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Journal of Virology, May 2006, p. 4227-4241, Vol. 80, No. 9
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.9.4227-4241.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

An Alternative Splice Product of IB Kinase (IKK), IKK-, Differentially Mediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-B Activation

Department of Internal Medicine,¹ Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas 77555-1060,³ Laboratory of Molecular Microbiology, Molecular Virology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0460,² Department of Microbiology, Tokyo Medical and Dental University, School of Medicine, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan,⁴ Department of Pathology, Creighton University, Omaha, Nebraska 68131⁵

Received 28 November 2005/ Accepted 8 February 2006

	ABSTRACT

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NF-B is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IB kinase (IKK) "signalsome". The core IKK consists of two catalytic serine kinases, IKK and IKKß, and a noncatalytic subunit, IKK. IKK is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKK mRNA isoform, encoding an in-frame deletion of exon 5, termed IKK-. Using a specific reverse transcription-PCR assay, we find that IKK- is widely expressed in cultured human cells and normal human tissues. Because IKK- protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-B activity. Coimmunoprecipitation and confocal colocalization assays indicate IKK- has strong homo- and heterotypic association with wild-type (WT) IKK and, like IKK WT, associates with the IKKß kinase. Similarly, IKK- mediates IKK kinase activity and downstream NF-B-dependent transcription in response to tumor necrosis factor (TNF) and the NF-B-inducing kinase-IKK signaling pathway. Surprisingly, however, in contrast to IKK WT, IKK- is not able to mediate HTLV-1 Tax-induced NF-B-dependent transcription, even though IKK- binds and colocalizes with Tax. These observations suggest that IKK- is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKK- expression, therefore, may affect signal transduction cascades coupling to IKK.

	INTRODUCTION

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Nuclear factor-B (NF-B) is an inducible transcription factor that controls the expression of inducible inflammatory and antiapoptotic genes (49, 51). NF-B responds to a diverse series of inflammatory activators, including UV light, double-stranded RNA, cytokines, vasoactive peptides, and viral oncogenes through several distinct intracellular pathways (reviewed in reference 36). Because of its central role as an integrator of stress and inflammatory stimuli, the pathways controlling NF-B have been intensively investigated.

Currently, it is thought that NF-B activation is controlled by two distinct pathways, termed the canonical (23) and noncanonical pathways (9, 43). The canonical pathway controls nuclear translocation of the prototypical NF-B complex, composed of 65-kDa Rel A-50-kDa NF-B1 heterodimers. Under normal conditions, the Rel A · NF-B1 complex is sequestered and inactivated in the cytoplasm by the IB inhibitors, proteins which inactivate Rel A DNA binding and nuclear translocation by masking its nuclear localization sequence (reviewed in reference 1). NF-B activators induce IB phosphorylation on serine residues 32 and 36 on its NH₂-terminal regulatory domain (reviewed in reference 23). Phospho-IB is then specifically bound by the Skp1-Cullin-F-Box-type E3 ubiquitin ligase, E3RS, initiating IB ubiquitination and proteolysis through the proteasome (4, 23, 24) and calpain pathways (15). Nuclear translocated NF-B binds high-affinity chromatin sites and activates the expression of a diverse gene network (50) by inducing assembly of active promoters (2) and recruiting coactivators to target gene promoters (44).

The cytoplasmic IB kinase (IKK), also known as the "signalsome," is the rate-limiting kinase responsible for inducible IB phosphorylation (23) and is composed of core catalytic kinases and scaffolding proteins. The catalytic core contains the ubiquitous helix-loop-helix proteins IKK and IKKß (33, 54), associated with the noncatalytic regulatory protein, IKK, in a precise stoichiometric relationship of 2 catalytic subunits (either an IKKß homodimer or an IKK-IKKß heterodimer) and 2 subunits of IKK (32). In spite of significant sequence similarity between the two /ß catalytic kinase subunits, IKKß has 30-fold higher activity toward IB (17, 54) and its phosphorylation is the final common step in IKK activation (11, 33). IKK, known also as the NF-B essential modulator (NEMO) (57), the IKK-associated protein (32), and the 14.7K interacting protein (27), is essential for inducible IKK activity, as IKK-deficient cells are unable to activate the canonical NF-B pathway in response to all stimuli tested, including interleukin-1, tumor necrosis factor (TNF), phorbol myristate acetate, double-stranded RNA, or human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein (21, 40, 41, 57). IKK plays multiple roles in IKK activation through its ability to organize the assembly of IKKs into the activated high-molecular-weight complex (38, 56) and to serve as an adapter molecule to recruit upstream kinases that phosphorylate the catalytic subunits (40, 58, 59). Through these activities, IKK forms a molecular bridge between IKK, its upstream activators, and its substrate.

Consistent with its role as a signaling integrator, IKK activity is induced by several discrete mechanisms. In response to the type I cytokine TNF, IKK recruits inactive cytosolic IKK to a submembranous complex formed on the cytoplasmic effector domains of the liganded TNF receptor (59). Here, an ordered activation process is initiated by the upstream mitogen-activated protein kinase kinase kinases (MAP3Ks). The MAP3Ks that activate IKK include the NF-B-inducing kinase (NIK) (28, 30, 34), a kinase that activates IKKß in a directional manner through IKK (35), and the transforming growth factor ß-associated kinase-1. In a separate mechanism, the Tax oncogenic protein from HTLV-1 directly associates with IKK, resulting in Tax recruitment into the IKK signalsome and kinase activation (21). Together, these observations indicate that oligomerization and MAP3K-induced sequential IKK/ß phosphorylation are important processes regulating IKK activity.

