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

Human T cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia. HTLV-1 encodes a trans-activating protein, Tax, which is largely responsible for the oncogenic properties of the virus. Tax promotes T cell transformation by deregulating the activity of various cellular factors, including the transcription factor NF-κB. Tax activates the IκB kinase (IKK) via physical interaction with the regulatory subunit, IKKγ, although it is unknown precisely how Tax activates the IKK complex. Here we show that Tax modulates the cellular localization of the IKK complex. The IKKs relocalize from a broad distribution in the cytoplasm to concentrated perinuclear “hot spots” in both HTLV-1-transformed lines and in Tax-expressing Jurkat cells. Relocalization of IKK is not observed with Tax mutants unable to activate NF-κB, suggesting that only activated forms of IKK are relocalized. However, relocalization of IKK is strictly dependent on Tax expression because it does not occur in ATL cell lines that lack Tax expression or in Jurkat cells treated with phorbol 12-myristate 13-acetate and ionomycin. Furthermore, IKKγ is required for redistribution because cells lacking IKKγ were unable to relocalize IKKα upon expression of Tax. We also find that Tax ubiquitination likely regulates IKK relocalization because mutation of three critical lysine residues in Tax renders it unable to relocalize IKK and activate the canonical and noncanonical NF-κB pathways. Finally, we have observed that the perinuclear IKK in Tax-expressing cells colocalizes with the Golgi, and disruption of Golgi with either nocodazole or brefeldin A leads to a redistribution of IKK to the cytoplasm. Together, these results demonstrate that Tax induces relocalization of the IKK complex in a ubiquitin-dependent manner, and dynamic changes in the subcellular localization of the IKK complex may be critical for Tax function.

The human T cell leukemia virus type I (HTLV-1) 3 is associated with adult T cell leukemia (ATL), an aggressive malignancy of CD4 ϩ T cells, and a neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (1). The HTLV-1 genome encodes a regulatory protein Tax in the pX region that plays a central role in HTLV-1associated disease (2). Tax acts as a trans-activating protein that is critical for the expression of viral genes and also deregulates the expression of cellular genes. Tax regulates the expression of cellular genes by modulating signaling pathways such as NF-B, AP-1, CREB, and nuclear factor of activated T cells (3). Tax activation of NF-B is required for immortalization of T lymphocytes (4). Further, pharmacologic inhibition of NF-B leads to apoptosis of HTLV-1 transformed T cell lines and ATL cells (5).
NF-B represents a family of transcription factors that regulate a wide variety of genes controlling cell survival, development, differentiation, and activation. NF-B family members include RelA (p65), c-Rel, RelB, p50, and p52, all of which contain a 300-amino acid Rel homology domain that mediates DNA binding, dimerization, and nuclear localization (6). In quiescent cells, heterodimeric NF-B complexes are held in the cytoplasm as latent transcription factors because of an interaction with members of the inhibitory IB proteins. In response to cytokine stimulation, antigen stimulation, stress, or infection, the IB proteins are phosphorylated on two N-terminal serine residues by a large kinase complex consisting of the catalytic subunits IKK␣, IKK␤, and the regulatory protein IKK␥ (also known as NEMO) (7). Phosphorylated IBs are ubiquitinated and targeted to the 26 S proteasome for degradation, thus releasing NF-B from inhibition, allowing it to enter the nucleus and activate target genes (6). In the canonical NF-B pathway, IKK␤ and IKK␥ are essential for the phosphorylation of IB␣ and IB␤, leading to the liberation of heterodimers composed of p50 and RelA or c-Rel (7). In the noncanonical pathway, the NF-B inducing kinase and IKK␣ play important roles in the phosphorylation and processing of the p52 precursor protein p100 (8,9). The noncanonical NF-B pathway reg-ulates the development of lymphoid organs as well as B cell maturation and survival (10).
