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Distinct Roles of the IκB Kinase α and β Subunits in Liberating Nuclear Factor κB (NF-κB) from IκB and in Phosphorylating the p65 Subunit of NF-κB

Distinct Roles of the IκB Kinase α and β Subunits in Liberating Nuclear Factor κB (NF-κB) from... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 6, Issue of February 8, pp. 3863–3869, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Distinct Roles of the IB Kinase  and  Subunits in Liberating B (NF-B) from IB and in Phosphorylating the p65 Nuclear Factor B* Subunit of NF- Received for publication, November 2, 2001, and in revised form, November 30, 2001 Published, JBC Papers in Press, December 3, 2001, DOI 10.1074/jbc.M110572200 Nywana Sizemore‡, Natalia Lerner‡, Nicole Dombrowski‡, Hiroaki Sakurai§, and George R. Stark‡¶ From the ‡Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the §Biology and Pharmacology Department, Discovery Research Laboratory, Tanabe Seiyaku Company Limited, Osaka 532-8505, Japan Phosphatidylinositol 3-kinase (PI3K) and the serine/ IB and the consequent liberation of NF-B are not sufficient to threonine kinase AKT have critical roles in phosphoryl- activate NF-B-dependent transcription, which also relies on a ating and transactivating the p65 subunit of nuclear second pathway, which leads to the stimulus-induced phospho- B (NF-B) in response to the pro-inflammatory factor rylation of the p65/RelA, RelB, and c-Rel subunits of NF-B cytokines interleukin-1 (IL-1) and tumor necrosis factor (6 –15). (TNF). Mouse embryo fibroblasts (MEFs) lacking either Our laboratory (13) and others (7, 14) have shown that the or  subunit of IB kinase (IKK) were deficient in the pro-inflammatory cytokines IL-1 and TNF induce the phospho- B-dependent transcription following treatment NF- rylation and activation of the p65 subunit of NF-B, a pathway -null with IL-1 or TNF. However, in contrast to IKK distinct from the one leading to IB degradation and NF-B -null MEFs were not substantially defective MEFs, IKK nuclear translocation. Additionally, phosphatidylinositol 3-ki- or in the in the cytokine-stimulated degradation of I nase (PI3K) and the serine/threonine kinase AKT play critical B. The IKK complexes nuclear translocation of NF- roles in this pathway (13, 16, 17). Recently, an additional -orIKK-null MEFs were both deficient in from IKK function for IL-1-stimulated PI3K/AKT activation has been PI3K-mediated phosphorylation of the transactivation reported: phosphorylation of the NF-B p50 subunit in re- B in response to IL-1 domain of the p65 subunit of NF- sponse to these kinases increases the DNA-binding capacity of and TNF, and constitutively activated forms of PI3K or the NF-B complex (18). AKT did not potentiate cytokine-stimulated activation Targeted gene disruptions have demonstrated that IKK B in either IKK-orIKK-null MEFs. Collec- of NF- (but not IKK) is largely responsible for cytokine-induced IB tively, these data indicate that, in contrast to IKK B liberation and p65 degradation and NF-B nuclear translocation (19 –24). How- which is required for both NF- is required solely for the cyto- ever, IKK-null mouse embryo fibroblasts (MEFs) are deficient phosphorylation, IKK kine-induced phosphorylation and activation of the p65 in inducing several NF-B-dependent mRNAs in response to B that are mediated by the PI3K/AKT subunit of NF- IL-1 and TNF (21). Activated AKT interacts with IKK upon pathway. cytokine stimulation and induces the phosphorylation of thre- onine 23 (25). These findings raise the interesting possibility that, although IKK is dispensable for IB degradation and The NF-B family of transcription factors consists of binary NF-B nuclear translocation, it may be required in the PI3K/ complexes of subunits with related promoter-binding and AKT pathway that leads to the phosphorylation and activation transactivation properties. The p65/RelA, RelB, and c-Rel sub- of NF-B. Therefore, we have investigated the roles of the IKK units stimulate transcription, whereas the p50 and p52 sub- and  subunits in the IL-1- and TNF-mediated phosphoryl- units serve primarily to bind to DNA (1). The prototypical ation and activation of the p65 subunit of NF-B. NF-B complex is the p65-p50 heterodimer (2). NF-Bisse- questered in a latent form in the cytoplasm through its inter- EXPERIMENTAL PROCEDURES action with the inhibitory IB proteins. In response to signals, Biological Reagents and Cell Culture—Recombinant human IL-1 IB kinase is activated, and IB is phosphorylated and de- was from NCI, National Institutes of Health. Recombinant human TNF graded, releasing NF-B, which enters the nucleus and binds to was from Preprotech (Rocky Hill, NJ). LY 294002 was from Sigma. Polyclonal anti-IKK, anti-IKK, anti-IKK, anti-p65/RelA, and anti- DNA (2–5). However, the phosphorylation and degradation of IB antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho- * The costs of publication of this article were defrayed in part by the AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho- payment of page charges. This article must therefore be hereby marked RSK RSK p90 , anti-p90 , anti-phospho-JNK1, and anti-JNK antibodies “advertisement” in accordance with 18 U.S.C. Section 1734 solely to were from Cell Signaling Technologies (Beverly, MA). Protein A-Sepha- indicate this fact. rose and glutathione-agarose beads were from Amersham Biosciences, To whom correspondence should be addressed: Lerner Research Inc. (Buckinghamshire, United Kingdom). Wild-type and IKK- and Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH IKK-null MEFs, kindly provided by Dr. Inder Verma (21), were main- 44195. Tel.: 216-444-3900; Fax: 216-444-3279; E-mail: [email protected]. 1 tained in Dulbecco’s modified Eagle’s medium supplemented with 10% The abbreviations used are: NF-B, nuclear factor B; IL, interleu- fetal calf serum, 100 g/ml penicillin G, and 100 g/ml streptomycin. kin; TNF, tumor necrosis factor; PI3K, phosphatidylinositol 3-kinase; For all experiments, unless otherwise indicated, cells at 80% confluence IKK, IB kinase; MEFs, mouse embryo fibroblasts; MAPK, mitogen- on 100-mm dishes were preincubated with the PI3K inhibitor LY activated protein kinase; ERK, extracellular signal-regulated kinase; 294002 (20 M) for 30 min at 37 °C prior to stimulation with IL-1 (2 RSK, ribosomal S6 kinase; JNK, c-Jun NH -terminal kinase; IP10, ng/ml) or TNF (25 ng/ml) at 37 °C for the indicated time periods. All interferon-inducible protein 10; EMSA, electrophoretic mobility shift assay; TAD, transactivation domain; GST, glutathione S-transferase. results shown are typical of at least three independent experiments. This paper is available on line at http://www.jbc.org 3863 This is an Open Access article under the CC BY license. 3864 IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF Northern Analyses—The cells were stimulated with either IL-1 or TNF for 4 h or, where indicated, preincubated with LY 294002 for 30 min prior to stimulation with IL-1 or TNF for 4 h. Total RNA was isolated using the TRIzol reagent (Invitrogen). RNA was fractionated by electrophoresis on a formaldehyde gel and transferred to Hybond-N, a positively charged nylon membrane, according to the procedures pro- vided by Amersham Biosciences, Inc. cDNA probes for murine IL-6, murine interferon-inducible protein 10 (IP10), and human glyceralde- hyde-3-phosphate dehydrogenase mRNAs were made using the random priming kit from Amersham Biosciences, Inc. Probe hybridization and washing were performed according to procedures provided by Amer- sham Biosciences, Inc., and signals were visualized by autoradiogra- phy. The murine probes for IL-6 and IP10 were kindly provided by Dr. Thomas Hamilton (Cleveland Clinic Foundation). Transfection and Reporter Assay—The NF-B-dependent reporter plasmid p5XIP10B, a kind gift of Dr. Bryan Williams (Cleveland Clinic Foundation), contains five tandem copies of the NF-B site from the IP10 gene. For reporter assays, wild-type and IKK- and IKK-null MEFs were stably transfected using Lipofectin (Invitrogen) with 10 g of p5XIP10B and 1 g of pBABEPuro. Pools of stably transfected cells were selected with and maintained in puromycin. In separate experi- ments, the NF-B reporter cells were transiently transfected using Lipofectin with 0.5 g of pSV2-gal and 5 g of either vector or con- struct expressing wild-type or lipid phosphatase-deficient mutants of PTEN, kindly provided by Dr. Kenneth Yamada (26); constitutively PI3K activated p110 , kindly provided by Drs. Doreen Cantrell and Karin Reif (27); constitutively activated AKT, kindly provided by Dr. Julian Downward (28); wild-type p65/RelA, kindly provided by Dr. Dean Bal- lard (29); wild-type IKK, kindly provided by Dr. David Donner (25); or FIG.1. IKK- and IKK-null MEFs are both deficient in IL-1- wild-type IKK, kindly provided by Dr. Zhaodan Cao (30). Cells were and TNF-stimulated induction of NF-B-dependent transcrip- divided into the appropriate number of plates for treatment 8 h follow- tion and promoter activation. A, transcription. Cells were stimu- ing transfection. After 24 h, the cells were harvested. The cells were lated with IL-1 (IL) or TNF (T) for 4 h, and total RNA was isolated. stimulated with either IL-1 or TNF for 4 h or, where indicated, prein- Equal amounts of RNA were electrophoresed and subjected to Northern cubated with LY 294002 for 30 min prior to stimulation with IL-1 or analysis with probes for murine IL-6 and IP10 and human glyceralde- TNF for 4 h. Luciferase or galactosidase activity was determined