NF-kappa B-inducing Kinase and Ikappa B Kinase Participate in Human T-cell Leukemia Virus I Tax-mediated NF-kappa B Activation*

Mark UhlikDagger §, LiFeng GoodDagger §, Gutian XiaoDagger , Edward W. HarhajDagger parallel , Ebrahim Zandi**Dagger Dagger , Michael Karin**, and Shao-Cong SunDagger §§

From the Dagger  Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and the ** Department of Pharmacology, Laboratory of Gene Regulation and Signal Transduction, University of California at San Diego, La Jolla, California 92093-0636

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The tax gene product of human T-cell leukemia virus I induces aberrant expression of various cellular genes, which contributes to transformation of host cells. Induction of many Tax target genes is mediated through transcription factor NF-kappa B. Here we show that Tax triggers activation of cellular protein kinases, Ikappa B kinase alpha  (IKKalpha ) and IKKbeta , which phosphorylate the NF-kappa B inhibitory protein Ikappa Balpha , resulting in its degradation and NF-kappa B activation. Constitutive IKK activation occurs in both Tax-transfected and human T-cell leukemia virus I-infected T cells. We further demonstrate that Tax-mediated NF-kappa B signaling also requires the NF-kappa B-inducing kinase (NIK). Consistently, inactive forms of either IKKs or NIK attenuate Tax-mediated NF-kappa B activation. Therefore, Tax activates NF-kappa B by targeting cellular signaling molecules, including both IKKs and NIK.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The type I human T-cell leukemia virus (HTLV-I)1 is the etiologic agent of an acute T-cell malignancy, termed adult T-cell leukemia (1-3). HTLV-I encodes a regulatory protein, Tax, which plays a central role in HTLV-I-induced host cell transformation (4-6). Tax not only serves as a transactivator of the HTLV-I viral long terminal repeat (LTR) but also alters the expression of a large number of cellular genes (7). Deregulation of cellular genes appears to contribute to HTLV-I-induced host cell transformation (7, 8).

Lacking DNA binding activity, Tax induces the target genes indirectly by modulating the activity of specific host transcription factors (7, 9). Tax activates HTLV-I LTR by physical interaction with the CREB/ATF family of proteins, which specifically bind to the cAMP response element-like sequences present in HTLV-I LTR (10-12). Tax interacts with the basic leucine zipper region of the CREB/ATF factors, enhancing their dimerization and DNA binding activities (13-15). Induction of many cellular genes by Tax is mediated through the cellular transcription factor NF-kappa B (7), a key regulator of genes involved in cell activation and proliferation (16). NF-kappa B activity is normally regulated through its cytoplasmic retention by specific inhibitors, including Ikappa Balpha and related proteins (17). Activation of NF-kappa B by cytokines is mediated by signal transduction cascades, leading to activation of the Ikappa B kinases, IKKalpha and IKKbeta (18-22). These kinases phosphorylate the Ikappa Bs, a modification that triggers their ubiquitination and proteolysis, allowing the released NF-kappa B dimers to enter the nucleus and activate target genes (23). In HTLV-I-infected cells or cells transfected with Tax cDNA expression vector, NF-kappa B is constitutively nuclear (24). The deregulated nuclear expression of NF-kappa B is essential for Tax-induced host cell transformation (25). Although precisely how Tax induces the nuclear translocation of NF-kappa B is not fully understood, recent studies have shown that Tax induces the phosphorylation and degradation of Ikappa Bs (26-33).

In this paper, we show that Tax triggers activation of the IKKs, and this action of Tax requires the recently identified NF-kappa B-inducing kinase (NIK) (34).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Cultures-- Jurkat T cells (ATCC) and Jurkat cells expressing the SV40 large T antigen (Jurkat-Tag; Ref. 35) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete RPMI). C8166 (36), HUT102 (37), MT-2 (38), and SLB-1 (39) are IL-2-independent HTLV-I-transformed T-cell lines. E55, N1185, and N1186 are HTLV-I-infected human T cells that require IL-2 for growth (40). Tax1 is a human T-cell clone expressing Tax in the context of a herpes saimiri vector (6). Human peripheral blood T cells were prepared as described (41). In vitro infection of T cells with HTLV-I was performed by cocultivating the target cells with gamma -irradiated HTLV-I donor cells (MT-2) (42, 43).