HTLV-1-transformed cell lines and leukemic cells from ATL patients exhibit persistent activation of the canonical and noncanonical NF-B pathways (11)(12)(13). We and others have shown that Tax interacts with IKK␥, and this interaction is essential for Tax-mediated activation of IKK and NF-B (14 -16). However, it is unclear exactly how Tax binding to IKK␥ triggers the catalytic activity of the IKKs. Several recent studies have reported that ubiquitination and sumoylation are important post-transcriptional regulators of Tax-mediated NF-B activation (17,18). Ubiquitinated Tax resides in the cytoplasm and activates NF-B, whereas sumoylated Tax is located in nuclear bodies together with RelA (17,18). Ubiquitination and sumoylation of Tax occur on overlapping lysine residues of which lysines 263, 280, and 284 are critical for NF-B activation (17,18). In this report, we have monitored the subcellular localization of the individual endogenous IKK subunits in Tax-expressing T cells as well as HTLV-1-transformed cells. We found that Tax expression triggers a dynamic relocalization of IKK into concentrated perinuclear "hot spots" that reside within or in close proximity to the Golgi apparatus. Tax-mediated relocalization of IKK␣ is dependent on IKK␥ because this event does not occur in IKK␥-deficient T cells. The relocalization is likely dependent on a direct interaction of IKK␥ with Tax because a Tax mutant that does not bind to IKK␥ is unable to relocalize IKK. Further, we find that mutation of lysines 263, 280, and 284 within Tax that are important for Tax ubiquitination are essential for Tax and IKK targeting to the Golgi. Thus, Tax-mediated NF-B activation is associated with ubiquitin-dependent relocalization of IKK to specific compartments within the cell.
Retroviral Infection-Retroviral infections were performed with the pCLXSN system from Dr. I. Verma (23) as previously described (13). Briefly, 293-T cells were transfected with 1 g of pCL-ampho, 0.15 g of VSV-g and either 1 g of pCLXSN, pCLXSN-Tax WT, M22, M47, or K263R,K280R,K284R mutants, or pCLXSN-GFP using FuGENE 6 (Roche Applied Science). 48 h post-transfection, the viral supernatant was filtered through 0.45-m polysulfone filters specialized for low protein binding (Pall Life Sciences, Ann Arbor, MI). Viral supernatant supplemented with fresh media and 8 g/ml polybrene was used to infect 1 ϫ 10 6 Jurkat cells. The cells were centrifuged at 1800 rpm for 45 min to increase infection efficiency. Viral supernatant was removed 24 h post-infection, and normal growth medium was replenished. The cells were subjected to experimentation 72 h post-infection.
Immunostaining-The cells were seeded onto 12-mm poly-L-lysine-coated coverslips (BD Biosciences, Bedford, MA) and were briefly centrifuged prior to fixation. The cells were washed twice with PBS and fixed in 1% paraformaldehyde for 10 min at room temperature. The fixed cells were permeabilized with PBS containing 0.2% Triton X-100, and nonspecific binding was prevented by a 1-h incubation in PBS containing 10% normal donkey serum (Jackson Immunoresearch, West Grove, PA) and 0.1% Triton X-100. The antibodies were diluted in PBS containing 0.1% Triton X-100. Primary antibody incubations were for 1 h, followed by five washes in PBS containing 0.1% Triton X-100. Secondary antibody incubations were for 1 h, followed by three washes in PBS containing 0.1% Triton X-100. The coverslips were mounted onto slides using AquaPolymount (Polysciences, Warrington, PA) and were analyzed with a Zeiss LSM-510 confocal laser scanning microscope. In each experiment, the controls were stained only with secondary antibody to confirm specificity of the primary antibody. Some of the images were adjusted using Adobe Photoshop.
EMSA-Nuclear extracts were prepared from transfected 293-T cells as described previously (24). EMSA was performed essentially as described (24). Double-stranded oligonucleotides were generated representing a consensus NF-B site derived from the interleukin-2R␣ promoter. The probe was radiolabeled with [ 32 P]␣-dCTP in a fill-in reaction with dNTPs (Invitrogen) and Klenow (Promega, Madison, WI). Equal amounts of nuclear extracts (4 g) were incubated with radio-labeled probe and poly(dI-dC) (1 g), dithiothreitol (1 mM), and buffer (25 mM HEPES, pH 7.9, 10% glycerol, 100 mM KCl, 0.1 mM EDTA) for 15 min at room temperature. The DNA-protein complexes were resolved on a 5% polyacrylamide gel in 1ϫ TBE buffer. The gel was dried and subjected to autoradiography.
