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The Human T-cell Leukemia Virus Type-1 Tax Protein Regulates the Activity of the IκB Kinase Complex

1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês

10.1074/jbc.274.48.34417

ISSN

1083-351X

Autores

Xiaohua Li, Kathleen M. Murphy, Kevin T. Palka, Rama Mohan Surabhi, Richard B. Gaynor,

Tópico(s)

Animal Disease Management and Epidemiology

Resumo

Two cytokine-inducible kinases, IKKα and IKKβ, are components of a 700-kDa kinase complex that specifically phosphorylates IκB. Phosphorylation of IκB by IKK leads to its ubiquitination and subsequent degradation, resulting in the nuclear translocation of NF-κB. The oncogenic protein Tax, encoded by human T-cell leukemia virus type-1 (HTLV-1), stimulates IKK activity to result in constitutive nuclear levels of NF-κB. In an attempt to gain insights into the mechanism by which Tax mediates constitutive activation of the NF-κB pathway, we analyzed the chromatographic distribution of IKK proteins using cellular extracts prepared from three T lymphocytes either lacking or containing Tax. IKK kinase activity and the distribution of proteins in the IKK complex were characterized. In extracts prepared from cells containing Tax, the activity of both IKKα and IKKβ present in the 700-kDa IKK complex were increased. Surprisingly, cell lines expressing Tax also contained an additional peak of IKKβ, but not IKKα activity, that migrated at 300 kDa rather than at 700 kDa. We noted that extracts containing Tax had extremely low levels of IκBβ, but not IκBα, and contained predominantly a truncated form of the MAP3K MEKK1. These results suggest that Tax may target several components of the NF-κB pathway leading to constitutive activation of this important regulator of cellular gene expression. Two cytokine-inducible kinases, IKKα and IKKβ, are components of a 700-kDa kinase complex that specifically phosphorylates IκB. Phosphorylation of IκB by IKK leads to its ubiquitination and subsequent degradation, resulting in the nuclear translocation of NF-κB. The oncogenic protein Tax, encoded by human T-cell leukemia virus type-1 (HTLV-1), stimulates IKK activity to result in constitutive nuclear levels of NF-κB. In an attempt to gain insights into the mechanism by which Tax mediates constitutive activation of the NF-κB pathway, we analyzed the chromatographic distribution of IKK proteins using cellular extracts prepared from three T lymphocytes either lacking or containing Tax. IKK kinase activity and the distribution of proteins in the IKK complex were characterized. In extracts prepared from cells containing Tax, the activity of both IKKα and IKKβ present in the 700-kDa IKK complex were increased. Surprisingly, cell lines expressing Tax also contained an additional peak of IKKβ, but not IKKα activity, that migrated at 300 kDa rather than at 700 kDa. We noted that extracts containing Tax had extremely low levels of IκBβ, but not IκBα, and contained predominantly a truncated form of the MAP3K MEKK1. These results suggest that Tax may target several components of the NF-κB pathway leading to constitutive activation of this important regulator of cellular gene expression. human T-cell leukemia virus type-1 amino acids cAMP response element-binding protein cadmium C-terminal domain glutathione S-transferase IKK complex-associated protein IKK-associated protein 1 inhibitor of NF-κB IκB kinase mitogen-activated protein kinase kinase kinase mitogen- activated protein kinase/extracellular signal-regulated kinase kinase 1 NF-κB essential modulator nuclear factor-κB NF-κB-inducing kinase tumor necrosis factor-α Human T-cell leukemia type-1 (HTLV-1)1 is a retrovirus responsible for the development of human adult leukemia/lymphoma, an aggressive and often fatal malignancy of activated CD4-positive T lymphocytes (1Poiesz B.J. Ruscetti R.W. Gazdar A.F. Bunn P.A. Minna J.D. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7415-7419Crossref PubMed Scopus (4204) Google Scholar, 2Yoshida M. 