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J. Biol. Chem., Vol. 279, Issue 36, 37452-37460, September 3, 2004
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-IKK
Subcomplexes in Monocytic Cells and Characterization of Associated Signaling*
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From the
Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technische Universität München, 81675 München, Germany and the ¶Department of Dermatology and Allergology, Biederstein, Technische Universität München, 80802 München, Germany
Received for publication, November 5, 2003 , and in revised form, June 25, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B kinase (IKK) complex is one major step in the regulation of the NF-B/Rel system that is involved in inflammatory and immune responses as well as in proliferation and apoptosis. At present it is not clear whether besides the "classical" IKK
-IKK
-IKK
configuration additional complexes exist in vivo that solely contain IKK and IKK (without IKK). In the current study we were able to demonstrate in monocytic cells that endogenous complexes, which only include IKK as the kinase-active molecule do indeed exist in vivo and that these complexes contain IKK as an additional component. Furthermore, we showed that these IKK-IKK complexes are involved in mainstream NF-B activation cascades because they can be activated by tumor necrosis factor. In contrast, these subcomplexes appear not to participate in NIK-dependent pathways. As a next step we showed that exogenous IKK-IKK complexes can be formed in an intact cell by overexpression and that these artificial complexes fulfill the requirement for participation in regular signaling. Finally, in the absence of IKK we found a retarded proteolysis of IB, but not of IB
, which is associated with a reduced IKK activity. Differential pathways represented by various IKK subcomplexes may open attractive possibilities in treatment of inflammation or cancer allowing specific therapeutic intervention. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B/Rel system is the IKK1 complex (1, 2). Activation of this high molecular weight assembly complex is achieved via signaling pathways induced by numerous molecules or conditions, e.g. cytokines like TNF, bacterial, and viral products as well as several forms of cellular stress (1, 3, 4). Such stimulation leads to the phosphorylation of proteins of the NF-B inhibitor family IB, for example IB, -, and -, which subsequently undergo ubiquitin-dependent proteasomal degradation (5–7). This frees the NF-B dimer, most commonly p50/p65 (RelA), which is trapped in an inactive state in the cytosol by IB (5–7). Following stimulation, NF-B translocates to the nucleus, where it binds to promoters or enhancers of genes, e.g. coding for cytokines, chemokines, adhesion molecules, proteases, and procoagulatory proteins involved in inflammatory and immune responses as well as in proliferation and apoptosis (7, 8). Differential signaling pathways leading to the activation of NF-B/Rel transcription factors as well as targeted gene expression are only partly understood and may be attractive targets for specific therapeutic approaches for inflammation or cancer (9–12).
The core IKK complex is composed of three subunits: IKK, IKK, and IKK (also called NF-B essential modulator, NEMO; IKK-associated protein 1, IKKAP1) (1, 2, 13, 14). IKK and IKK are the two kinase-active components of this complex. IKK is essential for activation of the IKK complex by various proinflammatory stimuli (15–18), whereas IKK appears to be involved in specific signaling associated with proliferation/differentiation (19–22). It should also be mentioned that it is now evident that IKK, although dispensable for IKK activation in response to proinflammatory stimuli, is essential for IKK activation by an alternative set of signals that do not affect the IKK subunit (1). Furthermore, IKK has been suggested to represent an adaptor or scaffold protein, which stabilizes the high molecular weight complex and/or regulates its kinase activity (1, 2, 23–26).
The upstream signaling pathways, which lead to the activation of the IKK complex, are still only partly understood (2, 27). A relatively well studied example is the TNF signaling pathway: Engagement of TNF with TNF receptor (TNFR) 1 results in the formation of a multiprotein signaling complex containing the cytosolic adaptor proteins TNFR-associated death domain protein, TNFR-associated factor 2, and receptor-interacting protein (3, 28, 29). These adaptor proteins in turn recruit IKK to the TNFR1 complex where it becomes activated, finally initiating downstream processes leading to NF-B activation (1, 28). Additional kinases have been suggested to activate the IKK complex such as TAK1, which is associated with Toll-like receptor/IL-1 receptor-associated signaling pathways (1, 30, 31). The IKK signaling event may also be mediated, dependent on the stimulus, by protein kinase R, mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1) or MEKK3 (1, 27). One of the first kinases proposed to activate the IKK complex was the NF-B-inducing kinase (NIK), but further studies indicated that it plays a function in specific pathways, e.g. processing of p100 and depends on the catalytic activities of downstream acting IKK (13, 32, 33).