Reverse Transcription-PCR-Total RNA was isolated from cells infected with empty vector pCLXSN or pCLXSN Tax using the RNeasy Kit (Qiagen). RNA was converted to cDNA and used for PCRs to amplify either Tax or GAPDH. The primer sequences are available upon request.
Reporter Gene Assays-Jurkat cells (1 ϫ 10 6 ) were transfected with pCMV4-Tax (0.25 g), pCMV4-Tax K263R,K280R,K284R (0.25 g), or empty vector together with B-TATA Luc (0.1 g). The cell lysates were subjected to a luciferase assay (Promega, Madison, WI) as recommended by the manufacturer. Activation of the reporter by Tax or Tax mutants was calculated as fold induction compared with empty vector.

Tax Induces Relocalization of IKK into Perinuclear Hot
Spots-A hallmark of receptor-mediated signal transduction is the concentration of signaling components in specific membrane microdomains known as lipid rafts (25). It is less clear how intracellular molecules, such as Tax, initiate signal transduction. Nevertheless, strong evidence suggests that Tax-mediated IKK activation does not involve cellular receptors or upstream signaling components (26). To understand the mechanism of IKK activation by Tax, we examined whether Tax alters the subcellular localization of individual IKK subunits in T lymphocytes. We first employed a frequently used T cell line model, Jurkat, to analyze IKK␥ localization upon Tax expression. Jurkat T cells were infected with a Tax-expressing retroviral vector or empty vector, stained with anti-IKK␥ and anti-Tax and subjected to confocal microscopy. In cells infected with the empty vector, IKK␥ was diffusely expressed throughout the cytoplasm (Fig. 1, A and C). Interestingly, however, Tax expression resulted in marked relocalization of IKK␥ to cytoplasmic hot spots that appeared to reside in a perinuclear region (Fig. 1, D and F). Tax was predominantly colocalized with IKK␥ in these hot spots (Fig. 1, E and F). IKK␥ was also relocalized in Tax-inducible JPX-9 cells (data not shown). Importantly, these IKK␥ hot spots were also observed in human T cells transformed by HTLV-1 (C8166 cells; Fig. 1G), and Tax was partially colocalized with IKK␥ in these cells (Fig. 1I).
Because IKK␥ exists largely in a trimeric kinase complex that also contains IKK␣ and IKK␤, we hypothesized that Tax induces aggregation of the IKK holoenzyme. To examine this idea, we analyzed the subcellular localization of IKK␣ and IKK␤. As seen with IKK␥, IKK␣ and IKK␤ were diffusely expressed in the cytoplasm in Jurkat cells (Fig. 2, A and E), although some punctate IKK␤ staining was also observed (Fig.  2, E and F). In C8166 cells, IKK␣ (Fig. 2C) was present in perinuclear hot spots similar to those observed in Fig. 1 with IKK␥. IKK␤ also appeared to be relocalized in C8166 cells compared with Jurkat cells (Fig. 2, E-H), although not to the same extent as seen with IKK␣ and IKK␥. Thus, Tax expression appears to be associated with the movement of the IKKs to a concentrated perinuclear region within the cell.

IKK␥ Is Required for Tax-induced Relocalization of the IKK Complex-
We have previously shown that IKK␥ functions as an adaptor for recruiting Tax to the IKK complex, which is essential for NF-B activation (14,27). Therefore, we examined whether Tax required IKK␥ for inducing the relocalization of IKK complex. For this purpose, we used IKK␥-deficient Jurkat cells (JM4.5.2) that we had previously generated by somatic mutagenesis (20). JM4.5.2 cells or JM4.5.2 cells stably reconstituted with IKK␥ (JM4.5.2-IKK␥) were infected with recombinant retroviruses either expressing Tax or empty vector control. Tax had no effect on the distribution of IKK␣ in IKK␥-deficient Jurkat cells (Fig. 3C), although Tax was expressed as determined by reverse transcription-PCR (panel I). Furthermore, this defect was rescued when the IKK␥-deficient cells were reconstituted with exogenous IKK␥ (Fig. 3G). Therefore, Tax requires IKK␥ to modulate the subcellular localization of IKK␣.