In this study, we have identified a 43-kDa IKK alternate-splice product that results in an in-frame deletion of exon 5, encoding a protein that we term IKK-, that is widely expressed all cell types examined. IKK- strongly associates with IKK wild type (WT) in coimmunoprecipitation assays; reasoning that enhanced self-association could influence its response to IKK inducing signals, we explored whether there were functional differences between these two IKK isoforms. Experiments involving coexpression of the catalytic kinases IKK/ß and the MAP3K NIK indicate that IKK- efficiently mediates IKK and NF-B activation. In striking contrast, IKK- is unable to mediate Tax-inducible IKK activation, even though it associates with Tax. These findings suggest that IKK- is a functionally distinct alternate-splice product, which is HTLV Tax resistant and unable to form productive Tax-IKK complexes with cellular proteins.

	MATERIALS AND METHODS

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Cell culture, treatment, and transfections. Human HepG2, HeLa S3, A549, K562, U937, and Hep3B cells were obtained from ATCC and cultured as recommended by the supplier (ATCC, Rockville, MD). mRNA was isolated from normal human tissues collected from discarded surgical specimens in accordance with UTMB IRB-approved protocol. Human CD4⁺ lymphocytes were isolated from Ficoll gradient-purified peripheral mononuclear cells using commercial anti-CD4⁺ magnetic beads (Miltenyi Biotech). Tax-transformed, IKK-deficient 5R cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, and 15% filtered conditioned medium (57). 8321 Jurkat T-cell lines were cultured in Iscove's modified Dulbecco's medium, supplemented with 10% FBS (20% FBS for the stables), 50 µM ß-mercaptoethanol, and 15 µg/ml of gentamicin (16). IKK-deficient mouse embryonic fibroblasts (E8i cells) were cultured as described previously (42). Cells were transiently transfected using Lipofectamine (Invitrogen) into triplicate 60-mm plates with indicated plasmids. The total amount of DNA was kept constant by inclusion of empty vector pcDNA3. After 48 h, cells were stimulated with TNF- (30 ng/ml, 6 h) and harvested for the measurement of reporter activity. Relative induction was calculated by dividing normalized reporter treatment values by those of the control.

Plasmid construction. The NF-B-LUC reporter consists of the trimerized angiotensinogen NF-B sequences driving the expression of the firefly luciferase reporter gene (19). The bacterial expression vector encoding glutathione S-transferase (GST)-IB (1 to 51) was constructed by PCR amplification of the human IB cDNA using the upstream primer 5'-GTG ATA GGA ATT CTC CAG GCG GCC GAG CGC CCC-3', and the downstream primer 5'-ACC TAA GCT TCT AGA GGC GGA TCT CC TGC AGC-3'to incorporate EcoRI and HindIII restriction sites (underlined). IB (1 to 51) was restricted with EcoRI and HindIII and cloned into pGEX-KG restricted with the same sites (12). The eukaryotic expression vector pcDNA3-FLAG containing an N-terminal FLAG epitope downstream of a strong Kozak initiation sequence was produced by ligating a duplex oligonucleotide, 5'-AGC TCG TCT ACC ATG GAC TAC AAA GAC GAT GAC GAT AAG GGA TCC AAG GAA AAG CTT GAT ATC GATC-3' encoding the initiator methionine (underlined) upstream of the FLAG epitope and unique downstream restriction sites into the Hind III/XbaI-digested pcDNA3 vector (Invitrogen). pcDNA-FLAG-IKK (WT) and pcDNA-FLAG-IKK were constructed by PCR using the upstream primer 5'-TAA GGGA TCC ATG AAT AGG CAC CTC TTG GAA GAG CC-3' and the downstream primer 5'-ATA TCAA GCT TCT ACT CAA TGC ACT CCA TGA CAT GTA TCT GC-3' to amplify the IKK and IKK- cDNAs and incorporate BamHI and HindIII restriction sites (underlined) flanking the initiation and stop codons (bold), respectively. The cDNAs were digested with BamHI/HindIII, purified, and cloned into pcDNA3-FLAG. To construct pEF6-FLAG-IKK-, the T7 and SP6 primers were used to PCR amplify pcDNA-FLAG-IKK-. The purified PCR product was then TA cloned into the pEF6/V5-His-TOPO vector (Invitrogen). Plasmids were purified by ion-exchange chromatography prior to transfection; all constructions were confirmed by automated sequencing. The eukaryotic expression vectors pRK-IKK, pRK-IKKß, pRK-IKKß (K44A), pRKmyc-NIK, and pRKmyc-NIK (T559A) were gifts of D. Goeddel, Tularik, South San Francisco, CA (54). pCMV-Tax was a gift of Warner Greene, The Gladstone Foundation, San Francisco, CA (46). The hemagglutinin (HA)-tagged IKK WT expression vector was previously described (21).

Stable transfectants. For HeLa stable transfectants, cells were transfected with 20 µg of pcDNA-FLAG-IKK or pcDNA-FLAG-IKK expression plasmid and selected for antibiotic resistance to G418 (400 µg/ml). For 5R cell stable transfectants, 5R cells were transfected with 20 µg of pEF6-IKK expression plasmid DNA and selected for antibiotic resistance in the presence of 10 µg/ml blasticidin S (Invitrogen Corp., San Diego, CA). 8321 is a CD3⁺ derivative of Jurkat T-cell clone 3T8 generated by ICR191 mutagenesis and subsequent negative enrichment for cells that do not respond to phorbol myristate acetate that also lack functional IKK expression (16). To make 8321 cells stably expressing either IKK or IKK- stable cell line, 8321 cells were transfected with pEF6-FLAG-IKK and pEF6-FLAG-IKK- and selected for blasticidin S (12 µg/ml) resistance. Transfectants were cloned and identified by screening with Western immunoblots using horseradish peroxidase-conjugated anti-FLAG antibody.