with hyde-3-phosphate dehydrogenase (GAPDH). B, reporter assay. Wild- the luciferase assay system or chemiluminescent reagents (both from type (WT) and IKK- and IKK-null MEFs, stably transfected with an Promega, Madison, WI). Luciferase activity was normalized to -galac- NF-B-dependent luciferase reporter construct containing five copies of tosidase activity to control for transfection efficiency. The viability of the NF-B consensus site from the IP10 gene, were either unstimulated each transfected cell population was measured at the time of harvesting or stimulated with IL-1 or TNF for 4 h and lysed. Equal amounts of by trypan blue exclusion. protein were assayed for luciferase activity. C, control. Gel Electrophoretic Mobility Shift Assays—For electrophoretic mobil- ity shift assays (EMSAs), where indicated, the cells were preincubated with LY 294002 prior to stimulation with IL-1 or TNF for 20 min. The M ATP, 20 mM -glycerophosphate, 20 mM disodium p-nitrophenyl NF-B-binding site (5-GAGCAGAGGGAAATTCCGTAACTT-3) from phosphate, 0.1 mM sodium orthovanadate, 3 Ci of [- P]ATP, and 10 the IP10 gene was used as a probe. Briefly, complementary oligonucleo- mM reduced glutathione). The cells were stimulated with either IL-1 or tides, end-labeled with polynucleotide kinase and [- P]ATP, were TNF for 20 min or, where indicated, preincubated with LY 294002 for annealed by slow cooling. Approximately 20,000 cpm of probe were used 30 min prior to stimulation with IL-1 or TNF for 20 min. Nuclear and per reaction mixture. Nuclear and cytoplasmic extracts were prepared cytoplasmic extracts were prepared as described previously (31); or the in binding reaction buffer as described previously (31). The binding cells were lysed, and anti-IKK antibody was used to immunoprecipi- reaction was carried out with nuclear extracts containing equal tate the IKK complex from each sample. In vitro phosphorylation was amounts of protein at room temperature for 30 min in a total volume of performed using 1 g of the p65 TAD/GST or IB-(1–54)/GST fusion 20 l. The DNANF-B complexes were separated on 5% polyacryl- protein as a substrate with either cytoplasmic or nuclear extracts (3 g amide gels by electrophoresis in low ionic strength Tris borate/EDTA of protein) or the immunoprecipitated IKK complex as the kinase in buffer. The gels were dried, and the labeled complexes were visualized kinase buffer at 30 °C for 30 min (12). Following the kinase reaction, by autoradiography. phosphorylation of either substrate was analyzed by SDS-PAGE, fol- Immunoblotting and Immunoprecipitation—Cells were washed once lowed by autoradiography. In separate experiments, wild-type MEFs with phosphate-buffered saline and lysed for 30 min at 4 °Cin1mlof were transiently transfected using Lipofectin with 5 g of either vector 0.5% Nonidet P-40 lysis buffer as described previously (32). Cellular or construct expressing constitutively activated AKT (28). The trans- debris was removed by centrifugation at 16,000  g for 15 min. For fected cells were divided into three plates for treatment 8 h following immunoblotting, cell extracts were fractionated directly by SDS-PAGE transfection. After 24 h, the cells were stimulated with either IL-1 or and transferred to nitrocellulose membranes. Immunoblot analysis was TNF for 20 min. The cells were lysed, and anti-IKK antibody was used performed with the indicated primary antibodies, which were visual- to immunoprecipitate the IKK complex from each sample. In vitro ized with horseradish peroxidase-coupled goat anti-rabbit or anti- phosphorylation of the p65 TAD/GST fusion protein was performed as mouse immunoglobulins using the ECL Western blotting detection described above with the immunoprecipitated IKK complex. system (PerkinElmer Life Sciences). For immunoprecipitations, cell extracts were incubated with 3 g of primary antibody for 4 h, followed RESULTS by incubation for 1 h with 50 l of protein A-Sepharose beads (50% IL-1- and TNF-induced NF-B-dependent Transcription Is suspension). The beads were washed three times with lysis buffer, and Deficient in Both IKK- and IKK-null MEFs, but Only IKK- samples were analyzed by SDS-PAGE and autoradiography. null MEFs Are Deficient in NF-B Liberation and Nuclear Analysis of the Phosphorylation of the p65 Transactivation Domain and IB-(1–54)—A fragment of p65 (residues 354 –551) representing Translocation—The complete activation of NF-B requires two the transactivation domain (TAD) was used (12). The IB fragment pathways leading to the liberation of NF-B and to the activa- (residues 1–54) was kindly provided by Dr. Joseph DiDonato (Cleveland tion of the transcription function of NF-B. Studies from our Clinic Foundation). These proteins were expressed as glutathione S- laboratory (13) and by Madrid et al. (16, 34) have demonstrated transferase (GST) fusions in bacteria and purified using glutathione- a critical role of the PI3K/AKT pathway in activating NF-Bin agarose beads after sonication at 4 °C in 0.5% Nonidet P-40 lysis buffer response to the pro-inflammatory cytokines IL-1 and TNF by (33). The GST fusion proteins were eluted from the beads in kinase buffer (20 mM HEPES (pH 7.6), 20 mM MgCl ,2mM dithiothreitol, 20 phosphorylation of the carboxyl-terminal transactivation do- 2 IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF 3865 FIG.2. IL-1- and TNF-stimulated phosphorylation and activation of RSK AKT, ERK, JNK, p90 , and p38 are intact in both IKK- and IKK-null MEFs. Cells were exposed to either IL-1 or TNF for the times indicated. Total cell extracts were prepared, and equal amounts of protein were analyzed by SDS-PAGE and Western blotting for acti- vated, phosphorylated AKT (pAKT), ERK RSK (pERK), JNK (pJNK), p90 (pRSK), and p38 (pp38). No defects were detected in any of these pathways in IKK-or IKK-null MEFs. WT, wild-type MEFs. main of p65. IKK-null MEFs were reported to be deficient in NF-B-dependent transcription following stimulation with IL-1 and TNF (21), even though IKK seemed to be dispensable for cytokine-induced IB degradation and NF-B nuclear translocation (19 –24). We have now investigated the roles of both IKK and IKK in the activation of NF-B through the PI3K/AKT pathway. Both IKK- and IKK-null MEFs were deficient in IL-1- and TNF-stimulated induction of the NF-B- dependent endogenous genes IL-6 and IP10 (Fig. 1A). Both were also deficient in activating an NF-B-dependent reporter (Fig. 1B), confirming that the defects are at the level of NF-B function. To explore why IL-1 and TNF fail to activate NF-Bin IKK- and IKK-null MEFs, we investigated known pathways leading to NF-B activation and the signaling deficiencies re- sponsible for causing the null MEFs to be unresponsive to cytokine stimulation. Wild-type and IKK- and IKK-null MEFs were exposed to either IL-1 or TNF, and total cell ex- tracts were analyzed for phosphorylated AKT, ERK, JNK, RSK p90 , and p38. No defects in the induction of these kinase pathways by IL-1 and TNF were detected (Fig. 2). Interest- FIG.3. IKK-null MEFs (but not IKK-null MEFs) are substan- ingly, the down-regulation of these kinase activities appeared tially deficient in IL-1- and TNF-stimulated IB degradation, to be more sustained in IKK- and IKK-null MEFs compared p65 nuclear translocation, and NF-B DNA binding. A,IB with wild-type MEFs (Fig. 2). The Western blots were stripped degradation. Cells were exposed to either IL-1 or TNF for the times indicated, and total cell extracts were prepared. Equal amounts of and reprobed to determine total levels of each of the above protein were analyzed by SDS-PAGE and Western blotting. B, EMSAs. kinases, which were the same in the different cell lines (data Cells were exposed to either IL-1 (IL) or TNF (T) for 20 min, and nuclear not shown). The data of Fig. 2 demonstrate that the induction extracts were prepared. Equal amounts of each extract were analyzed of these kinase pathways was completely intact in response to by EMSA for the ability of NF-B to bind to a labeled NF-B consensus IL-1 and TNF in both IKK- and IKK-null MEFs. However, site from the IP10 gene. WT, wild-type MEFs; C, control. IKK-null MEFs were deficient in cytokine-stimulated IB degradation. In contrast, IKK-null MEFs were as efficient as TAD/GST). The results of this experiment indicated that the wild-type MEFs in degrading IB in response to IL-1 and TNF majority of the IL-1- and TNF-induced kinase activity for this (Fig. 3A). There were no substantial defects in the ability of substrate resided in the cytoplasmic fraction of wild-type MEFs NF-B to translocate to the nucleus and to bind to DNA follow- and was absent in both IKK- and IKK-null MEFs (data not ing IL-1 or TNF stimulation in IKK-null MEFs, but IKK-null shown). In view of these results, we assayed the ability of MEFs were deficient in cytokine-stimulated NF-B nuclear immunoprecipitated IKK complexes from wild-type and IKK- translocation and DNA binding (Fig. 3B). and IKK-null MEFs to phosphorylate p65 TAD/GST. The cells IKK- and IKK-null MEFs Are Both Deficient in IL-1- and were treated with IL-1 or TNF and, where indicated, incubated TNF-stimulated Phosphorylation of the Transactivation Do- with LY 294002 before treatment to inhibit the cytokine-stim- main of the p65 Subunit of NF-B, but Only IKK-null MEFs ulated PI3K/AKT pathway. The composition of the immunopre- Are Deficient in IL-1- and TNF-stimulated Phosphorylation of cipitated IKK complex from each cell type is shown in Fig. 4A. IB—Although there was no substantial defect in the degra- The IKK complex from IKK-null MEFs was unable to