INTRODUCTION

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The NF-B/Rel family of mammalian transcription factors represents a focal point for understanding how extracellular signals induce the expression of specific genes, which are involved in processes as diverse as cell division, inflammation, and apoptosis (programmed cell death) (3-5, 18, 31, 47, 49, 50). The Rel protein family can be classified into two structurally related groups. The first consists of p50 and p52, the products of the NF-B1 and NF-B2 genes, respectively (48). These proteins contain a 300-amino-acid sequence known as the Rel homology domain, which contains the information required for dimerization, nuclear translocation, and DNA binding (2, 42). The second group of Rel proteins includes RelA (p65), RelB, and c-Rel (the cellular homologue to the product of the v-Rel oncogene, isolated from the reticuloendotheliosis virus) (18). In addition to a Rel homology domain, these proteins have a transcriptional transactivation domain and form homo- and heterodimers with p50 and p52. The most common form of NF-B is a heterodimer composed of p50 and RelA subunits.

NF-B is anchored in the cytoplasm of most nonstimulated cells by a noncovalent interaction with an inhibitory protein, IB (1). The principal IB-like proteins are IB, -, and - (3, 17). Additionally, the p105 and p100 products of the NF-B1 and NF-B2 genes can exert inhibitory effects on NF-B (3, 48). Exposure of cells to proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-) or interleukin-1 (IL-1), promotes the dissociation of IB from NF-B, unmasking the NF-B nuclear localization signal, thereby allowing its nuclear translocation to upregulate specific gene expression (3, 48). The ability of TNF receptors to induce NF-B activation requires the serine-threonine kinase RIP (receptor-interacting protein) (21) and adapter proteins belonging to the TRAF (TNF-receptor-associated factor) family (39), which lack enzymatic activity and share sequence homology at their C-terminal receptor-binding regions. In transfection studies, TRAF2 and RIP may mediate the activation of NF-B in response to TNF-, whereas TRAF6, MyD88, and IRAK are required for activation of NF-B in response to IL-1 (7, 8, 13, 34, 51). However, studies using cells derived from RIP and TRAF2 knockout (KO) mice have shown that RIP is essential for NF-B induction whereas TRAF2 is required for c-Jun N-terminal kinase (JNK) activation by TNF- (21, 24, 54).

It has long been appreciated that the major regulatory step in NF-B activation is the phosphorylation of IB on two serine residues near the N terminus (serines 32 and 36 of IB) (6, 46). These two phosphorylation events target the IB subunit for ubiquitination and subsequent degradation by the 26S proteosome, thus liberating NF-B from its inhibitory constraint (9, 41). Initial attempts to identify the kinase(s) responsible demonstrated specific IB kinase activity to be present in an ~700-kDa cytoplasmic complex (10). Activation of IB kinase activity within this complex can be mediated by MAP-ERK kinase kinase 1 (MEKK-1), although the precise mechanism of this pathway remains to be established (19, 22, 23, 35, 36). Attempts to identify the mechanism by which TRAF2 activates the NF-B pathway led to the isolation of the NF-B-inducing kinase (NIK) (30). NIK, like MEKK1 is a serine-threonine kinase of the MAPKK family. Phosphorylation of IB in response to TNF- requires NIK enzymatic activity (30, 43). However, NIK does not phosphorylate NF-B directly but via two NIK-interacting kinases called IKK and IKK (IB kinases  and ) (38, 52). IKK was independently cloned in a yeast two-hybrid screen with NIK as bait (38) and also by conventional biochemical purification of the major IB kinase activity induced by TNF- stimulation of HeLa cells (15, 33). IKK had also been cloned previously in a search for myc-like genes and was termed CHUK (conserved helix-loop-helix [HLH] ubiquitous kinase) (12). The CHUK gene was shown to encode a 745-amino-acid polypeptide with an amino-terminal serine-threonine kinase catalytic domain, a carboxy-terminal HLH domain, and a leucine zipper-like (LZip) amphipathic -helix juxtaposed in between the HLH and kinase domains (12). IKK, a structurally homologous kinase, was cloned by copurification with IKK (33, 56) and by database-assisted searches for IKK-related expressed sequence tags (52).

Two regulatory components of the 700-kDa cytoplasmic complex have also been identified: NEMO (NF-B essential modulator) (53) (also termed IKK and IKKAP1) (32, 40) and IKAP (IKK complex-associated protein) (11). The former is a 50-kDa protein with a putative leucine zipper motif which can bind to IKK and IKK complexes (perhaps via direct interactions with IKK) and appears to be essential for agonist-mediated stimulation of NF-B. NEMO was isolated both by genetic complementation of an NF-B activation-defective cell line (53) and by purification from the IB kinase complex (32, 40). IKAP was isolated from affinity-purified IB kinase complexes as a 150-kDa protein which binds to both NIK and the IKKs, presumably via one or more of its WD protein interaction domains (11). As both NEMO and IKAP have no discernible kinase or other enzyme activity, they appear to function as "scaffold," or coordinating, proteins which may be required for correct formation of the IB kinase complex and regulated interaction between the IKK complex and other upstream activators, like NIK and perhaps RIP.

IKK and - both possess all the hallmarks of IB kinases, specifically phosphorylating serines 32 and 36 of IB, with both sites requiring phosphorylation in vivo to target IB for destruction. Initial studies demonstrated that activation of IKK and - occurred in response to NF-B-activating agents and that mutant, catalytically inactive IKK and - blocked NF-B stimulation by cytokines. Coexpression studies suggested that IKK and - can form both homo- and heterodimers via their LZip domains and that an IKK-IKK heterodimer may be the functional IKK unit (33, 52, 56). Recombinant IKK and IKK were shown to specifically phosphorylate IB substrates in vitro, proving that they are indeed direct IB kinases (23, 25, 55). Interestingly, site-directed mutation of the HLH domain in IKK severely impaired its kinase activity without significantly reducing its interaction with IKK (56). Similarly, deletion of the IKK HLH domain failed to modify its interaction with either IKK or NIK (52). Recent experiments indicate that the HLH domain of IKK functions as an essential positive effector of the kinase's amino-proximal catalytic domain (14). Furthermore, targeted inactivation of the IKK and IKK genes in mice have revealed that only IKK is essential for mediating NF-B activation by inflammatory-response cytokines (27, 28, 45). In contrast, IKK was not required for activation of IKK or NF-B by proinflammatory stimuli but was instead essential for keratinocyte differentiation (20, 26, 44). It remains to be determined if the essential role of IKK in the differentiation of epidermal keratinocytes is in keeping with its role as an IB kinase or if other, unknown IKK substrates are involved in this developmental pathway. In addition, it remains unclear if other cellular kinases are complementing the loss of IKK to activate NF-B in response to proinflammatory signals and if the HLH domain of IKK is essential for its functional activation, akin to IKK.

