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

Abstract

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. 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) 3The abbreviations used are: HTLV-1, human T cell leukemia virus type 1; IKK, IκB kinase; ATL, adult T cell leukemia; CREB, cAMP-responsive elementbinding protein; DAPI, 4′,6′-diamino-2-phenylindole; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; RRX, rhodamine red-X. is associated with adult T cell leukemia (ATL), an aggressive malignancy of CD4+ T cells, and a neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (1Yoshida M. Annu. Rev. Immunol. 2001; 19: 475-496Crossref PubMed Scopus (394) Google Scholar). The HTLV-1 genome encodes a regulatory protein Tax in the pX region that plays a central role in HTLV-1-associated disease (2Grant C. Barmak K. Alefantis T. Yao J. Jacobson S. Wigdahl B. J. Cell. Physiol. 2002; 190: 133-159Crossref PubMed Scopus (87) Google Scholar). 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-κB, AP-1, CREB, and nuclear factor of activated T cells (3Hall W.W. Fujii M. Oncogene. 2005; 24: 5965-5975Crossref PubMed Scopus (87) Google Scholar). Tax activation of NF-κB is required for immortalization of T lymphocytes (4Akagi T. Ono H. Nyunoya H. Shimotohno K. Oncogene. 1997; 14: 2071-2078Crossref PubMed Scopus (80) Google Scholar). Further, pharmacologic inhibition of NF-κB leads to apoptosis of HTLV-1 transformed T cell lines and ATL cells (5Mori N. Yamada Y. Ikeda S. Yamasaki Y. Tsukasaki K. Tanaka Y. Tomonaga M. Yamamoto N. Fujii M. Blood. 2002; 100: 1828-1834Crossref PubMed Scopus (256) Google Scholar). NF-κB represents a family of transcription factors that regulate a wide variety of genes controlling cell survival, development, differentiation, and activation. NF-κB 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 (6Rothwarf D.M. Karin M. Sci. STKE. 1999; 5: RE1Google Scholar). In quiescent cells, heterodimeric NF-κB complexes are held in the cytoplasm as latent transcription factors because of an interaction with members of the inhibitory IκB proteins. In response to cytokine stimulation, antigen stimulation, stress, or infection, the IκB proteins are phosphorylated on two N-terminal serine residues by a large kinase complex consisting of the catalytic subunits IKKα, IKKβ, and the regulatory protein IKKγ (also known as NEMO) (7Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3372) Google Scholar). Phosphorylated IκBs are ubiquitinated and targeted to the 26 S proteasome for degradation, thus releasing NF-κB from inhibition, allowing it to enter the nucleus and activate target genes (6Rothwarf D.M. Karin M. Sci. STKE. 1999; 5: RE1Google Scholar). In the canonical NF-κB pathway, IKKβ and IKKγ are essential for the phosphorylation of IκBα and IκBβ, leading to the liberation of heterodimers composed of p50 and RelA or c-Rel (7Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3372) Google Scholar). In the noncanonical pathway, the NF-κB inducing kinase and IKKα play important roles in the phosphorylation and processing of the p52 precursor protein p100 (8Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 9Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1137) Google Scholar). The noncanonical NF-κB pathway regulates the development of lymphoid organs as well as B cell maturation and survival (10Beinke S. Ley S.C. Biochem. J. 2004; 382: 393-409Crossref PubMed Scopus (507) Google Scholar). HTLV-1-transformed cell lines and leukemic cells from ATL patients exhibit persistent activation of the canonical and noncanonical NF-κB pathways (11Harhaj E.W. Harhaj N.S. IUBMB Life. 2005; 57: 83-91Crossref PubMed Scopus (38) Google Scholar, 12Sun S.C. Yamaoka S. Oncogene. 2005; 24: 5952-5964Crossref PubMed Scopus (194) Google Scholar, 13Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). We and others have shown that Tax interacts with IKKγ, and this interaction is essential for Tax-mediated activation of IKK and NF-κB (14Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 15Chu Z.L. Shin Y.A. Yang J.M. DiDonato J.A. Ballard D.W. J. Biol. Chem. 1999; 274: 15297-15300Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 16Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T. J. Biol. Chem. 1999; 274: 17402-17405Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). However, it is unclear exactly how Tax binding to IKKγ 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-κB activation (17Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (119) Google Scholar, 18Nasr R. Chiari E. El-Sabban M. Mahieux R. Kfoury Y. Abdulhay M. Yazbeck V. Hermine O. de The H. Pique C. Bazarbachi A. Blood. 