Reverse transcription (RT)-PCR cloning of IKK. Total RNA was isolated using RNAqueous (Ambion, Austin, TX). First-strand cDNA synthesis was performed using 5 µg HeLa S3 total RNA, 0.5 µg oligo(dT)_12-18, and 50 units of SuperScript^II reverse transcriptase (Invitrogen) in a final volume of 20 µl of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 5 mM MgCl₂, 10 mM dithiothreitol, and 0.5 mM concentrations of each deoxynucleoside triphosphate. PCR amplification of IKK cDNA was performed using 100 pmol of each primer (5'-ATGAATAGGCACCTCTGGAAGAGC-3', 5'-CTACTCAATGCACTCCATGACATG-3') in a final volume of 100 µl of Tris-HCl, pH 8.4, 1.5 mM MgCl₂, 50 mM KCl, 0.2 mM concentrations of deoxynucleoside triphosphates, and 2.5 U of AmpliTaq polymerase (Applied Biosystems). After an initial denaturation at 95°C for 2 min, the reaction mixture was subjected to 40 cycles of the following program: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and primer extension for 8 min at 72°C. Two amplified DNAs (1,107 bp and 1,260 bp) were purified by polyacrylamide gel electrophoresis and TA cloned into pCRII (Invitrogen). Nucleotide sequences for representative clones were determined using fluorescent tagged terminator cycle sequencing and analyzed on an Applied Biosystems model 310 Genetic Analyzer.

IKK exon 5 assay. Normal human tissue was obtained from the UTMB Tumor Bank representing discarded material from surgical specimens obtained with our IRB-approved protocol. Total RNA was isolated using Totally RNA (Ambion). First-strand cDNA synthesis was performed using 5 µg of total RNA as described above. PCR amplification was performed using Fail Safe buffer D (Epicenter) and 45 pmol of each primer (5'-AGCCCAGGTGACGTCCTTGCTC-3', 5'-CTTCAGCTTATCGATCACCTCCTG-3'). After denaturation at 95°C for 2 min, the reaction mixtures were incubated for 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 4 min. The last primer extension was continued for 10 min at 72°C to ensure completion of DNA synthesis. Amplified DNA (wild type, 427 bp; exon 5 deletion, 274 bp) was analyzed by electrophoresis on polyacrylamide gels. Primers for detection of human GAPDH (5'-GTCATCCATGACAACTTTGGTATCG-3', 5'-CAGGTTTTTCTAGACGGCAGGTC-3'), and human polymerase beta (5'-CGGGGGAATCACCGACATGCTC-3', 5'-TCCAGTTTACGTAATTTTCCAGTTGC-3') were included as positive controls. The respective amplified target DNAs were 269 bp for GAPDH and 222 bp (wild type) and 166 bp (exon 2 deletion) for polymerase beta (7).

2D electrophoresis (2DE). Isoelectric focusing (IEF) was performed with 11-cm precast IPG strips (pH 3 to 10, or 5 to 8 as indicated; Bio-Rad). Two-hundred-microliter aliquots of protein were loaded onto an IPG strip and allowed to rehydrate overnight. IEF was performed at 20°C with the following parameters: 50 V, 11 h; 250 V, 1 h; 500 V, 1 h; 1,000 V, 1 h; 8,000 V, 2 h; 8,000 V, 6 h. After IEF, the IPG strip was stored at –80°C until the two-dimensional (2D) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). For the 2D SDS-PAGE, the IPG strips were incubated in 4 ml of equilibration buffer (6 M urea, 2% SDS, 50 mM Tris-HCl, pH 8.8, 20% Glycerol) plus 10 µl/ml Tris(2-carboxyethyl) phosphine for 15 min at 22°C with shaking. The samples were then incubated in equilibration buffer with 25 mg/ml iodoacetamide for 15 min at 22°C with shaking. Electrophoresis was performed at 150 V for 2.25 h at 4°C with precast 8 to 16% polyacrylamide gels in Tris-glycine buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes and used for Western immunoblots.

Preparation of subcellular extracts. S100 cytosolic and particulate fractions were prepared as previously described (14, 15). In brief, cells were incubated in hypotonic buffer (20 mM HEPES, pH 7.4, 10 mM potassium acetate, 1.5 mM magnesium acetate) for 5 min on ice. Lysis was completed in a Dounce homogenizer and verified by microscopic examination. Nuclei and unbroken cells were removed by low-speed centrifugation. The low-speed supernatants, containing cytoplasm and membrane proteins were centrifuged at 100,000 x g for 1 h at 4°C in a Beckman SW 55Ti rotor. The resultant S100 supernatants were taken as cytosolic extract, and pellets (the particulate) were resuspended in hypotonic buffer with 1% (vol/vol) IGEPAL. Assay of enrichment of cytoplasmic and nuclear markers is shown in Results (see Fig. 6A). All extracts (cytosol, particulate, cytoplasmic, and nuclear extracts) were normalized for protein amounts determined by Coomassie G-250 staining (Bio-Rad, Hercules, CA).