Luciferase Assay-- Jurkat T cells (5 × 106) were transfected using DEAE-dextran (44) with the indicated luciferase reporters and cDNA expression vectors. After 40 h, luciferase activity was determined as described (45).

Immunoblotting Assays-- Human 293 kidney carcinoma cells were seeded in 0.1% gelatin-treated 24-well plates (2.5 × 104 cells/well) and transfected using DEAE-dextran (44) with 0.12 µg of HA-Ikappa Balpha and other indicated expression vectors. Jurkat-Tag cells (5 × 106) were transfected using the same protocol with 1.6 µg of HA-Ikappa Balpha and other indicated expression vectors. After 40 h, whole-cell extracts were prepared and analyzed by Western blotting (46).

Immune Complex Kinase Assays (KA)-- Jurkat-Tag cells were transfected with HA-IKKs together with either the wild type (WT) or mutant forms of Tax. After 40 h, cells were lysed in a lysis buffer, containing 20 mM Hepes (pH 7.6), 250 mM NaCl, 0.5% Nonidet P-40, 20 mM beta -glycerophosphate, 1 mM EDTA, 20 mM p-nitrophenyl phosphate, 0.1 mM Na3VO4, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and a previously described protease inhibitor mixture (47). HA-IKKalpha and HA-IKKbeta were isolated by IP using anti-HA antibody. Immune complexes were washed three times with lysis buffer, once with lysis buffer supplemented with 1 M urea, and twice with a kinase buffer (20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM beta -glycerophosphate, 1 mM EDTA, 2 mM p-nitrophenyl phosphate, and 2 mM dithiothreitol). KA were performed as described (20) using GST-Ikappa Balpha -(1-54) or the mutant GST-Ikappa Balpha -(1-54)32A/36A as substrate. IKK proteins in the immune complexes were detected by immunoblotting. Endogenous IKK activity in Tax-expressing and HTLV-I-infected T cells was determined by a similar strategy but using anti-IKKalpha antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in the immune precipitation.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Catalytically Inactive IKKs Inhibit Tax-mediated NF-kappa B Activation-- To determine whether the recently cloned IKKs are required for activation of NF-kappa B by Tax, we examined the effects of catalytically inactive forms of IKKalpha (IKKalpha (K44M)) and IKKbeta (IKKbeta (K44A)) (20, 21) on Tax-mediated transactivation of a kappa B-driven luciferase reporter gene (kappa B-TATA-luc). The kappa B-TATA-luc reporter gene was transfected into Jurkat T cells either alone or together with a Tax expression vector along with increasing amounts of IKKalpha (K44M) or IKKbeta (K44A). As expected, Tax potently activated the kappa B enhancer, resulting in marked induction of luciferase activity (Fig. 1A, column 2). More importantly, expression of increasing amounts of catalytically inactive IKKalpha or IKKbeta led to a dose-dependent inhibition of reporter induction (columns 3-6). At the concentrations used, the inactive IKKs only modestly affected RelA-mediated kappa B-TATA-luc transactivation (Fig. 1B). Thus, the IKKs appeared to be required for Tax-mediated NF-kappa B activation but not the nuclear function of NF-kappa B. Parallel experiments performed with the WT forms of IKKalpha and IKKbeta revealed that IKKalpha potentiated Tax-mediated induction of kappa B-dependent transcription in a dose-dependent manner (Fig. 1C, columns 2-4). At low concentrations, IKKbeta also significantly enhanced Tax-mediated transactivation (columns 5 and 6), although expression of higher amounts of IKKbeta caused a reduction in reporter gene expression (column 7). At the amounts transfected, neither IKKalpha nor IKKbeta significantly affected reporter expression in Tax's absence (columns 8 and 9). These results further suggested that these Ikappa B kinases participate in Tax-mediated NF-kappa B activation. The different dose requirement for IKKalpha and IKKbeta to potentiate Tax activity may be due to differential expression of the endogenous IKKs in Jurkat cells. However, it may also be due to their different properties (19, 21, 22).


