Title: Human T-cell Lymphotropic Virus Type 1 Tax Induction of Biologically Active NF-κB Requires IκB Kinase-1-mediated Phosphorylation of RelA/p65
Abstract: Activation of the NF-κB/Rel family of transcription factors proceeds through a catalytic complex containing IκB kinase (IKK)-1 and IKK2. Targeted disruption of each of the IKK genes suggests that these two kinases may mediate distinct functions in the activation pathway. In our studies of the human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein, we have uncovered a new function of IKK1 required for complete activation of the NF-κB transcriptional program. In IKK1–/– murine embryonic fibroblasts (MEFs), Tax normally induced early NF-κB activation events. However, NF-κB induced by Tax in these IKK1–/– cells was functionally impaired. In IKK1–/– (but not wild-type) MEFs, Tax failed to activate several different κB reporter constructs or to induce the endogenous IκBα gene. In contrast, Tax normally activated the cAMP-responsive element-binding protein/activating transcription factor pathway, leading to full stimulation of an HTLV-1 long terminal repeat reporter construct in IKK1–/– cells. Furthermore, reconstitution of IKK1–/– cells with kinase-proficient (but not kinase-deficient) forms of IKK1 restored the Tax induction of full NF-κB transactivation. We further found that the defect in NF-κB action in IKK1–/– cells correlated with a failure of Tax to induce phosphorylation of the RelA/p65 subunit of NF-κB at Ser529 and Ser536. Such phosphorylation of RelA/p65 was readily detected in wild-type MEFs. Phosphorylation of Ser536 was required for a complete response to Tax expression, whereas phosphorylation of Ser529 appeared to be less critical. Together, these findings highlight distinct roles for the IKK1 and IKK2 kinases in the activation of NF-κB in response to HTLV-1 Tax. IKK2 plays a dominant role in signaling for IκBα degradation, whereas IKK1 appears to play an important role in enhancing the transcriptional activity of NF-κB by promoting RelA/p65 phosphorylation. Activation of the NF-κB/Rel family of transcription factors proceeds through a catalytic complex containing IκB kinase (IKK)-1 and IKK2. Targeted disruption of each of the IKK genes suggests that these two kinases may mediate distinct functions in the activation pathway. In our studies of the human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein, we have uncovered a new function of IKK1 required for complete activation of the NF-κB transcriptional program. In IKK1–/– murine embryonic fibroblasts (MEFs), Tax normally induced early NF-κB activation events. However, NF-κB induced by Tax in these IKK1–/– cells was functionally impaired. In IKK1–/– (but not wild-type) MEFs, Tax failed to activate several different κB reporter constructs or to induce the endogenous IκBα gene. In contrast, Tax normally activated the cAMP-responsive element-binding protein/activating transcription factor pathway, leading to full stimulation of an HTLV-1 long terminal repeat reporter construct in IKK1–/– cells. Furthermore, reconstitution of IKK1–/– cells with kinase-proficient (but not kinase-deficient) forms of IKK1 restored the Tax induction of full NF-κB transactivation. We further found that the defect in NF-κB action in IKK1–/– cells correlated with a failure of Tax to induce phosphorylation of the RelA/p65 subunit of NF-κB at Ser529 and Ser536. Such phosphorylation of RelA/p65 was readily detected in wild-type MEFs. Phosphorylation of Ser536 was required for a complete response to Tax expression, whereas phosphorylation of Ser529 appeared to be less critical. Together, these findings highlight distinct roles for the IKK1 and IKK2 kinases in the activation of NF-κB in response to HTLV-1 Tax. IKK2 plays a dominant role in signaling for IκBα degradation, whereas IKK1 appears to play an important role in enhancing the transcriptional activity