2006; 107: 4021-4029Crossref PubMed Scopus (93) Google Scholar). Ubiquitinated Tax resides in the cytoplasm and activates NF-κB, whereas sumoylated Tax is located in nuclear bodies together with RelA (17Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (119) Google Scholar, 18Nasr R. Chiari E. El-Sabban M. Mahieux R. Kfoury Y. Abdulhay M. Yazbeck V. Hermine O. de The H. Pique C. Bazarbachi A. Blood. 2006; 107: 4021-4029Crossref PubMed Scopus (93) Google Scholar). Ubiquitination and sumoylation of Tax occur on overlapping lysine residues of which lysines 263, 280, and 284 are critical for NF-κB activation (17Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (119) Google Scholar, 18Nasr R. Chiari E. El-Sabban M. Mahieux R. Kfoury Y. Abdulhay M. Yazbeck V. Hermine O. de The H. Pique C. Bazarbachi A. Blood. 2006; 107: 4021-4029Crossref PubMed Scopus (93) Google Scholar). 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 IKKα is dependent on IKKγ because this event does not occur in IKKγ-deficient T cells. The relocalization is likely dependent on a direct interaction of IKKγ with Tax because a Tax mutant that does not bind to IKKγ 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-κB activation is associated with ubiquitin-dependent relocalization of IKK to specific compartments within the cell. Antibodies, Plasmids, and Other Reagents—Rabbit anti-IKKγ (FL419) and mouse anti-IKKα (B8) were from Santa Cruz (Santa Cruz, CA). Rabbit IKKα and IKKβ were from Cell Signaling (Beverly, MA). Giantin antibody was purchased from Abcam (Cambridge, MA). GM-130 antibody was purchased from BD Transduction Laboratories (San Diego, CA). Anti-p100/p52 antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The Tax hybridoma (168B17-46-34) was obtained from the AIDS Research and Reference Program, NIAID, National Institutes of Health. RRX-conjugated donkey anti-rabbit F(ab′)2 IgG, Cy5-conjugated donkey anti-mouse F(ab′)2 IgG, RRX-conjugated donkey anti-mouse F(ab′)2 IgG, and Cy5-conjugated donkey anti-rabbit F(ab′)2 IgG were from Jackson Immunoresearch (West Grove, PA). Alexa Fluor 555-conjugated donkey anti-mouse IgG was purchased from Molecular Probes/Invitrogen (Carlsbad, CA). DAPI was purchased from EMD Biosciences (San Diego, CA). pCMV4 Tax and pCMV4-p100 were previously described (8Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 13Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). Tax K263R,K280R,K284R was constructed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). PMA, ionomycin, and nocodazole were purchased from Sigma. Brefeldin A solution was purchased from eBioscience (San Diego, CA). Cell Lines and Transient Transfections—Jurkat and C8166 cells were previously described (19Uhlik M. Good L. Xiao G. Harhaj E.W. Zandi E. Karin M. Sun S.C. J. Biol. Chem. 1998; 273: 21132-21136Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). JM4.5.2 cells are a mutant cell line derivative from Jurkat SVT.35 cells that lack IKKγ (20Harhaj E.W. Good L. Xiao G. Uhlik M. Cvijic M.E. Rivera-Walsh I. Sun S.C. Oncogene. 2000; 19: 1448-1456Crossref PubMed Scopus (92) Google Scholar), and JM4.5.2-IKKγ cells are reconstituted with IKKγ (21Rivera-Walsh I. Cvijic M.E. Xiao G. Sun S.C. J. Biol. Chem. 2000; 275: 25222-25230Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). ED40515(-), MT-1, and TL-OM1 cells are clones of leukemic cells derived from ATL patients, kindly provided by Dr. Michiyuki Maeda (Kyoto University). These cells lack Tax expression but exhibit constitutive NF-κB activation (22Miura H. Maeda M. Yamamoto N. Yamaoka S. Exp. Cell Res. 2005; 308: 29-40Crossref PubMed Scopus (8) Google Scholar). All of the cells were cultured in RPMI medium containing 10% fetal bovine serum, 2 mm l-glutamine, and penicillin-streptomycin. 293-T kidney carcinoma cells (ATCC, Manassas, VA) were cultured in Dulbecco's medium containing 10% fetal bovine serum, 2 mm l-glutamine, and penicillin-streptomycin. 