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Fig. 1.   Effects of WT and catalytically inactive IKKs on Tax-induced kappa B transactivation A and B, inhibition of Tax- or RelA-mediated kappa B activation by catalytically inactive forms of IKKalpha and IKKbeta . Jurkat cells were transfected with 2 µg of either empty vector (pCMV4) or expression vectors encoding Tax (A) or RelA (B) together with the indicated amounts of IKKalpha (K44M) or IKKbeta (K44A). All transfections included 0.4 µg of kappa B-TATA-luc. Luciferase activity is presented as -fold induction relative to the basal level measured in cells transfected with pCMV4 (8128 cpm). The values shown in A are means ± S.E. from three independent experiments, and values shown in B are representative of four independent experiments. C, effect of WT IKKs on Tax-mediated kappa B transactivation. Jurkat cells were transfected with Tax (2 µg) and the indicated expression vectors. Luciferase activity was determined and expressed as in A.

Inactive IKKs Interfere with Tax-induced Ikappa Balpha Phosphorylation-- To determine whether the effect of the transdominant IKK mutants on Tax-mediated NF-kappa B activation was due to the inhibition of Ikappa B phosphorylation, immunoblotting was used to examine phosphorylation of Ikappa Balpha in transiently transfected cells. When coexpressed with Ikappa Balpha , Tax induced the appearance of a slower migrating form of Ikappa Balpha (Fig. 2, lane 2), which upon incubation with alkaline phosphatase regained faster electrophoretic mobility (data not shown). As demonstrated (29, 48), this electrophoretic mobility change of Ikappa Balpha resulted from its phosphorylation at serines 32 and 36, since mutation of these two serines within Ikappa Balpha abolished this change (data not shown). We then examined the effect of the inactive IKKs on Tax-induced Ikappa Balpha phosphorylation. Expression of either IKKalpha (K44M) or IKKbeta (K44A) led to a dose-dependent inhibition of the Tax-induced appearance of the phosphorylated form of Ikappa Balpha , Ikappa Balpha -P (Fig. 2, lanes 3-9, middle panel). Under these conditions, the level of Tax expression was not significantly influenced (lower panel), suggesting that the dominant negative effect of the inactive IKKs was specifically on Tax-induced signal transductions. These findings strongly suggest that IKKalpha and IKKbeta are key components of Tax-mediated induction of both Ikappa Balpha phosphorylation and subsequent NF-kappa B activation.


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Fig. 2.   Inhibition of Tax-induced Ikappa Balpha phosphorylation by catalytically inactive IKKs. Jurkat-Tag cells were transfected with either pCMV4 (lane 1) or Tax expression vector (2 µg) in the absence (lanes 2 and 3) or presence (lanes 4-11) of the indicated amounts of HA-tagged IKKalpha (K44M) or IKKbeta (K44A) vectors. The cells were also transfected with an HA-tagged Ikappa Balpha expression vector (1.6 µg). Whole-cell extracts were subjected to immunoblotting with anti-HA antibody (upper and middle panels) or anti-Tax antiserum (lower panel). The basal and phosphorylated forms of HA-Ikappa Balpha (Ikappa Balpha and Ikappa Balpha -P, respectively), HA-IKKalpha and HA-IKKbeta (IKKs), and Tax proteins are indicated. The bands labeled with an arrowhead are nonspecific, since they were also detected from untransfected cells (data not shown).

Tax Activates IKKs in both Transfected and HTLV-I-infected Human T Cells-- To examine whether Tax causes IKK activation, we transfected Jurkat T cells with cDNA expression vectors encoding hemagglutinin (HA)-tagged IKKalpha or IKKbeta in the presence or absence of the Tax expression vector. The transiently expressed IKKalpha and IKKbeta were immunoprecipitated using an anti-HA monoclonal antibody, and their kinase activity was measured by an in vitro kinase assay (20, 21) using a substrate containing the N-terminal portion of Ikappa Balpha fused to glutathione S-transferase (GST-Ikappa Balpha -(1-54)). When expressed alone, IKKalpha exhibited low basal kinase activity (Fig. 3A, lane 1, top). However, Tax coexpression led to marked enhancement in IKKalpha catalytic activity (lane 2, top). Additionally, Tax enhanced autophosphorylation of IKKalpha (lane 2, top). We similarly examined whether IKKbeta also serves as a target of Tax. As previously reported (21, 22), IKKbeta exhibited higher levels of basal kinase activity than IKKalpha (Fig. 3A, lane 3, top). However, this isoform of IKK appeared to be less responsive to Tax than IKKalpha . Nevertheless, expression of Tax in the cells did result in significant enhancement of IKKbeta catalytic activity (lane 4).