of NF-κB by promoting RelA/p65 phosphorylation. Over the past 2 decades, an ever increasing body of work has indicated that the NF-κB/Rel family of transcription factors lies at the crux of such diverse cellular processes as proliferation, differentiation, and death (1Beg A.A. Baldwin Jr., A.S. Genes Dev. 1993; 7: 2064-2070Google Scholar, 2Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Google Scholar, 3Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar). Initially identified as pivotal regulators of immune and inflammatory responses, the actions of this family of transcription factors have now expanded to encompass many physiological and pathological conditions, such as fetal development, neuronal function and degeneration, angiogenesis, ischemia, and tumorigenesis (4Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Google Scholar, 5Denk A. Wirth T. Baumann B. Cytokine Growth Factor Rev. 2000; 11: 303-320Google Scholar). Principally found in the cytoplasm under basal conditions, latent NF-κB·IκB complexes are poised to rapidly respond to a variety of external and internal signals, including TNF-α 1The abbreviations used are: TNF-α, tumor necrosis factor-α; HTLV-1, human T-cell lymphotropic virus type 1; NIK, NF-κB-inducing kinase; IKK, IκB kinase; MSK1, mitogen- and stress-activated protein kinase-1; IFN, interferon; MEFs, murine embryo fibroblasts; WT, wild-type; siRNA, small interfering RNA; GST, glutathione S-transferase; CREB, cAMP-responsive element-binding protein; LTR, long terminal repeat. and interleukin-1, and bacterially and virally derived products, such as lipopolysaccharide, HTLV-1 Tax, and UV light (6Thanos D. Maniatis T. Cell. 1995; 80: 529-532Google Scholar, 7Rothwarf D.M. Karin M. Science's STKE.http://stke.sciencemag.org/cgi/content/full/sigtrans;1999/5/re1Date: 1999Google Scholar). The activation of this NF-κB/Rel family of transcription factors, all of whom share an N-terminal Rel homology domain, is rapid and occurs in the absence of protein synthesis (3Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar). As such, NF-κB must be tightly regulated at multiple levels within the cell, including its release from the inhibitor protein, its mobilization from the cytoplasm into the nucleus, and its engagement of cognate κB enhancers, leading to changes in target gene expression. The activation of NF-κB can be viewed as occurring in two discrete phases. The first phase comprises the proximal events that lead to the degradation of the IκBα inhibitor and the translocation of the NF-κB complex into the nuclear compartment. This initial phase of NF-κB activation proceeds through a kinase cascade culminating in the site-specific phosphorylation, ubiquitylation, and subsequent degradation of the IκBα inhibitor by the 26 S proteasome. Although the panel of kinases that are recruited after the detection of a particular stimulus includes NIK, TAK-1 (Transforming growth factor-β-activated kinase), and MEKK1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1), they all appear to converge at a common point in the pathway, viz. the activation of a large catalytic complex termed the signalsome (8Karin M. Oncogene. 1999; 18: 6867-6874Google Scholar, 9Senftleben U. Karin M. Crit. Care Med. 2002; 30: S18-S26Google Scholar). In turn, the signalsome catalyzes the phosphorylation of the inhibitor protein at two specific N-terminal serines (Ser32 and Ser36 in IκBα and Ser19 and Ser23 in IκBβ) (10Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. 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Cell. 1997; 91: 243-252Google Scholar, 18Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Google Scholar, 19Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Google Scholar). These two highly related kinases appear to act in concert to transmit the activation signal in a directional manner, leading to the phosphorylation of IκBα (20O'Mahony A. Lin X. Geleziunas R. Greene W.C. Mol. Cell. Biol. 2000; 20: 1170-1178Google Scholar, 21May M.J. Ghosh S. Science. 