[1] 10w A cytokine-responsive IB kinase that activates the transcription factor NF-B

[1] 91w Nuclear transcription factors of the NF-B/Rel family are inhibited by IB proteins, which inactivate NF-B by trapping it in the cell cytoplasm. Phosphorylation of IBs marks them out for destruction, thereby relieving their inhibitory effect on NF-B. A cytokine-activated protein kinase complex, IKK (for IB kinase), has now been purified that phosphorylates IBs on the sites that trigger their degradation. A component of IKK was molecularly cloned and identified as a serine kinase. IKK turns out to be the long-sought-after protein kinase that mediates the critical regulatory step in NF-B activation.

[1] 218w To investigate whether phosphorylation of IB␣ at S32/36 in response to TNF is due to activation of an IB kinase specific for these sites, we fractionated extracts of non-stimulated and TNFstimulated HeLa cells on a Mono-Q column. Column fractions were tested for their ability to phosphorylate a glutathione S-transferase (GST)-IB␣ fusion protein containing the N-terminal regulatory domain (residues 1 to 54). The specificity of the kinase and its physiological relevance were examined by using the mutant substrate GST-IB␣(1-54; TT), in which serines 32 and 36 are substituted with threonines 7 . In intact cells this substitution decreases the efficiency of IB␣ phosphorylation, indicating that the relevant kinase strongly prefers serines over threonines 7 . As shown in Fig. 1a, TNF stimulation of HeLa cells resulted in rapid activation of a kinase activity that phosphorylates GST-IB␣(1-54). This activity did not phosphorylate GST-IB␣(1-54; TT). Fractionation of extracts of non-stimulated and TNF-stimulated HeLa cells by gel filtration revealed a TNF-stimulated IB␣(1-54) kinase activity that eluted with an apparent M r of ϳ900K (Fig. 1b). This activity was not detected when GST-IB␣(1-54; TT) was used as a substrate and also did not phosphorylate GST-IB␣(1-54; AA), in which serines 32 and 36 were substituted with alanines (Fig. 1c). This kinase activity was activated rapidly, peaking 5-10 min after TNF addition (data not shown).

[2] 252w We purified this kinase activity from large-scale cultures of HeLa cells stimulated with TNF. Cytosolic extracts (S-100 fraction) were chromatographed on Q-Sepharose and the active fractions were pooled and chromatographed through a ␥-phosphate-linked ATP-Sepharose 16 affinity column. Bound proteins were eluted with ATP and the active fractions pooled and chromatographed on a Superose-6-gel-filtration column. This procedure resulted in ϳ25,000fold enrichment of IB␣ kinase activity (Table 1). The purified kinase, which we call IB kinase (IKK), has the same substrate specificity as the TNF-stimulated activity already described (Fig. 2c). To examine the polypeptide composition of IKK, we analysed the Superose-6 column fractions by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Only two polypeptides, of 85K and 87K, clearly co-eluted with IKK activity at this stage (Fig. 2a). As even the most active fraction contained a number of polypeptides, we applied the pooled Superose-6 IKK fractions to an affinity column of a GST-IB␣(1-54; AA) 8× fusion protein covalently linked to agarose. After extensive washing, the column was developed with a NaCl gradient. This resulted in a further fourfold enrichment of IKK activity (Table 1). The composition of the material retained on the affinity column is shown in Fig. 2b. Although this fraction contains several polypeptides, we consistently detected enrichment of the 85K and 87K band, as well as of the 64K band. It is not clear yet whether the other bands represent additional IKK components or contaminants. Gel-filtration analysis of this material indicated that it continued to elute as a very large (ϳ900K) complex.