In this report, we describe the structure and properties of two novel cellular isoforms of IKK which are produced by alternative mRNA maturation. The first, IKK-H, is strictly identical to IKK from its N terminus until amino acid 576 and thereafter lacks the HLH-like domain present in IKK and IKK. The second isoform, termed IKK-LH, contains an internal deletion removing the LZip domain and generating a premature stop codon in its stead, thereby removing the remainder of the carboxy terminus, including the HLH domain. Unlike IKK, IKK-H and IKK-LH are differentially expressed in various cell lines and normal tissues and predominate over IKK in activated T lymphocytes and the brain. Remarkably, both of these carboxy-terminally truncated forms of IKK appropriately phosphorylate IB in response to TNF- signaling with kinetics analogous to those of full-length IKK, indicating that, unlike those of IKK, the HLH and LZip domains of IKK are not essential for its functional activation.

	MATERIALS AND METHODS

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Cell culture. Human embryonic kidney cells (HEK293) and HeLa cells were cultivated in Dulbecco's modified Eagle's medium (Gibco/BRL) containing 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin sulfate (50 µg/ml). Explanted BALB/c thymocytes were cultured in RPMI 1640 medium supplemented with penicillin, streptomycin, and 10% fetal bovine serum (Hyclone Inc.). In some experiments, T-cell cultures were stimulated with either 10 ng of the phorbol ester phorbol myristate acetate (PMA) (Sigma)/ml plus 100 ng of the calcium ionophore A23187 (Calbiochem)/ml or 100 ng of the T-cell mitogen concanavalin A (Amersham Pharmacia Biotech)/ml for 7 days prior to harvesting of total cellular RNAs.

cDNA library screening. An MPC-11 mouse myeloma cDNA library was prepared in -ZapII(XR) (Stratagene Inc.) and screened with IKK-specific probes along with a BALB/c lung -Zap II library (Stratagene Inc.) and a BXSB mouse spleen -gt-10 library (kindly provided by Konrad Huppi) as previously described (12).

Plasmids. Murine IKK was amplified by PCR from pBluescript KS(+) (Stratagene) and cloned into pcDNA3.1 (Invitrogen, Carlsbad, Calif.) in frame with a C-terminal hemagglutinin (HA) epitope tag to generate pcDNA-IKK-HA, Myc-NIK, IKK-T7, NF-B-dependent luciferase, and Rous sarcoma virus long terminal repeat-driven -galactosidase (RSV--Gal) reporter plasmids were all as previously described (16). The kinase-deficient IKK-(K44A)-HA mutant was generated by PCR (16). pcDNA3.1 FLAG-IKK and FLAG-NIK expression vectors were kind gifts of Randy Noelle. IKK-LH and IKK-H were cloned by PCR from pBluescript KS(+) in frame with a carboxy-terminal V5 epitope tag in pcDNA3.1/V5/His-TOPO as described by the manufacturer (Invitrogen Inc.). IKK-Cm (amino acids 1 to 451 of IKK), a recombinant derivative of IKK-LH lacking its unique 20-amino-acid C-terminal tail, was also cloned by PCR in frame with the C-terminal V5 epitope of pcDNA3.1/V5/His-TOPO. IKK-Cm (amino acids 1 to 454; structurally analogous to IKK-Cm) was amplified from a human IKK construct with the primer pair 5'-TAGAGAACCGCACTGCTTACTGGCT-3' and 5'-GGCGGCTCGCTGTCCCTGCT-3' into pcDNA3.1/V5/His-TOPO. IKK-Km (amino acids 1 to 345; specifying the kinase catalytic domain) was amplified from a human IKK expression vector (a kind gift of Steven Pullen) with the primer pair 5'-CCGATGGACTACAAAGACGA-3' and 5'-TCAAGTTTCACGCTCAATACGAG-3' into pcDNA3.1/V5/His-TOPO. A complete NEMO coding sequence (53) was cloned by reverse transcriptase (RT)-PCR with the primer pair 5'-ACACTGTCCTGTTGGATGAA-3' and 5'-CTCTATGCATCCATGACAT-3' from the EL4 murine T-cell line. Two independent 1.3-kb full-length clones yielded a sequence identical to that previously published (53) except for one base change (C38T) converting amino acid 13 from threonine to methionine. The NEMO cDNA was subcloned into pcDNA3.1(+) in frame with a carboxy-terminal Myc epitope tag coding sequence. -NEMO (an N-terminal truncation, leaving amino acids 235 to 419) was amplified from a full-length cDNA clone with the primer pair 5'-CCAACTCTTAGACTACGACAG-3' and 5'-CTCTATGACCTCCATGACAT-3', initially cloned into the TA cloning vector pCR2.1 (Invitrogen) and subsequently released by EcoRI digestion and recloned in frame with an N-terminal M45 epitope tag into the CMX mammalian expression vector (37).