1999; 284: 271-273Google Scholar). The second phase in the activation of NF-κB involves the post-translational modification of the NF-κB subunits themselves by both phosphorylation and acetylation (22Okazaki T. Sakon S. Sasazuki T. Sakurai H. Doi T. Yagita H. Okumura K. Nakano H. Biochem. Biophys. Res. Commun. 2003; 300: 807-812Google Scholar, 23Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. 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Although the former phosphorylation events on IκBα are required for release of NF-κB, the latter modifications on RelA/p65 appear to enhance its transcriptional activity and the duration of the response (32Chen L.-F. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Google Scholar). In contrast to the phosphorylation of IκBα, the events that culminate in the modification of RelA/p65 are less well defined. Moreover, the end result appears to vary with respect to cell type, the stimulus evoked, the kinase recruited, and the target residue modified. However, irrespective of these differences, it is clear that the phosphorylation of RelA/p65 augments the transactivation potential of the NF-κB transcription factor complex. Kinases and their target residues that have been implicated in RelA/p65 modification include protein kinase A (on Ser276) (22Okazaki T. Sakon S. Sasazuki T. Sakurai H. Doi T. Yagita H. Okumura K. Nakano H. Biochem. Biophys. Res. Commun. 2003; 300: 807-812Google Scholar, 33Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Google Scholar), MSK1 (on Ser276) (26Vermeulen L. De Wilde G. Van Damme P. Vanden Berghe W. Haegeman G. EMBO J. 2003; 22: 1313-1324Google Scholar), casein kinase II (on Ser529) (27Wang D. Westerheide S.D. Hanson J.L. Baldwin Jr., A.S. J. Biol. Chem. 2000; 275: 32592-32597Google Scholar), IKK2 (on Ser536) (23Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Google Scholar), and NIK and IKK1 (on Ser536) (34Jiang X. Takahashi N. Ando K. Otsuka T. Tetsuka T. Okamoto T. Biochem. Biophys. Res. Commun. 2003; 301: 583-590Google Scholar). In this study, we demonstrate that IKK1 plays a key role in the activation of NF-κB by the HTLV-1 protein Tax (35Geleziunas 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: 5157-5165Google Scholar, 36Jeang K.T. Cytokine Growth Factor Rev. 2001; 12: 207-217Google Scholar). In addition to Tax activation of the IKK1·IKK2·NEMO signalsome complex, which leads to IκBα phosphorylation and degradation, Tax also activates IKK1-mediated phosphorylation of RelA/p65, which enhances the transcriptional activity of the NF-κB complex. Thus, Tax appears to regulate NF-κB in both the proximal and distal phases of the activation pathway. Unlike the transient release of cytokines, Tax is continually expressed in HTLV-1-infected cells, leading to constitutive NF-κB action (37Chu Z.L. DiDonato J.A. Hawiger J. Ballard D.W. J. Biol. Chem. 1998; 273: 15891-15894Google Scholar). The ability of Tax to promote increased transcriptional activity of NF-κB through the phosphorylation of the C-terminal transactivation domain in RelA/p65 likely plays a role in sustaining the NF-κB transcription response and contributes to leukemogenesis induced by HTLV-1 infection. Expression Vectors and Biological Reagents—Plasmids pCMV4Tax and pCMV4TaxM22 have also been reported elsewhere (20O'Mahony A. Lin X. Geleziunas R. Greene W.C. Mol. Cell. Biol. 2000; 20: 1170-1178Google Scholar, 35Geleziunas 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: 5157-5165Google Scholar, 38Beraud C. Sun S.C. Ganchi P. Ballard D.W. Greene W.C. Mol. Cell. Biol. 1994; 14: 1374-1382Google Scholar). κB reporter plasmids including 5x-κB-Luc, IFN-κB-Luc, and E-selectin-κB-Luc have been described previously (35Geleziunas 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: 