View larger version (24K): [in this window] [in a new window]   FIG. 6. IKK- mediates TNF-induced target gene expression. (A) IKK expression in IKK-deficient 8321 cells. 8321 cells stably transfected with empty vector (8321EMPTY), IKK WT (8321IKK-WT), or IKK- (8321IKK-) were isolated, and IKK expression was confirmed by Western blotting. Top panel, Western blot using anti-FLAG (-FLAG); Middle panel, Western blot using anti-IKK (-IKK); bottom panel, Western blot using anti-ß-actin (-ßActin). 8321EMPTY cells are deficient in IKK expression, whereas 8321IKK-WT and 8321IKK- express similar amounts of epitope-tagged IKK isoform. (B) Northern blot hybridization of NF-B-dependent gene expression in IKK-complemented 8321 cells. 8321EMPTY, 8321IKK-WT, or 8321IKK- cells were stimulated with TNF (20 ng/ml, T) for 1.5 h. Graph represents hybridization intensities quantitated by exposure to PhosphorImager cassette where IB intensity normalized to that of thymosin ß is plotted. Inset, autoradiogram of Northern blot hybridization to IB probe (IB) or Thymosin ß (Thy). The experiment was repeated twice with similar results. C, control; ––, absent. (C) HeLa cells stably expressing IKK WT or IKK- were TNF stimulated as in Fig. 5B. Shown is Northern blot hybridization to IB probe (top) or thymosin B (bottom). IB mRNA is induced to a greater degree in HeLa IKK--expressing cells.

Northern blots. Total cellular RNA was extracted by acid guanidium-phenol extraction (Tri Reagent; Sigma). RNA (20 µg) was denatured, fractionated by electrophoresis on a 1.2% agarose-formaldehyde gel, capillary transferred to a nitrocellulose membrane (Zeta-ProbeGT; Bio-Rad), and prehybridized as described previously (48). The IB and thymosin B probes were produced by asymmetric PCR with plasmid templates as previously described (14, 60). The probes were purified by size exclusion chromatography on MicroSpin TMG-25 columns (Amersham Biosciences). The membrane was hybridized with 1 x 10⁶ to 2 x 10⁶ cpm/ml probe at 60°C overnight in 5% SDS hybridization buffer (50). The membrane was washed with a buffer containing 5% SDS and 1x saline-sodium citrate (0.15 M NaCl and 0.015 M sodium citrate) for 20 min at room temperature followed by 30 min at 60°C. The membrane was exposed to BioMax film and quantified by exposure to a PhosphorImager cassette.

Confocal colocalization microscopy. Cells cultured on 25-mm coverslips (Thomas Scientific) were transfected with 0.5 µg of plasmid DNA. Twenty-four hours later, cells were fixed with 3.7% formaldehyde and then permeabilized with phosphate-buffered saline containing 0.1% Triton X-100. Where indicated, cells were stained with monoclonal anti-HA, anti-FLAG antibodies (Abs), followed with anti-mouse Alexa Fluor 594 or Alexa Fluor 488 (Molecular Probes). Subcellular localization of green fluorescent protein (GFP)-Tax was revealed by direct laser excitation. Coverslips were mounted onto glass slides with VECTASHIELD mounting medium with 4',6'-diamidino-2-phenylindole (DAPI) (Roche) and examined with a Zeiss Axiovert 135 laser-scanning microscope.

	RESULTS

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IKK- encodes a deletion in the NH₂-terminal coiled-coil domain and is widely expressed. IKK is essential for inducible regulation of the IKK complex through its ability to induce oligomeric association of the catalytic IKK and ß subunits and couple them to upstream activators. IKK cDNA sequences independently cloned by three groups identified the predicted amino acid sequence to be of 419 amino acids (aa) encoded by an open reading frame of 1,257 bp (32, 40, 57). We therefore were surprised that RT-PCR using primers spanning exons 2 to 10 encoding the IKK open reading frame produced two bands under a variety of stringent conditions: one corresponding to the predicted 1.26-kb size and another of 1.1 kb. Multiple independent clones of the smaller product were sequenced and found to contain a 153-nucleotide (nt) "in-frame" deletion exactly spanning exon 5 of the human IKK gene (45), encoding a protein we term IKK-. Exon 5 encodes amino acid residues 174 to 224 that form a COOH-terminal portion of the central IKK coiled-coil domain, a secondary structure motif responsible for protein-protein association, including its self-association (5, 29, 58).

To determine whether the IKK- expression was unique to HeLa, we next examined its expression patterns in cultured cells and normal human tissues. First, Western immunoblots were performed on cytoplasmic lysates of cultured cells to determine the distribution of IKK- expression and the relative steady-state abundances of the isoforms. Because no antibody will uniquely identify IKK- without also recognizing IKK WT, the abundance of IKK - was determined by its unique migration in one-dimensional SDS-PAGE and 2DE (Fig. 1A, B, respectively). In HepG2 cells, two isoforms of IKK were identified, each comigrating with a respective transiently expressed epitope-tagged standard of 48 and 43 kDa (Fig. 1A, top panel). From this experiment, we estimated that IKK- was expressed at a 1:4 ratio with the IKK WT isoform in HepG2 cells. Moreover, in K562, HeLa, A549, and U937 cells, the 43-kDa IKK- isoform could be detected at various ratios with IKK WT, from <1:4 in A549 cells to 1:2 in U937 cells (Fig. 1A, lower panel). To more definitively separate the IKK isoforms, Western immunoblots of cytoplasmic proteins were performed after 2DE. Consistent with its multiple posttranslational modifications (6, 39, 47), IKK WT fractionated into three distinct isoforms based on pI (Fig. 1B). Interestingly, the IKK- isoform focused as a single spot at a distinctly higher pI than the IKK WT, suggesting that it lacked posttranslational modifications similar to IKK WT (even though the identified IKK phosphoacceptor sites are outside the occluded exon 5).