RT-PCRs of IKK isoforms. As indicated in Results, expression of IKK, IKK-H, and IKK-LHa and -b transcripts were distinguished by RT-PCR assays. Total cellular RNAs (5 µg) were extracted from various cell lines and tissues with triazol reagent (Roche Molecular Biochemicals) and reverse transcribed into cDNAs in a 20-µl RT reaction. The RNAs were preincubated with 10 pmol of an anchored oligo(dT) primer, 5'-AGCTCCGGAATTCGGTTTTTTTTTTTTVN-3', in up to 12 µl of sterile, distilled H₂O at 70°C for 10 min and quick chilled on ice. After a brief centrifugation, the RT reactions were performed with a SUPERSCRIPT II RT kit (BRL Life Technologies) as recommended by the manufacturer. Briefly, the reaction was initially supplemented with 4 µl of 5× first-strand buffer (BRL Life Technologies), 2 µl of 0.1 M dithiothreitol and 1 µl of a 10 mM mixture of all four dinucleoside triphosphates. After a second preincubation at 42°C for 2 min, 1 µl (200 U) of SUPERSCRIPT II, a mutant form of Moloney murine leukemia virus RT lacking RNase activity, was added, and the reaction was allowed to proceed at 42°C for 50 min followed by inactivation at 70°C for 15 min. The resultant cDNAs were used directly in 40-µl PCRs containing 20 pmol of each primer, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.01% Triton X-100, 1.5 mM MgCl₂, and 2 U of Taq polymerase (Promega Inc.). All RT-PCRs were performed with a 5' amplimer present in all forms of IKK (, 5'-ACCATTTGCATCCAGAAGTTTATC-3'; bp 1241 to 1264) and one of four 3' primers: (i) , 5'-CAGGAGGTCTGTGCTTTAGCTG-3' (1,761 to 1,782 bp in all forms of IKK), (ii) , 5'-TGCTCAGGTGACCAAACAGCT-3' [1,861 to 1,881 bp of IKK and CHUK(LHa)], (iii) , 5'-GCAAAAAGAATACCAAAACAGGAT-3' (1,879 to 1,902 bp of IKK-H and IKK-LHb), and (iv) , 5'-GATAACCAATGACACCAACCTC-3' (1,620 to 1,641 bp in all forms of IKK). In some PCRs (see Fig. 1 and 3), 20 pmol of 5'  was mixed with 10 pmol each of  and . PCRs were submitted to a hot-start protocol (AmpliWax Gems; Perkin-Elmer Inc.) followed by a 4-min preincubation at 94°C and 26 cycles (30 s at 94°C, 1 min at 62°C, and 1 min at 72°C). The reaction products were resolved by 6% polyacrylamide gel electrophoresis (PAGE).

Luciferase reporter assays. HEK293 cells were transfected by the calcium phosphate method essentially as previously described (16) with 4 µg of expression plasmid, together with 0.5 and 0.25 µg of NF-B luciferase reporter and RSV--Gal reference vectors, respectively. The total DNA concentrations in each transfection were kept constant by the inclusion of appropriate empty vector. Twenty-four hours posttransfection, the cells were stimulated where appropriate with TNF- (10 ng/ml) for 6 h prior to cell lysis. Luciferase and -Gal assays were performed as detailed in the Promega assay kit. Luciferase activity was found to vary over a threefold range, although activities for individual plasmid preparations were qualitatively identical.

Antibodies and recombinant proteins. Anti-T7 and Anti-V5 antibodies were obtained from Novagen and Invitrogen, respectively, and recombinant TNF- was from GIBCO-BRL. GST-IB(1-62) was produced as previously described (16) and purified by standard procedures.

Immune complex kinase assays. HEK293 cells (2.5 × 10⁶ in 10-cm-diameter plates) were transfected with 10 µg of kinase expression plasmid by the calcium phosphate method and stimulated 24 h later in Dulbecco's modified Eagle's medium with the appropriate agonist at 37°C for the times indicated. The cells were washed with ice-cold phosphate-buffered saline and lysed with Triton X-100 lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM -glycerophosphate, 200 µM sodium orthovanadate, 10⁴ M phenylmethylsulfonyl fluoride, 1 mg of leupeptin/ml, 1 µM pepstatin A, 1% Triton X-100). Proteins from the lysates (500 µg) were incubated with specific anti-HA (12CA5) or V5 epitope (Invitrogen Inc.) antibodies preadsorbed to protein A-Sepharose-coated beads for 2 h at 4°C. The immune complexes were washed three times with Triton X-100 lysis buffer and twice with kinase assay buffer (20 mM HEPES, pH 7.4, 20 mM MgCl₂, 1 mM dithiothreitol, 10 mM p-nitrophenylphosphate). IKK activity was assayed by resuspending the final pellet in 40 µl of kinase buffer containing 50 µM [-³²P]ATP (5,000 cpm/pmol) (Amersham) and 0.25 mg of glutathione-S-transferase GST-IKK(1-62)/ml. The reaction was incubated for 10 min at 30°C and stopped with Laemmli sample buffer. Samples were resolved by sodium dodecyl sulfate (SDS)-PAGE (10% acrylamide), and phosphorylation was determined by exposure in a PhosphorImager (Molecular Dynamics). Kinase activity was found to vary over a twofold range, although activities for individual plasmid preparations were qualitatively identical.

Immunoblotting. Cell lysates were prepared in Triton X-100 lysis buffer as described above for the kinase assays. The proteins in cellular lysates were separated by SDS-7% PAGE and electroblotted onto Hybond-C Extra membranes (Amersham). The protein blots were exposed to specific primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, which were subsequently detected by enhanced-chemiluminescence immunodetection (Amersham) by standard procedures.

In vitro translation. Constructs in pcDNA3.1 were translated in a Promega rabbit reticulocyte in vitro translation kit either with [³⁵S]methionine (Amersham) or with unlabeled methionine according to the manufacturer's instructions.