5157-5165Google Scholar). Expression plasmids encoding wild-type and kinase-deficient constructs of IKKα and NIK have been described previously (20O'Mahony A. Lin X. Geleziunas R. Greene W.C. Mol. Cell. Biol. 2000; 20: 1170-1178Google Scholar). Recombinant human TNF-α was purchased from Pierce Biotechnology Inc. (Rockford, IL). The epitope-specific reagents used were obtained from the indicated manufacturers: anti-NEMO, anti-IκBα, anti-RelA/p65, anti-IKK1, and anti-IKK2 antibodies (Santa Cruz Biotechnology); protein A beads and FuGENE 6 (Roche Applied Science); and the κB probe for electrophoretic mobility shift assay (Promega). Cultured Cells—The IKK1–/– and IKK2–/– MEF lines were generously provided by Dr. Michael Karin (University of California, San Diego) and Dr. Inder Verma (Salk). WT MEF cultures were established from embryonic day 14–16 mouse embryos. Reconstituted p65–/– MEFs (WT p65, p65-S529A, p65-S536A, and p65-S529A/S536A) were generously supplied by Dr. Hiroyasu Nakano (Juntendo University School of Medicine, Tokyo, Japan). All MEF lines and 293 and HeLa epithelial cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. Cell lysates from these lines were prepared and probed with anti-IKK1 or anti-1KK2 antibodies to confirm the phenotype. Luciferase Reporter Assays—IKK1–/– MEFs, IKK2–/– MEFs, WT MEFs, 293 cells, HeLa cells, and reconstituted p65–/– MEFs were transiently transfected using FuGENE 6 with either Tax or NIK expression plasmids (0.05, 0.1, and 0.5 μg/well) or empty vector and the reporter construct indicated in the figure legends. After 12 h, TNF-α was added to select cultures, and the cells were incubated for an additional 6–8 h. Cells were lysed with diluted 5× passive lysis buffer (Promega), and the resulting lysates were assessed for luciferase activity using a luciferase detection kit (Promega) and a multiwell luminometer (Wallac). Cells were also cotransfected with an expression plasmid encoding Renilla luciferase that is not regulated by NF-κB as a control to normalize for differences in transfection efficiency. For experiments involving siRNA, oligonucleotides (Dharmacon, Lafayette, CO) corresponding to a portion of the coding region of the IKK1 open reading frame or nonspecific scrambled oligonucleotides were transfected into cells with OligofectAMINE (Invitrogen) 12 h prior to transfection with the expression plasmids listed above. Results are expressed as relative luciferase units adjusted for transfection efficiency and represent the means of triplicate wells from six independent experiments. In Vitro Kinase Assays—Cells were transfected with either Tax or NIK expression vectors as described above. At 24 h post-transfection, the indicated cells were stimulated with TNF-α (10 ng/ml) for 7–10 min. Cells were lysed in buffer containing 1% Nonidet-P-40, 250 mm NaCl, 50 mm HEPES (pH 7.4), and 1 mm EDTA supplemented with a mixture of protease inhibitors (Roche Applied Science), 1 mm phenylmethylsulfonyl fluoride, 50 μm dithiothreitol, and 50 μm Na3VO4 (added fresh before used). Lysates were immunoprecipitated with either anti-NEMO antibodies alone or with anti-IKK1 antibodies conjugated to protein A beads. The immunoprecipitates were then incubated with 1 μCi of [γ-32P]ATP and 1 μg of various recombinant p65 protein substrates, including His-tagged full-length RelA/p65 and GST-tagged RelA/p65 N- and C-terminal regions at 30 °C for 30 min. Reactions were stopped by the addition of 2× SDS-PAGE sample buffer and boiling for 5 min. Products were analyzed by SDS-PAGE, followed by electrophoretic transfer to nitrocellulose membranes and exposure to Hyperfilm MP (Amersham Biosciences). Lysates were probed with anti-Tax antibodies to confirm the expression of Tax. Electrophoretic Mobility Shift Assay—Cells were transfected with a Tax expression vector and incubated for 24 h or were stimulated with TNF-α for 20 min as described above. Cells