phos- dation of IB or in the nuclear translocation and DNA binding phorylate p65 TAD/GST efficiently (Fig. 4B, upper panel). of NF-BinIKK-null MEFs (Fig. 3), these cells were deficient Phosphorylation of p65 TAD/GST by the wild-type IKK com- in IL-1- and TNF-stimulated activation of NF-B-dependent plex depends on activation of the PI3K/AKT pathway by IL-1 endogenous genes and an NF-B-dependent reporter construct and TNF, as preincubation with LY 294002 almost completely (Fig. 1). We analyzed of the ability of cytoplasmic and nuclear blocked phosphorylation in wild-type MEFs (Fig. 4B, upper extracts from wild-type and IKK- and IKK-null MEFs ex- panel). Interestingly, IKK-null MEFs were also deficient in posed to either IL-1 or TNF to phosphorylate a GST fusion phosphorylating p65 TAD/GST (Fig. 4B, upper panel). How- protein containing the transactivation domain of p65 (p65 ever, the IKK complex from IKK-null MEFs was not defective 3866 IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF FIG.4. The IKK complex from IKK- and IKK-null MEFs is deficient in both the IL-1- and TNF-stimulated phosphorylation of the transactivation domain of the p65 subunit of NF-B, but only the IKK complex from IKK-null MEFs is defective in phosphorylating IB. Cells were stimulated with IL-1 (IL) or TNF (T) for 20 min or, where indicated, preincubated with LY 294002 (LY) for 30 min prior to stimulation with IL-1 or TNF for 20 min. Cells were lysed, and anti-IKK antibody was used to immunoprecipitate the IKK complex from each sample. A, Western analysis of the composition of the IKK complex from each cell type. B, in vitro phosphorylation. Approximately 1 gofp65 TAD/GST (upper panel)orIB-(1–54)-GST (lower panel) was used as substrate, and the immunoprecipitated IKK complex was the kinase. Following the kinase reaction, phosphorylation of p65 TAD/GST was analyzed by SDS-PAGE, followed by autoradiography. C, in vitro phospho- rylation of p65 TAD/GST. Wild-type MEFs (WT) were transiently transfected with 5 g of either vector or activated AKT construct. After 8 h, transfected cells were divided into three plates each. After 24 h, each transfected sample was left unstimulated or was stimulated with either IL-1 or TNF for 20 min. Cells were lysed; anti-IKK antibody was used to immunoprecipitate the IKK complex from each sample; and the p65 TAD in vitro kinase assay was performed with the IKK complexes as described for B. The phosphorylation of p65 TAD/GST was analyzed by SDS-PAGE, followed by autoradiography. C, control. in the IL-1- and TNF-stimulated phosphorylation of IB-(1– indicate a separate, essential function of IKK, distinct from 54)/GST, as was the IKK complex from IKK-null MEFs (Fig. that of IKK, in the activation of NF-B. 4B, lower panel) (21). Phosphorylation of IB-(1–54)/GST does IL-1- and TNF-dependent Activation of NF-B through the not depend on activation of the PI3K/AKT pathway by IL-1 and PI3K/AKT Pathway Requires IKK Independently of IB TNF, as preincubation with LY 294002 had no effect on the Degradation—We tested the ability of constitutively activated phosphorylation of this substrate by the IKK complex (Fig. 4B, PI3K and AKT to enhance IL-1- and TNF-stimulated NF-B- lower panel). To test whether IKK is capable of phosphoryl- dependent promoter activation in wild-type and IKK- and ating p65 in AKT-activated cells, the effect of overexpressing IKK-null MEFs. These cells, stably transfected with the NF- constitutively activated AKT on IKK phosphorylation of p65 B-dependent luciferase reporter construct p5XIP10B, were TAD/GST was investigated. Wild-type MEFs were transiently transiently transfected with vector alone, activated p110, acti- transfected with either vector alone or activated AKT. 8 h after vated AKT, or wild-type p65. 8 h after transfection, the cells transfection, the cells were divided into three plates each. After were divided into three plates each. After 24 h, the cells were 24 h, the cells were left unstimulated or were stimulated with left unstimulated or were stimulated with IL-1 (Fig. 5A)or IL-1 or TNF for 20 min, and the immunoprecipitated IKK TNF (Fig. 5B) for 4 h and assayed for luciferase activity. Trans- complex from each sample was assayed for p65 TAD kinase fection of wild-type p65 weakly increased both IL-1-induced activity by the in vitro kinase assay. Phosphorylation of p65 (IKK  5.4-fold and IKK  3.8-fold) and TNF-induced TAD/GST by the wild-type IKK complex was highly induced by (IKK  3.9-fold and IKK  3.0-fold) NF-B-dependent pro- activated AKT compared with the vector-transfected control moter activation in IKK- and IKK-null MEFs, but not nearly and could not be significantly further induced by either IL-1 or as well as in wild-type MEFs (IL-1  23.9-fold and TNF TNF (Fig. 4C). Therefore, one reason that IKK-null MEFs are 17.9-fold), over their respective untreated p65-transfected con- unable to activate NF-B in response to IL-1 and TNF is trols (Fig. 5, A and B). Transfection of p65 alone with no because they fail to phosphorylate and activate the p65 subunit cytokine treatment resulted in an 8-fold increase in wild-type of NF-B in response to cytokine-stimulated PI3K and AKT. MEFs, a 3-fold increase in IKK-null MEFs, and a 2-fold in- Our data also indicate a role for IKK in this pathway in crease in IKK-null MEFs over their respective vector-trans- addition to its role in phosphorylating IB. Only reconstitution fected controls (data not shown), indicating that both basal and with IKK (and not IKK) can restore cytokine-stimulated cytokine-induced p65-dependent transactivation is diminished NF-B-dependent promoter and endogenous gene activation in in both IKK- and IKK-null MEFs compared with wild-type IKK-null MEFs (data not shown). All together, these data MEFs. However, constitutively activated PI3K and AKT both IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF 3867 B-dependent luciferase reporter p5XIP10B. The cells were transiently transfected with either vector or wild-type PTEN. After 48 h, cells were preincubated with either vehicle or LY 294002 for 30 min prior to stimulation with IL-1 or TNF, and luciferase activity was measured (Fig. 6D). Blockade of IL-1- and TNF-induced PI3K activity with either wild-type PTEN or LY 294002 inhibited both IL-1 and TNF stimulation of the NF-B-dependent promoter. We also compared the effects of wild-type PTEN with those of three different mutants that lack lipid phosphatase activity. Only wild-type PTEN suppressed NF-B activation (data not shown). Therefore, the ability of PTEN to suppress NF-B-dependent transcription driven by IL-1 and TNF depends on its lipid phosphatase activity. These data confirm our previous results showing that the PI3K/AKT pathway functions to stimulate p65 activation independently of the degradation of IB and activation of the ability of NF-B to bind to DNA (13). DISCUSSION IKK and IKK Are Both Required for PI3K- and AKT- mediated Activation of NF-B in Response to IL-1 and TNF—We have investigated the roles of IKK and IKK in the FIG.5. Constitutively activated PI3K and AKT are unable to activation of NF-B through the PI3K/AKT pathway in re- enhance IL-1- or TNF-stimulated activation of a reporter gene sponse to IL-1 and TNF. IKK is required for the PI3K/AKT in the absence of either IKK or IKK. Wild-type (WT) and IKK- pathway to phosphorylate and transactivate the p65 subunit of and IKK-null MEFs, stably transfected with an NF-B-dependent luciferase reporter construct controlled by five copies of the NF-B NF-B. In addition to the role of IKK as the IB kinase, it consensus site from the IP10 gene, were transiently transfected with 5 also plays a role in the PI3K/AKT-mediated phosphorylation g of either vector (Vec) or construct encoding activated p110, activated and activation of p65. Our previous findings indicated that AKT, or the p65 subunit of NF-B plus 0.5 g of pSV2-gal. After 8 h, activation of the PI3K/AKT pathway is not sufficient to activate transfected cells were divided into three plates each. After 24 h, each transfected sample was left unstimulated or was stimulated with either NF-B-dependent transcription, but is necessary for IL-1 and IL-1 (A) or TNF (B) for 4 h and lysed. Equal amounts of protein were TNF to activate NF-B together with the pathway that leads to assayed for luciferase and -galactosidase activities. Luciferase activity IB degradation and nuclear translocation of NF-B (13). Our was normalized to -galactosidase activity to control for transfection present results (Figs. 4B, lower panel; and 6) agree with these efficiency. Data are expressed as the fold induction, the ratio of the findings and with the studies of others, indicating that the luciferase activity of each IL-1- or TNF-treated transfected sample to the same activity of the untreated transfected control. The data shown IL-1- and TNF-stimulated PI3K/AKT pathway functions to are representative of at least three separate independent experiments. stimulate the transactivation potential of NF-B and does not participate in the pathway leading to IB phosphorylation, failed to enhance IL-1- or TNF-stimulated NF-B-dependent IB degradation, and NF-B liberation (13, 16, 34). We also promoter activation in IKK- and IKK-null MEFs in contrast demonstrated that the endogenous antagonist of the PI3K/AKT to wild-type MEFs (Fig. 5, A and B). As we reported previously pathway, the tumor suppressor PTEN, inhibits the IL-1- and (13), neither activated PI3K nor AKT can induce NF-B acti- TNF-stimulated activation of NF-B in a manner that depends vation alone, presumably because a