	RESULTS

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Structural comparisons of murine IKK, IKK-H, and IKK-LHa and -b cDNA clones. An RT-PCR strategy, devised to clone novel proteins sharing domains with the c-MYC protein, resulted in the isolation of a cDNA clone encoding a new member of the HLH and leucine zipper gene families (12). This novel gene was originally named the CHUK gene, for conserved HLH ubiquitous kinase (12), and was subsequently shown to be the IKK component of the IB kinase complex (15, 33, 38). Consequently, we refer to the full-length murine form of the protein as IKK throughout this report. Subsequent screening of several murine cDNA libraries (BALB/c lung, BXSB spleen, and MPC-11 mouse myeloma libraries) with IKK-specific probes produced multiple isolates of three other IKK cDNAs with overlapping and different structural features. Thus, alternative IKK transcripts are expressed by different cell types. As shown in Fig. 1 and 2, IKK-H is a unique isoform which is identical to IKK until residue 576 (nucleotide 1782), where the former cDNA has a novel 3' noncoding sequence (NCS). The presence of a translation stop, after eight additional codons in IKK-H, truncates the polypeptide chain 24 amino acids upstream of the HLH domain, replacing the remainder of the protein with a short, 8-amino-acid carboxy-terminal extension (Fig. 2A). In addition, the alternative 3' NCS in IKK-H exhibited significant homology with the sequence of the HLH domain, indicating that this 3' NCS is likely specified by an alternative splice to a duplicated exon which has undergone extensive sequence divergence (Fig. 2B). IKK-LHa and IKK-LHb are two other isoforms of the full-length IKK transcript, both bearing the same 152-bp deletion of nucleotides 1408 to 1559. This deletion excises the LZip domain downstream of residue 451 and then switches the reading frame to generate a translation stop codon after adding a short 20-amino-acid carboxy-terminal tail (Fig. 1). The remainder of the IKK-LHa mRNA is structurally identical to full-length IKK mRNA, while the related IKK-LHb mRNA isoform possesses the same 3' NCS as IKK-H, again at nucleotide 1782 (Fig. 1).

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Structural comparisons of four IKK mRNAs and an RT-PCR assay for their relative detection. Comparative diagrams of four different IKK cDNA clones, each obtained as multiple isolates from three independent cDNA libraries (murine BXSB spleen, BALB/c lung, and MPC-11 plasma cell tumor libraries). A 152-nucleotide internal deletion in the IKK-LHa and -b isoforms, removing nucleotides 1408 to 1559, deletes the LZip domain and inserts a translation stop codon after a unique 20-amino-acid carboxy-terminal tail downstream of IKK alanine 451. A novel 3' NCS in the IKK-H isoform is shown to replace all IKK coding sequences downstream of nucleotide 1782, terminating the translation reading frame at a new stop codon eight unique amino acids after IKK proline 576. IKK-LHb contains the same 3' NCS as IKK-H. The locations of PCR primer pairs used in RT-PCRs discussed throughout the text are shown in each cDNA. The PCR primers , , and  anneal to sequences in all four mRNAs, as opposed to primers  and , which only anneal to sequences in the IKK HLH domain or the novel 3' NCS in isoforms IKK-H and IKK-LHb, respectively. At the bottom, a representative RT-PCR assay of BALB/c thymus total RNA reveals that all four IKK mRNAs can be reliably detected and quantitated by employing a mixture of primer pairs , , and . Varying the input of total cDNA template reveals that the relative intensities of bands in each lane of the ethidium bromide-stained gel are comparable to the relative abundance of their specific mRNA species. The identity of each band was confirmed by restriction enzyme mapping and DNA sequencing. The ratios of expression of each mRNA species in individual lanes were derived by densitometric scanning of individual lanes with the NIH Image program followed by the appropriate length corrections.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Nucleotide sequence comparisons of IKK and IKK-H. The site of insertion (A) and primary structure (B) of a novel 3' NCS in IKK-H are shown. Nucleotides conserved between IKK and IKK-H are boxed. The novel 3' NCS in IKK-H is also present at the same location in the IKK-LHb isoform (see maps in Fig. 1).

Unlike IKK, IKK-H and IKK-LHa and -b are differentially expressed. We employed RT-PCR to investigate the expression patterns of the four IKK transcripts in a variety of cell types and normal murine tissues. An RT-PCR strategy was designed to coamplify all four isoforms and to distinguish their PCR products on a 6% polyacrylamide gel. We paired a 5' Pan IKK amplimer, which is conserved in all four sequences (nucleotides 1241 to 1264; see the location of  primer in Fig. 1), with four different 3' primers: (i) 3' Pan IKK/CHUK 1761-1782, which is between the LZip and HLH domains and present in all four sequences, ( in Fig. 1); (ii) 3' IKK 1861-1881 in the HLH domain ( in Fig. 1); (iii) 3' IKK-H 1879-1902 in the 3' NCS of IKK-H and IKK-LHb ( in Fig. 1); and (iv) 3' IKK 1620-1641, which, like primer , is between the LZip and HLH domains and is present in all four sequences ( in Fig. 1). PCR amplification of anchored oligo(dT)-primed cDNAs with  versus  produced IKK- and IKK-LHa-specific bands of 640 and 488 bp (Fig. 1 and 3). RT-PCR performed with  versus  yielded IKK-H and IKK-LHb bands of 661 and 509 bp (Fig. 1 and 3). Amplifications with a mixture of all three primers produced all four bands with similar relative intensities (Fig. 1 and 3B). The identities of the four bands were confirmed by restriction digestion and DNA sequencing (data not shown). The IKK-H and IKK-LHa bands were 21 bp larger than the IKK and IKK-LHb species, since the distances between  and  versus  and  differed by 21 bp (see the sequence comparisons of IKK and IKK-H [Fig. 2]). PCRs performed with increasing doses of cDNA templates indicated that the relative intensities of the individual bands in each amplification were close approximations of the relative quantities of their mRNAs (see the cDNA dose response analyses of thymus, brain, and 70Z3 lines [Fig. 1 and 3B]). To independently determine the relative amounts of the IKK-LHa and -b isoforms in comparison to IKK and IKK-H, RT-PCRs were performed with primer pairs conserved in all four isoforms (IKK 5' and 3' Pan primers) which flanked the site of the 152-bp (LZip) deletion in the IKK-LHa and -b isoforms (see the locations of primers , , and  in Fig. 1 and 4). As shown in Fig. 4, these results are in good agreement with those of the RT-PCRs shown in Fig. 1 and 3.