were lysed in nuclear extraction buffer (20 mm Tris-HCl (pH 7.8), 125 mm NaCl, 5 mm MgCl2, 0.2 mm EDTA, 12% (w/v) glycerol, and 0.1% (w/v) Nonidet P-40) supplemented with 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol. Protein-matched samples were incubated with a κB enhancer construct labeled with [γ-32P]ATP. Nucleoprotein·κB complexes were separated on nondenaturing gels and visualized by autoradiography. Tax Fails to Induce NF-κB-mediated Transactivation in IKK1-deficient Cells—To date, many studies have employed NF-κB translocation into the nucleus or the induced appearance of κB-specific DNA binding activity as a signature of NF-κB activation (8Karin M. Oncogene. 1999; 18: 6867-6874Google Scholar). In actuality, complete activation of this transcription factor culminates in its ability to regulate the expression of various target genes. One experimental approach to detect this functional end point is to assess the expression of a reporter gene placed under the control of a κB enhancer element. Accordingly, we first tested the ability of a transiently expressed HTLV-1 Tax protein to induce NF-κB transactivation of the IFN-κB-Luc reporter gene in IKK1–/– MEFs versus WT MEFs. Although Tax induced robust luciferase reporter activity in WT MEFs, we observed only minimal luciferase activity in IKK1–/– MEFs over a range of transfected Tax expression vector DNAs (Fig. 1, A and B). To rule out the possibility that these results reflect a selective failure to activate the IFN-κB enhancer, we tested a second reporter plasmid containing the κB enhancer from the E-selectin gene promoter. A similar defect in Tax induction of luciferase activity was observed with this E-selectin-κB-Luc construct in IKK1-deficient cells expressing Tax (Fig. 1, A and B). Comparable expression of Tax in these IKK1–/– and WT MEFs was confirmed by Western blotting of the lysates with anti-Tax antibodies (Fig. 1E). In contrast to the defect observed with Tax as the agonist, the expression of NIK induced a dose-dependent activation of both κB-Luc reporters. Similarly, stimulation with TNF-α induced luciferase activity with both reporter constructs, although the response observed with IFN-κB was moderately impaired. This functional defect in the Tax response is not attributable to the embryonic stage of development of these IKK1–/– cells because Tax normally activated both of these κB reporters in similarly derived WT MEFs. Next, we examined the effects of Tax activation of κB-Luc reporters in MEFs lacking IKK2 and in 293 fibroblasts. Loss of IKK2 leads to a reduced, but demonstrable induction of NF-κB-mediated transactivation in response to Tax expression. Although the response to Tax was attenuated in IKK2–/– MEFs, we observed greater κB reporter activity in IKK2–/– MEFs than in IKK1–/– cells over a range of Tax expression levels. This induction of NF-κB activity by Tax in IKK2-deficient cells likely involves IKK1-mediated kinase activity, resulting in IκBα phosphorylation, degradation, DNA binding, and transactivation by the NF-κB complex (Fig. 1C). In addition, strong Tax responses were observed in 293 fibroblasts (Fig. 1D). The expression of Tax in these cells was confirmed by Western blotting of the lysates with anti-Tax antibodies (Fig. 1E). To further explore the role of IKK1 in Tax activation of NF-κB, we employed siRNAs to "knock down" the expression of IKK1 in HeLa cells. As shown in Fig. 1 (F and G), transfection of HeLa cells with siRNA specific for IKK1 both inhibited IKK1 protein expression and impaired the induction of κB-Luc activity by Tax, but not by TNF-α. Conversely, transfection of these cells with scrambled siRNA with the same nucleotide composition did not inhibit IKK1 expression or the Tax response. In addition to the induction of NF-κB, Tax expression also leads to the activation of the CREB/activating transcription factor pathway, which mediates