[3] 106w We purified the 85K polypeptide associated with IKK to homogeneity by preparative SDS-PAGE and subjected it to microsequen-cing. In addition to using oligonucleotide primers based on the obtained sequence, we searched Genebank for related sequences and found that both peptides were contained within a partial sequence of a putative human serine/threonine kinase with unknown function named CHUK 17 . Polymerase chain reaction (PCR) and library screening were used to isolate the complete complementary DNA. Nucleotide sequencing revealed the presence of an open reading frame for a 744-amino-acid polypeptide (Fig. 3; Genebank accession number AF009225). The N-terminal half of the predicted polypeptide contains a 301-amino-acid protein-kinase domain,

[4] 149w articles NATURE | VOL 388 | 7 AUGUST 1997 549 Table 1 Purification of IKK activity Step Protein yield (mg) Total IKK activity* S-100 900 -Q-Sepharose 40 100% ATP-Sepharose 0.5 170% Superose 6 0.035 230% IB␣-affinity 0.009 160% .............................................................................................................................................................................. The yields and relative activities for the various steps involved in IKK purification are shown from a typical 15-litre HeLa cell suspension culture. * The total IKK activity is approximate because of the difficulty in estimating IKK activity in the starting material owing to the presence of other kinase activities. Also, owing to removal of inhibitors, the kinase activity increases instead of decreases during most of the steps. whereas its C-terminal half, as previously reported 17 , contains several protein-interaction motifs, including a leucine zipper and a helix-turn-helix motif. In the light of its function, we renamed the protein encoded by this cDNA as IKK␣ (for ␣ subunit of IKK).

[5] 136w Cell-free translation of in vitro generated IKK␣ transcripts resulted in production of a polypeptide of the expected size (Fig. 4a; cell-free-translated IKK␣ migrates more slowly than the p85 IKK subunit owing to the presence of a haemagglutinin(HA) epitope). We tested this protein for IB␣ kinase activity. HA-IKK␣ protein was immunoprecipitated with HA antibody, extensively washed in urea at concentrations of up to 3 M, and the immune complexes incubated with various GST-IB proteins or GST-c-Jun in the presence of [␥-32 ]ATP. This resulted in the efficient phosphorylation of either GST-IB␣(1-54) or full-length GST-IB␣ (Fig. 4b). Phosphorylation of either GST-IB␤(1-44) or full-length GST-IB␤ was less efficient, and GST-IB␣(1-54; TT), GST-IB␣(1-54; AA), GST-IB␤(1-44; AA) or GST-c-Jun(1-79) were not phosphorylated. This profile of substrate specificity is identical to that of native IKK purified from TNF-treated HeLa cells (Fig. 2c).

[6] 172w The most efficient NF-B activators are the proinflammatory cytokines interleukin (IL)-1 and TNF 13,18 . These cytokines are also efficient inducers of IB␣ phosphorylation and degradation 7 . We examined the effect of TNF and IL-1 on IKK␣-associated kinase activity. HA-IKK␣ was transiently expressed in HeLa cells. As a control, we transiently transfected HeLa cells with a HA-JNK1 expression vector. After 36 h, these cells were stimulated with TNF or IL-1 for 5 min and the HA-tagged protein kinases immunoprecipitated. HA-IKK␣ immunoprecipitates from TNF-or IL-1stimulated cells phosphorylated GST-IB␣(1-54) but did not phosphorylate GST-c-Jun(1-79) (Fig. 5a). By contrast, HA-JNK1 phosphorylated GST-c-Jun(1-79) but not GST-IB␣(1-54). The extent of IKK␣ activation by TNF or IL-1 was similar to the extent of JNK activation by these cytokines. Time-course analysis indicated that stimulation of IKK␣ activity occurred rapidly (Fig. 5b). Maximal IB␣ kinase activity was reached within 5 min of TNF or IL-1 addition. After 15-30 min, this activity rapidly declined. IKK␣ activity was also moderately stimulated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate acetate (TPA; data not shown).

[7] 46w We investigated the substrate specificity of HA-IKK␣ immune complexes isolated from TNF-stimulated cells and found it to be identical to that of purified IKK (data not shown). Phosphopeptide mapping on Tris-Tricine gels 7 revealed that phosphorylation of GST-IB␣(1-54) occurred on the peptide that contains S32 and