View larger version (29K): [in this window] [in a new window]   FIG. 1. IKK- protein expression. (A) Western immunoblot. Cytoplasmic lysates were fractionated by one-dimensional SDS-PAGE and blotted onto PVDF membranes. Epitope-tagged IKK WT and IKK- stably expressed in HeLa cells serve as markers and were identified by anti-FLAG in a Western blot (left panel). Two distinct IKK isoforms are identified in HepG2 cells that comigrate with the 48-kDa IKK WT and 43-kDa IKK- standards (right). The blot was reprobed with ß-actin as a loading control. Bottom panel, cytoplasmic lysates from K562, A549, and U937 cells were blotted with anti-IKK. The relative migration of IKK WT and IKK- are indicated. (B) Western immunoblot in 2DE. Cytoplasmic lysates were subjected to isoelectric focusing over the pH range 5 to 8 and fractionated by 12 to 20% gradient SDS-PAGE in the second dimension. The proteins were blotted onto PVDF membranes and probed with anti-IKK antibody. The relative migration of IKK WT and IKK- was determined in HeLa cell stable transfectants (data not shown). IKK- focuses as a single spot at a more basic pI in a 1:4 abundance ratio with the IKK WT isoforms.

View larger version (70K): [in this window] [in a new window]   FIG. 2. IKK- mRNA expression. (A) IKK RT-PCR assay. Ethidium bromide staining of RT-PCR products fractionated by PAGE. Top panel, RT-PCR assay of various cellular RNA with IKK exon 4 and 6 primers. Bottom panel, housekeeping mRNA controls amplified from same source, GAPDH and DNA ß-polymerase (ß-Pol) are indicated. Lane 1, A549 human alveolar carcinoma cells; lane 2, human erythroleukemia (HL-60 cells) expressing Bcr-Abl; lane 3, human hepatocarcinoma Hep3B; lane 4, K562 erythroleukemia; lane 5, HL-60; lane 6, HeLa S3. (B) IKK expression in normal human tissues. Ethidium bromide staining of RT-PCR products fractionated by PAGE. Top panel, PCR products using IKK primers as in Fig. 2B. Bottom panel, PCR products for GAPDH and ß-Pol. Lane 1, breast; lane 2, cervix; lane 3, kidney; lane 4, liver; lane 5, testicle; lane 6, adrenal gland; lane 7, colon; lane 8, lung; lane 9, pancreas.

Effect of exon 5 exclusion on IKK heterotypic association.

View larger version (35K): [in this window] [in a new window]   FIG. 3. IKK- is heterotypic association competent. (A) Enhanced IKK- heterotypic association. Eukaryotic expression vectors encoding FLAG epitope-tagged IKK WT were cotransfected into HepG2 cells with either Myc-tagged IKK WT or Myc-IKK-. Forty-eight hours later, cells were lysed and fractionated by SDS-PAGE for Western immunoblotting (IB) with anti-Myc (-Myc) antibody (top panel). Cytosolic extracts were then immunoprecipitated (IP) with anti-Myc. Immune complexes were then fractionated and association with FLAG-IKK WT was determined by Western immunoblotting with anti-FLAG (-FLAG) antibody (bottom panel). Although FLAG-IKK WT was detected in both lanes, IKK- immunoprecipitates contained a greater abundance of FLAG-IKK WT. (B) IKK- heterotypic association in IKK-deficient background. E8i cells were transfected with HA-tagged IKK WT or FLAG-IKK- as indicated. Lanes 1 to 3, lysates; lanes 4 to 6, IP with anti-HA Ab. Shown is a Western blot (WB) using anti-IKK Ab. ++, present;––, absent. (C) Confocal colocalization microscopy. Top, FLAG-IKK- and HA-IKK WT were cotransfected into E8i cells cultured on coverslips. Cells were stained with monoclonal anti-HA or polyclonal anti-FLAG Abs, followed by Alexa Fluor 594 anti-mouse Ab and Alexa Fluor 488 anti-rabbit Ab. Bottom, FLAG-IKK- and HA-IKK WT were cotransfected into HeLa cells and stained as described above. Merged image (yellow in panels a and d) shows cytoplasmic colocalization.

To exclude potential artifacts induced by biochemical fractionation, confocal colocalization experiments were performed using HA-tagged IKK WT and FLAG-IKK-. Expression vectors encoding HA-IKK WT and FLAG-IKK- were cotransfected into IKK^–/–-deficient E8i cells (42). Cells were stained with monoclonal anti-HA or rabbit anti-FLAG Abs followed by Alexa Fluor 594 anti-mouse and Alexa Fluor 488 anti-rabbit Abs, producing red and green fluorescence, respectively. Consistent with previous studies, IKK WT was primarily distributed in a cytoplasmic distribution, with some nuclear staining (53). The IKK- distribution was similar, being primarily cytoplasmic, with an apparently greater fraction distributed in the nucleus. The merged image shows strong cytoplasmic colocalization (Fig. 3C). Similar results were found in HeLa cells (Fig. 3C, bottom panel). Together, these data indicate that IKK- is capable of homo- and heterotypic association in a cell type-independent manner.