293-T cells were transfected with FuGENE 6 (Roche Applied Science) as recommended by the manufacturer. Approximately 40 h after transfections, the cells were harvested in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 1% Igepal CA-630, 0.1% SDS, and 0.5% deoxycholic acid). Retroviral Infection—Retroviral infections were performed with the pCLXSN system from Dr. I. Verma (23Naviaux R.K. Costanzi E. Haas M. Verma I.M. J. Virol. 1996; 70: 5701-5705Crossref PubMed Google Scholar) as previously described (13Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar). Briefly, 293-T cells were transfected with 1 μg of pCL-ampho, 0.15 μg of VSV-g and either 1 μg 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-μm polysulfone filters specialized for low protein binding (Pall Life Sciences, Ann Arbor, MI). Viral supernatant supplemented with fresh media and 8 μg/ml polybrene was used to infect 1 × 106 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 (24Harhaj E.W. Harhaj N.S. Grant C. Mostoller K. Alefantis T. Sun S.C. Wigdahl B. Virology. 2005; 333: 145-158Crossref PubMed Scopus (25) Google Scholar). EMSA was performed essentially as described (24Harhaj E.W. Harhaj N.S. Grant C. Mostoller K. Alefantis T. Sun S.C. Wigdahl B. Virology. 2005; 333: 145-158Crossref PubMed Scopus (25) Google Scholar). Double-stranded oligonucleotides were generated representing a consensus NF-κB site derived from the interleukin-2Rα promoter. The probe was radiolabeled with [32P]α-dCTP in a fill-in reaction with dNTPs (Invitrogen) and Klenow (Promega, Madison, WI). Equal amounts of nuclear extracts (4 μg) were incubated with radiolabeled probe and poly(dI-dC) (1 μg), 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 1× 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 × 106) were transfected with pCMV4-Tax (0.25 μg), pCMV4-Tax K263R,K280R,K284R (0.25 μg), or empty vector together with κB-TATA Luc (0.1 μg). 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 (25He H.T. Lellouch A. Marguet D. Semin. Immunol. 2005; 17: 23-33Crossref PubMed Scopus (75) Google Scholar). 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 (26Sun S.C. Ballard D.W. Oncogene. 1999; 18: 6948-6958Crossref PubMed Scopus (167) Google Scholar). 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 IKKγ localization upon Tax expression. Jurkat T cells were infected with a Tax-expressing retroviral vector or empty vector, stained with anti-IKKγ and anti-Tax and subjected to confocal microscopy. In cells infected with the empty vector, IKKγ was diffusely expressed throughout the cytoplasm (Fig. 1, A and C). Interestingly, however, Tax expression resulted in marked relocalization of IKKγ to cytoplasmic hot spots that appeared to reside in a perinuclear region (Fig. 1, D and F). Tax was predominantly colocalized with IKKγ in these hot spots (Fig. 1, E and F). IKKγ was also relocalized in Tax-inducible JPX-9 cells (data not shown). Importantly, these IKKγ hot spots were also observed in human T cells transformed by HTLV-1 (C8166 cells; Fig. 1G), and Tax was partially colocalized with IKKγ in these cells (Fig. 1I). Because IKKγ exists largely in a trimeric kinase complex that also contains IKKα and IKKβ, we hypothesized that Tax induces aggregation of the IKK holoenzyme. To examine this idea, we analyzed the subcellular localization of IKKα and IKKβ. As seen with IKKγ, IKKα and IKKβ were diffusely expressed in the cytoplasm in Jurkat cells (Fig. 2, A and E), although some punctate IKKβ staining was also observed (Fig. 2, E and F). In C8166 cells, IKKα (Fig. 2C) was present in perinuclear hot spots similar to those observed in Fig. 1 with IKKγ. IKKβ 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 IKKα and IKKγ. Thus, Tax expression appears to be associated with the movement of the IKKs to a concentrated perinuclear region within the cell. IKKγ Is Required for Tax-induced Relocalization of the IKK Complex—We have previously shown that IKKγ functions as an adaptor for recruiting Tax to the IKK complex, which is essential for NF-κB activation (14Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Xiao G. Harhaj E.W. Sun S.C. J. Biol. Chem. 2000; 275: 34060-34067Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Therefore, we examined whether Tax required IKKγ for inducing the relocalization of IKK complex. For this purpose, we used IKKγ-deficient Jurkat cells (JM4.5.2) that we had previously generated by somatic mutagenesis (20Harhaj E.W. Good L. Xiao G. Uhlik M. Cvijic M.E. Rivera-Walsh I. Sun S.C. Oncogene. 