[8] 234w articles 550 NATURE | VOL 388 | 7 AUGUST 1997 S36 (Fig. 6). The alanine-substitution mutant GST-IB␣(1-54; AA) was not phosphorylated at all (data not shown; but see Figs 2c and 4c). The use of single-substitution mutants, IB␣(A32) or IB␣(A36), indicated that both purified IKK and HA-IKK␣ immune complexes phosphorylated both S32 and S36 with comparable efficiency (Fig. 6b, c). However, phosphopeptide mapping of full-length IB␣ phosphorylated by either purified IKK or HA-IKK␣ immune complexes revealed that, in addition to the peptide containing S32 and S36, a smaller amount of 32 P was incorporated into a peptide spanning amino acids 264 to 314 (Fig. 6a, c). When full-length IB␣(A32/36) was used as a substrate, all of the 32 P was incorporated into that C-terminal peptide. Phosphoaminoacid analysis indicated that phosphorylation occurred at serine residues. Previously we have localized the constitutive phosphorylation sites of IB␣ to the same peptide near its C terminus 7 Q R intact cells, we generated a stably transfected pool of HT-29 cells expressing HA-IKK␣. Previous studies indicated that TNF induces partial IB␣ degradation in these cells with slower kinetics than in other cell lines, especially HeLa (J.A.D., unpublished results). Expression of HA-IKK␣ in HT-29 cells markedly accelerated IB␣ phosphorylation (indicated by the appearance of a slower migrating form 7 ) and degradation in response to TNF (Fig. 7). The kinetics of IB␣ degradation correlated with the kinetics of IKK activation.

[9] 165w We tested the effect of raised or reduced IKK␣ expression on NF-B activation by transient transfection. A 2 ϫ NF-B-Luc reporter 12 was cotransfected with an empty expression vector, an HA-IKK␣ expression vector, or an antisense (AS) IKK␣ vector into HeLa cells. Expression of HA-IKK␣ potentiated basal reporter gene activity and its stimulation by TNF or IL-1 (Fig. 8a). By contrast, cotransfection with AS-IKK␣ blocked induction of 2 ϫ NF-B reporter activity by IL-1 or TNF. In other experiments, AS-IKK␣ inhibited induction of 2 ϫ NF-B-Luc by TPA (data not shown). The inhibition was specific as it was not seen upon cotransfection of either JNKK1 or MKK3 antisense vectors. In addition, the inhibitory effect of antisense IKK␣ was reversed upon cotransfection of the HA-IKK␣ vector (Fig. 8b). Furthermore, immunoblot analysis indicated that AS-IKK␣ inhibited expression of HA-IKK␣ but not HA-JNK1 (data not shown). The stimulatory effect of HA-IKK␣ on NF-B reporter activity was specific as it was not detected using a ␤-actin-LacZ reporter (Fig. 8c).

[10] 179w The identification of a cytokine-activated IB kinase suggests that the activation of NF-B and induction of IB␣ phosphorylation by the PP2A-inhibitor okadaic acid 14,15 could be due to negative regulation of IKK itself by PP2A. Such an interpretation is consistent with the rapid inactivation of IKK activity following its stimulation by TNF or IL-1. To test this possibility, we performed the experiment shown in Fig. 9a. Whereas okadaic acid did not affect IB␣ phosphorylation by IKK purified from TNF-treated HeLa cells, inclusion of PP2A in the reaction or preincubation of IKK with PP2A diminished IB␣ phosphorylation. This effect was inhibited by okadaic acid. To determine the target for PP2A action, we preincubated purified IKK enzyme with PP2A and then performed the kinase reaction in the presence of okadaic acid, which caused a large decrease in IB␣ phosphorylation. When PP2A was added during the kinase reaction together with okadaic acid, no effect on IB␣ phosphorylation was seen. Hence, IKK activity is sensitive to PP2A. We also found that, once phosphorylated by IKK, IB␣ was not dephosphorylated by PP2A (Fig. 9b).

[11] 104w The IB kinase activity associated with IKK␣ is also sensitive to PP2A. HA-IKK␣ immune complexes from transiently transfected non-stimulated or TNF-stimulated cells were treated or not with PP2A and tested for IB␣ phosphorylation in the absence or presence of okadaic acid. Like the purified enzyme, the activity of HA-IKK␣ was sensitive to PP2A (Fig. 9c). Consistent with these results, we find that incubation of HeLa or HT-29 cells, stably expressing HA-IKK␣, with okadaic acid caused a slow but considerable increase in IKK activity (Fig. 9d). In addition, cotransfection with the AS-IKK␣ vector inhibited induction of 2 ϫ NFB-Luc by okadaic acid (data not shown).

