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• W2076596105 abstract "Upon T cell activation, IκB kinases (IKKs) are transiently recruited to the plasma membrane-associated lipid raft microdomains for activation of NF-κB in promoting T cell proliferation. Retroviral Tax proteins from human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are capable of activating IKK, yet only HTLV-1 infection causes T cell leukemia, which correlates with persistent activation of NF-κB induced by Tax1. Here, we show that the Tax proteins exhibit differential modes of IKK activation. The subunits of IKK are constitutively present in lipid rafts in activated forms in HTLV-1-infected T cells that express Tax. Disruption of lipid rafts impairs IκB kinase activation by Tax1. We also show that the cytoplasmic Tax1 protein persistently resides in the Golgi-associated lipid raft microdomains. Tax1 directs lipid raft translocation of IKK through selective interaction with IKKγ and accordingly, depletion of IKKγ impairs Tax1-directed lipid raft recruitment of IKKα and IKKβ. In contrast, Tax2 activates NF-κB in a manner independent of lipid raft recruitment of IKK. These findings indicate that Tax1 actively recruits IKK to the lipid raft microdomains for persistent activation of NF-κB, thereby contributing to HTLV-1 oncogenesis. Upon T cell activation, IκB kinases (IKKs) are transiently recruited to the plasma membrane-associated lipid raft microdomains for activation of NF-κB in promoting T cell proliferation. Retroviral Tax proteins from human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are capable of activating IKK, yet only HTLV-1 infection causes T cell leukemia, which correlates with persistent activation of NF-κB induced by Tax1. Here, we show that the Tax proteins exhibit differential modes of IKK activation. The subunits of IKK are constitutively present in lipid rafts in activated forms in HTLV-1-infected T cells that express Tax. Disruption of lipid rafts impairs IκB kinase activation by Tax1. We also show that the cytoplasmic Tax1 protein persistently resides in the Golgi-associated lipid raft microdomains. Tax1 directs lipid raft translocation of IKK through selective interaction with IKKγ and accordingly, depletion of IKKγ impairs Tax1-directed lipid raft recruitment of IKKα and IKKβ. In contrast, Tax2 activates NF-κB in a manner independent of lipid raft recruitment of IKK. These findings indicate that Tax1 actively recruits IKK to the lipid raft microdomains for persistent activation of NF-κB, thereby contributing to HTLV-1 oncogenesis. Human T cell leukemia virus type 1 (HTLV-1) 3The abbreviations used are: HTLV-1, human T cell leukemia virus type 1; Tak1, transforming growth factor β-activated kinase 1; RFP, red fluorescence protein; EMSA, electrophoretic mobility shift assay; MβCD, methyl-β-cyclodextrin; CREB, cAMP-response element-binding protein; TNFα, tumor necrosis factor α; GST, glutathione S-transferase; HA, hemagglutinin; WT, wild type; ERK, extracellular signal-regulated kinase; HEK, human embryonic kidney; CAV1, caveolin-1; GalT, galactosyltransferase. is an oncogenic retrovirus that causes human adult T cell leukemia. The viral oncoprotein Tax1, encoded by the HTLV-1 genome, is the molecular determinant for transformation of T lymphocytes (1Tanaka A. Takahashi C. Yamaoka S. Nosaka T. Maki M. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1071-1075Crossref PubMed Scopus (368) Google Scholar). Tax1 modulates the activity of the transcriptional factor NF-κB to promote T cell proliferation, and it also inactivates tumor suppressor p53 (2Sun S.C. Ballard D.W. Oncogene. 1999; 18: 6948-6958Crossref PubMed Scopus (165) Google Scholar, 3Pise-Masison C.A. Choi K.S. Radonovich M. Dittmer J. Kim S.J. Brady J.N. J. Virol. 