Earlier studies indicate that IKK is a large complex with a size varying between 700 and 900 kDa, suggesting the presence of additional components (1, 13, 14, 34). One possibility that has been suggested is that some of the kinases or adaptors mentioned above in the previous paragraph, as well as additional signaling molecules, temporarily associate with the core complex (1). Alternatively, it may be possible that besides the "classical" components, different subunits are present as permanent components in the IKK complex. In this context it should be mentioned that two recent reports identified cell division control (Cdc) 37 and heat shock proteins (Hsp) 90 as well as 27 as further components of a TNF-inducible IKK complex (35, 36). In addition, it has been shown that Hsp70 is able to form a supramolecular structure with the IKK subunit (25).
At present it is not clear if besides the classical IKK-IKK-IKK configuration additional complexes exist in vivo that solely contain IKK and IKK (without IKK) as suggested earlier (24). Interestingly, in genetic experiments IKK–/– mice display a significant IKK activation in response to proinflammatory stimuli and also induction of NF-B DNA binding activity, presumably because of formation of artificial (since IKK is missing), but still functional IKK-IKK complexes (19–22). Recombinant IKK and IKK are able to form homo- or heterodimers, which suggests that similar IKK complexes may also exist in vivo (34). In addition, complete recombinant reconstitution of the IKK in yeast revealed that the core IKK complex is composed of 112, but also 22 (37).
The current study was designed to examine in vivo in monocytic cells whether endogenous complexes do indeed exist which solely contain IKK and no IKK and if these complexes contain IKK. If so, we wanted to investigate whether these IKK-IKK complexes participate in regular signaling and can be activated by certain stimuli, e.g. TNF, and to investigate which IB proteolysis patterns are induced during this activation.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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knockout murine embryonic fibroblast (MEF) cells (gift from Prof. Michael Karin and Dr. Antonio Rossi, University of California, San Diego, CA and the Institute of Neurobiology and Molecular Medicine, Rome, Italy) were cultured in DMEM high glucose medium with stable glutamine (PromoCell, Heidelberg, Germany) (7% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin). Endotoxin contamination was screened by the limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD). A potential toxicity of the cell culture conditions applied was monitored by cell morphology/count, trypan blue dye exclusion, and the WST-1 test (Roche Diagnostics, Mannheim, Germany).
PAGE and Western Blot Analysis—Cytosolic and nuclear extracts were isolated as described (4, 39), and electrophoresis was performed with 12.5% polyacrylamide gels. The proteins were transferred to nitrocellulose membranes using the wet blot technique. After transfer, the membranes were incubated with antibodies against IKK (Alexis Biochemicals, Carlsbad), IKK (Biocarta, San Diego), IKK (BD Biosciences, Palo Alto, CA), IB, IB, NIK (Santa Cruz Biotechnology, Heidelberg, Germany), phospho-IB (Calbiochem, Darmstadt, Germany), FLAG (Stratagene, Amsterdam, Netherlands), or actin (Sigma). This was followed by the appropriate horseradish peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany). The proteins were visualized on x-ray film using the Chemiluminescent Reagent Plus (PerkinElmer Life and Analytical Sciences). The obtained signals were analyzed densitometrically, and the values corrected for the loading control for each protein.