[1] 152w The proinflammatory cytokines TNF and IL-1 exert many of their effects through activation of transcription factors AP-1 and NF-B, which mediate induction of genes encoding other cytokines and various proteins involved in the inflammatory response [18][19][20] . We understand the immediate events involved in TNF (refs 21-23) and IL-1 (refs 24, 25) signalling and how they lead to activation of the Jun N-terminal kinase (JNK) and p38 MAPK cascades that mediate induction of AP-1 activity 26 (Z. G. Liu, unpublished results), but not how these early signalling events lead to NF-B activation. Although IB phosphorylation is critical for cytokine-mediated NF-B activation, the protein kinase(s) responsible was previously unidentified. It was even suggested that increased IB phosphorylation following cytokine treatment was due to inhibition of a phosphatase. We have now described the purification and molecular cloning of a component of the cytokine-activated protein kinase complex IKK which is responsible for inducible IB phosphorylation.

[2] 34w Several lines of evidence indicate that IKK is the critical protein kinase mediating NF-B activation by TNF or IL-1. First, IKK activity is rapidly stimulated by TNF, IL-1 or TPA and its kinetics of

[3] 161w articles 552 NATURE | VOL 388 | 7 AUGUST 1997 activation match those of IB␣ phosphorylation and degradation in intact cells. Second, IKK phosphorylates IB␣ and ␤ on the same residues, S32/36 and S19/23, respectively, that are phosphorylated in intact cells in response to TPA, IL-1 or TNF 7 . Phosphorylation of these serines triggers the polyubiquitination and degradation of IB [3][4][5][6][7][8] . Third, IKK does not phosphorylate mutants of IB␣ in which these serines are replaced with threonines. Such mutants are poorly phosphorylated in intact cells and are refractory to cytokineinduced degradation 7 . Fourth, elevated expression of IKK␣, the subunit of IKK that we have cloned, accelerates IB␣ degradation and potentiates NF-B activation, whereas expression of an antisense IKK␣ construct blocks NF-B activation by TNF or IL-1. Fifth, IKK activity is sensitive to PP2A, whereas the specific PP2A inhibitor, okadaic acid, activates NF-B 14,15 and IKK. On the other hand, PP2A does not dephosphorylate IB␣ previously phosphorylated by IKK.

[4] 259w IKK purifies as a very large and stable species composed of several polypeptides. The exact polypeptide composition of IKK remains to be determined and the protein kinase we have cloned, IKK␣, is only one subunit of that complex. Although the size of the IKK complex is similar (but not identical) to that of a previously reported IB kinase 15 , the two activities may not be related. The activity of the enzyme studied by Chen et al. 15 depends on its ubiquitination, but we find no evidence that ubiquitination is required for IKK activation. Rather, the critical modification for IKK activation, based on its sensitivity to PP2A, appears to be phosphorylation. In addition, the previously described IB kinase activity appears to be constitutive 15 , whereas IKK activity is rapidly stimulated by cytokines. Nevertheless, IKK isolated from unstimulated cells has a basal activity, amounting to 5-10% of that of the activated enzyme. Indeed, overexpression of IKK␣ can enhance NF-B activity even in unstimulated cells. In addition, the upstream inputs that lead to IKK activation are weakly active in unstimulated cells. Inhibition of PP2A, the phosphatase that inactivates IKK, results in slow but considerable IKK activation. The nature of the input responsible for basal IKK activity remains to be identified. It has been shown 27 that the ubiquitination-dependent IB kinase can be further activated in vitro by MEKK1; however, expression of a catalytically inactive MEKK1 mutant inhibits JNK activation without affecting NF-B activation 26 and so far we have been unable to activate IKK in vitro with MEKK1 (unpublished results).

[5] 145w As IKK␣ is a serine/threonine kinase by sequence and its cell-free translation produces an IB kinase activity with the same substrate specificity as native IKK, it is likely that it is a catalytic subunit of this complex. IKK␣, which is essentially identical in sequence to the putative serine/threonine kinase CHUK, whose function and mechanism of regulation were previously unknown 17 , contains protein-interaction motifs in its C-terminal half, including a leucine zipper and a helix-turn-helix. These motifs could mediate interaction of IKK␣ with other subunits of the IKK complex. Although the IBs are not part of this complex, IKK binds to an IB␣-affinity column. The IKK subunit responsible for this interaction remains to be identified. Previously, by studying the interaction of JNK with c-Jun, we demonstrated that docking of a signal-regulated protein kinase to its substrate is important for rapid and specific phosphorylation 28 .