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Fig. 3.   Activation of IKKs by Tax and HTLV-I. A, activation of IKKs by transiently expressed Tax. Jurkat-Tag cells were transfected with the indicated cDNA expression vectors (1 µg for Tax, 1 µg for IKKalpha , and 0. 2 µg for IKKbeta ). After 40 h, cell extracts were prepared and analyzed by immune complex KA. The phosphorylated GST-Ikappa Balpha substrate (Ikappa Balpha -P) and autophosphorylated IKKalpha and IKKbeta (IKK-P) in the KA (upper panel) are indicated. IKKalpha and IKKbeta were detected by immunoblotting (IB) with anti-HA (lower panel). B, control Jurkat cells or HTLV-I-infected C8166 cells were either not treated (lanes 1, 2, and 4) or incubated for 20 min with 50 µM tosylphenylalanyl chloromethyl ketone (lane 3). Endogenous IKKalpha was immunoprecipitated from whole-cell extracts using anti-IKKalpha (Santa Cruz Biotechnology) or a preimmune serum (PI) followed by KA. The higher intensity of a background band (indicated by ns) in the immunoblot (IB) of lane 4 was probably due to the use of a crude preimmune serum in this control immunoprecipitation. C, endogenous IKK activity in various Tax-expressing and HTLV-I-infected human T cells. KA was performed as described for B. The cells are described under "Materials and Methods." D, activation of IKKalpha in human peripheral blood T lymphocytes by mitogen and HTLV-I. IKKalpha activity was detected from normal or HTLV-I-infected human peripheral blood T cells, either not treated (lanes 1 and 3) or stimulated with phorbol 12-myristate 13-acetate (10 ng/ml, lane 2).

To assess the physiologic relevance of our findings with transfected cells, we next examined whether IKKs are also activated in HTLV-I-infected human T cells. For these studies, we used cell lines from different stages of HTLV-I-induced transformation, including E55, N1185, N1186, HUT102, SLB-1, C8166, and MT-2. The E55, N1185, and N1186 cells are infected with HTLV-I but have not progressed to the IL-2-independent stage, and the growth of these cells requires IL-2 (40). The HUT102, SLB-1, C8166, and MT-2 cells are IL-2-independent HTLV-I-transformed T cells (36-39). Both the IL-2-dependent and IL-2-independent HTLV-I-infected cell lines express high levels of Tax, which is associated with constitutive NF-kappa B activation (26, 28, 33).2 The C8166 cells are particularly interesting, since these cells carry a defective HTLV-I that selectively expresses Tax but not other viral proteins (36). To examine whether IKK was activated in these Tax-expressing cells, endogenous IKKalpha was immunoprecipitated from equal amounts of protein extracts of either control Jurkat cells or the C8166 cells. In vitro KA revealed that while IKKalpha from Jurkat cells had no detectable kinase activity, IKKalpha isolated from C8166 cells exhibited substantial kinase activity (Fig. 3B, middle) as well as autophosphorylation activity (top). The presence of constitutive IKKalpha kinase activity in C8166 cells was not due to an elevated IKKalpha expression. In fact, the C8166 IKKalpha immune complex contained significantly less IKKalpha than the Jurkat IKKalpha immune complex (Fig. 3B, bottom). Interestingly, when C8166 cells were incubated with tosylphenylalanyl chloromethyl ketone, an alkylating agent that inhibits Tax-induced Ikappa Balpha phosphorylation and NF-kappa B activation (26), IKKalpha activity was largely abolished (lane 3, middle). Similar KA demonstrated that IKK was also constitutively activated in all of the other HTLV-I-infected cell lines (Fig. 3C) and a T-cell clone expressing Tax in the context of a herpes saimiri vector (Fig. 3C, lane 2, Tax1; Ref. 6). Immunoblotting revealed that all of these Tax-expressing cell lines had a lower IKK protein level than the Jurkat cells (data not shown), suggesting that the IKK activity induction associated with Tax expression was due to activation of the kinase. To further confirm that constitutive IKK activation resulted from HTLV-I infection, we infected human peripheral blood T cells with HTLV-I in vitro and compared the IKKalpha activity between the uninfected and the bulk HTLV-I-infected T cells. As expected, the uninfected human T cells exhibited undetectable levels of IKKalpha activity, which was activated upon mitogen stimulation (Fig. 3D, lanes 1 and 2). Remarkably, upon HTLV-I infection, the IKKalpha kinase activity was dramatically induced (lane 3). Together, these results demonstrate that Tax activates the IKKs in both Tax-expressing and HTLV-I-infected T cells.