second signal is needed for on its lipid phosphatase function (Fig. 6 and data not shown). IB degradation. As there was no defect in the cytokine- Our finding that wild-type PTEN is able to inhibit NF-B-de- stimulated phosphorylation and degradation of IB in IKK- pendent transcriptional activation agrees with two previous null MEFs, a major role of IKK in cytokine-dependent signal- reports. In one, PTEN suppressed both the activation of IKK ing must be in the phosphorylation and activation of the p65 and the ability of NF-B to bind to DNA (35), whereas in the subunit of NF-B. Blockade of IL-1- and TNF-induced PI3K second, PTEN affected the ability of NF-B to bind to DNA by activity with LY 294002 inhibited IL-1- and TNF-stimulated inhibiting the phosphorylation of the p50 subunit of NF-B induction of IL-6 and IP10 mRNAs (Fig. 6A) as well as NF-B- independently of IB degradation (18). We found no substan- dependent promoter activation (Fig. 6D). Despite dramatically tial difference in IL-1- and TNF-stimulated NF-B DNA bind- inhibiting NF-B-dependent endogenous gene transcription ing between wild-type and IKK-null MEFs (Fig. 3B). How- and promoter activation (Fig. 6, A and D), neither IL-1- or ever, the total levels of induced NF-B DNA binding in IKK- TNF-induced IB degradation (Fig. 6B, upper panel) nor null MEFs are 2-fold lower than those in wild-type MEFs NF-B DNA binding (Fig. 6C) was affected by inhibiting PI3K. (Fig. 3B). This may be due to reduced phosphorylation of p50 in However, the phosphorylation and activation of AKT in re- these cells and remains to be investigated. Neither constitu- sponse to IL-1 and TNF were dramatically inhibited by PI3K tively activated PI3K nor AKT is able to enhance the activation blockade (Fig. 6B, lower panel). The IL-1- and TNF-stimulated of NF-B by IL-1 or TNF in either IKK-orIKK-null MEFs RSK phosphorylation of ERK, JNK, p90 (Fig. 5). These data indicate that both IKK and IKK are , and p38 was unaffected by pretreatment with LY 294002 (data not shown). The pro- required for the PI3K/AKT pathway to activate NF-B. duction of the phospholipid second messenger phosphatidyl- IKK, Unlike IKK, Is Dispensable for NF-B Liberation, but inositol 3,4,5-trisphosphate by PI3K could be inhibited directly Both Are Required for the PI3K/AKT-mediated Phosphoryla- by its endogenous antagonist, the tumor suppressor PTEN, a tion of p65—Targeted gene disruption studies have demon- dual-specificity phosphatase that dephosphorylates phosphati- strated that IKK (but not IKK) is largely responsible for dylinositol 3,4,5-trisphosphate. We explored the effects of in- cytokine-induced IB degradation and translocation of NF-B hibiting IL-1- and TNF-stimulated PI3K activity by LY 294002 to the nucleus in response to IL-1 and TNF. In this respect, the as well as by expressing PTEN on NF-B-dependent promoter failure of PI3K and AKT to activate NF-BinIKK-null MEFs activation in wild-type MEFs stably transfected with the NF- may be due in part to the defective liberation of NF-B in these 3868 IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF FIG.6. Inhibition of PI3K blocks NF-B-dependent transcription independently of NF-B liberation and DNA binding. A, analysis of IL-6 transcription. Wild-type MEFs were preincubated with either vehicle or 20 M LY 294002 (LY) for 30 min before being stimulated with IL-1 (IL) or TNF (T) for 4 h, and total RNA was isolated. Equal amounts of RNA were electrophoresed and subjected to Northern analysis with probes for murine IL-6 and IP10 and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, analysis of IB and phosphorylated AKT (pAkt). Cells were preincubated with either vehicle or 20 M LY 294002 for 30 min before stimulation with either IL-1 or TNF for 20 min. Total cell extracts were prepared, and equal amounts of protein were analyzed by SDS-PAGE and Western blotting. C, EMSAs. Cells were preincubated with either vehicle or 20 M LY 294002 for 30 min prior to stimulation with either IL-1 or TNF for 20 min, and nuclear extracts were prepared. Equal amounts of each extract were analyzed by EMSA for the binding of NF-BtoanNF-B consensus probe. D, analysis of the activation of an NF-B-dependent reporter. Wild-type MEFs stably transfected with an NF-B-dependent luciferase reporter construct were transiently transfected with 5 gof either vector (VEC) or construct encoding wild-type PTEN plus 0.5 g of pSV2-gal. After 24 h, each cell population was split into three plates. After an additional 24 h, cells were preincubated with either vehicle or 20 M LY 294002 for 30 min before being stimulated with IL-1 or TNF for 4 h. After lysis, equal amounts of protein were assayed for luciferase activity, which was normalized to -galactosidase activity to control for transfection efficiency. C, control. cells (Fig. 3). However, the diminished capacity of overex- activation of p38 and the other kinases observed in IKK- and pressed p65 to drive both basal and cytokine-induced NF-B IKK-null MEFs compared with wild-type MEFs is an inter- promoter activation in IKK-null MEFs also indicates a role of esting observation (Fig. 2). A recent report suggests that both IKK in the phosphorylation and activation of NF-B p65 (Fig. IKK- and p65-null MEFs demonstrate a sustained JNK acti- 5). Both IKK and IKK are required for IL-1 and TNF to vation in response to TNF compared with wild-type MEFs, activate NF-B-dependent transcription (Fig. 1). However, the contributing to TNF-induced apoptosis, which can be reversed IKK complex from IKK-null MEFs is not deficient in IB by overexpression of NF-B-induced XIAP (X-chromosome- phosphorylation (Fig. 4B, lower panel), IB degradation, or linked inhibitor of apoptosis) (38). A second report demon- NF-B liberation, but is deficient in activating NF-B-depend- strated that inhibition of NF-B activation by the super-repres- ent transcription in response to IL-1 and TNF (Figs. 1 and 3, A sor IB(A32/36) mutant elicits sustained JNK activation and B). There are no defects in the IL-1- or TNF-dependent without inhibiting MAPK phosphatase-1, a JNK phosphatase RSK phosphorylation and activation of AKT, ERK, JNK, p90 ,or (39). These reports suggest that NF-B target genes may down- p38 in either IKK-orIKK-null MEFs (Fig. 2). Our demon- regulate JNK activation. Our results indicate that inhibition of stration that the phosphorylation and activation of p38 in re- NF-B and NF-B target genes may have a broader impact on sponse to IL-1 are intact in IKK-null MEFs differs from a the down-regulation of the IL-1- and TNF-stimulated MAPKs recent report indicating that IL-1 activates NF-B by inducing and remain to be further investigated. Our studies demon- p38 activity in a manner dependent on AKT and IKK (34). We strate a novel role of IKK in the phosphorylation and activa- have no explanation for this discrepancy other than that the tion of p65 by the PI3K/AKT pathway. We have demonstrated IKK-null MEFs utilized in the study of Madrid et al. (34) were that the IKK complex from IKK-null MEFs is unable to phos- obtained from a different source (19). However, our demonstra- phorylate the transactivation domain of p65 efficiently (Fig. tion that the absence of IKK (Fig. 2) or inhibition of PI3K 4B, upper panel). The phosphorylation of the p65 transactiva- (data not shown) does not affect p38 phosphorylation and acti- tion domain by the IKK complex depends on the activation of vation in response to IL-1 and TNF, but does inhibit AKT the PI3K/AKT pathway by IL-1 and TNF, as preincubation activation and NF-B-dependent transcription (Fig. 6), indi- with LY 294002 almost completely blocked this phosphoryla- cates that cytokine-mediated p38 activation is independent of tion in wild-type MEFs (Fig. 4B, upper panel). Our study dem- the IKK and PI3K/AKT pathways. However, we cannot ex- onstrates that the IKK complex from IKK-null MEFs is also clude a separate role of p38 in the activation of NF-Bas deficient in phosphorylating the transactivation domain of p65 suggested by Madrid et al. (34) and corroborated by previous (Fig. 4B, upper panel). The deficiency of IKK-null MEFs in reports of NF-B modulation by p38 (36, 37). The sustained phosphorylating p65 is in agreement with the study of Madrid IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF 3869 J. Biol. Chem. 272, 32606 –32612 et al. (34), demonstrating that AKT targets the transactivation 8. Diehl, J. A., Tong, W., Sun, G., and Hannink, M. (1995) J. Biol. Chem. 270, function of p65 in a manner that is dependent on IKK, and 2703–2707 9. Martin, A. G., and Fresno, M. (2000) J. Biol. Chem. 275, 24383–24391 studies demonstrating the direct phosphorylation of p65 by 10. Martin, A. G., San Antonio, B., and Fresno, M. (2001) J. Biol. Chem. 276, IKK (12). Therefore, both IKK and IKK are required for the 15840 –15849 IL-1- and TNF-stimulated PI3K/AKT pathway to induce the 11. Naumann, M., and Scheidereit, C. (1994) EMBO J. 13, 4597– 4607 12. Sakurai, H., Chiba, H., Miyoshi, H., Sugita, T., and Toriumi, W. (1999) J. Biol. phosphorylation and transactivation potential of p65. How- Chem. 274, 30353–30356 ever, the inability of IKK to substitute for IKK in restoring 13. Sizemore, N., Leung, S., and Stark, G. R. (1999) Mol. Cell. Biol. 19, 4798 – 4805 NF-B activation to IKK-null MEFs (data not shown) indi- 14. Wang, D., and Baldwin, A. S. (1998) J. Biol. Chem. 273, 29411–29416 15. Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell 1, 661– 671 cates an essential function of IKK separate from that of IKK 16. Madrid, L. V., Wang, C. Y., Guttridge, D. C., Schottelius, A. J., Baldwin, A. S., in mediating the cytokine-stimulated activation of p65. Jr., and Mayo, M. W. (2000) Mol. Cell. Biol. 20, 1626 –1638 17. Tang, Y., Zhou, H., Chen, A., Pittman, R. N., and Field, J. (2000) J. Biol. Chem. Is the IB Kinase the PI3K- and AKT-activated p65 Ki- 275, 9106 –9109 nase?—Our data that the IKK complex from wild-type MEFs 18. Koul, D., Yao, Y., Abbruzzese, J. L., Yung, W. K., and Reddy, S. A. (2001) J. Biol. Chem. 276, 11402–11408 expressing activated AKT displays significantly increased ki- 19. Hu, Y., Baud, V., Delhase, M., Zhang, P., Deerinck, T., Ellisman, M., Johnson, nase activity for the p65 TAD (Fig. 4C) indicate that the IKK R., and Karin, M. (1999) Science 284, 316 –320 complex phosphorylates the p65 TAD in response to activated 20. Li, Q., Estepa, G., Memet, S., Israel, A., and Verma, I. M. (2000) Genes Dev. 14, 1729 –1733 AKT. We plan to investigate whether IKK and IKK require 21. Li, Q., Lu, Q., Hwang, J. Y., Buscher, D., Lee, K.-F., Izpisua-Belmonte, J. C., their kinase activities to stimulate the pathway leading to p65 and Verma, I. M. (1999) Genes Dev. 13, 1322–1328 activation. Reconstitution of IKK/IKK double knockout 22. Li, Q., Van Antwerp, D., Mercurio, F., Lee, K.-F., and Verma, I. M. (1999) Science 284, 321–325 MEFs with combinations of wild-type and kinase-dead mutants 23. 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Distinct Roles of the IκB Kinase α and β Subunits in Liberating Nuclear Factor κB (NF-κB) from IκB and in Phosphorylating the p65 Subunit of NF-κB

Sizemore, Nywana; Lerner, Natalia; Dombrowski, Nicole; Sakurai, Hiroaki; Stark, George R.
Journal of Biological Chemistry – Feb 1, 2002
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 6, Issue of February 8, pp. 3863–3869, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Distinct Roles of the IB Kinase  and  Subunits in Liberating B (NF-B) from IB and in Phosphorylating the p65 Nuclear Factor B* Subunit of NF- Received for publication, November 2, 2001, and in revised form, November 30, 2001 Published, JBC Papers in Press, December 3, 2001, DOI 10.1074/jbc.M110572200 Nywana Sizemore‡, Natalia Lerner‡, Nicole Dombrowski‡, Hiroaki Sakurai§, and George R. Stark‡¶ From the ‡Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the §Biology and Pharmacology Department, Discovery Research Laboratory, Tanabe Seiyaku Company Limited, Osaka 532-8505, Japan Phosphatidylinositol 3-kinase (PI3K) and the serine/ IB and the consequent liberation of NF-B are not sufficient to threonine kinase AKT have critical roles in phosphoryl- activate NF-B-dependent transcription, which also relies on a ating and transactivating the p65 subunit of nuclear second pathway, which leads to the stimulus-induced phospho- B (NF-B) in response to the pro-inflammatory factor rylation of the p65/RelA, RelB, and c-Rel subunits of NF-B cytokines interleukin-1 (IL-1) and tumor necrosis factor (6 –15). (TNF). Mouse embryo fibroblasts (MEFs) lacking either Our laboratory (13) and others (7, 14) have shown that the or  subunit of IB kinase (IKK) were deficient in the pro-inflammatory cytokines IL-1 and TNF induce the phospho- B-dependent transcription following treatment NF- rylation and activation of the p65 subunit of NF-B, a pathway -null with IL-1 or TNF. However, in contrast to IKK distinct from the one leading to IB degradation and NF-B -null MEFs were not substantially defective MEFs, IKK nuclear translocation. Additionally, phosphatidylinositol 3-ki- or in the in the cytokine-stimulated degradation of I nase (PI3K) and the serine/threonine kinase AKT play critical B. The IKK complexes nuclear translocation of NF- roles in this pathway (13, 16, 17). Recently, an additional -orIKK-null MEFs were both deficient in from IKK function for IL-1-stimulated PI3K/AKT activation has been PI3K-mediated phosphorylation of the transactivation reported: phosphorylation of the NF-B p50 subunit in re- B in response to IL-1 domain of the p65 subunit of NF- sponse to these kinases increases the DNA-binding capacity of and TNF, and constitutively activated forms of PI3K or the NF-B complex (18). AKT did not potentiate cytokine-stimulated activation Targeted gene disruptions have demonstrated that IKK B in either IKK-orIKK-null MEFs. Collec- of NF- (but not IKK) is largely responsible for cytokine-induced IB tively, these data indicate that, in contrast to IKK B liberation and p65 degradation and NF-B nuclear translocation (19 –24). How- which is required for both NF- is required solely for the cyto- ever, IKK-null mouse embryo fibroblasts (MEFs) are deficient phosphorylation, IKK kine-induced phosphorylation and activation of the p65 in inducing several NF-B-dependent mRNAs in response to B that are mediated by the PI3K/AKT subunit of NF- IL-1 and TNF (21). Activated AKT interacts with IKK upon pathway. cytokine stimulation and induces the phosphorylation of thre- onine 23 (25). These findings raise the interesting possibility that, although IKK is dispensable for IB degradation and The NF-B family of transcription factors consists of binary NF-B nuclear translocation, it may be required in the PI3K/ complexes of subunits with related promoter-binding and AKT pathway that leads to the phosphorylation and activation transactivation properties. The p65/RelA, RelB, and c-Rel sub- of NF-B. Therefore, we have investigated the roles of the IKK units stimulate transcription, whereas the p50 and p52 sub- and  subunits in the IL-1- and TNF-mediated phosphoryl- units serve primarily to bind to DNA (1). The prototypical ation and activation of the p65 subunit of NF-B. NF-B complex is the p65-p50 heterodimer (2). NF-Bisse- questered in a latent form in the cytoplasm through its inter- EXPERIMENTAL PROCEDURES action with the inhibitory IB proteins. In response to signals, Biological Reagents and Cell Culture—Recombinant human IL-1 IB kinase is activated, and IB is phosphorylated and de- was from NCI, National Institutes of Health. Recombinant human TNF graded, releasing NF-B, which enters the nucleus and binds to was from Preprotech (Rocky Hill, NJ). LY 294002 was from Sigma. Polyclonal anti-IKK, anti-IKK, anti-IKK, anti-p65/RelA, and anti- DNA (2–5). However, the phosphorylation and degradation of IB antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho- * The costs of publication of this article were defrayed in part by the AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho- payment of page charges. This article must therefore be hereby marked RSK RSK p90 , anti-p90 , anti-phospho-JNK1, and anti-JNK antibodies “advertisement” in accordance with 18 U.S.C. Section 1734 solely to were from Cell Signaling Technologies (Beverly, MA). Protein A-Sepha- indicate this fact. rose and glutathione-agarose beads were from Amersham Biosciences, To whom correspondence should be addressed: Lerner Research Inc. (Buckinghamshire, United Kingdom). Wild-type and IKK- and Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH IKK-null MEFs, kindly provided by Dr. Inder Verma (21), were main- 44195. Tel.: 216-444-3900; Fax: 216-444-3279; E-mail: [email protected]. 1 tained in Dulbecco’s modified Eagle’s medium supplemented with 10% The abbreviations used are: NF-B, nuclear factor B; IL, interleu- fetal calf serum, 100 g/ml penicillin G, and 100 g/ml streptomycin. kin; TNF, tumor necrosis factor; PI3K, phosphatidylinositol 3-kinase; For all experiments, unless otherwise indicated, cells at 80% confluence IKK, IB kinase; MEFs, mouse embryo fibroblasts; MAPK, mitogen- on 100-mm dishes were preincubated with the PI3K inhibitor LY activated protein kinase; ERK, extracellular signal-regulated kinase; 294002 (20 M) for 30 min at 37 °C prior to stimulation with IL-1 (2 RSK, ribosomal S6 kinase; JNK, c-Jun NH -terminal kinase; IP10, ng/ml) or TNF (25 ng/ml) at 37 °C for the indicated time periods. All interferon-inducible protein 10; EMSA, electrophoretic mobility shift assay; TAD, transactivation domain; GST, glutathione S-transferase. results shown are typical of at least three independent experiments. This paper is available on line at http://www.jbc.org 3863 This is an Open Access article under the CC BY license. 