[6] 128w Although in Jurkat cells TNF induces the degradation of IB␣ but not IB␤ 29 , in several other cell lines we found that the main difference between the two IBs was in the rate of their degradation: IB␣ is degraded faster than IB␤ 7 . This could be caused by a limiting amount of a protein kinase with a higher affinity for IB␣ than for IB␤. IKK exhibits this property in vitro, phosphorylating IB␣ more efficiently than IB␤. Also, if IB␤ degradation contributes to NF-B activation during a 3-hour stimulation in HeLa cells, the fact that antisense IKK␣ RNA blocks NF-B activation indicates that IKK may also be an IB␤ kinase. But it remains to be seen whether overexpression or activation of IKK accelerates IB␤ phosphorylation and degradation.

[7] 67w Our results establish IKK as the long-sought-after protein kinase that phosphorylates at least IB␣ in response to proinflammatory cytokines. The identification of IKK and the molecular cloning of one of its subunits will lead to a better understanding of the signalling pathways originating at the TNF and IL-1 receptors that depend on recruitment of TRAF mediators [21][22][23][24][25] to elicit activation of NF-B and the inflammatory gene-induction response.

[1] 603w Protein purification. Suspension cultures of HeLa S3 cells were stimulated with 20 ng ml -1 TNF (R&D Systems) for 5 min at 5 ϫ 10 6 cells ml Ϫ 1 . Cells were collected and lysed in buffer A (20 mM Tris-Cl, 20 mM NaF, 20 mM ␤glycerophosphate, 19 mM PNPP, 500 M Na 3 VO 4 , 2.5 mM metabisulphite, 5 mM benzamidine, 1 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 10% glycerol, pH 7.6) supplemented with 20 g ml -1 aprotonin, 2.5 g ml -1 leupeptin, 8.3 g ml -1 bestatin, 1.7 g ml -1 pepstatin and 0.05% NP-40, by using a glass Dounce homogenizer. After centrifugation at 12,000 r.p.m. for 20 min in a Beckman SS34 rotor, the supernatant was recentrifuged at 38,000 r.p.m. for 80 min in a Beckman Ti 50.1 rotor, all at 4 ЊC. The supernatant (S-100) was flash-frozen and stored at -80 ЊC. Thawed S-100 fractions were purified over a 56 ml Q-Sepharose FF column equilibrated with buffer A and 0.1% Brij-35 (buffer B). After washing with buffer B plus 100 mM NaCl, the column was eluted with a linear 100-300 mM NaCl gradient. Fractions containing peak IKK activity were pooled, diluted 1:4 and applied to a 5-ml Hi Trap-Q column (Pharmacia). After washing with buffer B, the column was eluted in buffer B plus 300 mM NaCl. IKK-containing fractions were pooled and diluted1:1 with ATP-column buffer 16 . The material was passed 4 times over a 4-ml ␥phosphate-linked ATP-affinity column 16 . After washing with 10 ml ATP column buffer, 0.05% Brij-35, and with 10 ml ATP-column buffer, 0.05% Brij-35, 250 mM NaCl, the column was eluted with ATP-column buffer, 0.05% Brij-35, 250 mM NaCl, 10 mM ATP. After 1:3 dilution with buffer B, the eluate was applied to a 1-ml Hi-Trap-Q column. The column was eluted with buffer B plus 300 mM NaCl. IKK-containing fractions were pooled, concentrated and applied to a Superose-6 column, equilibrated and eluted in the same buffer. IKK-containing fractions were pooled and applied to an affinity column of a GST-IB␣(1-54; AA) 8× (eight repeats of the N-terminal domain) covalently linked to Sepharose after 1:4 dilution in buffer A. After washing in buffer A the column was eluted with a NaCl gradient. IKK assay, immunoprocipitation and immunoblotting. Kinase activity was assayed in 20 mM HEPES, 20 mM ␤-glycerophosphate, 10 mM MgCl 2 , 10 mM PNPP, 100 M Na 3 VO 4 , 2 mM DTT, 20 M ATP, 10 g ml -1 approtonin, 50-200 mM NaCl, pH 7.5, and (1-10 Ci)[␥-32 P]ATP at 30 ЊC for 30 min. IBsubstrate proteins were expressed and purified from E. coli 7 . HA-IKK␣ immune complexes were isolated as described 30 and washed in kinase buffer containing 3M urea before determining kinase activity. Immunoblotting was done as described 7 . Peptide sequencing and mapping. Polypeptides to be sequenced were purified by preparative SDS-PAGE. The 85K IKK␣ band was digested in situ with endoprotease Lys-C. The resultant peptides were eluted and purified by reverse-phase chromatography on an ABI 173 microblotter. Their sequence was determined on an ABI Procise Protein MicroSequencer. Phosphopeptide mapping on Tris-Tricine gels and amino-acid analysis were done as described 7 . Plasmids, cell culture and transfections. The various expression vectors were constructed using standard recombinant DNA procedures. The ␤-actin promoter 31 was used to drive expression of both HA-IKK␣ and antisense IKK␣. Transient transfections were done as described 26,28 . Stably transfected cell lines were generated as described 7,31 and identified by immunoblot screening of individual clones or pools of clones. Luciferase and ␤-galactosidase assays have also been described 12,26 .