Immunoprecipitation—Cytosolic extracts were subjected to immunoprecipitation in TNT buffer (200 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1 mM dithiothreitol, 0.5 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride, leupeptin, antipain, aprotinin, pepstatin A, chymostatin 0.75 µg/ml each; Sigma) (4, 39). Immunoprecipitation was carried out using 35 µl of 6% protein A- or G-agarose (Roche Applied Science) for 2 h at 4 °C with 1 µg of anti-IKK (BD Biosciences), IKK (QED Bioscience), IKK (Santa Cruz Biotechnology), or anti-FLAG antibody. Specificity of the antibodies was tested by Western blot analyses after separating the IKK and - bands. After washing three times with TNT buffer and three times with kinase buffer (20 mM HEPES, pH 8.0, 10 mM MgCl2, 100 µM Na3VO4, 20 mM -glycerophosphate, 50 mM NaCl, 2 mM dithiothreitol, 0.5 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride, leupeptin, antipain, aprotinin, pepstatin A, chymostatin 0.75 µg/ml each) the precipitated proteins were either analyzed by Western blot or kinase assay.
Immunodepletion—For immunodepletion cytosolic extracts were subjected to serial immunoprecipitation 4x for 1 h at 4 °C with 20 µgof specific non-cross-reactive anti-IKK or IKK antibody and 250 µl of 6% protein A-agarose in TNT buffer as described above. This procedure was followed by a normal immunoprecipitation for 1 h at 4 °C with 1 µg of antikinase antibody as described above.
Kinase Assay—IP was carried out as described above and followed by the kinase assay (11). The kinase reaction was performed in kinase buffer for 30 min at 30 °C in the presence of 5 µCi of [-32P]ATP (PerkinElmer Life Sciences) and 500 ng of the substrate GST-IB (Santa Cruz Biotechnology). Proteins were analyzed on 12.5% polyacrylamide gels (0.1% SDS), and following Western blot the transferred proteins were visualized by autoradiography. Densitometric analysis was performed for the obtained signals, and the values corrected for the loading control for each protein.
Overexpression Experiments—The plasmids coding for IKK, IKK (both wild type, FLAG-tagged) as well as NIK wild type and mutated (KK429-430AA) were obtained from Tularik Inc., South San Francisco, and the overexpression plasmid for IKK (NEMO) was a gift from Prof. Alain Israël and co-workers, Institut Pasteur, Paris, France. RcCMV (Invitrogen) containing no insert was used as a negative control. These plasmids were transiently co-transfected into HeLa cells or IKK knockout MEFs using Superfect (Qiagen, Hilden, Germany) (38). After overnight culture, cells were left untreated or stimulated with TNF at 2 ng/ml, and cytosolic or nuclear extracts were isolated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-IKK in Vivo in Monocytic Cells—Initial experiments were performed with monocytic cells to demonstrate the presence of endogenous complexes, which contain IKK as well as IKK but no IKK. For this purpose THP-1 cells were incubated in the absence or presence of TNF (2 ng/ml) for 5 min, and cytosolic extracts were isolated. IKK-containing complexes or free IKK were removed by immunodepletion using an antibody against the IKK protein, and this procedure was repeated up to 4x for each sample. The remaining IKK complexes were then precipitated by anti-IKK, and the nature of the precipitated IKK proteins was monitored by Western blot analysis. These experiments demonstrated that virtually no IKK (associated with IKK) remained in the cellular extracts following the IKK depletion procedure (Fig. 1A). In contrast, in the IKK-depleted samples a significant level of IKK and, as expected, IKK was detected in the precipitate (Fig. 1A). Comparable results were observed in the absence or presence of TNF. The densitometric analysis of three experiments showed that during repeated immunoprecipitations with the anti-IKK antibody the level of IKK went down essentially to zero (Fig. 1B). Under the same conditions, a decrease of IKK down to 
46% of the undepleted control was observed, which was comparable to the decrease in IKK (31%) at the same time (Fig. 1B). Essentially the same results were obtained when, following the IKK immunodepletion, the anti-IKK antibody was used instead of anti-IKK to precipitate the remaining complexes for further analysis (data not shown). These experiments demonstrate that IKK complexes exist in vivo in monocytic cells, whose major components are IKK as well as IKK. In the present article these complexes are designated as IKK-IKK complexes.