Tax Mutants Defective in NF-B Activation Fail to Induce IKK Relocalization-To assess the functional significance of Tax-induced IKK relocalization in NF-B activation, we examined the ability of two well characterized Tax mutants, M22 and M47, in the relocalization of IKK␥. The Tax mutant M22 is deficient in NF-B activation but remains competent in CREB activation, whereas M47 is deficient in CREB activation but active in NF-B activation (28). Jurkat cells were infected with recombinant retroviruses expressing either Tax, Tax M22, Tax M47, or empty vector. The cells were stained with anti-IKK␥ and anti-Tax and subjected to confocal microscopy. As expected, the normally diffusely localized IKK␥ (Fig. 4A) was IKK␥ was detected using RRX-conjugated donkey anti-rabbit F(abЈ) 2 IgG, Tax was detected using Cy5-conjugated donkey anti-mouse F(abЈ) 2 IgG, and nuclei were stained with DAPI. Tax was pseudocolored green using Zeiss LSM software. These confocal images depict the extended projection of the z stack. Bar, 10 m. This experiment was performed a minimum of three times with similar results. FEBRUARY 9, 2007 • VOLUME 282 • NUMBER 6 JOURNAL OF BIOLOGICAL CHEMISTRY 4187 relocalized into hot spots upon expression of wild type Tax (Fig.  4D). Similar results were obtained with cells infected with Tax M47 (Fig. 4J), suggesting the dispensability of the CREB-activating function of Tax in IKK relocalization. In sharp contrast, the M22 mutant failed to induce the relocalization of IKK␥ to perinuclear hot spots (Fig. 4G) despite its strong expression in the infected cells (Fig. 4H). Thus, Tax-mediated IKK relocalization is correlated with activation of IKK/NF-B signaling. Because the M22 mutant is defective in Tax binding (14), these results also imply that Tax may require a direct interaction with IKK␥ to mediate the redistribution of IKK from a diffuse cytoplasmic pattern to concentrated perinuclear hot spots.

IKK Relocalization Is Dependent on Tax Expression-Thus
far, we have observed that the relocalization of IKK was tightly linked to Tax-mediated NF-B activation. However, it was important to determine whether IKK relocalization was dependent on Tax expression or simply a result of IKK activation. To address this question, we examined IKK␥ localization  in three different ATL cell lines that lack Tax expression but maintain activated NF-B (22). IKK␥ was expressed diffusely throughout the cytoplasm and on top of the nucleus in the ATL cell lines MT-1, TL-OM1, and ED40515(Ϫ) (Fig. 5, E-J). As expected IKK␥ was present in hot spots in C8166 cells (Fig. 5, C and D) but not in Jurkat cells (Fig. 5, A and B). Next, we treated Jurkat cells with PMA and ionomycin for either 15 min or 2 h to activate IKK. As observed in Fig. 5 (K-P), PMA and ionomycin did not alter the diffuse staining of IKK␣. The treatment was effective because RelA translocated to the nucleus at both time points (data not shown). Also, treatment of cells with TNF␣ did not trigger the relocalization of IKK␣ (data not shown). These data strongly suggest that relocalization of IKK to perinuclear hot spots is critically dependent on Tax expression. Tax Promotes IKK Relocalization to the Golgi Apparatus-We next wanted to characterize the perinuclear hot spots containing IKKs. For this purpose, we used antibodies recognizing different cellular markers and organelles. C8166 cells were costained with anti-IKK␥ and markers for either endosomal, lysosomal, lipid rafts, or Golgi apparatus. IKK␥ did not colocalize with endosomes, lysosomes, or lipid rafts (data not shown). However, we did observe a partial colocalization of IKK␣ and IKK␥ with the Golgi apparatus when cells were costained with either anti-Giantin or anti-Golgi matrix protein 130 (GM-130) antibodies (Fig. 6, C and F). To further demonstrate IKK␥ localization to the Golgi, we treated C8166 cells with nocodazole and brefeldin A to disrupt the Golgi. As expected, treatment with nocodazole resulted in a relocalization of GM-130 to multivesicular bodies dispersed throughout the cytoplasm (Fig.  6K). Interestingly, IKK␥ localization changed from perinuclear to a more broadly distributed vesicular staining (Fig. 6J). Much of IKK␥ remained colocalized with GM-130, suggesting that IKK␥ was tightly associated with the Golgi (Fig. 6L). Treatment of C8166 cells with brefeldin A also led to a dramatic relocalization of IKK␥ from a perinuclear area to vesicular staining throughout the cytoplasm (Fig. 6M). Thus, it appears that IKK is localized within or is associated with the Golgi apparatus in HTLV-1 transformed cells. In support of these observations, a recent study has demonstrated that Tax is localized, at least partially, in the cis-Golgi stacks in transfected cells as well as naturally infected T lymphocytes from HTLV-1-associated myelopathy/tropical spastic paraparesis patients (29). Because the Golgi apparatus is a major site of signal transduction (30), the Tax-mediated IKK relocalization to this site may be an important mechanism of IKK activation.