1998; 72: 1165-1170Crossref PubMed Google Scholar). These molecular bases are thought to be crucial for Tax1-induced transformation of T lymphocytes. Moreover, vast amounts of experimental data have shown that Tax1 regulates a wide variety of cellular factors in addition to NF-κB and p53, which may play distinct roles at various stages of oncogenesis (4Matsuoka M. Jeang K.T. Nat. Rev. Cancer. 2007; 7: 270-280Crossref PubMed Scopus (622) Google Scholar). Persistent activation of NF-κB is one of the characteristic features of adult T cell leukemia. Tax1 is essential to establish proliferative growth of T cells at least in the early stage of infection, and Tax1 immortalizes primary human T cells in a manner highly dependent on its ability to activate NF-κB (2Sun S.C. Ballard D.W. Oncogene. 1999; 18: 6948-6958Crossref PubMed Scopus (165) Google Scholar). Tax2, the Tax protein from HTLV-2, shares a significant amino acid sequence homology with Tax1 and is able to activate NF-κB and to transactivate the long terminal repeat of both HTLV-1 and HTLV-2 by enhancing the activity of CREB (5Feuer G. Green P.L. Oncogene. 2005; 24: 5996-6004Crossref PubMed Scopus (133) Google Scholar). Although the viral genome can be isolated from some hairy cell leukemia patients, HTLV-2 infection is not causally linked to T cell leukemia (5Feuer G. Green P.L. Oncogene. 2005; 24: 5996-6004Crossref PubMed Scopus (133) Google Scholar). Furthermore, unlike Tax1, Tax2 rarely transforms primary cells although it can immortalize primary human T cells. The underlying mechanism for the differential effect of Tax1 and Tax2 in transformation of T cell remains poorly understood. The precise mechanism of Tax activation of NF-κB remains elusive. It has been demonstrated that Tax1 induces activation of NF-κB via several distinct mechanisms. First, Tax1 modulates the IκB kinase complex, the key regulator of NF-κB signaling, by directly stimulating the activity of the catalytic subunits, IKKα and IKKβ, through interaction with the non-catalytic subunit, IKKγ or NEMO (6Geleziunas R. Ferrell S. Lin X. Mu Y. Cunningham Jr., E.T. Grant M. Connelly M.A. Hambor J.E. Marcu K.B. Greene W.C. Mol. Cell. Biol. 1998; 18: 5157-5165Crossref PubMed Google Scholar, 7Li X.H. Murphy K.M. Palka K.T. Surabhi R.M. Gaynor R.B. J. Biol. Chem. 1999; 274: 34417-34424Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 8Chu Z.L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 9Jin 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, 10Chu 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 (159) Google Scholar, 11Harhaj E.W. Sun S.C. J. Biol. Chem. 1999; 274: 22911-22914Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Indeed, Tax1 fails to activate IκB kinases in NEMO/IKKγ-deficient cells (12Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israël A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar), indicating that IKKγ is one of the key targets for Tax1-mediated activation of IKK. Second, Tax1 was shown to modulate the activity of upstream kinases of IKK, which include mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEKK1) and NF-κB-inducing kinase (13Yin M.J. Christerson L.B. Yamamoto Y. Kwak Y.T. Xu S. Mercurio F. Barbosa M. Cobb M.H. Gaynor R.B. Cell. 1998; 93: 875-884Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 14Uhlik 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). It has been recently shown that Tax1 stimulates the activity of transforming growth factor β-activated kinase 1 (Tak1), an upstream kinase of IKK, and promotes interaction of Tak1 with IKKγ (15Wu X. Sun S.C. EMBO Rep. 2007; 8: 510-515Crossref PubMed Scopus (64) Google Scholar). Depletion of Tak1 abrogates the activation of IKK induced by Tax1, suggesting a critical role of Tak1 in this activation process. Furthermore, Tax1 was shown to interfere with the noncanonical pathway of NF-κB signaling by inducing processing of NF-κB2/p100 through up-regulation of IKKα in T cells (16Xiao 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 (251) Google Scholar). These evidences implicate that Tax1-mediated activation of NF-κB involves a complex process. The mechanism of the persistent activity of NF-κB by Tax1 is yet to be addressed. In T lymphocytes, activation of TCR elicits tyrosine phosphorylation cascades and induces membrane lipid raft recruitment and activation of signaling molecules such as ZAP70, phosphatidylinositol 3-kinase, and PKCθ (17Drevot P. Langlet C. Guo X.J. Bernard A.M. Colard O. Chauvin J.P. Lasserre R. He H.T. EMBO J. 2002; 21: 1899-1908Crossref PubMed Scopus (277) Google Scholar, 18Bi K. Tanaka Y. Coudronniere N. Sugie K. Hong S. van Stipdonk M.J. Altman A. Nat. Immunol. 