View larger version (70K): [in this window] [in a new window]   FIG. 3.   RT-PCR analysis of murine tissues and cell lines reveals four IKK isoforms. (A) Following reverse transcription with an anchored oligo(dT) primer (as described in Materials and Methods), PCRs were performed on 5 ng of total cDNAs with a mixture of primer pairs , , and . (B) Comparisons of IKK RT-PCR products in a cDNA dose-response analysis of brain, 70Z3(35.15wt) and 70Z3(1.3E2 mutant) cells. The PCR products were separated on a 6% polyacrylamide gel and stained with ethidium bromide. The locations and sequences of PCR primers are provided in Fig. 1 and in Materials and Methods, respectively.

View larger version (44K): [in this window] [in a new window]   FIG. 4.   RT-PCR analysis to compare the expression of the IKK-LH isoforms to those of IKK-H and IKK. (A) PCRs were performed with 5 ng of the indicated total cDNAs using the primer pair  and , which produce a band of 541 bp for IKK and IKK-H and a band of 388 bp for IKK-LHa and -b. (B) PCRs were performed with 5 ng of cDNA from unstimulated, in vitro-cultured T cells or from T cells after 7 days of tissue culture with the indicated mitogens (as described in Materials and Methods). Primer pairs  and  produce a band of 400 bp for IKK-H and IKK and a band of 288 bp for IKK-LHa and -b. The PCR products were analyzed on a 6% polyacrylamide gel and revealed by ethidium bromide staining as described in the legend to Fig. 3, but the gel was run 1 h longer to achieve greater fragment resolution. The locations of PCR primer pairs are provided in adjacent diagrams of the amplified portions of the IKK isoforms, and their sequences are given in Materials and Methods. +, present; , absent.

Polypeptides encoded by IKK-H and IKK-LH upregulate NF-B. Activation of NF-B can be readily detected in transient-transfection assays using an NF-B-dependent reporter gene construct. We next investigated whether the IKK-H and IKK-LH proteins, like IKK and IKK, would activate NF-B and also potentiate its induction by TNF-. Cotransfection of IKK leads to a twofold increase in TNF--stimulated luciferase activity, with little difference in basal NF-B-driven luciferase activity. As shown in Fig. 5A and B, IKK-H and IKK-LH also increase the ability of TNF- to stimulate NF-B-dependent luciferase activity. As the amount of plasmid encoding each IKK isoform was increased, the TNF--induced luciferase activity increased correspondingly in a fashion similar to that of IKK (Fig. 5B). Western blot experiments conducted on HEK293 cells transfected with each of the IKK isoforms revealed similar levels of protein expression throughout the dose-response analysis (data not shown). Given that each expression vector is limiting at its lowest DNA input but relative activities remain comparable throughout, these observations are not due to differences attributable to overexpression. Hence, in comparison to IKK and IKK, the two new smaller IKK isoforms are comparably efficient at potentiating NF-B activation in response to TNF-. To verify that the short carboxy-terminal extensions of IKK-H and IKK-LH had no unanticipated effects on their activities, we removed the 20-amino-acid tail of the smaller IKK-LH protein. As shown in Fig. 5 and other figures below, IKK-Cm, a recombinant form of IKK-LH lacking its 20-amino-acid tail, was equally capable of enhancing TNF- stimulation of the NF-B luciferase reporter. However, further deletion of the remaining 106 amino acids of IKK separating the amino-proximal kinase and LZip domains inactivated the protein kinase assays (Fig. 7A, and data not shown). IKK was constitutively active and could enhance the activity of the NF-B-driven luciferase reporter independently of cytokine stimulation, in agreement with another report (33). In sharp contrast to IKK-Cm, IKK-Cm (amino acids 1 to 454), a structurally analogous recombinant form of IKK (amino acids 1 to 451), was inactive in the NF-B reporter assay (Fig. 5A). Thus, IKK does not appear to have the same activation constraints as IKK.

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Truncated IKK isoforms and full-length IKK activate NF-B with similar potencies. (A) HEK293 cells were transiently transfected with plasmids encoding IKK, IKK-Cm, IKK-LH, IKK-H, IKK, or IKK-Cm as indicated, together with reporter plasmid (NF-B-luciferase). Control cells (CON) received only reporter plasmid. Twenty-four hours posttransfection, the cells were stimulated or not for 6 h with TNF- (10 ng/ml) prior to cell lysis and luciferase activity measurement as described in Materials and Methods. The error bars indicate standard deviations. +, present; , absent. (B) HEK293 cells were transiently transfected with increasing concentrations of vectors encoding HA epitope-tagged IKK and IKK-Cm or V5 epitope-tagged IKK-LH and IKK-H, along with reporter (NF-B-luciferase) and reference control (RSV--Gal) plasmids. Twenty-four hours posttransfection, the cells were stimulated for 6 h with TNF- (10 ng/ml) prior to lysis and luciferase measurements. The data represent the increase in activity above that obtained with TNF- alone and are the means of two experiments (n = 2). Error bars are not shown for clarity, but the error was less than 4% of the mean in all cases. Variations in transfection efficiencies were corrected against a cotransfected RSV--Gal control vector, which is not subject to regulation by NF-B (16).

View larger version (33K): [in this window] [in a new window]   FIG. 6.   IKK and IKK-Cm activate NF-B by overlapping pathways. (A) HEK293 cells were transiently transfected with plasmids encoding IKK, IKK-Cm, or IKK as indicated, either separately (4 µg) or in combination (2 µg each), together with reporter (NF-B-luciferase) and reference control (RSV--Gal) plasmids as in Fig. 5. Control cells (CON) received only reporter plasmid. Twenty-four hours posttransfection, the cells were stimulated or not for 6 h with TNF- (10 ng/ml) prior to cell lysis and luciferase activity measurement as described in Materials and Methods. +, present; , absent. (B) HEK293 cells were transiently transfected with plasmid encoding IKK or IKK-Cm (1 µg per well) (top) or, in an independent experiment, IKK-LH and IKK-H (bottom) together with reporter plasmid (NF-B-luciferase; 0.5 µg per well) and increasing concentrations of plasmid encoding catalytically inactive IKK (IKK-K44A). Control cells (NS) received only reporter plasmid. Twenty-four hours after transfection, the cells were stimulated or not (NS) for 6 h with TNF- (10 ng/ml) prior to cell lysis and luciferase activity measurement. Luciferase activity is expressed as arbitrary units normalized to -Gal activity. The data are means ± range of duplicates from a single experiment representative of three performed.