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Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1177) Google Scholar). The discovery of two IκB kinases, IKKα and IKKβ, whose activity is markedly increased by cytokine treatment of cells (32Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 33Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar, 34Zandi E. Chen Y. Karin M. Science. 1998; 281: 1360-1363Crossref PubMed Google Scholar, 35Didonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1930) Google Scholar, 36Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1073) Google Scholar) or the presence of the HTLV-1 Tax protein (37Yin M.-J. Christerson L.B. Yamamoto Y. Kwak Y.-T. Xu S. 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Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar) and NIK (43Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (272) Google Scholar); a scaffold protein, IKAP (43Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (272) Google Scholar); and a regulatory protein, NEMO/IKKγ/IKKAP1 (44Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 45Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 46Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (857) Google Scholar). Several lines of evidence suggest that MEKK1 and NIK are upstream activators of IKKs and function to mediate cytokine induction of the NF-κB pathway (32Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar,47Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar, 48Ling L. Cao Z. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3792-3797Crossref PubMed Scopus (454) Google Scholar, 49Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (474) Google Scholar). IKK activity is induced in cells overexpressing either MEKK1 or NIK (47Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar, 49Nakano H. Shindo M. Sakon S. Nishinaka S. Mihara M. Yagita H. Okumura K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3537-3542Crossref PubMed Scopus (474) Google Scholar). In addition, NIK associates with IKK both in vivo and in vitro (32Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 36Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1073) Google Scholar, 43Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (272) Google Scholar). Finally, recombinant MEKK1 induces in vitro phosphorylation of IKKα and IKKβ (37Yin 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, 47Lee F.S. Peters R.T. Dang L.C. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9319-9324Crossref PubMed Scopus (360) Google Scholar). These results suggest that NIK and MEKK1 kinases can modulate IKK activity. The exact role of NEMO/IKKγ/IKKAP1 is not clear. However, the direct interaction of this protein with IKKβ and its ability to activate IKK kinase activity suggest that this protein mediates association of IKK with the upstream activators of the NF-κB pathway (44Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 45Mercurio F. Murray B.W. Shevchenko A. Bennett B.L. Young D.B. Li J.W. Pascual G. Motiwala A. Zhu H. Mann M. Manning A.M. Mol. Cell. Biol. 1999; 19: 1526-1538Crossref PubMed Google Scholar, 46Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (857) Google Scholar). IKAP may be a scaffold protein that binds to NIK and IKKs to assemble them into an active kinase complex (43Cohen L. Henzel W.J. Baeuerle P.A. Nature. 1998; 395: 292-296Crossref PubMed Scopus (272) Google Scholar). Tax-mediated nuclear translocation of NF-κB is likely controlled by at least two distinct mechanisms. Tax is able to bind directly to the p100 and p105 precursors of NF-κB that function as cytoplasmic inhibitors of the NF-κB protein, facilitating NF-κB nuclear translocation (50Kanno T. Brown K. Franzoso G. Siebenlist U. Mol. Cell. Biol. 1994; 14: 6443-6451Crossref PubMed Google Scholar). More importantly, Tax increases the activity of cellular signal transduction pathways that increase the phosphorylation of IκBα and IκBβ at N-terminal serine residues (29McKinsey T.A. Brockman J.A. Scherer D.C. Al-Murrani S.W. Green P.L. Ballard D.W. Mol. Cell. Biol. 1996; 16: 2083-2090Crossref PubMed Google Scholar, 39Geleziunas 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). Recent studies demonstrate that Tax interaction with MEKK1 (37Yin 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), IKKs (38Chu Z.