2001; 2: 556-563Crossref PubMed Scopus (265) Google Scholar). The lipid rafts are glycosphingolipid- and cholesterol-enriched, detergent-resistant microdomains that play crucial roles in signaling transduction (19He H.T. Marguet D. EMBO Rep. 2008; 9: 525-530Crossref PubMed Scopus (46) Google Scholar, 20Gupta N. DeFranco A.L. Mol. Biol. Cell. 2003; 14: 432-444Crossref PubMed Scopus (133) Google Scholar). The lipid rafts are assembled in the Golgi and can be recycled between the plasma membrane and Golgi (21Rocks O. Peyker A. Kahms M. Verveer P.J. Koerner C. Lumbierres M. Kuhlmann J. Waldmann H. Wittinghofer A. Bastiaens P.I. Science. 2005; 307: 1746-1752Crossref PubMed Scopus (637) Google Scholar). Carma1, PKCθ, and Bcl10 are the key players that direct the plasma membrane lipid raft recruitment of the IKK complex, leading to transient activation of IKK and nuclear translocation of NF-κB upon T cell activation (22Gaide O. Favier B. Legler D.F. Bonnet D. Brissoni B. 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Aside from TCR-directed signaling events, tumor necrosis factor α (TNFα) also induces lipid raft translocation of IKK, together with the IKK-associated chaperone protein, Hsp90 (34Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). It remains largely unclear whether Tax1-mediated activation of NF-κBis involved in lipid raft translocation of IκB kinases. It has been shown that cytoplasmic Tax1 mediates activation of IκB kinases. Indeed, a portion of Tax1 was found to reside in the Golgi and to recruit the IKK complex to this subcellular location (35Lamsoul I. Lodewick J. Lebrun S. Brasseur R. Burny A. Gaynor R.B. Bex F. Mol. Cell. Biol. 2005; 25: 10391-41006Crossref PubMed Scopus (119) Google Scholar, 36Nejmeddine 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 (117) Google Scholar, 37Harhaj N.S. Sun S.C. Harhaj E.W. J. Biol. Chem. 2007; 282: 4185-4192Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In the present study, we show that the Tax1 protein accumulates in the Golgi-associated lipid rafts in directing translocation of IKK to the microdomains by primarily targeting IKKγ. This process is crucial for Tax1 activation of NF-κB in both T cells and non-lymphoid cells. In contrast, Tax2 activates NF-κB in a manner independent of the lipid raft recruitment of IKK as seen in Tax2-immortalized T cells. These differential modes of IKK activation by the Tax proteins may have implications on the pathogenesis of T cell leukemia. Cell Lines, Antibodies, and Reagents-Human T cell lines including MT1, MT2, MT4, SupT1, and Jurkat were cultured in RMPI1640 medium supplemented with 10% fetal bovine serum plus antibiotics at 37 °C, 5% CO2. SupT1 and Jurkat E6-1 cell lines were obtained from ATCC (Manassas, VA), MT1 and MT4 were kindly provided by Drs. Atsushi Koito and Takeo Ohsugi (Center for AIDS Research and Institute of Resource Development and Analysis, Kumamoto University, Japan). MT2 cells were obtained from Dr. Douglas Richman (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health). Antibodies for IκBα, Tak1, Hsp90, and HA epitope were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IKKα, IKKβ, IKKγ, and serine-phosphorylated IκBα were purchased from IMGENEX (San Diego, CA). Anti-LAT was from Upstate Biotechnology (Charlottesville, VA). Anti-β-actin, anti-FLAG M2 monoclonal antibodies, horseradish peroxidase-conjugated cholera toxin β subunit, methyl-β-cyclodextrin (MβCD), protease, and phosphatase inhibitor mixtures were obtained from Sigma. The proteasome inhibitor MG-132 was purchased from Calbiochem, and anti-Tax1 antibody was acquired from the AIDS Research and Reference Reagent Program. Mammalian Expression Plasmids, DNA Transfection, and GST Pulldown-The expression plasmids for GST-tagged IKKα, IKKβ, and IKKγ as well as HA-tagged Tax1 were described previously (38Cheng H. Cenciarelli C. Nelkin G. Tsan R. Fan D. Cheng-Mayer C. Fidler I.J. Mol. Cell. Biol. 2005; 25: 44-59Crossref PubMed Scopus (37) Google Scholar). To generate Tax1 mutants, PCR-based site-directed mutagenesis was performed to construct the fragments with mutations at the positions illustrated in Fig. 3A. The fragments of Tax1 mutants were inserted into expression vector pCEF with a C-terminal HA or GST tag and verified with DNA sequencing. Tax2 was constructed in the same vector as Tax1. To construct lipid raft-targeted IKKγ, a myristoylation signal from Lck was attached to the full-length of IKKγ to generate the myristoylated IKKγ fusion fragment (Myr-IKKγ), which was inserted into the lentivirus vector. GFP-GalT (galactosyltransferase) was purchased from Addgene Inc. (MA). To construct RFP-tagged caveolin-1, the full-length of the caveolin-1 (CAV1) cDNA was amplified from a human cDNA library, and C-terminal tagged with the RFP (monomeric red fluorescence protein) fragment. The CAV1-RFP fusion fragment was inserted into vector pLCEF8 and CAV1 was sequence-validated. To examine the interaction of IKKγ with Tax1 and its mutants, the FLAG-IKKγ expression plasmid was co-transfected with GST-tagged wild type Tax1, the Tax1 mutants, or IKKβ into HEK cells using SuperFect transfection reagent (Qiagen, Alencia, CA). 24 h post-transfection, the cells were lysed in the Buffer A containing 1% Triton X-100, 40 mm Tris-Cl (pH 7.5), 150 mm NaCl, 2 mm MgCl2, 0.5 mm dithiothreitol and protease inhibitor mixture at 15 °C for 30 min. Glutathione-Sepharose beads were added into the soluble supernatants and incubation was at room temperature for 2 h. The beads were then washed three times with the lysis buffer and subjected for SDS-PAGE plus Western blot analysis using anti-FLAG M2 monoclonal antibody to detect FLAG-IKKγ. To evaluate the interaction of endogenously expressed Tak1 with IκB kinases, GST-tagged IKKα, IKKβ, or IKKγ was transfected into HEK cells in the presence of either the empty vector or Tax1-HA. GST pulldown assay and immunoblot analysis were performed as described above. Lentivirus Vector and Transduction-The full-length fragment of enhanced green fluorescence protein was fused with IKKβ and IKKγ to generate GFP-IKKβ and GFP-IKKγ fusion genes, which were constructed in the lentivirus vector pLCEF8, a modified vector of pLL3.7 (39Rubinson D.A. Dillon C.P. Kwiatkowski A.V. Sievers C. Yang L. Kopinja J. Rooney D.L. Zhang M. Ihrig M.M. McManus M.T. Gertler F.B. Scott M.L. Van Parijs L. Nat. Genet. 2003; 33: 401-406Crossref PubMed Scopus (1336) Google Scholar) in which human elongation factor 1α promoter replaced U6 promoter. The Tax1-GFP fusion fragment was also constructed in pLCEF8. The lentivirus production and transduction in T cell lines were performed as described previously (38Cheng H. Cenciarelli C. Nelkin G. Tsan R. Fan D. Cheng-Mayer C. Fidler I.J. Mol. Cell. Biol. 2005; 25: 44-59Crossref PubMed Scopus (37) Google Scholar), and ∼10 multiplicity of infection was used for transduction of T cells. Transduction efficiency was verified with fluorescence imaging and immunoblot. Generation of Tax2 Immortalized T Cell Lines-Primary CD4+ T lymphocytes from healthy donors were isolated with Dynal beads conjugated with anti-CD4 antibody (Invitrogen). The CD4+ cells were activated with phytohemagglutinin (1 μg/ml) and recombinant interleukin-2 (100 units/ml) for 5 to 7 days prior to transduction with the lentivirus expressing Tax2-GFP fusion protein. Following one month of in vitro culture of the transduced cells in the