Activation of IKKs by Tax Correlates with Its Ability to Activate NF-kappa B-- Prior studies led to identification of Tax mutants that are either defective or effective in NF-kappa B activation (49). To correlate Tax-mediated activation of IKKalpha with induction of NF-kappa B, we examined the effect of two well characterized Tax mutants, M22 and M47, on IKKalpha activity. Consistent with its deficiency in NF-kappa B activation (49), M22 did not activate IKKalpha (Fig. 4, lane 3). In contrast, M47, which is fully effective in NF-kappa B activation (49), significantly stimulated IKKalpha activity and autophosphorylation (lane 4). Of note, neither the autophosphorylation nor phosphorylation of Ikappa Balpha -(1-54) was detected with a catalytically inactive IKKalpha mutant, IKKalpha (K44M), even when it was coexpressed with the wild type Tax (lane 5), suggesting the requirement of IKKalpha catalytic activity in these phosphorylation events. Furthermore, Tax-induced IKKalpha activity was specific for serine 32 and serine 36 of Ikappa Balpha , since substitution of these two serines with alanines in the substrate abolished 32P incorporation (Fig. 4, lane 6, middle). Similar results were obtained with IKKbeta , whose catalytic activity was enhanced by both WT Tax and TaxM47 but not influenced by TaxM22 (lanes 8-10). We noticed that TaxM47 consistently induced stronger IKK activity than WT Tax (compare the middle part of lanes 2 and 8 and lanes 4 and 10). While the underlying mechanism remains to be further investigated, this result is consistent with the finding that M47 is a stronger NF-kappa B activator compared with WT Tax (49). Parallel immunoblotting assays revealed that the kinase activity of IKKs was well correlated with phosphorylation of Ikappa Balpha in intact cells, indicated by its retarded electrophoretic mobility (data not shown).


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Fig. 4.   Activation of IKKs by WT and Mutant Forms of Tax. Jurkat-Tag cells were transfected with the indicated cDNA expression vectors (1 µg for WT and mutant forms of Tax; 0.25 µg for IKKalpha and IKKalpha (K44M); 0.1 µg for IKKbeta ). After 40 h, cell extracts were prepared and analyzed by KA using GST-Ikappa Balpha -(1-54) or GST-Ikappa Balpha -(1-54)32A/36A. The phosphorylated GST-Ikappa Balpha substrate (Ikappa Balpha -P) and autophosphorylated IKKalpha and IKKbeta (IKK-P) are indicated. IKKalpha and IKKbeta proteins were detected by immunoblotting (IB) with anti-HA (bottom).

NIK Participates in Tax-induced Ikappa Balpha Phosphorylation and NF-kappa B Activation-- Recent studies suggested that a MAP3K-related kinase, NIK, participates in NF-kappa B activation induced by cytokines (34). NIK was suggested to physically interact and activate the IKKs (18, 19). To investigate the role of NIK in Tax-mediated NF-kappa B signaling, we examined the effect of a catalytically inactive form of NIK (NIK(K429A/K430A)) (34) on Tax-induced Ikappa Balpha phosphorylation (Fig. 5A). At moderate doses (0.2-0.5 µg), NIK(K429A/K430A) significantly inhibited Tax-induced generation of the slower migrating phosphorylated Ikappa Balpha (Ikappa Balpha -P) (Fig. 5A, lanes 3 and 4). Reporter gene assays demonstrated that NIK(K429A/K430A) also inhibited Tax-mediated kappa B-TATA-luc activation (Fig. 5B). Thus, NIK appears to play an important role in Tax-induced NF-kappa B activation.