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-IKK Complexes by TNF—In the following we wanted to evaluate whether the endogenous IKK-IKK complexes play a role in the regular NF-B signaling pathway, and whether these can be activated by known stimuli of this system. For this purpose, THP-1 monocytic cells were incubated with TNF (2 ng/ml) and the cytosolic extracts were subjected to an immunodepletion of IKK as described above. The supernatants containing the IKK-IKK complexes as well as undepleted samples were subjected to immunoprecipitation with an anti-IKK antibody, and the obtained precipitate was added to a kinase assay. The experiments demonstrated a reduced baseline of IKK activity in unstimulated IKK-depleted cells (Fig. 2A). However, exposure to TNF still significantly increased IKK activity in IKK-depleted cells, albeit with a lower absolute peak level compared with normal, undepleted cytosolic extracts. Following the evaluation of the kinase activity the complete depletion of IKK protein as well as the remaining amount of IKK- and IKK proteins was verified by Western blot analyses of the kinase assay precipitate (Fig. 2A). These experiments were further extended by a kinetic study in which THP-1 cells were incubated with TNF for 2.5, 5, 7.5, and 10 min. As described above, kinase activity of IKK-precipitated complexes was determined followed by densitometric analysis. These data show a transient activation of IKK-IKK complexes by TNF, with fold induction values of kinase activity lowered to 60% compared with non-depleted samples (Fig. 2B). Taken together, these results indicate that the IKK-IKK complexes in monocytic cells can be stimulated by a classical stimulus such as TNF and are therefore likely to be involved in mainstream NF-B activation cascades potentially displaying a slightly different signaling dynamic.
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-IKK Complexes—Recently, it has been shown that NIK together with IKK- or IKK-containing complexes is involved in specific pathways, e.g. p100 processing (32). In our experiments, we wanted to test if the IKK-IKK complexes also participate in NIK-induced signaling. To investigate this we overexpressed either wild type or the dominant negative form of NIK together with the CMV vector alone as a control in HeLa cells. Western blot analysis of cytosolic extracts of these cells revealed an overexpression of NIK both in the wild-type and mutated form (Fig. 3A). NIK-induced IKK activity was monitored in cytosolic extracts of these cells by kinase assay following an immunoprecipitation using an antibody against IKK or IKK. These experiments demonstrated significantly increased kinase activity in HeLa cells overexpressing wild-type NIK with a more intense absolute peak level following the anti-IKK immunoprecipitation compared with the immunoprecipitation using an antibody against IKK (Fig. 3B). As a next step to investigate the role of IKK the above samples were subjected to an immunodepletion using an anti-IKK antibody. Following two and four steps of depletion, the samples were added to an immunoprecipitation using an anti-IKK antibody and subjected to a kinase assay. Remarkably, NIK-induced kinase activity was reduced after two steps of immunodepletion and totally abolished following four steps of IKK immunodepletion (Fig. 3C). Western blot analysis revealed, similar as described above, that IKK was completely depleted while there were still IKK-IKK complexes left. These results indicate that the signaling induced by NIK overexpression cannot be mediated by IKK-IKK complexes and that the removal of IKK abolishes the inducbility by NIK.