media supplemented with interleukin-2 (50 units/ml), virtually all of the viable cells were green fluorescence positive and the cells were maintained in culture for more than 6 months. Western Blot Analysis-Cells were collected and lysed in lysis buffer B containing 40 mm Tris-Cl (pH 7.6), 1% Triton X-100, 1% deoxycholate, 150 mm NaCl plus protease and phosphatase inhibitor mixtures at 4 °C for 30 min. Equal amounts of cellular proteins were analyzed by SDS-PAGE, followed by immunoblot. Anti-β-actin blot was used for the protein loading control. In Vitro Kinase Assay and NF-κB Reporter Assay-In vitro kinase assay to detect the activity of IκB kinases and the NF-κB reporter assay were performed as previously reported (38Cheng H. Cenciarelli C. Nelkin G. Tsan R. Fan D. Cheng-Mayer C. Fidler I.J. Mol. Cell. Biol. 2005; 25: 44-59Crossref PubMed Scopus (37) Google Scholar). Electrophoretic Mobility Shift Assay (EMSA)-Nuclear extracts were prepared from various T cell lines with or without TNFα stimulation (10 ng/ml for 10 min at 37 °C), using NE-PER nuclear and cytoplasmic extraction reagents (Pierce). The sequence of the oligonucleotide corresponding to the κB element from the interleukin-2Rα gene was 5′-gatcCGGCAGGGGAATCTCCCTCTC-3′. The underlined sequence is the κB cis-element. The oligonucleotide was 5′-end labeled with biotin (Integrated DNA Technologies, Coralville, IA) and annealed to its complementary strand. The NF-κB binding activity to the κB element was examined by EMSA using the LightShift Chemiluminescent EMSA Kit (Pierce). In brief, 5 μg of the nuclear extracts were preincubated in a 20-μl total volume containing 100 mm Tris (pH 7.5), 500 mm KCl, 10 mm dithiothreitol, 200 mm EDTA (pH 8.0), 50% glycerol, and 1 μg of polydeoxyinosinic-deoxycytidylic acid and 2 μl of biotin-labeled probe (20 fmol) for 20 min at room temperature. The reactions were mixed with 5 μl of 5× loading buffer and run on a 6% non-denaturing polyacrylamide gel in 0.5× TBE (1× TBE: 89 mm Tris borate, 2 mm EDTA, pH 8.3) for 90 min at 100 V on ice, and transferred to nylon membranes (Amersham Biosciences) at 380 mA for 1 h in 0.5× TBE on ice. The membrane was optimally UV light cross-linked. Biotin-labeled DNA was detected by streptavidin-horseradish peroxidase, and followed by chemiluminescence. Lipid Raft Fractionation by OptiPrep Density Gradient Ultracentrifugation-Cells (4 × 107 for T cells and 1 × 107 cells for HEK cells) were lysed in 2 ml of extraction buffer (20 mm Tris-Cl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100 plus protease inhibitor mixture). Lysates were sheared by 20 passages through a 22-gauge needle, incubated for 20 min on ice before mixing with the OptiPrep density gradient medium (Iodixanol solution, final concentration, 40% v/v; AXIS-SHIELD PoC AS, Oslo, Norway), and placed at the bottom of a 12-ml tube. By overlaying 4 ml of 30% and 4 ml of 5% of OptiPrep medium, a discontinuous OptiPrep gradient was formed. Ultracentrifugation was performed at 100,000 × g for 4 h at 4 °C in an SW41 rotor. 1 ml of each fraction from the top to bottom was collected and subjected to Western blot analysis. Depletion of plasma and intracellular membrane cholesterol by MβCD in T cells was performed by pretreatment of the cells (4 × 107 cells/each sample) with 10 mm MβCD for 45 min at 37 °C in Hanks’ balanced salt solution. Following this step, the cells were subjected to density gradient ultracentrifugation for lipid raft fractionation analysis. Fluorescence Imaging-MT2 cells were transduced with GFP, Tax1-GFP, GFP-IKKβ, or GFP-IKKγ using the lentivirus-mediated gene delivery system. Lipid raft was labeled with the Alexa Fluor 594 Lipid Raft Labeling Kit (Molecular Probes, Eugene, OR). Cells were centrifuged and gently resuspended in chilled, complete culture media. Following centrifugation, the cell pellets were gently resuspended in 500 μl of Alexa 594-conjugated cholera toxin B (6 μg/ml) at 4 °C for 20 min. After this