transactivation of the HTLV-1 LTR (39Li X.H. Gaynor R.B. Gene Expr. 1999; 7: 233-245Google Scholar). To determine whether the lack of IKK1 impacts on this Tax signaling pathway, we tested the ability of Tax to induce a CREB-regulated HTLV-1-LTR-Luc reporter in IKK1–/– cells. In contrast to the functional defect observed in the NF-κB pathway, Tax normally activated the CREB/activating transcription factor pathway, leading to full stimulation of the HTLV-1 LTR reporter gene in IKK1–/– cells (Fig. 2). Comparable induction of HTLV-1 LTR reporter gene expression was observed in IKK1–/– and WT MEFs. As expected, the coexpression of NIK or stimulation with TNF-α failed to activate the CREB-regulated reporter construct while inducing a robust κB enhancer-dependent response. Together, these functional studies highlight a key role for IKK1 in Tax-mediated induction of κB-dependent gene expression. Initial NF-κB Induction Events Are Intact in IKK1–/– Cells Expressing Tax—To further define this essential role of IKK1 in Tax signaling, we investigated various steps in the NF-κB signaling axis in IKK1–/– versus WT MEFs and various IKK1-expressing cell lines (Fig. 3A). Of note, Tax functioned normally during the initial stages of NF-κB activation, promoting the activation of IKK (Fig. 3B), degradation of IκBα (Fig. 3C), and nuclear translocation of RelA/p65 (Fig. 3D). These findings underscore the notion that homodimeric IKK2 complexes are fully competent to mediate the phosphorylation events associated with the initial phase of NF-κB activation (Fig. 3B). Tax expression did not induce detectable in vitro phosphorylation of IκBα with NEMO-immunoprecipitated signalsomes from IKK2–/– MEFs; however, we did observe IκBα degradation in IKK2–/– MEFs, albeit with slower kinetics than seen in either WT or IKK1–/– MEFs (Fig. 3C). In contrast, the levels of NF-κB p65 remained unchanged over the same time course in IKK2–/– MEFs. This decrease in IκBα levels likely results from IKK1-mediated phosphorylation targeting IκBα for proteasome-mediated degradation. Finally, we did not observe any defect in the binding of NF-κB to a κB DNA probe using nuclear extracts from IKK1-deficient cells expressing Tax (Fig. 3E). Interestingly, in IKK2–/– MEFs, NF-κB DNA binding was induced in response to Tax expression; however, loss of IKK2 resulted in significantly diminished NF-κB DNA binding in response to TNF-α (Fig. 3E). Together, these results demonstrate that the proximal events in the NF-κB signaling pathway occur normally in IKK1–/– cells stimulated with HTLV-1 Tax. Nonetheless, NF-κB induced by Tax in these IKK1–/– cells is functionally impaired as evidenced by its failure to activate several different κB reporter constructs (Fig. 1, A and C). Reconstitution with Kinase-proficient (but Not Kinase-deficient) IKK1 Restores Tax Induction of Biologically Active NF-κB—To determine whether the defect in NF-κB transcriptional activity in IKK1–/– MEFs induced with Tax was due solely to the absence of IKK1, we reconstituted IKK1–/– cells with WT IKK1 or kinase-deficient IKK1-K44M vector DNA (Fig. 4A). In WT IKK1-reconstituted cells, the defect in Tax signaling was repaired in the κB reporter assay as well as in the resynthesis of the NF-κB target gene IκBα (Fig. 4B). In contrast, when IKK1–/– cells were reconstituted with kinase-deficient IKK1, no increase in κB-Luc activity or IκBα resynthesis was observed. Thus, the failure of NF-κB to mediate transcription in response to Tax in IKK1–/– cells relates specifically to the loss of IKK1 catalytic activity. In addition, reconstitution of IKK1–/– MEFs with WT (but not kinase-deficient) IKK1 enhanced the binding of NF-κB to its cognate enhancer probe in a gel shift experiment (Fig. 4C). Recently, IKK1 was shown to be required for the signal-coupled induction of p100 processing to p52, a transactivation partner of RelA/p65 and RelB (40Senftleben 