-L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar,40Uhlik 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), or NIK (40Uhlik 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) stimulates IKK activity (37Yin 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, 38Chu Z.-L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 39Geleziunas 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, 40Uhlik 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). NEMO or IKKγ, a component of the 700-kDa IKK complex, is critical for modulating IKK activity, and has recently been demonstrated to function in mediating Tax activation of the NF-κB pathway (44Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 51Chu 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 (162) Google Scholar, 52Jin 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 (199) Google Scholar). These results suggest that the same set of signaling components that are necessary for the rapid cytokine-induced phosphorylation of IκB are also likely utilized for the persistent activation of the NF-κB pathway by Tax. In contrast to the effects of most cytokines, which lead to a rapid but transient increase in IKK activity, Tax leads to a persistent increase in IKK activity (38Chu Z.-L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). This leads to the constitutive accumulation of NF-κB in the nucleus of HTLV-1-transformed T lymphocytes (53Sun S.-C. Elwood J. Béraud C. Greene W.C. Mol. Cell. Biol. 1994; 14: 7377-7384Crossref PubMed Google Scholar). Persistent decreases in IκBβ levels by Tax have been suggested to be a potential mechanism by which Tax leads to constitutive activation of the NF-κB pathway (29McKinsey T.A. Brockman J.A. Scherer D.C. Al-Murrani S.W. Green P.L. Ballard D.W. Mol. Cell. Biol. 1996; 16: 2083-2090Crossref PubMed Google Scholar). IκBβ gene expression, in contrast to that of IκBα, is not up-regulated by NF-κB (29McKinsey T.A. Brockman J.A. Scherer D.C. Al-Murrani S.W. Green P.L. Ballard D.W. Mol. Cell. Biol. 1996; 16: 2083-2090Crossref PubMed Google Scholar, 54Suyang H. Phillips R. Douglas I. Ghosh S. Mol. Cell. Biol. 1996; 16: 5444-5449Crossref PubMed Google Scholar), which may explain the ability of Tax to lead to persistent decreases in IκBβ but not IκBα protein levels. In an attempt to better understand the role of Tax on constitutive activation of IKK kinase activity, we analyzed IKK activity and protein composition following chromatographic fractionation of extracts prepared from cells either containing or lacking Tax. We demonstrate that in HTLV-1-infected T- cells, IKK activity in the high molecular weight IKK complex is significantly increased compared with its activity in cells lacking Tax. In addition, we detected a lower molecular mass complex that contains increased IKKβ activity in cells containing Tax. Tax was also found to modify the composition of several other components that regulate the NF-κB pathway. These results suggest that Tax mediates activation of the NF-κB pathway by affecting several components of this important regulatory loop. SLB (55Koeffler H.P. Chen I.S.Y. Golde D.W. Blood. 1984; 64: 482-490Crossref PubMed Google Scholar) and JPX-9 cells (56Nagata K. Ohtani K. Nakamura M. Sugamura K. J. Virol. 1989; 68: 3220-3226Crossref Google Scholar) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone Laboratories), 2 mml-glutamine, and antibiotics (penicillin-streptomycin). Jurkat cells were maintained in the same medium, except that the serum (HyClone) containing low levels of lipopolysaccharide were used. Cytoplasmic extracts were prepared according to Dignam (57Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (10033) Google Scholar) with slight modifications. Cells were harvested from culture medium by centrifugation for 10 min at 2000 rpm (Beckman bench-top centrifuge, CH3.7 rotor). Pelleted cells were washed twice with cold phosphate-buffered saline and were resuspended in 5 volumes of buffer A (10 mm Hepes (pH 7.9), 1.5 mm MgCl, 10 mm KCl, 0.5 mmdithiothreitol) supplemented with phosphatase inhibitors (50 mm NaF, 50 mm glycerophosphate, 0.125 μm okadaic acid, 1 mm sodium orthovanadate) and