IKK- partitions into the nucleus and membrane compartment. Previously, it has been shown that IKK is a dynamic molecule undergoing cytoplasmic-nuclear translocation (52). Inspection of our confocal microscopy experiments indicated that IKK- apparently distributed into the nuclear compartment to a greater degree than IKK WT (Fig. 3C). To confirm this finding, and exclude artifactual distribution produced by transient overexpression, stable transfectants of epitope-tagged IKK WT and IKK- in HeLa cells were generated. The cells were TNF stimulated and fractionated into cytoplasmic (100,000 x g supernatant), membrane-enriched particulate (100,000 x g pellet), or sucrose cushion-purified nuclear fractions using well-established protocols (14, 15). These subcellular fractions were first assayed for the presence of specific cytosolic (IB) membrane (ß-catenin) and nuclear (lamin B) markers by Western immunoblotting. ß-Catenin was chosen as a specific membrane marker because this protein forms intercellular adhesion complexes with E-cadherin in cell membranes (37). As seen in Fig. 4A, anti-ß-catenin strongly stained the particulate fraction but not the cytoplasmic or nuclear subcellular fractions. Conversely, the cytoplasmic marker (IB) selectively stained the cytoplasmic compartment, and the nuclear marker (ß-lamin) stained the nuclear fraction (Fig. 4A). Together, these observations indicate that the subcellular fractions were enriched in their respective marker proteins and could be used to determine relative partitioning of the IKK isoforms.

View larger version (75K): [in this window] [in a new window]   FIG. 4. IKK- partitions into nuclear and membrane compartments. (A) Characterization of subcellular fractionation. Unstimulated HeLa cells were fractionated into cytosolic (100,000 x g supernatant) (Cyto), membrane (100,000 x g pellet) (Partic.), and nuclear (Nuc) fractions as previously described (14). The abundance of specific cytoplasmic (IB), membrane (cytokeratin), and nuclear (lamin B) markers were determined by Western blotting. For example, the membrane fraction stains selectively with cytokeratins, not IBa or lamin B, indicating that it is relatively free of detectable cytoplasmic or nuclear contamination. (B) Partitioning of IKK -. Stably transfected HeLa cells expressing pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- were isolated, stimulated with TNF for indicated times (top, in min) and fractionated into cytosolic, particulate (membrane), and nuclear fractions. Shown is a Western immunoblot of equivalent cell amounts using anti-FLAG antibody. Top panel, IKK--expressing cells. Middle panel, IKK WT-expressing cells. Bottom panel, Rel B as a protein loading control. Rel B equally distributes into the cytoplasmic, membrane, and nuclear compartments (15). Relative to IKK WT, IKK- has a greater distribution into the particulate fraction and shows distinct nuclear redistribution after TNF stimulation.

IKK- efficiently couples with the core signalsome kinase. Previous studies have shown that IKK forms complexes with IKKß, the major catalytic activity in the IKK responsible for IB phosphorylation (26, 38, 40). We next determined whether there were differences in the functional interaction of IKK WT and IKK- with IKKß. IKK-deficient 5R cells were transfected with NF-B-LUC in the presence of either pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- and increasing amounts of IKKß expression vector. In the presence of IKK WT, cotransfection of IKKß increased reporter activity 2.3-fold at the lowest amount of IKKß, which was maintained over a broad concentration range (Fig. 5A). Note that approximately equivalent expression levels of the FLAG-IKK WT and FLAG-IKK- proteins are produced by these expression vectors (cf. Fig. 2A). In contrast, in the presence of IKK-, IKKß increased reporter activity 5-fold relative to its control value, with values consistently above those produced by IKK WT at any dose. These data suggested that IKK- efficiently couples with IKKß kinase to activate NF-B-dependent transcription.

View larger version (37K): [in this window] [in a new window]   FIG. 5. IKK- couples with IKKß catalytic kinases. (A) IKK - potentiates the effect of IKKß. 5R cells were transfected with NF-B-LUC in the presence of either pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- and increasing amounts of IKKß expression vector. For each transfection condition, normalized reporter activity was converted to relative activation over the control. Open boxes, no IKKß; shaded boxes, IKKß expression vector at the indicated doses. Results were repeated a minimum of three times. Although the relative activation is shown, similar findings were obtained for the raw luciferase values, where IKK- induced greater absolute amounts of NF-B-LUC activity for all concentrations of IKKß (not shown). *, P < 0.05, two-tailed t test. (B) IP-kinase assays of IKKß-IKK isoform complexes. Top panel, autoradiogram of IP-kinase assay. Cytoplasmic lysates of 5R cells transiently transfected with pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- in the absence (––) or presence (++) of IKKß were immunoprecipitated with anti-FLAG antibodies and used to phosphorylate GST-IB (1 to 51) in vitro. Radiolabeled GST-IB (1 to 51) was fractionated by SDS-PAGE and exposed for autoradiography. A doublet is seen due to NH2-terminal proteolysis of the GST molecule during preparation. Middle panel, anti-FLAG Western immunoblot (WB). Immunoprecipitates were subjected to Western immunoblotting to control for IKK recovery in the immunoprecipitation. Relative location of IKK isoforms are indicated at the right. Bottom panel, anti-IKKß Western immunoblot. Indistinguishable amounts of IKKß were detected in the immunoprecipitates, indicating that the kinase activity was increased. (C) IKK- potentiates the effect of IKKß in the HepG2 background. HepG2 cells were transfected with NF-B-LUC in the presence of either pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- and increasing amounts of IKKß expression vector as in Fig. 5A. *, P < 0.05, two-tailed t test. (D) IKK IP-kinase assay. Top panel, autoradiogram of IP-kinase assay. Cytoplasmic lysates of HepG2 cells transiently transfected with IKKß in the presence of either pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- were assayed for IKK activity by IP-kinase as described for Fig. 5B. Bottom panel, the blots were probed with anti-FLAG. con, control.