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: 1495-1499Google Scholar, 41Xiao G. Cvijic M.E. Fong A. Harhaj E.W. Uhlik M.T. Waterfield M. Sun S.C. EMBO J. 2001; 20: 6805-6815Google Scholar). In IKK1–/– MEFs, both Tax and NIK fail to induce the proteasome-mediated processing of p100 to p52, rendering the cells deficient in this pathway of NF-κB signaling. To investigate whether this defect could explain the transactivation defect observed in IKK1–/– cells expressing Tax, we transiently transfected IKK1–/– cells with an expression plasmid encoding p52. Despite reconstituting p52 levels in these cells, the defect in κB reporter transactivation in response to Tax was not repaired, suggesting that IKK1 mediates an additional role required for Tax induction of NF-κB action (Fig. 5A). Furthermore, examination of the nuclear complexes induced in WT MEFs expressing Tax revealed that the major species of NF-κB elicited contained p50 and RelA/p65. Thus, failure to induce p52 processing does not appear to explain the transactivation defect observed in IKK1–/– MEFs stimulated with Tax (Fig. 5B). Tax Fails to Induce RelA/p65 Phosphorylation in IKK1-deficient MEFs—The observation that IKK1 kinase activity was required for full activation of NF-κB action by Tax supports the notion that the phosphorylation of a substrate is a key component of this response. Recently, several groups identified IKK2 as a kinase capable of phosphorylating the RelA/p65 subunit of the NF-κB complex (22Okazaki T. Sakon S. Sasazuki T. Sakurai H. Doi T. Yagita H. Okumura K. Nakano H. Biochem. Biophys. Res. Commun. 2003; 300: 807-812Google Scholar, 23Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Google Scholar, 26Vermeulen L. De Wilde G. Van Damme P. Vanden Berghe W. Haegeman G. EMBO J. 2003; 22: 1313-1324Google Scholar, 27Wang D. Westerheide S.D. Hanson J.L. Baldwin Jr., A.S. J. Biol. Chem. 2000; 275: 32592-32597Google Scholar, 28Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin Jr., A.S. J. Biol. Chem. 2001; 276: 18934-18940Google Scholar, 29Jiang X. Takahashi N. Matsui N. Tetsuka T. Okamoto T. J. Biol. Chem. 2003; 278: 919-926Google Scholar, 33Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Google Scholar). Because we observed a defect in Tax signaling in IKKI–/– cells, the ability of Tax to induce the phosphorylation of the RelA/p65 subunit was assessed in an in vitro kinase assay. NEMO-containing complexes were immunoprecipitated from IKK1–/– MEFs, WT MEFs, 293 cells, or IKK2–/– MEFs transfected with Tax or stimulated with TNF-α. In both WT MEFs and 293 cells, we observed that Tax and TNF-α induced robust phosphorylation of a full-length RelA/p65 substrate in vitro. In addition, using immunoprecipitated IKK1·NEMO complexes, we observed that both Tax and TNF-α induced p65 phosphorylation in IKK2–/– MEFs. In contrast, although TNF-α induced RelA/p65 phosphorylation in IKK1-deficient MEFs, Tax did not (Fig. 6A). Of note, reconstitution of IKK1–/– MEFs with kinase-proficient (but not kinase-deficient) IKK1 restored the phosphorylation of RelA/p65 in Tax-activated cells (Fig. 6B). Thus, although IKK2 is capable of phosphorylating RelA/p65 in vitro, this activity is not sufficient to support the Tax-mediated response in IKK1–/– cells. With respect to TNF-α stimulation, we did not observe a defect in either IKK1–/– or WT MEFs, suggesting the involvement of kinase(s) other than IKK1 as catalytic responders to TNF-α. This finding is in agreement with other reports implicating IKK2, casein kinase II, and MSK1 as mediators of RelA/p65 phosphorylation (22Okazaki T. Sakon S. Sasazuki T. Sakurai H. Doi T. Yagita H. Okumura K. Nakano H. Biochem. Biophys. Res. Commun. 2003; 300: 807-812Google Scholar, 23Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Google Scholar, 26Vermeulen L. De Wilde G. Van Damme P. Vanden Berghe W. Haegeman G. 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