2000; 19: 1448-1456Crossref PubMed Scopus (92) Google Scholar). JM4.5.2 cells or JM4.5.2 cells stably reconstituted with IKKγ (JM4.5.2-IKKγ) were infected with recombinant retroviruses either expressing Tax or empty vector control. Tax had no effect on the distribution of IKKα in IKKγ-deficient Jurkat cells (Fig. 3C), although Tax was expressed as determined by reverse transcription-PCR (panel I). Furthermore, this defect was rescued when the IKKγ-deficient cells were reconstituted with exogenous IKKγ (Fig. 3G). Therefore, Tax requires IKKγ to modulate the subcellular localization of IKKα. Tax Mutants Defective in NF-κB Activation Fail to Induce IKK Relocalization—To assess the functional significance of Tax-induced IKK relocalization in NF-κB activation, we examined the ability of two well characterized Tax mutants, M22 and M47, in the relocalization of IKKγ. The Tax mutant M22 is deficient in NF-κB activation but remains competent in CREB activation, whereas M47 is deficient in CREB activation but active in NF-κB activation (28Smith M.R. Greene W.C. Genes Dev. 1990; 4: 1875-1885Crossref PubMed Scopus (347) Google Scholar). Jurkat cells were infected with recombinant retroviruses expressing either Tax, Tax M22, Tax M47, or empty vector. The cells were stained with anti-IKKγ and anti-Tax and subjected to confocal microscopy. As expected, the normally diffusely localized IKKγ (Fig. 4A) was 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 IKKγ 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-κB signaling. Because the M22 mutant is defective in Tax binding (14Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), these results also imply that Tax may require a direct interaction with IKKγ 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-κB 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 IKKγ localization in three different ATL cell lines that lack Tax expression but maintain activated NF-κB (22Miura H. Maeda M. Yamamoto N. Yamaoka S. Exp. Cell Res. 2005; 308: 29-40Crossref PubMed Scopus (8) Google Scholar). IKKγ was expressed diffusely throughout the cytoplasm and on top of the nucleus in the ATL cell lines MT-1, TL-OM1, and ED40515(-) (Fig. 5, E-J). As expected IKKγ 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 IKKα. The treatment was effective because RelA translocated to the nucleus at both time points (data not shown). Also, treatment of cells with TNFα did not trigger the relocalization of IKKα (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-IKKγ and markers for either endosomal, lysosomal, lipid rafts, or Golgi apparatus. IKKγ did not colocalize with endosomes, lysosomes, or lipid rafts (data not shown). However, we did observe a partial colocalization of IKKα and IKKγ 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 IKKγ 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, IKKγ localization changed from perinuclear to a more broadly distributed vesicular staining (Fig. 6J). Much of IKKγ remained colocalized with GM-130, suggesting that IKKγ was tightly associated with the Golgi (Fig. 6L). Treatment of C8166 cells with brefeldin A also led to a dramatic relocalization of IKKγ 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 (29Nejmeddine M. Barnard A.L. Tanaka Y. Taylor G.P. Bangham C.R. J. Biol. Chem. 2005; 280: 29653-29660Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Because the Golgi apparatus is a major site of signal transduction (30Mor A. Philips M.R. Annu. Rev. Immunol. 2006; 24: 771-800Crossref PubMed Scopus (325) Google Scholar), 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-κB activation (17Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (119) Google Scholar, 18Nasr R. Chiari E. El-Sabban M. Mahieux R. Kfoury Y. Abdulhay M. Yazbeck V. Hermine O. de The H. Pique C. Bazarbachi A. Blood. 2006; 107: 4021-4029Crossref PubMed Scopus (93) Google Scholar), 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 (31Chiari E. Lamsoul I. Lodewick J. Chopin C. Bex F. Pique C. J. Virol. 