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Fig. 5.   Effects of NIK and NIK(K429A/K430A) on Tax-induced Ikappa Balpha phosphorylation and NF-kappa B activation. A, inhibition of Tax-induced Ikappa Balpha phosphorylation by NIK(K429A/K430A). Jurkat-Tag cells were transfected with Tax (1 µg) together with HA-Ikappa Balpha (1.6 µg) and the indicated amounts of NIK(K429A/K430A). Cell extracts were prepared and analyzed by immunoblotting with anti-HA. The phosphorylated and basal forms of Ikappa Balpha as well as two nonspecific bands (ns) are indicated. B, inhibition of Tax-mediated kappa B reporter gene activation by NIK(K429A/K430A). Jurkat cells were transfected with indicated empty vector or expression vectors and then subjected to luciferase assay. C, cooperation of Tax with NIK and IKKbeta . Human 293 cells were transfected in 24-well plates with the indicated amounts of cDNA expression vectors. Cell extracts were prepared and analyzed by immunoblotting with anti-HA. The phosphorylated and nonphosphorylated forms of Ikappa Balpha are indicated.

To further investigate the role of NIK in Tax-mediated NF-kappa B signaling, we analyzed the effect of WT NIK on Tax-induced Ikappa Balpha phosphorylation. In 293 cells, Tax induced only a modest increase in Ikappa Balpha phosphorylation (Fig. 5C, lane 2). As previously reported (19), overexpression of IKKbeta or IKKbeta plus NIK produced certain levels of Ikappa Balpha phosphorylation activity (data not shown). However, at lower levels, neither IKKbeta alone nor IKKbeta plus NIK induced Ikappa Balpha phosphorylation (Fig. 5C, lanes 5 and 6). Furthermore, when coexpressed with Tax, IKKbeta induced only weak Ikappa Balpha phosphorylation (lane 4), suggesting the lack of additional factors. Interestingly, when Tax was cotransfected with both IKKbeta and NIK, a markedly higher level of Ikappa Balpha phosphorylation was detected (lane 8). Similar results were obtained with IKKalpha (data not shown). These results strongly suggest that Tax-induced Ikappa Balpha phosphorylation involves both IKKs and NIK.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have shown that Tax activation of NF-kappa B is mediated by an indirect mechanism, which requires cellular signaling machinery. This mechanism differs from the one that mediates Tax activation of the CREB/ATF factors, which involves direct interaction between Tax and these basic leucine zipper-containing cellular DNA binding proteins (13-15). Our data demonstrate that the recently cloned Ikappa B kinases, IKKalpha and IKKbeta , are among the cellular signaling molecules participating in Tax-induced NF-kappa B activation. Catalytically inactive IKKs interfere with both the phosphorylation of Ikappa Balpha and subsequent NF-kappa B activation induced by Tax (Figs. 1 and 2). Consistently, Tax triggers the activation of both IKKalpha and IKKbeta , and this is tightly associated with the site-specific phosphorylation of Ikappa Balpha in intact cells. The importance of IKK for Tax-mediated NF-kappa B induction is further supported by experiments performed with Tax mutants. A mutant form of Tax (M22), defective in NF-kappa B activation, is unable to trigger IKK activation. On the other hand, M47, which harbors a mutation selectively affecting the CREB/ATF pathway, is fully active upon IKK activation (Fig. 4). Thus, Tax-mediated IKK activation is well correlated with its ability to activate NF-kappa B. Notably, IKK activation occurs not only in Tax-transfected cells but also in various HTLV-I-infected human T-cell lines as well as in human peripheral blood T cells freshly infected with HTLV-I (Fig. 3), thus confirming the physiological relevance of our findings. Our findings support the previous reports that Tax may induce the phosphorylation and degradation of Ikappa Bs (26-31). We believe that this is a major pathway by which Tax activates NF-kappa B.