A Ubiquitination-defective Tax Mutant Does Not Mediate IKK Relocalization-Because ubiquitination of Tax likely regulates its subcellular localization and NF-B activation (17,18), we wanted to determine the role of Tax ubiquitination in IKK relocalization. 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. Therefore, we performed site-directed mutagenesis to generate the Tax K263R,K280R,K284R mutant. We then examined the function of this mutant for activation of the canonical and noncanonical NF-B pathways. In agreement with others (17,18), Tax K263R,K280R,K284R was defective in NF-B activation as assessed by EMSA (Fig. 7B) and luciferase assays (Fig. 7A). This mutant was also defective in the noncanonical pathway because p100 processing to p52 was diminished compared with wild type Tax (Fig. 7C). We also mutated lysines 263, 280, and 284 to arginines in the context of the pCLXSN-Tax retroviral vector. Jurkat cells were infected with recombinant retroviruses expressing the Tax K263R,K280R,K284R mutant. The cells were stained with anti-Tax and anti-IKK␥ and subjected to confocal microscopy. In contrast to wild type Tax, the Tax mutant did not relocalize IKK␥ to perinuclear hot spots in infected cells (Fig. 7D). Interestingly, Tax was also not present in the perinuclear hot spots. Thus, ubiquitination of Tax and specifically lysines 263, 280, and 284 likely regulates coordinated Tax-IKK␥ movement to the Golgi.

DISCUSSION
In this report, we demonstrate that Tax modulates the subcellular localization of endogenous IKK subunits in T lymphocytes. In the absence of Tax, the IKKs are present diffusely throughout the cytoplasm; however, in Tax-expressing T lymphocytes and HTLV-1 transformed cell lines, IKK␣ and IKK␥  E, H, and K). IKK␥ was detected with Cy5-conjugated donkey anti-rabbit F(abЈ) 2 IgG, and Tax was detected with RRX-conjugated donkey anti-mouse F(abЈ) 2 IgG. Merged images with DAPI-stained nuclei are shown in C, F, I, and L. IKK␥ was pseudocolored green using Zeiss LSM software. These confocal images depict single slices in the z stack. Bar, 5 m. This experiment was performed two times with similar results. FEBRUARY 9, 2007 • VOLUME 282 • NUMBER 6 are predominantly localized within perinuclear hot spots that colocalize with the Golgi apparatus. The redistribution of IKK to the Golgi appears to be important for IKK activation, because this cellular event was not observed with Tax mutants unable to activate IKK/NF-B. This idea is further supported by the finding that IKK␥, an essential adaptor of Tax in IKK activation, is required for Tax-mediated relocalization of IKK. Furthermore, the relocalization of IKK is dependent on Tax expression, indicating that IKK activation is not sufficient for movement to the Golgi. We also provide evidence that lysines 263, 280, and 284 are critical for Tax activation of the canonical and noncanonical pathways. These lysines are also essential for the redistribution of IKK. Thus, ubiquitination of Tax may modulate the interaction with IKK and the movement of Tax-IKK complexes within the cell.