3864 IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF Northern Analyses—The cells were stimulated with either IL-1 or TNF for 4 h or, where indicated, preincubated with LY 294002 for 30 min prior to stimulation with IL-1 or TNF for 4 h. Total RNA was isolated using the TRIzol reagent (Invitrogen). RNA was fractionated by electrophoresis on a formaldehyde gel and transferred to Hybond-N, a positively charged nylon membrane, according to the procedures pro- vided by Amersham Biosciences, Inc. cDNA probes for murine IL-6, murine interferon-inducible protein 10 (IP10), and human glyceralde- hyde-3-phosphate dehydrogenase mRNAs were made using the random priming kit from Amersham Biosciences, Inc. Probe hybridization and washing were performed according to procedures provided by Amer- sham Biosciences, Inc., and signals were visualized by autoradiogra- phy. The murine probes for IL-6 and IP10 were kindly provided by Dr. Thomas Hamilton (Cleveland Clinic Foundation). Transfection and Reporter Assay—The NF-B-dependent reporter plasmid p5XIP10B, a kind gift of Dr. Bryan Williams (Cleveland Clinic Foundation), contains five tandem copies of the NF-B site from the IP10 gene. For reporter assays, wild-type and IKK- and IKK-null MEFs were stably transfected using Lipofectin (Invitrogen) with 10 g of p5XIP10B and 1 g of pBABEPuro. Pools of stably transfected cells were selected with and maintained in puromycin. In separate experi- ments, the NF-B reporter cells were transiently transfected using Lipofectin with 0.5 g of pSV2-gal and 5 g of either vector or con- struct expressing wild-type or lipid phosphatase-deficient mutants of PTEN, kindly provided by Dr. Kenneth Yamada (26); constitutively PI3K activated p110 , kindly provided by Drs. Doreen Cantrell and Karin Reif (27); constitutively activated AKT, kindly provided by Dr. Julian Downward (28); wild-type p65/RelA, kindly provided by Dr. Dean Bal- lard (29); wild-type IKK, kindly provided by Dr. David Donner (25); or FIG.1. IKK- and IKK-null MEFs are both deficient in IL-1- wild-type IKK, kindly provided by Dr. Zhaodan Cao (30). Cells were and TNF-stimulated induction of NF-B-dependent transcrip- divided into the appropriate number of plates for treatment 8 h follow- tion and promoter activation. A, transcription. Cells were stimu- ing transfection. After 24 h, the cells were harvested. The cells were lated with IL-1 (IL) or TNF (T) for 4 h, and total RNA was isolated. stimulated with either IL-1 or TNF for 4 h or, where indicated, prein- Equal amounts of RNA were electrophoresed and subjected to Northern cubated with LY 294002 for 30 min prior to stimulation with IL-1 or analysis with probes for murine IL-6 and IP10 and human glyceralde- TNF for 4 h. Luciferase or galactosidase activity was determined with hyde-3-phosphate dehydrogenase (GAPDH). B, reporter assay. Wild- the luciferase assay system or chemiluminescent reagents (both from type (WT) and IKK- and IKK-null MEFs, stably transfected with an Promega, Madison, WI). Luciferase activity was normalized to -galac- NF-B-dependent luciferase reporter construct containing five copies of tosidase activity to control for transfection efficiency. The viability of the NF-B consensus site from the IP10 gene, were either unstimulated each transfected cell population was measured at the time of harvesting or stimulated with IL-1 or TNF for 4 h and lysed. Equal amounts of by trypan blue exclusion. protein were assayed for luciferase activity. C, control. Gel Electrophoretic Mobility Shift Assays—For electrophoretic mobil- ity shift assays (EMSAs), where indicated, the cells were preincubated with LY 294002 prior to stimulation with IL-1 or TNF for 20 min. The M ATP, 20 mM -glycerophosphate, 20 mM disodium p-nitrophenyl NF-B-binding site (5-GAGCAGAGGGAAATTCCGTAACTT-3) from phosphate, 0.1 mM sodium orthovanadate, 3 Ci of [- P]ATP, and 10 the IP10 gene was used as a probe. Briefly, complementary oligonucleo- mM reduced glutathione). The cells were stimulated with either IL-1 or tides, end-labeled with polynucleotide kinase and [- P]ATP, were TNF for 20 min or, where indicated, preincubated with LY 294002 for annealed by slow cooling. Approximately 20,000 cpm of probe were used 30 min prior to stimulation with IL-1 or TNF for 20 min. Nuclear and per reaction mixture. Nuclear and cytoplasmic extracts were prepared cytoplasmic extracts were prepared as described previously (31); or the in binding reaction buffer as described previously (31). The binding cells were lysed, and anti-IKK antibody was used to immunoprecipi- reaction was carried out with nuclear extracts containing equal tate the IKK complex from each sample. In vitro phosphorylation was amounts of protein at room temperature for 30 min in a total volume of performed using 1 g of the p65 TAD/GST or IB-(1–54)/GST fusion 20 l. The DNANF-B complexes were separated on 5% polyacryl- protein as a substrate with either cytoplasmic or nuclear extracts (3 g amide gels by electrophoresis in low ionic strength Tris borate/EDTA of protein) or the immunoprecipitated IKK complex as the kinase in buffer. The gels were dried, and the labeled complexes were visualized kinase buffer at 30 °C for 30 min (12). Following the kinase reaction, by autoradiography. phosphorylation of either substrate was analyzed by SDS-PAGE, fol- Immunoblotting and Immunoprecipitation—Cells were washed once lowed by autoradiography. In separate experiments, wild-type MEFs with phosphate-buffered saline and lysed for 30 min at 4 °Cin1mlof were transiently transfected using Lipofectin with 5 g of either vector 0.5% Nonidet P-40 lysis buffer as described previously (32). Cellular or construct expressing constitutively activated AKT (28). The trans- debris was removed by centrifugation at 16,000  g for 15 min. For fected cells were divided into three plates for treatment 8 h following immunoblotting, cell extracts were fractionated directly by SDS-PAGE transfection. After 24 h, the cells were stimulated with either IL-1 or and transferred to nitrocellulose membranes. Immunoblot analysis was TNF for 20 min. The cells were lysed, and anti-IKK antibody was used performed with the indicated primary antibodies, which were visual- to immunoprecipitate the IKK complex from each sample. In vitro ized with horseradish peroxidase-coupled goat anti-rabbit or anti- phosphorylation of the p65 TAD/GST fusion protein was performed as mouse immunoglobulins using the ECL Western blotting detection described above with the immunoprecipitated IKK complex. system (PerkinElmer Life Sciences). For immunoprecipitations, cell extracts were incubated with 3 g of primary antibody for 4 h, followed RESULTS by incubation for 1 h with 50 l of protein A-Sepharose beads (50% IL-1- and TNF-induced NF-B-dependent Transcription Is suspension). The beads were washed three times with lysis buffer, and Deficient in Both IKK- and IKK-null MEFs, but Only IKK- samples were analyzed by SDS-PAGE and autoradiography. null MEFs Are Deficient in NF-B Liberation and Nuclear Analysis of the Phosphorylation of the p65 Transactivation Domain and IB-(1–54)—A fragment of p65 (residues 354 –551) representing Translocation—The complete activation of NF-B requires two the transactivation domain (TAD) was used (12). The IB fragment pathways leading to the liberation of NF-B and to the activa- (residues 1–54) was kindly provided by Dr. Joseph DiDonato (Cleveland tion of the transcription function of NF-B. Studies from our Clinic Foundation). These proteins were expressed as glutathione S- laboratory (13) and by Madrid et al. (16, 34) have demonstrated transferase (GST) fusions in bacteria and purified using glutathione- a critical role of the PI3K/AKT pathway in activating NF-Bin agarose beads after sonication at 4 °C in 0.5% Nonidet P-40 lysis buffer response to the pro-inflammatory cytokines IL-1 and TNF by (33). The GST fusion proteins were eluted from the beads in kinase buffer (20 mM HEPES (pH 7.6), 20 mM MgCl ,2mM dithiothreitol, 20 phosphorylation of the carboxyl-terminal transactivation do- 2 IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF 3865 FIG.2. IL-1- and TNF-stimulated phosphorylation and activation of RSK AKT, ERK, JNK, p90 , and p38 are intact in both IKK- and IKK-null MEFs. Cells were exposed to either IL-1 or TNF for the times indicated. Total cell extracts were prepared, and equal amounts of protein were analyzed by SDS-PAGE and Western blotting for acti- vated, phosphorylated AKT (pAKT), ERK RSK (pERK), JNK (pJNK), p90 (pRSK), and p38 (pp38). No defects were detected in any of these pathways in IKK-or IKK-null MEFs. WT, wild-type MEFs. main of p65. IKK-null MEFs were reported to be deficient in NF-B-dependent transcription following stimulation with IL-1 and TNF (21), even though IKK seemed to be dispensable for cytokine-induced IB degradation and NF-B nuclear translocation (19 –24). We have now investigated the roles of both IKK and IKK in the activation of NF-B through the PI3K/AKT pathway. Both IKK- and IKK-null MEFs were deficient in IL-1- and TNF-stimulated induction of the NF-B- dependent endogenous genes IL-6 and IP10 (Fig. 1A). Both were also deficient in activating an NF-B-dependent reporter (Fig. 1B), confirming that the defects are at the level of NF-B function. To explore why IL-1 and TNF fail to activate NF-Bin IKK- and IKK-null MEFs, we investigated known pathways leading to NF-B activation and the signaling deficiencies re- sponsible for causing the null MEFs to be unresponsive to cytokine stimulation. Wild-type and IKK- and IKK-null MEFs were exposed to either IL-1 or TNF, and total cell ex- tracts were analyzed for phosphorylated AKT, ERK, JNK, RSK p90 , and p38. No defects in the induction of these kinase pathways by IL-1 and TNF were detected (Fig. 2). Interest- FIG.3. IKK-null MEFs (but not IKK-null MEFs) are substan- ingly, the down-regulation of these kinase activities appeared tially deficient in IL-1- and TNF-stimulated IB degradation, to be more sustained in IKK- and IKK-null MEFs compared p65 nuclear translocation, and NF-B DNA binding. A,IB with wild-type MEFs (Fig. 2). The Western blots were stripped degradation. Cells were exposed to either IL-1 or TNF for the times indicated, and total cell extracts were prepared. Equal amounts of and reprobed to determine total levels of each of the above protein were analyzed by SDS-PAGE and Western blotting. B, EMSAs. kinases, which were the same in the different cell lines (data Cells were exposed to either IL-1 (IL) or TNF (T) for 20 min, and nuclear not shown). The data of Fig. 2 demonstrate that the induction extracts were prepared. Equal amounts of each extract were analyzed of these kinase pathways was completely intact in response to by EMSA for the ability of NF-B to bind to a labeled NF-B consensus IL-1 and TNF in both IKK- and IKK-null MEFs. However, site from the IP10 gene. WT, wild-type MEFs; C, control. IKK-null MEFs were deficient in cytokine-stimulated IB degradation. In contrast, IKK-null