2004; 78: 11823-11832Crossref PubMed Scopus (71) Google Scholar), and mutation of these lysines to arginine residues results in a defect in NF-κB activation (17Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (119) Google Scholar). 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-κB pathways. In agreement with others (17Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (119) Google Scholar, 18Nasr R. Chiari E. El-Sabban M. Mahieux R. Kfoury Y. Abdulhay M. Yazbeck V. Hermine O. de The H. Pique C. Bazarbachi A. Blood. 2006; 107: 4021-4029Crossref PubMed Scopus (93) Google Scholar), Tax K263R,K280R,K284R was defective in NF-κB 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-IKKγ and subjected to confocal microscopy. In contrast to wild type Tax, the Tax mutant did not relocalize IKKγ 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-IKKγ movement to the Golgi. 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, IKKα and IKKγ 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-κB. This idea is further supported by the finding that IKKγ, 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. 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 IKKγ is emerging as an important role in the activation of the IKK complex. Forced oligomerization of IKKγ by fusion with the FKBP12 polypeptide leads to NF-κB activation and the redistribution of the IKK subunits from uniform to punctate cytoplasmic staining (32Poyet J.L. Srinivasula S.M. Lin J.H. Fernandes-Alnemri T. Yamaoka S. Tsichlis P.N. Alnemri E.S. J. Biol. Chem. 2000; 275: 37966-37977Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Tax also promotes the oligomerization of IKKγ through a direct interaction (33Huang G.J. Zhang Z.Q. Jin D.Y. FEBS Lett. 2002; 531: 494-498Crossref PubMed Scopus (30) Google Scholar). Thus, Tax likely triggers the oligomerization of IKKγ, 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 IKKγ subunit, and this interaction is critical for recruitment of Tax to the IKK complex and the activation of IKK and NF-κB (14Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 15Chu Z.L. Shin Y.A. Yang J.M. DiDonato J.A. Ballard D.W. J. Biol. Chem. 1999; 274: 15297-15300Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 16Jin D.Y. Giordano V. Kibler K.V. Nakano H. Jeang K.T. J. Biol. Chem. 1999; 274: 17402-17405Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Because Tax requires IKKγ for the relocalization of IKKα into the Golgi apparatus (Fig. 3), it is likely that Tax recruits IKKα to the Golgi via a direct interaction with IKKγ. Indeed, we have not observed Tax in the perinuclear area in the absence of IKKγ. Moreover, a Tax mutant (M22) defective in IKKγ binding fails to relocate IKK (Fig. 4). A recent study suggests that ubiquitination of Tax regulates binding to IKKγ, because a Tax mutant with multiple lysine to arginine substitutions does not interact with IKKγ (18Nasr R. Chiari E. El-Sabban M. Mahieux R. Kfoury Y. Abdulhay M. Yazbeck V. Hermine O. de The H. Pique C. Bazarbachi A. Blood. 2006; 107: 4021-4029Crossref PubMed Scopus (93) Google Scholar). We have found that a ubiquitination-defective Tax mutant (Tax K263R,K280R,K284R) is unable to relocalize IKKγ (Fig. 7D). However, this Tax mutant retains its ability to bind IKKγ, although a Tax mutant harboring arginine substitutions at all 10 lysines becomes defective in IKKγ 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 (17Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-10406Crossref PubMed Scopus (119) Google Scholar, 29Nejmeddine M. Barnard A.L. Tanaka Y. Taylor G.P. Bangham C.R. J. Biol. Chem. 2005; 280: 29653-29660Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 34Alefantis T. Mostoller K. Jain P. Harhaj E. Grant C. Wigdahl B. J. Biol. Chem. 2005; 280: 17353-17362Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 (29Nejmeddine M. Barnard A.L. Tanaka Y. Taylor G.P. Bangham C.R. J. Biol. Chem. 2005; 280: 29653-29660Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). 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 (29Nejmeddine M. Barnard A.L. Tanaka Y. Taylor G.P. Bangham C.R. J. Biol. Chem. 2005; 280: 29653-29660Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 35Igakura T. Stinchcombe J.C. Goon P.K. Taylor G.P. Weber J.N. Griffiths G.M. Tanaka Y. Osame M. Bangham C.R. Science. 