proteinase inhibitors at the level suggested by the manufacturer (Roche Molecular Biochemicals). After incubation for 15 min on ice, the cells were lysed with 40 strokes of a Kontes all-glass Dounce homogenizer (B type pestle). The nuclei were pelleted by centrifugation at 2000 rpm. The supernatant was mixed with 0.11 volume of buffer B (0.3 m Hepes (pH 7.9), 0.03 m MgCl, and 1.4m) and then centrifuged at for 60 min at 100,000 ×g. The supernatant was dialyzed for 5–8 h against 20 volumes of buffer D (20 mm HEPES (pH 7.9), 0.1m KCl, 0.5 mm dithiothreitol, 0.5 mm PMSF, 20% glycerol, and 0.2 mm EDTA). Cytoplasmic extracts were fractionated on a Q-Sepharose column and eluted stepwise with buffer D containing 0.2, 0.3, 0.4, 0.5, and 1 m KCl. Immune complexes precipitated from each fraction with IKK-specific antibody (Santa Cruz Biotechnology) were tested for IKK activity. Fractions eluted with 0.3 m KCl, which contained highest IKKα activity, were dialyzed, and the proteins were concentrated on a Q-Sepharose column. Equal amounts of concentrated proteins (2.5 mg) were further fractionated on a Superdex-200 column (Amersham Pharmacia Biotech). Protein markers (Sigma) used for the column include bovine thyroglobulin (669 kDa), horse spleen apoferritin (443 kDa), β-amylase (200 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.5 kDa). IKK complexes were immunoprecipitated from the Superdex-200 fractions and assayed for kinase activities as detailed (33Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar). Briefly, these fractions (50 μl) were incubated 2 h at 4 °C with 1–2 μg of the indicated antibodies (see figure legends for specifics) in 150 μl of PD buffer (40 mm Tris-HCl (pH 8.0), 500 mm NaCl, 0.1% Nonidet P-40, 6 mm EDTA, 6 mm EGTA, 10 mm β-glycerophosphate, 10 mm NaF, 300 μm Na3VO4, and protease inhibitors). Immune complexes were precipitated with protein A-agarose (Bio-Rad) for 1–3 h at 4 °C and subjected to kinase assays. Kinase reactions were carried out at 30 °C for 20 min in 25 μl of kinase buffer (20 mm Hepes (pH 7.6), 2 mmMgCl2, 2 mm MnCl2, 1 μm ATP, 1–3 μCi of [γ-32P]ATP, 10 mm β-glycerophosphate, 10 mm NaF, 300 μm Na3VO4 (pH 10.0), and protease inhibitors) in the presence of 5 μg of bacterially expressed GST fusion protein substrates. Phosphoproteins were resolved on SDS-polyacrylamide gels and visualized by autoradiography. The fusion proteins used included: GST/IκBα, which contains amino acids 1–54 of IκBα (33Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar); GST/IKKα (K44M), which contains kinase-inactive full-length IKKα with a K44M substitution to eliminate autophosphorylation (33Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Science. 1997; 278: 860-866Crossref PubMed Scopus (1861) Google Scholar); GST/MEK4Δ (a gift of Dr. Melanie Cobb), which contains the MEK4 with amino acids deleted from the N terminus (37Yin 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, 64Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. 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Before determining the effects of Tax on the activity and mobility of IKK complexes, we first analyzed the chromatographic distribution IKKα and IKKβ in extracts prepared from mock-treated and TNFα-stimulated Jurkat cells. Following Superdex-200 fractionation of these extracts, immunoprecipitation of column fractions with either IKKα- or IKKβ-specific antibodies was performed and kinase activity was assayed using a GST fusion with the N-terminal 54 amino acids of IκBα. Increased phosphorylation of IκB by IKKα (Fig.1 A) and IKKβ (Fig.1 C) was observed in fractions derived from TNFα-treated cells when compared with fractions derived from mock-treated cells (Fig. 1, A and C). There was no phosphorylation of a GST-IκBα fusion protein containing substitution of serine residues 32 and 36 with alanine (data not shown). The majority of this activity was found migrating between 600 and 700 kDa. There were similar levels of IKKα and IKKβ proteins in mock and TNFα-treated cells as determined by Western blot analysis (Fig. 1, B andD). These data confirm previous results that TNFα stimu

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