 in Tax-mediated NF-κB activation. Furthermore, we have provided genetic evidence that Tax requires Ubc13 for the induction of NF-κB-dependent genes, such as those for IL-6 and IL-2. Consistent with the results of our studies, Kfoury et al. recently demonstrated that Tax undergoes K63-linked polyubiquitination (31
Although previous studies have established Tax ubiquitination, the biochemical nature of the polyubiquitin chain linkages has remained elusive. Here, we describe Tax polyubiquitination as predominantly K63 linked. However, our findings do not rule out the ubiquitination of Tax through other types of ubiquitin linkages. K63-linked ubiquitination has emerged as an important regulator of a variety of different biological processes, including receptor trafficking and protein-protein interactions. The Kaposi's sarcoma-associated herpesvirus K3 gene product promotes the internalization of cell surface major histocompatibility complex class I molecules by K63-linked ubiquitination that is dependent on Ubc13 (10
). Similarly, TRAF6-dependent K63-linked ubiquitination of the nerve growth factor receptor TrkA and neurotrophin receptor interacting factor regulates the trafficking and localization of these proteins (13
). K63-linked ubiquitination may also regulate protein function, as recently demonstrated for interferon regulatory factor 7, which exhibits enhanced transcriptional activity when ubiquitinated (28
). In addition, p53 localization and activity are regulated by Ubc13- and K63-linked ubiquitination (32
). To our knowledge, Tax is the first viral oncoprotein identified that is regulated by K63-linked ubiquitination.
K63-linked polyubiquitin chains may function as scaffolds that promote the assembly of complexes containing kinases and other signaling proteins (1
). Thus, Tax polyubiquitination may nucleate a signaling complex that promotes the activation of IKK. Indeed, our results indicate that NEMO is likely an essential component of this complex. There are likely additional, yet-to-be-identified proteins within this complex, possibly including an E3 ligase specific for Tax. TRAF2, TRAF6, RNF5, RNF8, and CHFR are all E3 ligases capable of catalyzing K63-linked ubiquitination that is dependent on Ubc13 (3
). Our preliminary data indicate that TRAF6 is not required for Tax-mediated NF-κB activation; therefore, TRAF6 may not serve as the Tax E3 ligase (data not shown). A more remote possibility is that Tax itself functions as an E3 ligase since Tax interacts with Ubc13. However, it is not clear if Tax interaction with Ubc13 is direct, and Tax lacks a conserved domain typical of E3 ligases, such as HECT (46
) or RING (42
). Future studies will more precisely determine the requirements for Tax ubiquitination and NF-κB activation.