2003; 299: 1713-1716Crossref PubMed Scopus (576) Google Scholar). Based on our results, one possibility is that Tax may recruit IKK to the vicinity of the Golgi/microtubule 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-κB may be essential for this process (29Nejmeddine M. Barnard A.L. Tanaka Y. Taylor G.P. Bangham C.R. J. Biol. Chem. 2005; 280: 29653-29660Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). 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 IKKβ is not as pronounced (Fig. 2). Because IKKα and IKKγ are critical components for Tax-mediated induction of the noncanonical pathway involving p100 processing to p52 (13Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Crossref PubMed Scopus (254) Google Scholar), 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-κB pathways. Emerging evidence suggests that the Golgi apparatus is one of the intracellular sites of signal transduction (30Mor A. Philips M.R. Annu. Rev. Immunol. 2006; 24: 771-800Crossref PubMed Scopus (325) Google Scholar, 36Rios R.M. Bornens M. Curr. Opin. Cell Biol. 2003; 15: 60-66Crossref PubMed Scopus (136) Google Scholar). 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 (37Miura K. Jacques K.M. Stauffer S. Kubosaki A. Zhu K. Hirsch D.S. Resau J. Zheng Y. Randazzo P.A. Mol. Cell. 2002; 9: 109-119Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). 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 (38Abo A. Qu J. Cammarano M.S. Dan C. Fritsch A. Baud V. Belisle B. Minden A. EMBO J. 1998; 17: 6527-6540Crossref PubMed Scopus (316) Google Scholar). With regard to NF-κB signaling, the IKKγ-related coiled-coil protein NRP (NEMO-related protein) is a Golgi-resident protein, although its function in signaling is currently unknown (39Schwamborn K. Weil R. Courtois G. Whiteside S.T. Israel A. J. Biol. Chem. 2000; 275: 22780-22789Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). 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-κB signaling pathway. Tax has been demonstrated to be mono- and polyubiquitinated in transfected cells and HTLV-1-transformed cell lines (31Chiari E. Lamsoul I. Lodewick J. Chopin C. Bex F. Pique C. J. Virol. 2004; 78: 11823-11832Crossref PubMed Scopus (71) Google Scholar, 40Peloponese Jr., J.M. Iha H. Yedavalli V.R. Miyazato A. Li Y. Haller K. Benkirane M. Jeang K.T. J. Virol. 2004; 78: 11686-11695Crossref PubMed Scopus (81) Google Scholar). Tax also interacts with various subunits of the proteasome (31Chiari E. Lamsoul I. Lodewick J. Chopin C. Bex F. Pique C. J. Virol. 2004; 78: 11823-11832Crossref PubMed Scopus (71) Google Scholar, 41Hemelaar J. Bex F. Booth B. Cerundolo V. McMichael A. Daenke S. J. Virol. 2001; 75: 11106-11115Crossref PubMed Scopus (28) Google Scholar, 42Beraud C. Greene W.C. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 1996; 13: S76-S84Crossref PubMed Scopus (28) Google Scholar, 43Rousset R. Desbois C. Bantignies F. Jalinot P. Nature. 1996; 381: 328-331Crossref PubMed Scopus (126) Google Scholar). 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-κB activation via binding to IKKγ (18Nasr R. Chiari E. El-Sabban M. Mahieux R. Kfoury Y. Abdulhay M. Yazbeck V. Hermine O. de The H. Pique C. Bazarbachi A. Blood. 2006; 107: 4021-4029Crossref PubMed Scopus (93) Google Scholar). 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 Lys63 modulate subcellular localization and/or protein function (44Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1022) Google Scholar). Thus, it is likely that Tax ubiquitination consists of linkages other than Lys48, probably Lys63. Nevertheless, we have shown here that Tax ubiquitination may regulate Tax and IKKα and IKKγ localization to the Golgi apparatus. Future studies will determine additional roles polyubiquitination may play in Tax activation of NF-κB. We thank Dr. Warner Greene for the Tax and NF-κB expression vectors; Dr. Inder Verma for the retroviral vectors; Drs. Michiyuki Maeda, Shoji Yamaoka, and Gutian Xiao for the ATL cell lines; Matt Morrison and Cell Signaling Technologies for the IKK antibodies; and the AIDS Research and Reference Program (NIAID, National Institutes of Health) for the anti-Tax hybridoma. We also thank Dr. Beata Frydel and Brigitte Shaw (University of Miami imaging facility) for expert assistance with confocal microscopy.