incubation, the cells were washed twice with chilled 1× phosphate-buffered saline and resuspended in 500 μl of the chilled anti-cholera toxin B antibody (100-fold dilution working solution) for 15 min at 4 °C. The cells were washed twice with chilled 1× phosphate-buffered saline, and transferred to poly-l-lysine-coated dishes and fixed in 4% paraformaldehyde for 15 min. The Golgi-lipid raft association of Tax1 was assessed by co-transfection of Tax1-GFP with the Golgi lipid raft marker caveolin-1 tagged with RFP. The cells were then incubated with 300 nm 4′,6-diamidino-2-phenylindole (Sigma) to stain the nuclei. Microscopy was performed using a Leica TCS SP2 AOBS confocal microscope. Alkaline Phosphatase Activity Assay-p-Nitrophenyl phosphate phosphatase assay was carried out for 12 h at 37 °C in an assay mixture (100 μl) consisting of 100 mm Tris-Cl (pH 9.5), 100 mm NaCl, 5 mm MgCl2, 1 mg/ml p-nitrophenyl phosphate (Sigma). The reaction was started by adding 10 μl of each fraction, and terminated by adding 100 μl of 1 n NaOH. Alkaline phosphatase activity was measured by absorbance at 405 nm. IκB Kinases Are Constitutively Present in the Lipid Rafts in HTLV-1-infected T Cells That Express Tax-Five T cell lines, including non-HTLV-1-infected T cells (Jurkat and SupT1) and HTLV-1-infected T cells (MT1, MT2, and MT4) were assessed for Tax1 expression. MT2 cells, but not other T cell lines, produced a detectable level of Tax1 (Fig. 1A). A hyperphosphorylation of IκBα, a substrate for activated IκB kinase, was detected in MT2 cells when the cells were pretreated with the proteasome inhibitor MG-132 (Fig. 1B). Accordingly, MT2 cells exhibited a hyperactivity of the κB DNA binding, and TNFα stimulation did not further increase the κB binding activity (Fig. 1C), suggesting that NF-κB was maximally activated in these cells even in the absence of extracellular stimuli. IκBα was weakly phosphorylated in MT1 cells with a detectable basal activity of NF-κB, and this activity was further enhanced upon TNFα treatment (Fig. 1, B and C). In non-HTLV-1 infected, Jurkat T cells, the phosphorylation of IκBα was not detected and the basal activity of NF-κB activity was not observed, whereas the NF-κB activity was induced by TNFα (Fig. 1, B and C). These results implicate that a constitutive NF-κB activation is present in HTLV-1-infected T cells, and the expression of Tax1 leads to induction of full-scale activation of IκB kinases. To determine whether the activation of IκB kinase by Tax1 requires a process that involves lipid raft translocation of IKK, density gradient ultracentrifugation was applied for lipid raft fractionation analysis. As shown in Fig. 1D, portions of three subunits of the IKK complex, including IKKα, IKKβ, and IKKγ, were constantly present in the lipid raft fractions in MT2 cells, corresponding to lipid raft biomarkers LAT and GM1 (fractions 4 and 5), whereas ERK1 exclusively resided in the soluble fractions. In contrast, in the HTLV-1-infected, non-Tax1 expressing cell line MT1, IKKs constantly remained in the cytoplasmic, soluble fractions (Fig. 1E). We further tested another non-Tax1 expressing, HTLV-1-infected T cell line, TL-Om1, and found that IκB kinases were in the soluble fractions (data not shown). To verify if the lipid raft presence of the IKK complex correlates with Tax1 expression, we examined another HTLV-1-infected T cell line, SLB-1, that expresses Tax1 (40Richard V. Lairmore M.D. Green P.L. Feuer G. Erbe R.S. Albrecht B. D'Souza C. Keller E.T. Dai J. Rosol T.J. Am. J. Pathol. 