IKK-H and IKK-LH are TNF--inducible IB kinases. Release of NF-B from its IB inhibitor requires the latter's phosphorylation at serines 32 and 36 (6, 46). To assess the relative abilities of IKK, IKK-H, and IKK-LH to phosphorylate IB in response to TNF- stimulation, in-vitro kinase assays were performed with GST-IB(1-62) as a substrate in either anti-HA or anti-V5 immunoprecipitates of HEK293 cells transfected with HA epitope-tagged IKK and IKK-Cm or V5 epitope-tagged IKK-H and IKK-LH (Fig. 7A). HEK293 cells transiently transfected with each of the IKK isoforms expressed similar amounts of immunodetectable proteins with the expected molecular masses (Fig. 7A, top). TNF- stimulation of HEK293 cells transfected with each IKK isoform resulted in an increase in immunoprecipitable kinase activity towards GST-IB(1-62) (Fig. 7A, bottom). However, further truncation of IKK-Cm by removing its carboxy-terminal 106 amino acids to leave an intact amino-terminal kinase domain (IKK-Km [Fig. 7A]) inactivated its TNF--inducible IB kinase activity, implying that a block of amino acids residing between the kinase and LZip domains of IKK are part of a cytokine response domain. As anticipated from the NF-B reporter assay results, all experiments performed with IKK-H, IKK-LH, or the recombinant IKK-Cm produced comparable results, indicating that neither the LZip domain of IKK-H nor the short carboxy-terminal extensions of either short isoform had significant effects on IB phosphorylation in this assay. Indeed, time course experiments revealed that the activation profiles of IKK, IKK-Cm, and IKK-LH enzymatic activities in response to TNF- stimulation were superimposable (Fig. 7B). To determine whether IKK and its shorter isoforms function equivalently when exogenously expressed at different levels, dose-response kinase assays were performed (Fig. 7C). These experiments revealed that IKK and its shorter isoforms IKK-H and IKK-LH perform in similar TNF--activatable manners at different levels of expression. We next explored whether the IKK upstream activating kinase, NIK, would also activate IKK-Cm. Transfection of HEK293 cells with IKK-Cm, IKK-H, or IKK-LH and the upstream activating kinase, NIK, resulted in a similar increase in IB kinase activity (Fig. 7C and data not shown), further suggesting that IKK and its smaller isoforms have common upstream activators. In a separate set of experiments, HEK293 cells were transfected with IKK or IKK-LH together with MEKK-1. MEKK-1 failed to appreciably activate IKK and IKK-LH but activated JNK (data not shown). Control immune-complex kinase assays performed with a mutated GST-IB(1-62) (serines 32 and 36 mutated to alanines) failed to support phosphorylation (data not shown). Hence, similar to full-length IKK, its shorter cellular isoforms are IB kinases, which can be activated by both TNF- and NIK.

View larger version (29K): [in this window] [in a new window]   FIG. 7.   Truncated IKK isoforms are inducible IB kinases. (A) HEK293 cells were transiently transfected with plasmids encoding (+) either HA epitope-tagged IKK or IKK-Cm or V5 epitope-tagged IKK-LH, IKK-H, or IKK-Km, as indicated. Thirty hours posttransfection, the cells were stimulated for 5 min with TNF- (10 ng/ml) prior to lysis and subsequent HA-IKK or V5-IKK immunoprecipitation. The immunoprecipitates were then analyzed for IB kinase activity with GST-IB(1-62) as a substrate (bottom). Whole-cell lysates underwent immunoblotting to determine the level of expression of the various IKK constructs (top). (B) In a separate experiment, HEK293 cells were transiently transfected with HA-IKK, HA-IKK-Cm, or IKK-LH, and 24 h posttransfection, the cells were stimulated or not for the times indicated with TNF- (10 ng/ml) prior to cell lysis, HA-IKK immunoprecipitation, and determination of IKK activity. The data are expressed as the increase in 32P incorporation into the GST-IB(1-62) substrate relative to the basal activity and are means of single determinations pooled from three experiments (n = 3; the error bars represent ranges). (C) HEK293 cells were transiently transfected with increasing concentrations of plasmid encoding either HA-IKK, V5 epitope-tagged IKK-LH, or IKK-H. Twenty-four hours posttransfection, the cells were stimulated or not for 5 min with TNF- (10 ng/ml) prior to cell lysis, immunoprecipitation of exogenously expressed IKK, and determination of IKK activity. The data are expressed as the increase in 32P incorporation into the GST-IB(1-62) substrate relative to the basal activity and are means of single determinations pooled from two experiments (the error was less than 4%). (D) HEK293 cells were transiently transfected with plasmid encoding epitope-tagged IKK, IKK-Cm, or NIK as indicated (+), and 30 h posttransfection, the cells were lysed and IKK activity was determined as for panel A. Construct expression levels (top) were also determined.

Associations of the IKK isoforms with IKK and NIK. IKK possesses functional domains (Fig. 1) known to play roles in protein-protein association, while they are both absent in the IKK-LH isoform. Cotransfection assays demonstrate that IKK and IKK coimmunoprecipitate (33, 52, 56) and may also form homodimers (55). Even though the absence of both LZip and HLH domains in the IKK-LH isoform appears to have no effect on its TNF- activation, it may prevent its interactions with regulatory components of the IKK signalosome complex.