Tax has also been suggested to directly bind to NF-kappa B proteins and enhance their transactivation function in the nucleus (50). Although this proposal does not explain how Tax induces the nuclear translocation of NF-kappa B, such a mechanism may exist to amplify the cytoplasmic action of Tax, i.e. activation of IKKs and induction of NF-kappa B nuclear expression. We have noticed that the catalytically inactive IKKs and NIK have more dramatic inhibitory effects on Tax-induced Ikappa Balpha phosphorylation (Figs. 2 and 5A) than on Tax-mediated kappa B reporter activation (Figs. 1A and 5B). Although this difference could be due to the higher sensitivity of the luciferase assay compared with immunoblotting, it may also suggest that Tax has a direct transactivation function in the nucleus. Nevertheless, our results indicate that activation of IKKs plays a central and critical role in Tax-induced nuclear expression of NF-kappa B in both Tax-transfected and HTLV-I-infected T cells.

The mechanism by which Tax affects IKK activity remains to be further investigated. However, it appears that IKKs are not the sole target for Tax. The dominant-negative inhibition assays suggest that NIK is involved in Tax-induced IKK signaling functions. Recent studies suggest that NIK serves as an upstream kinase involved in IKK activation (18, 19, 51, 52). NIK physically interacts with IKKs and stimulates their kinase activity when these proteins are overexpressed together in 293 cells (19). It remains to be tested whether Tax enhances or stabilizes the interaction between NIK and IKKs. Nevertheless, the ability of Tax to induce sustained IKK activation clearly explains the basis for constitutive Ikappa B phosphorylation and NF-kappa B activation in Tax-expressing and HTLV-I-infected T cells. Given the critical role of NF-kappa B in cell growth and survival (53-57), the activation of these Ikappa B kinases may be an important mechanism by which HTLV-I transforms host cells. As such, Tax provides a useful model for how a retroviral regulatory protein transforms host cells by targeting cellular signal transduction pathways.

During the revision of this paper, similar findings were reported by Yin et al. (58). However, unlike our study, this report suggests that IKKbeta , but not IKKalpha , is required for Tax activation of NF-kappa B. This discrepancy is likely due to the different experimental strategies and conditions used in these two studies. For example, Yin et al. used HIV-1 LTR, instead of the kappa B enhancer, as an NF-kappa B reporter. Since the HIV-1 LTR also contains other enhancers, such as NF-AT, which may respond to Tax (59), the result obtained with this reporter may not exactly reflect the activity of NF-kappa B. Nevertheless, we have performed both reporter gene assays (using the kappa B reporter, Fig. 1) and in vivo and in vitro Ikappa Balpha phosphorylation assays (Figs. 2-4), which clearly demonstrate that both IKKalpha and IKKbeta are involved in Tax activation of NF-kappa B.

    ACKNOWLEDGEMENTS

We thank W. C. Greene and D. Wallach for cDNA expression vectors, R. Grassmann for Tax1 cells, and G. Franchini for HTLV-I-infected cells. The anti-Tax antiserum was kindly provided by the AIDS Research and Reference Program, NIAID, National Institutes of Health.

    FOOTNOTES

* This study was supported by U.S. Public Health Service Grant 1 R01 CA68471-03 (to S.-C. S.) and ES06376-05 (to M. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

Supported by National Institutes of Health (NIH) postdoctoral fellowship F32 CA 71108-01A1.

parallel Supported by NIH predoctoral training Grant 5 T32 CA 6039-5.

Dagger Dagger Supported by a postdoctoral fellowship from the American Cancer Society, California Division.

§§ A scholar of the American Society for Hematology. To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. Fax: 717-531-6522; Tel.: 717-531-4164; E-mail: sxs70{at}psu.edu.

The abbreviations used are: HTLV-I, human T-cell leukemia virus type I; LTR, long terminal repeat; IKK, Ikappa B kinase; NIK, NF-kappa B-inducing kinase; GST, glutathione S-transferaseCREB, cAMP response element-binding proteinIL, interleukinHA, hemagglutininKA, kinase assay(s)WT, wild type.

2 E. W. Harhaj and S. C. Sun, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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