2001; 158: 2219-2228Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The activity of NF-κB in SLB-1 cells was comparable with that in MT2 cells (Fig. 1F). Indeed, the IκB kinases, not ERK1, were found in the lipid raft fractions in SLB-1 cells, corresponding to GM1 fractions (Fig. 1G). The amounts of the IκB kinase proteins in lipid rafts were abundant, which were detected in both fractions 4 and 5. To verify whether the IκB kinases are associated with lipid rafts in HTLV-1-infected, Tax1-expressing T cells, we treated MT2 cells with MβCD (dissolved in water), a selective cholesterol inhibitor that impairs formation of lipid rafts. Both IKKβ and IKKγ were exclusively found in the soluble, non-lipid raft fractions, whereas the level of IKKα in lipid rafts was significantly reduced following MβCD treatment (Fig. 2A). In MT2 cells, IKKα was persistently phosphorylated in both the lipid raft fraction (highest intensity of the IKKα phosphorylation in the fraction 5) and non-lipid raft fractions (Fig. 2B). Under the identical immunoblot conditions, phosphorylation of IKKα was rarely detected in MT1 cells, primarily due to low affinity of the phospho-specific antibody, which was unable to detect weak phosphorylation at short exposure. These data indicate that Tax1 induces hyperphosphorylation and activation of IκB kinases, which in turn drives a full range of activation of NF-κBinT cells. Indeed, disruption of lipid rafts by MβCD inhibited NF-κB activity in MT2 cells (Fig. 2C), and prevented Jurkat cells from TNFα-induced activity of NF-κB (Fig. 2D). These results suggest that the lipid raft-translocated IKKs are in activated forms in Tax1-expressing T cells and that Tax1 is likely to be the causative factor to promote lipid raft translocation of IκB kinases. Tax1 Directs Lipid Raft Translocation of IKK Correlating with Its Ability to Activate IKK-To evaluate structural recruitments of Tax1 in activating IκB kinases, we constructed several mutants of Tax1. M22 contains two amino acid substitutions at amino acids 130–131 and abrogates the ability of Tax to activate IKK but retains a full capacity to induce viral gene transcription through HTLV-1 LTR. As depicted in Fig. 3A, two additional mutants of Tax1, ΔGG and ΔPXXP, with deletions at amino acids 33–34 and 73–79, respectively, were generated, as these two amino acid sequences are highly conserved among the Tax proteins from HTLV-1 and HTLV-2. We assessed the ability of wild type (WT) Tax1 and Tax1 mutants in inducing the activity of IKKβ and found that the WT Tax1, but not any of the Tax1 mutants, stimulated the kinase activity of IKKβ (Fig. 3B). Accordingly, Tax1, but not any of the mutants induced NF-κB driven β-galactosidase activity (Fig. 3C). Similar to the WT Tax1, M22 maintained the ability to activate HTLV-1 LTR, whereas the ΔGG and ΔPXXP mutants lost such ability (data not shown). Because the ΔGG and ΔPXXP mutants abolished the abilities in inducing the activities of NF-κB and CREB that transactivates HTLV-1 LTR, it is likely that these two mutants may be improperly folded. Tax1 activation of IκB kinases does not require lymphocyte-specific factors. To determine whether the lipid raft translocation of the IKK complex correlates with the ability of Tax1 to activate IKK, we transfected WT Tax1 and its mutants into a non-lymphoid cell line, HEK. The WT Tax1 induced the lipid raft translocation of IKKα, IKKβ, and IKKγ, corresponding to the GM1 fraction, whereas ERK1 remained in the soluble fractions (Fig. 3D, fraction 5). In contrast, all three Tax1 mutants lost the ability to drive the translocation of IκB kinases to lipid rafts (Fig. 3, E–G). Moreover, the IKK-associated chaperone protein Hsp90, an essential modulator of the IKK complex, and Tak1, an upstream kinase of IKK, were also able to translocate into the lipid raft fractions in Tax1-expressing HEK cells, but not in cel" @default.
• W2076596105 created "2016-06-24" @default.
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• W2076596105 date "2009-03-01" @default.
• W2076596105 modified "2023-10-18" @default.
• W2076596105 title "HTLV-1 Tax Is a Critical Lipid Raft Modulator That Hijacks IκB Kinases to the Microdomains for Persistent Activation of NF-κB" @default.
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• W2076596105 doi "https://doi.org/10.1074/jbc.m806390200" @default.
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