The composition of the IKK varies in a cell-type specific fashion where different abundances of IKK/ß are found (32). To confirm that the different behavior of the widely distributed IKK- was a general phenomenon, not restricted to the Tax-transformed 5R cells, we compared their activities in the well-studied HepG2 cells that exhibit highly TNF-inducible NF-B activation (3, 13, 14). However, in response to IKKß, IKK- mediated highly inducible NF-B transcription in a similar manner as that seen in the 5R cells (Fig. 5C). For example, 1 µg of pcDNA-FLAG-IKK- produced 28-fold activation of NF-B-dependent luciferase activity, whereas the same amount of pcDNA-FLAG-IKK-WT induced only a 5-fold activation (Fig. 5C). IP-kinase assays confirmed enhanced IKKß kinase activity in cells transfected with IKK- over that produced by IKK WT in both untreated and TNF--stimulated condition (Fig. 5D). Together, these findings indicate that IKK- effectively couples with IKKß, mediating both kinase activity and NF-B activation.

IKK- efficiently couples TNF stimulation to endogenous NF-B gene expression. The 8321 cell, a clonal derivative of 3T8 Jurkat cells, was generated by ICR191 mutagenesis and lacks functional IKK expression (16). To determine the signal transducing properties of IKK WT and IKK -, stably transfected 8321^IKK-WT and 8321^IKK- cells were isolated. The expression of the respective FLAG-IKK isoform was confirmed by Western blotting, where the FLAG- and IKK-reactive species exactly comigrated (Fig. 6A). The relative ability of the two IKK isoforms to mediate inducible NF-B activity was determined by Northern blot analysis of endogenous IB mRNA transcripts, a well-established NF-B-dependent gene (48, 49). Compared to 8321^EMPTY cells, we noted that basal IB expression was slightly (2-fold) higher in both 8321^IKK-WT and 8321^IKK- transfectants. Upon TNF stimulation of the 8321^EMPTY cells, cells that lack NF-B signaling, a rapid loss of both IB and internal control RNA transcripts was seen due to TNF's proapoptotic activity (Fig. 6B) (16). In cells stably expressing the IKK isoforms, TNF induced IB expression more strongly in 8321^IKK- cells than 8321^IKK-WT and 8321^EMPTY cells. In stably transfected HeLa cells expressing IKK WT and IKK -, TNF-inducible IB mRNA was also seen (Fig. 6C). Together, these data indicate that IKK - efficiently couples the TNF signaling pathway and the catalytic kinase, IKKß, to NF-B activation in a cell type-independent manner.

IKK- efficiently couples IKK and NIK pathways to NF-B activation. In certain cell types, the IKK signaling pathway to canonical NF-B activation is distinct from that of IKKß (25). To determine whether IKK- functionally coupled to the IKK signaling pathway, 5R cells were transfected with either IKK WT or IKK - in the presence of increasing concentrations of IKK (Fig. 7A). In the presence of IKK WT, IKK expression induced a maximum of two- to threefold activation in reporter activity. By contrast, in the presence of 0.5 µg (and greater) of IKK- expression vector, IKK induced a significantly increased five to sixfold increase in NF-B dependent reporter activity. More dramatic findings were observed in the HepG2 cellular background, where IKK produced a 38-fold activation in the presence of 2 µg transfected IKK- relative to a 12-fold activation in the presence of IKK WT (Fig. 7B). Together, these data suggest that the IKK- also couples IKK to IKK activation more efficiently than does IKK WT.

View larger version (24K): [in this window] [in a new window]   FIG. 7. IKK- couples with upstream IKK and NIK kinases. (A) IKK- efficiently couples with IKK. 5R cells were transfected with NF-B-LUC in the presence of either pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- and increasing amounts of IKK expression vector. Reporter activity and data analysis were as described for Fig. 5A. White bar, no IKK; black bar, IKK expression vector at the indicated amounts (bottom). *, P < 0.05, two-tailed t test. (B) IKK- couples with IKK in the HepG2 background. HepG2 cells were transfected with either pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- and IKK expression vector as described for Fig. 6A. (C) IKK - recruits IKK/ß into the membrane. Nondenaturing coimmunoprecipitation assays were performed with FLAG-IKK WT- and IKK--expressing cells. Membrane fractions were isolated and immunoprecipitated (IP) with anti-FLAG antibodies. Top panel, Western immunoblot (WB) of immune complexes probed with anti-IKKß and IKK antibodies. The relative migration of IKKß and IKK is indicated at right. Bottom panel, the immunoprecipitates were probed with anti-FLAG antibody. Equivalent amounts of IKK WT and IKK- are present in the immunoprecipitates. (D) IKK- mediates NIK-dependent NF-B activation in 5R. 5R cells were transfected with NF-B-LUC, pcDNA-FLAG-IKK-WT, or pcDNA-FLAG-IKK- and increasing amounts of NIK expression vector (amounts shown at bottom). NIK preferentially induces NF-B transcription in the presence of IKK-. (E) IKK- mediates NIK-dependent NF-B activation in the HepG2 background. HepG2 cells were transfected as described for Fig. 6C. At any concentration of NIK, NF-B transcriptional activity is greater in the presence of IKK- than that seen with IKK WT. +, present; ––, absent.