MEFs were as efficient as TAD/GST). The results of this experiment indicated that the wild-type MEFs in degrading IB in response to IL-1 and TNF majority of the IL-1- and TNF-induced kinase activity for this (Fig. 3A). There were no substantial defects in the ability of substrate resided in the cytoplasmic fraction of wild-type MEFs NF-B to translocate to the nucleus and to bind to DNA follow- and was absent in both IKK- and IKK-null MEFs (data not ing IL-1 or TNF stimulation in IKK-null MEFs, but IKK-null shown). In view of these results, we assayed the ability of MEFs were deficient in cytokine-stimulated NF-B nuclear immunoprecipitated IKK complexes from wild-type and IKK- translocation and DNA binding (Fig. 3B). and IKK-null MEFs to phosphorylate p65 TAD/GST. The cells IKK- and IKK-null MEFs Are Both Deficient in IL-1- and were treated with IL-1 or TNF and, where indicated, incubated TNF-stimulated Phosphorylation of the Transactivation Do- with LY 294002 before treatment to inhibit the cytokine-stim- main of the p65 Subunit of NF-B, but Only IKK-null MEFs ulated PI3K/AKT pathway. The composition of the immunopre- Are Deficient in IL-1- and TNF-stimulated Phosphorylation of cipitated IKK complex from each cell type is shown in Fig. 4A. IB—Although there was no substantial defect in the degra- The IKK complex from IKK-null MEFs was unable to phos- dation of IB or in the nuclear translocation and DNA binding phorylate p65 TAD/GST efficiently (Fig. 4B, upper panel). of NF-BinIKK-null MEFs (Fig. 3), these cells were deficient Phosphorylation of p65 TAD/GST by the wild-type IKK com- in IL-1- and TNF-stimulated activation of NF-B-dependent plex depends on activation of the PI3K/AKT pathway by IL-1 endogenous genes and an NF-B-dependent reporter construct and TNF, as preincubation with LY 294002 almost completely (Fig. 1). We analyzed of the ability of cytoplasmic and nuclear blocked phosphorylation in wild-type MEFs (Fig. 4B, upper extracts from wild-type and IKK- and IKK-null MEFs ex- panel). Interestingly, IKK-null MEFs were also deficient in posed to either IL-1 or TNF to phosphorylate a GST fusion phosphorylating p65 TAD/GST (Fig. 4B, upper panel). How- protein containing the transactivation domain of p65 (p65 ever, the IKK complex from IKK-null MEFs was not defective 3866 IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF FIG.4. The IKK complex from IKK- and IKK-null MEFs is deficient in both the IL-1- and TNF-stimulated phosphorylation of the transactivation domain of the p65 subunit of NF-B, but only the IKK complex from IKK-null MEFs is defective in phosphorylating IB. Cells were stimulated with IL-1 (IL) or TNF (T) for 20 min or, where indicated, preincubated with LY 294002 (LY) for 30 min prior to stimulation with IL-1 or TNF for 20 min. Cells were lysed, and anti-IKK antibody was used to immunoprecipitate the IKK complex from each sample. A, Western analysis of the composition of the IKK complex from each cell type. B, in vitro phosphorylation. Approximately 1 gofp65 TAD/GST (upper panel)orIB-(1–54)-GST (lower panel) was used as substrate, and the immunoprecipitated IKK complex was the kinase. Following the kinase reaction, phosphorylation of p65 TAD/GST was analyzed by SDS-PAGE, followed by autoradiography. C, in vitro phospho- rylation of p65 TAD/GST. Wild-type MEFs (WT) were transiently transfected with 5 g of either vector or activated AKT construct. After 8 h, transfected cells were divided into three plates each. After 24 h, each transfected sample was left unstimulated or was stimulated with either IL-1 or TNF for 20 min. Cells were lysed; anti-IKK antibody was used to immunoprecipitate the IKK complex from each sample; and the p65 TAD in vitro kinase assay was performed with the IKK complexes as described for B. The phosphorylation of p65 TAD/GST was analyzed by SDS-PAGE, followed by autoradiography. C, control. in the IL-1- and TNF-stimulated phosphorylation of IB-(1– indicate a separate, essential function of IKK, distinct from 54)/GST, as was the IKK complex from IKK-null MEFs (Fig. that of IKK, in the activation of NF-B. 4B, lower panel) (21). Phosphorylation of IB-(1–54)/GST does IL-1- and TNF-dependent Activation of NF-B through the not depend on activation of the PI3K/AKT pathway by IL-1 and PI3K/AKT Pathway Requires IKK Independently of IB TNF, as preincubation with LY 294002 had no effect on the Degradation—We tested the ability of constitutively activated phosphorylation of this substrate by the IKK complex (Fig. 4B, PI3K and AKT to enhance IL-1- and TNF-stimulated NF-B- lower panel). To test whether IKK is capable of phosphoryl- dependent promoter activation in wild-type and IKK- and ating p65 in AKT-activated cells, the effect of overexpressing IKK-null MEFs. These cells, stably transfected with the NF- constitutively activated AKT on IKK phosphorylation of p65 B-dependent luciferase reporter construct p5XIP10B, were TAD/GST was investigated. Wild-type MEFs were transiently transiently transfected with vector alone, activated p110, acti- transfected with either vector alone or activated AKT. 8 h after vated AKT, or wild-type p65. 8 h after transfection, the cells transfection, the cells were divided into three plates each. After were divided into three plates each. After 24 h, the cells were 24 h, the cells were left unstimulated or were stimulated with left unstimulated or were stimulated with IL-1 (Fig. 5A)or IL-1 or TNF for 20 min, and the immunoprecipitated IKK TNF (Fig. 5B) for 4 h and assayed for luciferase activity. Trans- complex from each sample was assayed for p65 TAD kinase fection of wild-type p65 weakly increased both IL-1-induced activity by the in vitro kinase assay. Phosphorylation of p65 (IKK  5.4-fold and IKK  3.8-fold) and TNF-induced TAD/GST by the wild-type IKK complex was highly induced by (IKK  3.9-fold and IKK  3.0-fold) NF-B-dependent pro- activated AKT compared with the vector-transfected control moter activation in IKK- and IKK-null MEFs, but not nearly and could not be significantly further induced by either IL-1 or as well as in wild-type MEFs (IL-1  23.9-fold and TNF TNF (Fig. 4C). Therefore, one reason that IKK-null MEFs are 17.9-fold), over their respective untreated p65-transfected con- unable to activate NF-B in response to IL-1 and TNF is trols (Fig. 5, A and B). Transfection of p65 alone with no because they fail to phosphorylate and activate the p65 subunit cytokine treatment resulted in an 8-fold increase in wild-type of NF-B in response to cytokine-stimulated PI3K and AKT. MEFs, a 3-fold increase in IKK-null MEFs, and a 2-fold in- Our data also indicate a role for IKK in this pathway in crease in IKK-null MEFs over their respective vector-trans- addition to its role in phosphorylating IB. Only reconstitution fected controls (data not shown), indicating that both basal and with IKK (and not IKK) can restore cytokine-stimulated cytokine-induced p65-dependent transactivation is diminished NF-B-dependent promoter and endogenous gene activation in in both IKK- and IKK-null MEFs compared with wild-type IKK-null MEFs (data not shown). All together, these data MEFs. However, constitutively activated PI3K and AKT both IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF 3867 B-dependent luciferase reporter p5XIP10B. The cells were transiently transfected with either vector or wild-type PTEN. After 48 h, cells were preincubated with either vehicle or LY 294002 for 30 min prior to stimulation with IL-1 or TNF, and luciferase activity was measured (Fig. 6D). Blockade of IL-1- and TNF-induced PI3K activity with either wild-type PTEN or LY 294002 inhibited both IL-1 and TNF stimulation of the NF-B-dependent promoter. We also compared the effects of wild-type PTEN with those of three different mutants that lack lipid phosphatase activity. Only wild-type PTEN suppressed NF-B activation (data not shown). Therefore, the ability of PTEN to suppress NF-B-dependent transcription driven by IL-1 and TNF depends on its lipid phosphatase activity. These data confirm our previous results showing that the PI3K/AKT pathway functions to stimulate p65 activation independently of the degradation of IB and activation of the ability of NF-B to bind to DNA (13). DISCUSSION IKK and IKK Are Both Required for PI3K- and AKT- mediated Activation of NF-B in Response to IL-1 and TNF—We have investigated the roles of IKK and IKK in the FIG.5. Constitutively activated PI3K and AKT are unable to activation of NF-B through the PI3K/AKT pathway in re- enhance IL-1- or TNF-stimulated activation of a reporter gene sponse to IL-1 and TNF. IKK is required for the PI3K/AKT in the absence of either IKK or IKK. Wild-type (WT) and IKK- pathway to phosphorylate and transactivate the p65 subunit of and IKK-null MEFs, stably transfected with an NF-B-dependent luciferase reporter construct controlled by five copies of the NF-B NF-B. In addition to the role of IKK as the IB kinase, it consensus site from the IP10 gene, were transiently transfected with 5 also plays a role in the PI3K/AKT-mediated phosphorylation g of either vector (Vec) or construct encoding activated p110, activated and activation of p65. Our previous findings indicated that AKT, or the p65 subunit of NF-B plus 0.5 g of pSV2-gal. After 8 h, activation of the PI3K/AKT pathway is not sufficient to activate transfected cells were divided into three plates each. After 24 h, each transfected sample was left unstimulated or was stimulated with either NF-B-dependent transcription, but is necessary for IL-1 and IL-1 (A) or TNF (B) for 4 h and lysed. Equal amounts