HTLV-1 Tax Promotes the Relocalization of IKK to the Golgi
The IKK-containing perinuclear hot spots that we have observed in Tax-expressing Jurkat cells and HTLV-1 transformed cell lines may represent highly concentrated, oligomerized forms of IKK. Oligomerization of IKK␥ is emerging as an important role in the activation of the IKK complex. Forced oligomerization of IKK␥ by fusion with the FKBP12 polypeptide leads to NF-B activation and the redistribution of the IKK subunits from uniform to punctate cytoplasmic staining (32). Tax also promotes the oligomerization of IKK␥ through a direct interaction (33). Thus, Tax likely triggers the oligomerization of IKK␥, through an undefined mechanism, leading to the oligomerization of associated IKKs and the redistribution to the perinuclear hot spots discovered in the present study.
We and others have reported that Tax physically interacts with the IKK␥ subunit, and this interaction is critical for recruitment of Tax to the IKK complex and the activation of IKK and NF-B (14 -16). Because Tax requires IKK␥ for the relocalization of IKK␣ into the Golgi apparatus (Fig. 3), it is likely that Tax recruits IKK␣ to the Golgi via a direct interaction with IKK␥. Indeed, we have not observed Tax in the perinuclear area in the absence of IKK␥. Moreover, a Tax mutant (M22) defective in IKK␥ binding fails to relocate IKK (Fig.  4). A recent study suggests that ubiquitination of Tax regulates binding to IKK␥, because a Tax mutant with multiple lysine to arginine substitutions does not interact with IKK␥ (18). We have found that a ubiquitination-defective Tax mutant (Tax K263R,K280R,K284R) is unable to relocalize IKK␥ (Fig. 7D). However, this Tax mutant retains its ability to bind IKK␥, although a Tax mutant harboring arginine substitutions at all 10 lysines becomes defective in IKK␥ binding (data not shown). Because the Tax K263R,K280R,K284R mutant itself fails to localize to the Golgi region, it suggests the possibility that ubiquitination of Tax plays a role in mediating its subcellular localization and, thereby, IKK distribution.
Our findings are supported by several previous reports indicating that a substantial fraction of Tax colocalizes with the Golgi apparatus (17,29,34). Tax appears to be localized partially in the cis-Golgi adjacent to the microtubule organizing center in naturally infected T lymphocytes from HTLV-1-associated myelopathy/tropical spastic paraparesis patients (29). It has been suggested that Tax promotes microtubule polymerization, in cooperation with ICAM-1 engagement, leading to the formation of the virological synapse that enables cell-to-cell spread of HTLV-1 (29,35). Based on our results, one possibility is that Tax may recruit IKK to the vicinity of the Golgi/micro- tubule organizing center to facilitate IKK-mediated phosphorylation of targets necessary for microtubule polymerization. However, Tax M47 was unable to mediate microtubule organizing center polymerization in Jurkat cells, suggesting that CREB but not NF-B may be essential for this process (29). Because Tax-mediated IKK relocalization is correlated with IKK activation, a more attractive hypothesis is that the IKK redistribution serves as a mechanism of IKK activation. Although Tax promotes the relocalization of all three IKK subunits, it appears that the effect of Tax on IKK␤ is not as pro-nounced (Fig. 2). Because IKK␣ and IKK␥ are critical components for Tax-mediated induction of the noncanonical pathway involving p100 processing to p52 (13), perhaps the relocalization may be more important for this specific function of Tax. Additional studies are required to more precisely determine the functional roles of Tax-mediated targeting of IKK to the Golgi in the canonical and/or noncanonical NF-B pathways.