IKK- efficiently couples with NIK. NIK/MEKK14, a member of the MAP3K family, has been reported to complex with the TNF receptor-associated factor 2, an early step in the IKK activation pathway downstream of the TNF and interleukin-1 receptors (30). To determine if IKK--containing IKK complexes mediated NIK-inducible NF-B activation, a eukaryotic NIK expression vector was transiently transfected into 5R cells in the presence of saturating concentrations of IKK WT and IKK-. In the presence of IKK WT, NIK activated NF-B-dependent reporter activity only 1.3-fold, whereas in the presence of IKK-, NIK potently activated NF-B-LUC activity by 13-fold, significantly higher than IKK WT at all concentrations tested (Fig. 7D). These findings were replicated in the HepG2 background to exclude cell type-specific effects on NIK activation, where similar differences between IKK- and IKK WT were found (Fig. 7E).

IKK- is unable to mediate IKK activity induced by HTLV-1 Tax. HTLV-1 Tax is a potent inducer of NF-B signaling and affects many steps in its activation pathway. Of these, IKK-mediated recruitment to the IKK complex plays an important role (36). Because earlier studies indicated that IKK amino acid residues 196 to 419, overlapping the region of the alternatively spliced exon 5, functioned as a dominant-negative inhibitor of Tax-induced IKK activity (18), we sought to determine the functional ability of IKK- in mediating Tax-dependent transcription. In this first experiment, Tax-transformed 5R cells were cotransfected with NF-B-LUC in the presence of increasing concentrations of pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- expression plasmids. As shown in Fig. 8A, consistent with earlier studies, a low level of pcDNA-FLAG-IKK-WT was a potent activator of NF-B-dependent reporter activity, producing a 17-fold induction of reporter activity (in presence of 10 ng of expression vector). At higher concentrations, reporter activity fell, a previously described phenomenon probably due to disruption of the precise stoichiometry of the IKK complex (57). Surprisingly, transfection of pcDNA-FLAG-IKK- produced a distinct response, being unable to mediate NF-B-dependent transcriptional activity, producing only twofold induction of NF-B-dependent reporter activity at 100 and 500 ng of transfected expression vector and returning to control values at 1 µg (Fig. 8A). These data indicated that, in contrast to cytokine-induced IKK activation, IKK- did not mediate Tax-inducible NF-B activity.

View larger version (27K): [in this window] [in a new window]   FIG. 8. IKK- is unable to mediate HTLV-1 Tax-induced IKK activity. (A) IKK alternate-splice forms differentially mediate Tax-induced IKK activation. Triplicate plates of Tax-transformed 5R cells were transfected with NF-B-LUC in the presence of increasing concentrations of pcDNA-FLAG-IKK-WT and pcDNA-FLAG-IKK-. The total amount of DNA was kept constant with empty expression vector. Normalized luciferase activity was calculated for each triplicate result and plotted. pcDNA-FLAG-IKK-WT activated NF-B-LUC activity 4-fold (5 ng), 17-fold (10 ng), 12-fold (100 ng), 3-fold (500 ng), and 1-fold (1000 ng). pcDNA-FLAG-IKK- activated NF-B-LUC activity 1-fold (5 ng), 0.8-fold (10 ng), 2-fold (100 ng), 2-fold (500 ng), and 1-fold (1,000 ng). The experiment was repeated three times with similar results. *, P < 0.05, two-tailed t test. (B) IKK WT, but not IKK-, mediates Tax-inducible IKK activation. Triplicate plates of E8i cells were transfected with NF-B-LUC in the presence of increasing amounts of either pcDNA-FLAG-IKK-WT or pcDNA-FLAG-IKK- and pCMV-Tax. Empty plasmid was used to maintain identical DNA concentrations. Normalized reporter activity was expressed as change relative to plates transfected with empty Tax expression vector (pcDNA). (C) Subcellular localization of IKK isoforms and Tax. Transfected E8i cells were fixed, permeabilized, and stained with either monoclonal anti-HA (HA-IKK [a, b]) or monoclonal anti-FLAG (FLAG-IKK- [d, e]) Ab followed with anti-mouse conjugated to Alexa Fluor 594 (red) (Molecular Probes). The subcellular localization of GFP-Tax (g, h) was directly revealed by laser excitation of its GFP tag (green). Nuclei were counterstained with DAPI (c, f, i) (blue). (D) Colocalization of IKK- and Tax. E8i cells were transfected with of HA-IKK (a, b) or FLAG-IKK- (d, e) (stained in red as described for panel A) with GFP-Tax (c, f) (green) resulted in trans-localization of GFP-Tax from nucleus to cytoplasm. Arrows indicate cytoplasmic colocalization of GFP-Tax and IKK or IKK-. (E) Coassociation of IKK- and Tax. HeLa cells were transfected with pCMV-Tax or pcDNA-FLAG-IKK- as indicated. Whole-cell lysates were immunoprecipitated with anti-FLAG Ab and immunoblotted (IB) with anti-Tax. Migration of Tax is shown at right. Tax is captured by FLAG Ab only in lysates cotransfected with IKK-. Bottom panel, lysates are immunoblotted with anti-Tax. +, present; –, absent; ––, absent.

IKK-