of protein were TNF to activate NF-B together with the pathway that leads to assayed for luciferase and -galactosidase activities. Luciferase activity IB degradation and nuclear translocation of NF-B (13). Our was normalized to -galactosidase activity to control for transfection present results (Figs. 4B, lower panel; and 6) agree with these efficiency. Data are expressed as the fold induction, the ratio of the findings and with the studies of others, indicating that the luciferase activity of each IL-1- or TNF-treated transfected sample to the same activity of the untreated transfected control. The data shown IL-1- and TNF-stimulated PI3K/AKT pathway functions to are representative of at least three separate independent experiments. stimulate the transactivation potential of NF-B and does not participate in the pathway leading to IB phosphorylation, failed to enhance IL-1- or TNF-stimulated NF-B-dependent IB degradation, and NF-B liberation (13, 16, 34). We also promoter activation in IKK- and IKK-null MEFs in contrast demonstrated that the endogenous antagonist of the PI3K/AKT to wild-type MEFs (Fig. 5, A and B). As we reported previously pathway, the tumor suppressor PTEN, inhibits the IL-1- and (13), neither activated PI3K nor AKT can induce NF-B acti- TNF-stimulated activation of NF-B in a manner that depends vation alone, presumably because a second signal is needed for on its lipid phosphatase function (Fig. 6 and data not shown). IB degradation. As there was no defect in the cytokine- Our finding that wild-type PTEN is able to inhibit NF-B-de- stimulated phosphorylation and degradation of IB in IKK- pendent transcriptional activation agrees with two previous null MEFs, a major role of IKK in cytokine-dependent signal- reports. In one, PTEN suppressed both the activation of IKK ing must be in the phosphorylation and activation of the p65 and the ability of NF-B to bind to DNA (35), whereas in the subunit of NF-B. Blockade of IL-1- and TNF-induced PI3K second, PTEN affected the ability of NF-B to bind to DNA by activity with LY 294002 inhibited IL-1- and TNF-stimulated inhibiting the phosphorylation of the p50 subunit of NF-B induction of IL-6 and IP10 mRNAs (Fig. 6A) as well as NF-B- independently of IB degradation (18). We found no substan- dependent promoter activation (Fig. 6D). Despite dramatically tial difference in IL-1- and TNF-stimulated NF-B DNA bind- inhibiting NF-B-dependent endogenous gene transcription ing between wild-type and IKK-null MEFs (Fig. 3B). How- and promoter activation (Fig. 6, A and D), neither IL-1- or ever, the total levels of induced NF-B DNA binding in IKK- TNF-induced IB degradation (Fig. 6B, upper panel) nor null MEFs are 2-fold lower than those in wild-type MEFs NF-B DNA binding (Fig. 6C) was affected by inhibiting PI3K. (Fig. 3B). This may be due to reduced phosphorylation of p50 in However, the phosphorylation and activation of AKT in re- these cells and remains to be investigated. Neither constitu- sponse to IL-1 and TNF were dramatically inhibited by PI3K tively activated PI3K nor AKT is able to enhance the activation blockade (Fig. 6B, lower panel). The IL-1- and TNF-stimulated of NF-B by IL-1 or TNF in either IKK-orIKK-null MEFs RSK phosphorylation of ERK, JNK, p90 (Fig. 5). These data indicate that both IKK and IKK are , and p38 was unaffected by pretreatment with LY 294002 (data not shown). The pro- required for the PI3K/AKT pathway to activate NF-B. duction of the phospholipid second messenger phosphatidyl- IKK, Unlike IKK, Is Dispensable for NF-B Liberation, but inositol 3,4,5-trisphosphate by PI3K could be inhibited directly Both Are Required for the PI3K/AKT-mediated Phosphoryla- by its endogenous antagonist, the tumor suppressor PTEN, a tion of p65—Targeted gene disruption studies have demon- dual-specificity phosphatase that dephosphorylates phosphati- strated that IKK (but not IKK) is largely responsible for dylinositol 3,4,5-trisphosphate. We explored the effects of in- cytokine-induced IB degradation and translocation of NF-B hibiting IL-1- and TNF-stimulated PI3K activity by LY 294002 to the nucleus in response to IL-1 and TNF. In this respect, the as well as by expressing PTEN on NF-B-dependent promoter failure of PI3K and AKT to activate NF-BinIKK-null MEFs activation in wild-type MEFs stably transfected with the NF- may be due in part to the defective liberation of NF-B in these 3868 IKK/IKK Mediate p65 Phosphorylation in Response to IL-1/TNF FIG.6. Inhibition of PI3K blocks NF-B-dependent transcription independently of NF-B liberation and DNA binding. A, analysis of IL-6 transcription. Wild-type MEFs were preincubated with either vehicle or 20 M LY 294002 (LY) for 30 min before being stimulated with IL-1 (IL) or TNF (T) for 4 h, and total RNA was isolated. Equal amounts of RNA were electrophoresed and subjected to Northern analysis with probes for murine IL-6 and IP10 and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, analysis of IB and phosphorylated AKT (pAkt). Cells were preincubated with either vehicle or 20 M LY 294002 for 30 min before stimulation with either IL-1 or TNF for 20 min. Total cell extracts were prepared, and equal amounts of protein were analyzed by SDS-PAGE and Western blotting. C, EMSAs. Cells were preincubated with either vehicle or 20 M LY 294002 for 30 min prior to stimulation with either IL-1 or TNF for 20 min, and nuclear extracts were prepared. Equal amounts of each extract were analyzed by EMSA for the binding of NF-BtoanNF-B consensus probe. D, analysis of the activation of an NF-B-dependent reporter. Wild-type MEFs stably transfected with an NF-B-dependent luciferase reporter construct were transiently transfected with 5 gof either vector (VEC) or construct encoding wild-type PTEN plus 0.5 g of pSV2-gal. After 24 h, each cell population was split into three plates. After an additional 24 h, cells were preincubated with either vehicle or 20 M LY 294002 for 30 min before being stimulated with IL-1 or TNF for 4 h. After lysis, equal amounts of protein were assayed for luciferase activity, which was normalized to -galactosidase activity to control for transfection efficiency. C, control. cells (Fig. 3). However, the diminished capacity of overex- activation of p38 and the other kinases observed in IKK- and pressed p65 to drive both basal and cytokine-induced NF-B IKK-null MEFs compared with wild-type MEFs is an inter- promoter activation in IKK-null MEFs also indicates a role of esting observation (Fig. 2). A recent report suggests that both IKK in the phosphorylation and activation of NF-B p65 (Fig. IKK- and p65-null MEFs demonstrate a sustained JNK acti- 5). Both IKK and IKK are required for IL-1 and TNF to vation in response to TNF compared with wild-type MEFs, activate NF-B-dependent transcription (Fig. 1). However, the contributing to TNF-induced apoptosis, which can be reversed IKK complex from IKK-null MEFs is not deficient in IB by overexpression of NF-B-induced XIAP (X-chromosome- phosphorylation (Fig. 4B, lower panel), IB degradation, or linked inhibitor of apoptosis) (38). A second report demon- NF-B liberation, but is deficient in activating NF-B-depend- strated that inhibition of NF-B activation by the super-repres- ent transcription in response to IL-1 and TNF (Figs. 1 and 3, A sor IB(A32/36) mutant elicits sustained JNK activation and B). There are no defects in the IL-1- or TNF-dependent without inhibiting MAPK phosphatase-1, a JNK phosphatase RSK phosphorylation and activation of AKT, ERK, JNK, p90 ,or (39). These reports suggest that NF-B target genes may down- p38 in either IKK-orIKK-null MEFs (Fig. 2). Our demon- regulate JNK activation. Our results indicate that inhibition of stration that the phosphorylation and activation of p38 in re- NF-B and NF-B target genes may have a broader impact on sponse to IL-1 are intact in IKK-null MEFs differs from a the down-regulation of the IL-1- and TNF-stimulated MAPKs recent report indicating that IL-1 activates NF-B by inducing and remain to be further investigated. Our studies demon- p38 activity in a manner dependent on AKT and IKK (34). We strate a novel role of IKK in the phosphorylation and activa- have no explanation for this discrepancy other than that the tion of p65 by the PI3K/AKT pathway. We have demonstrated IKK-null MEFs utilized in the study of Madrid et al. (34) were that the IKK complex from IKK-null MEFs is unable to phos- obtained from a different source (19). However, our demonstra- phorylate the transactivation domain of p65 efficiently (Fig. tion that the absence of IKK (Fig. 2) or inhibition of PI3K 4B, upper panel). The phosphorylation of the p65 transactiva- (data not shown) does not affect p38 phosphorylation and acti- tion domain by the IKK complex depends on the activation of vation in response to IL-1 and TNF, but does inhibit AKT the PI3K/AKT pathway by IL-1 and TNF, as preincubation activation and NF-B-dependent transcription (Fig. 6), indi- with LY 294002 almost completely blocked this phosphoryla- cates that cytokine-mediated p38 activation is independent of tion in wild-type MEFs (Fig. 4B, upper panel). Our study dem- the IKK and PI3K/AKT pathways. However, we cannot ex- onstrates that the IKK complex from IKK-null MEFs is also clude a separate role of p38 in the activation of NF-Bas deficient in phosphorylating the transactivation domain of p65 suggested by Madrid et al. (34) and corroborated by previous (Fig. 4B, upper panel). The deficiency of IKK-null MEFs in reports of NF-B modulation by p38 (36, 37). 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