Emerging evidence suggests that the Golgi apparatus is one of the intracellular sites of signal transduction (30,36). The small GTPases Rho, Rac, and Cdc42 are critical regulators of cytoskeleton reorganization and cell movement and can all localize to the Golgi. A signaling protein, ARAP1, resides in the Golgi and regulates Arf-, Rho-, and Cdc42-dependent cytoskeletal remodeling and cell movement (37). The p21-activated kinase (PAK)-related kinase PAK4, which has been implicated in cell transformation, is redistributed to the Golgi in the presence of activated Cdc42 (38). With regard to NF-B signaling, FIGURE 6. Tax promotes IKK relocalization to the Golgi. C8166 cells were costained for IKK␣, Giantin, and nuclei (A-C). IKK␣ was detected with Alexa Fluor-555-conjugated donkey anti-mouse IgG, and Giantin was detected with Cy5-conjugated donkey anti-rabbit F(abЈ) 2 IgG. Giantin was pseudocolored green using Zeiss LSM software. The nuclei were stained with DAPI. The arrow in C points to a region of colocalization between IKK␣ and Giantin. A-C depict the extended projection of the z stack. Bar, 5 m. This experiment was performed two times with similar results. C8166 cells were left untreated (D-F) or were treated with dimethyl sulfoxide (DMSO) for 2 h (G-I), nocodazole (10 m) for 2 h (J-L), or brefeldin A (1ϫ) for 1 h (M-O). The cells were immunostained for IKK␥, GM-130, and nuclei. IKK␥ was detected with RRXconjugated donkey anti-rabbit F(abЈ) 2 IgG, GM-130 was detected with Cy5conjugated donkey anti-mouse F(abЈ) 2 IgG, and nuclei were stained with DAPI. GM-130 was pseudocolored green using Zeiss LSM software. The arrows in F, I, and L point to regions of colocalization between IKK␥ and GM-130. D-O depict the extended projection of the z stack. Bar, 5 m.

FIGURE 7.
A Tax ubiquitination mutant that is defective in NF-B activation does not relocalize IKK to hot spots. A, Jurkat cells were cotransfected with plasmids containing B-TATA Luc and either empty vector, pCMV4 Tax, or pCMV4 Tax K263R,K280R,K284R. The cell lysates were subjected to a luciferase assay. Fold NF-B induction by Tax was calculated versus the empty vector. The graph depicts the results of one experiment with n ϭ 3. This experiment was performed two additional times with similar results. B, 293-T cells were transfected with empty vector, Tax, or Tax K263R,K280R,K284R. Nuclear extracts were isolated and subjected to EMSA for NF-〉. Tax expression in the nuclear extracts is shown as well. C, 293-T cells were cotransfected with pCMV4-p100 and empty vector, Tax, or Tax K263R,K280R,K284R. Whole cell extracts were immunoblotted (IB) for p100/p52 and Tax. This experiment was performed two times with similar results. D, Jurkat cells infected with Tax K263R,K280R,K284R were costained for IKK␥, Tax, and nuclei as in Fig. 5. This experiment was performed as part of the experiment in Fig. 5. Bar, 5 m. FEBRUARY 9, 2007 • VOLUME 282 • NUMBER 6 the IKK␥-related coiled-coil protein NRP (NEMO-related protein) is a Golgi-resident protein, although its function in signaling is currently unknown (39). We are currently trying to determine whether NRP plays any role in IKK relocalization to the Golgi. Nevertheless, the current findings provide an important insight into the mechanism by which Tax modulates the IKK/ NF-B signaling pathway.

HTLV-1 Tax Promotes the Relocalization of IKK to the Golgi
Tax has been demonstrated to be mono-and polyubiquitinated in transfected cells and HTLV-1-transformed cell lines (31,40). Tax also interacts with various subunits of the proteasome (31,(41)(42)(43). These results suggest that Tax ubiquitination may promote its degradation; however, several recent studies do not support this hypothesis. Rather, it appears that Tax ubiquitination may regulate its subcellular localization and NF-B activation via binding to IKK␥ (18). Ubiquitination mediates distinct functional roles depending on the exact lysine within ubiquitin that links the ubiquitin chains together. Whereas lysine 48-mediated ubiquitin linkages target proteins for proteasomal degradation, linkages supported by other lysines such as Lys 63 modulate subcellular localization and/or protein function (44). Thus, it is likely that Tax ubiquitination consists of linkages other than Lys 48 , probably Lys 63 . Nevertheless, we have shown here that Tax ubiquitination may regulate Tax and IKK␣ and IKK␥ localization to the Golgi apparatus. Future studies will determine additional roles polyubiquitination may play in Tax activation of NF-B.