View larger version (35K): [in this window] [in a new window]   FIG. 8.   Association of IKK isoforms and IKK in vivo. HEK293 cells were transiently transfected with plasmids encoding epitope-tagged versions of IKK, IKK-Cm, IKK-LH, or IKK-H together with FLAG-IKK as indicated. FL, full-length IKK. Thirty hours posttransfection, the cells were lysed and FLAG-IKK was immunoprecipitated. Samples were divided in two, and the presence of IKK (top) and IKK (bottom) in both the IKK immunoprecipitates (IP) and the total cell lysates (Lys) were revealed by immunoblotting (IB) with appropriate antibodies. -Flag, anti-FLAG antibody.

View larger version (25K): [in this window] [in a new window]   FIG. 9.   IKK-Cm associates with NIK in vivo and in vitro. (A) (Top) HEK293 cells were transiently transfected with plasmids encoding HA-IKK, HA-IKK-Cm, FLAG-IKK, Myc-NIK, or combinations thereof. Thirty hours posttransfection, NIK was immunoprecipitated and samples were resolved on SDS-9% PAGE gels, which were immunoblotted with appropriate primary antibodies. HA-p44MAPK was used in cotransfections as a negative control and did not associate with NIK (not shown). (Bottom) Comparable expression of each transfected construct was verified by immunoblotting of transfected cell lysates (50 µg of protein) with appropriate antibody. The data are from one experiment done twice. +, present; , absent. (B) HA-IKK-Cm and FLAG-NIK were translated in vitro with [35S]methionine and preincubated for 15 min at 30°C prior to immunoprecipitation with either anti-HA (IKK-Cm) or anti-FLAG (NIK) antibodies as indicated. Proteins from immunoprecipitates were resolved on SDS-PAGE (8% acrylamide gels), dried, and exposed in a phosphorimager.

IKK-H, IKK-LH, and IKK-Cm polypeptides fail to associate with NEMO. Complementation rescue of two cell types which were unresponsive to NF-B-activating agonists, along with purification of the IB kinase complex, resulted in the identification and cloning of NEMO (32, 40, 53). NEMO appears to be a prerequisite for activation of NF-B. It does not exhibit enzymatic activity but possesses a putative LZip domain and several coiled-coil motifs which may mediate interaction with other elements of the NF-B signaling cascade. In vitro-translated NEMO coimmunoprecipitates with IKK and to a lesser extent with IKK (40, 53). Cotransfection studies were performed to determine whether each IKK isoform interacted with NEMO. Transient transfection of HEK293 cells with IKK, IKK-H, IKK-LH, IKK-Cm, IKK, or NEMO followed by NEMO immunoprecipitation revealed that NEMO associated with both IKK and IKK in vivo but failed to associate with the smaller IKK isoforms (Fig. 10), indicating that the HLH domain of IKK was essential for interaction with NEMO.

View larger version (34K): [in this window] [in a new window]   FIG. 10.   Truncated IKK isoforms do not associate with NEMO. HEK293 cells were transiently transfected with plasmids encoding epitope-tagged versions of either IKK, IKK-Cm, IKK-LH, IKK-H, IKK-Cm, or IKK, together with NEMO-Myc. Thirty hours posttransfection, NEMO was immunoprecipitated and samples were resolved on SDS-PAGE (9% acrylamide gel) followed by immunoblotting (IB) with the appropriate primary antibody. (Top) Expression of all forms of IKK and IKK was verified in immunoprecipitates (IP) and whole-cell lysates (Lys). (Bottom) Protein expression of NEMO was verified in both immunoprecipitates and whole-cell lysates. The data are from a single experiment performed twice with similar results.

IKK-Cm, IKK-H, and IKK-LH rescue NF-B activity from the inhibitory effects of a dominant-negative NEMO mutant. To further elaborate functional distinctions between the activation pathways of IKK and those of its truncated isoforms, we assessed the effects of a dominant-negative mutant of NEMO on the abilities of IKK, IKK-Cm, IKK-H, and IKK-LH to activate NF-B in response to TNF-. In agreement with a previous report (32), an amino-terminally truncated mutant of NEMO inhibited the ability of IKK to stimulate TNF--inducible NF-B activation at all dosages (Fig. 11). In contrast, IKK-H, which lacks the HLH domain; IKK-LH; and IKK-Cm, which lack the IKK LZip and HLH domains and failed to interact with NEMO in vivo (Fig. 10), efficiently rescue NF-B induction from the inhibitory effects of the same NEMO mutant (Fig. 11). These and other results presented here support the view that the carboxy-terminally truncated IKK isoforms activate NF-B by an IKK- and NEMO-independent pathway.

View larger version (37K): [in this window] [in a new window]   FIG. 11.   Truncated IKK isoforms rescue NF-B activation from the inhibitory effects of a dominant-negative NEMO mutant, but IKK does not. HEK293 cells were transiently transfected with increasing concentrations of plasmid encoding either HA epitope-tagged IKK or IKK-Cm (top) or, in an independent experiment, V5 epitope-tagged IKK-LH or IKK-H (bottom) together with 1.5 µg of a -NEMO expression plasmid (an amino-terminally truncated, dominant-inhibitory form of NEMO) (32), 0.5 µg of NF-B-luciferase reporter, and 0.25 µg of RSV--Gal reference control plasmids. Control cells (CON) received only the reporter and reference control plasmids. Twenty-four hours posttransfection, the cells were stimulated for 6 h with TNF- (10 ng/ml) prior to lysis and luciferase activity measurement. Luciferase activity is expressed as arbitrary units normalized to -Gal activity, and the values are means ± range of duplicate determinations from an experiment performed three times with comparable results. The inset shows an immunoblot of -NEMO protein expression in lysates (100 µg of protein) from the IKK- and IKK-Cm-transfected cells.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The study of the cascade of events triggered by the binding of cytokines such as TNF- and IL-1 to their receptors, leading to the activation of the cytoplasmically anchored transcription factor NF-B, has recently seen considerable advances. A major breakthrough was the identification of IKK and subsequently IKK as cytoplasmic kinases which phosphorylate IB family members at physiologically relevant sites and thereby target them for proteasome-mediated degradation (15, 33, 38, 52, 55, 56). IKK and IKK proteins are 52% similar at the primary-sequence level and share two carboxy-proximal structural domains resembling LZip and HLH motifs (12). Since LZip and HLH domains are thought to play roles in protein-protein interactions, the IKKs may employ these domains to recruit proteins involved in their regulation or to facilitate binding to specific substrates. Recent experiments on the regulation of IKK activation suggest that the probable interaction of the carboxy-proximal HLH and amino-proximal catalytic domains are required for its cytokine-induced activation (14).