[1] 193w Transcription factors of the NF-B/Rel family are regulated through interactions with IBs, which are inhibitory proteins 1,2 . With the exception of mature B cells where they are consititutively nuclear, in all other cell types NF-B dimers are kept in the cytoplasm through association with the IBs, which mask their nuclear localization sequence 1,2 . In response to diverse extracellular stimuli, including proinflammatory cytokines, viral infection, oxidants, phorbol esters and ultraviolet irradiation, the IBs are rapidly phosphorylated at two serines within their amino-terminal regulatory domains. In two IB proteins, site-directed mutagenesis [3][4][5][6][7] and biochemical mapping 7 has identified these serines as S32 and S36 in IB␣ and S19 and S23 in IB␤. Phosphorylation at these sites triggers polyubiquitination of the IBs [7][8][9] and, as with other proteins 10 , targets them for rapid degradation by the 26S proteasome 7,8 . Proteasome inhibitors cause accumulation of IB, which in stimulated cells is N-terminally phosphorylated, and inhibit NF-B activation 11,12 . Substitution of two lysines in IB␣, K20 and K21, one of which is conserved in IB␤, with arginine residues decreases the rate of inducible IB␣ ubiquitination and degradation without affecting its phosphorylation 7 .

[2] 174w As N-terminal phosphorylation of the IBs occurs before and is necessary for their ubiquitination and degradation, it is likely to be the principal point of control through which diverse stimuli effect NF-B activation 13 . However, it is not yet clear whether increased Nterminal phosphorylation of the IBs is due to activation of an IB kinase or inhibition of an IB phosphatase 14,15 . Part of this doubt is due to the fact that a cytokine-responsive protein kinase(s) phosphorylating IB␣ at S32/36 or IB␤ at S19/23 has not been previously identified. In addition, okadaic acid, an inhibitor of protein-phosphatase 2A (PP2A), is a potent NF-B activator and an inducer of IB N-terminal phosphorylation 8,14,15 . In fact, an activity that phosphorylates IB␣ at S32/36 was detected in okadaic acidtreated cell extracts and had an apparent relative molecular mass (M r ) of 700K (ref. 15). It was not established, however, whether this activity is stimulated by proinflammatory cytokines or other agonists and, if so, whether its activation kinetics correlate with those of IB phosphorylation.

[3] 128w To understand better the signalling mechanism responsible for NF-B activation, we tested cell extracts for the presence of a protein kinase activity that is activated by tumour-necrosis factor (TNF) and phosphorylates IB␣ at S32/36. Such an activity was detected in extracts of TNF-treated HeLa cells and its substrate specificity and kinetics of activation correlated well with those of IB␣ phosphorylation in living cells 7 . We purified this activity and determined a partial peptide sequence for one of its components. Molecular cloning and functional analysis identified this subunit as a protein kinase whose associated IB kinase activity is rapidly stimulated by proinflammatory cytokines. This activity is inhibited upon dephosphorylation with PP2A. We demonstrate that this protein kinase, IKK␣, is critical for NF-B activation in response to proinflammatory cytokines.