Identification of novel, differentially expressed IKK isoforms. In this report, we describe the cloning and patterns of expression of three unique IKK mRNAs (IKK-H and IKK-LHa and -b) and the functional properties of their two encoded proteins. IKK-H is identical to IKK up to residue 576 (nucleotide 1782), where a novel 3' NCS replaces the rest of the full-length IKK mRNA. After adding 8 unique amino acids, a translation termination codon truncates the polypeptide encoded by IKK-H 24 amino acids upstream of the HLH domain (Fig. 1 and 2). Interestingly, the unique 3' NCS in IKK-H exhibits significant homology with the sequence of the HLH domain, perhaps indicating that the 3' NCS is specified by an alternative splice to a duplicated exon with extensive sequence divergence (Fig. 2A). In addition to IKK-H, IKK-LHa and IKK-LHb are isoforms of the full-length IKK transcript possessing the identical 152-bp deletion of IKK (nucleotides 1408 to 1559 [numbered according to reference 12]). This internal 152-bp deletion removes the LZip domain downstream of residue 451. The same deletion changes the remainder of the translation reading frame to encounter a termination codon after a short stretch of 20 unique amino acids (Fig. 1). The IKK-LHa transcript is otherwise identical to the full-length IKK mRNA, while its related IKK-LHb isoform also possesses the same 3' NCS as IKK-H, again inserted after IKK nucleotide 1782 (Fig. 1). Consequently, IKK-LHa and IKK-LHb encode the same 471-amino-acid carboxy-terminally truncated IKK polypeptide which lacks precisely the LZip and HLH domains. In surprising contrast to the full-length IKK transcript, RT-PCR analysis reveals that its three smaller isoforms are differentially expressed in normal murine tissues and established cell lines (Fig. 3 and 4). IKK is the predominant mRNA in most instances, except for the thymus and brain. In normal T lymphocytes, transcripts encoding the smaller IKK polypeptides predominate over full-length IKK, and the relative steady-state amounts of the IKK-LH isoforms are also preferentially increased by T-cell-mitogenic stimuli. Preferential expression of the IKK-LH isoforms are also observed for the EL4 mature T-cell lymphoma, while they are very weakly expressed in immature T cells, immature and mature B cells, and most other cell types (Fig. 3A and B and 4A). In the brain, the IKK-H isoform is the predominant IKK mRNA (Fig. 3B).

Like IKK, the IKK-H and IKK-LH polypeptides are TNF--inducible, NF-B-activating IB kinases. By a combination of NF-B-driven luciferase gene reporter assays, immune complex kinase assays, and coimmunoprecipitations with other known components of the ~700- to 900-kDa IKK complex, we find that the IKK-H- and IKK-LH-encoded proteins behave in a fashion similar to that of full-length IKK by several but not all criteria. First, plasmid dose-response curves reveal that all forms of IKK activate comparable levels of NF-B luciferase activity, even at limiting dosages (Fig. 5B). Second, each form of IKK correctly phosphorylates IB (on serines 32 and 36) in response to either TNF- signaling or overexpression of the upstream activator NIK (Fig. 7A and D). Third, IKK-Cm, a recombinant form of IKK-LH lacking its unique 20-amino-acid C-terminal tail, activates NF-B and phosphorylates IB like IKK-LH and IKK-H with an enzymatic time course superimposable on that of full-length IKK (Fig. 5 and 7). Fourth, the abilities of IKK-H, IKK-LH, and IKK-Cm to activate NF-B are not appreciably enhanced by coexpression with either IKK or IKK, while all of them are inhibited by a kinase-inactive, ATP-binding domain mutant of IKK (Fig. 6). We conclude that the smaller isoforms of IKK retain a number of functions of the complete polypeptide without its LZip and HLH domains. In addition, the unique, carboxy-terminal tail extremities of IKK-H and IKK-LH do not contribute to their functional activities in these assays.

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Modes of IKK-H and IKK-LH activation are not subject to the same constraints as full-length IKK and IKK: multiple signaling pathways for NF-B activation. Even though a number of functional attributes are shared among the IKK, IKK-H, and IKK-LH polypeptides, there are clear dissimilarities between the mechanisms and apparent requirements for activation of the smaller IKK isoforms and those for IKK and IKK. For instance, IKK-H associates with IKK and IKK, but IKK-LH and IKK-Cm do not (Fig. 8). Therefore, and in agreement with earlier reports (52, 56), we find that the LZip domain is required for IKK-IKK interaction but the HLH domain is not. However, interactions of the smaller isoforms of IKK (IKK-LH and IKK-Cm) with either IKK or IKK are not required for NF-B activation, given the comparable activities of IKK-H, IKK-LH, and IKK-Cm. In addition, unlike IKK and IKK, the smaller forms of IKK do not associate with NEMO (Fig. 10), which has been shown to be a common component of the ~700- to 900-kDa IKK complex and to be essential for IKK and IKK activation (32, 40, 53). We also show that IKK-Cm, IKK-H, and IKK-LH can each rescue the cytokine-inducible NF-B responsiveness of cells from the inhibitory effects of an amino-terminal deletion mutant of NEMO, while full-length IKK cannot (Fig. 11). Thus, the smaller forms of IKK neither dock with nor require NEMO for their functional activation, in contrast to IKK and IKK. Furthermore, the unique properties of the IKK-H isoform demonstrate that IKK and IKK association per se via their LZip domains does not ensure the functional docking of the kinase heterodimer to NEMO, unless each kinase molecule also contains an H-L-H domain (Fig. 8, 10, and 11). By analogy these observations also indicate that the HLH motifs of IKK and IKK appear to function like dominant effector domains, funneling them into NEMO-dependent complexes.