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-IKK by Overexpression and Activation of These Complexes—In these experiments we aimed to reconstitute exogenous IKK-IKK complexes by overexpression experiments. HeLa cells were used to overexpress different ratios of IKK and IKK or CMV vector alone. It should be mentioned that in this case only IKK has a FLAG tag whereas IKK is untagged. A total amount of 10 µg of DNA with different ratios was transfected, and the overexpression of the different proteins was detected by Western blot analysis (Fig. 4A). As a next step, the association of IKK with IKK was tested. For this, cytosolic extracts of HeLa cells overexpressing IKK and IKK in different ratios were subjected to an immunoprecipitation for either kinase, and the FLAG- or IKK-precipitated proteins were detected by Western blot analysis. The results demonstrate that the overexpressed IKK and IKK associate with each other and that exogenous IKK-IKK complexes can be reconstituted (Fig. 4B). For all of the following experiments a plasmid DNA ratio of 1:1 was used. To find out whether endogenous IKK protein binds to this artificial complex, the cytosolic extracts of either unstimulated or TNF-stimulated (2 ng/ml) HeLa cells overexpressing both IKK and IKK were immunoprecipitated using an anti-FLAG antibody, and the precipitates were analyzed. The experiments revealed that both IKK and IKK could be isolated under this condition whereas no significant amount of IKK is present in this artificial complex, and that TNF does not seem to have any effect on the formation of the complexes (Fig. 4C). Finally, we wanted to check the possibility of stimulating these complexes using cytosolic extracts of HeLa cells overexpressing IKK and IKK or CMV vector alone. Immunoprecipitations were carried out using an anti-FLAG antibody, and the precipitate was added to kinase assays. In these experiments no signal in cytosolic extracts of cells transfected with the CMV vector alone could be measured (Fig. 4D). In contrast, in cells overexpressing IKK as well as IKK a relatively high basal activity was detected as observed earlier (40), which was further increased by stimulation with TNF (Fig. 4D). Equal loading was confirmed by Western blot analyses following the kinase assay using FLAG-antibodies. These experiments revealed that exogenous IKK-IKK complexes appear to fulfill the minimal requirements to participate in regular signaling pathways.
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B but Not of IB Is Retarded in the Absence of IKK—The next question was to find out if the absence of IKK affects the proteolysis pattern of the inhibitory IB proteins. To examine this we used the model of IKK–/– MEF cells (41) in time course experiments in which the cells were stimulated with TNF (2 ng/ml). The proteolysis of IB and - was determined by Western blot analysis in wild type as well as in IKK knockout cells. As expected, TNF induced a rapid degradation of IB in wild-type cells (Fig. 5A). Interestingly, Western blot analysis indicated that, following TNF stimulation, IB was degraded more slowly in IKK–/– cells compared with the wild type. This effect could be reverted when an IKK overexpression plasmid was transfected into the IKK–/– cells (Fig. 5A). As a control, the level of actin was monitored, which remained unchanged during this experiment (data not shown). As a next step, data were analyzed by densitometry and IB values were normalized to actin levels. This analysis revealed that in wild-type cells a significant amount of proteolysis of IB was detected at 5 min following exposure to TNF with a maximal degradation at 15 min (Fig. 5B). In contrast, in knock-out fibroblasts a weak TNF-initiated degradation of IB was detected at 10 min, and a time point of maximal degradation was established at 20 min. When IKK was overexpressed in IKK knockout cells a significant amount of IB proteolysis could be already detected at 10 min after TNF stimulation with a maximal degradation at 15 min. In contrast, IB proteolysis seems to be similar in wild-type and IKK–/– cells (Fig. 5C). In both cases, initial IB degradation could be detected at 5–10 min after TNF stimulation reaching a maximum at 60 min (Fig. 5D).
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B Phosphorylation in the Absence of IKK—Because phosphorylation is the first step that marks IB proteins for degradation we decided to analyze the IKK activity as well as the phosphorylation status of IB in IKK knockout cells compared with wild-type MEFs. For this we performed kinase assays following IKK immunoprecipitation with extracts of wild-type or IKK–/– cells stimulated with TNF for 5 or 10 min, respectively. As a loading control, the level of immunoprecipitated IKK was detected by Western blot analysis. Autoradiography followed by densitometry revealed a significant TNF-induced IKK activity in both wild-type and knockout cells (Fig. 6A). However, in IKK–/– cells the IKK activity was reduced by 50% compared with the wild type (Fig. 6A), which is consistent with the data shown in Fig. 2B. Similar results were obtained when an anti-IKK antibody was used as the precipitation antibody for kinase assay (data not shown). As there seem to be differences in the kinase activity between wild-type and IKK knockout cells we decided to examine the phosphorylation status of IB following stimulation with TNF. Western blot analyses were performed using an anti-phospho-IB antibody or as a control for equal loading an anti-actin antibody. The data revealed a reduced phosphorylation of IB in the IKK–/– cells, which confirms the data obtained by kinase assays (Fig. 6B).
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B in the Presence of Overexpressed IKK-IKK—Finally, we reconstituted exogenous IKK complexes by overexpression (see also Fig. 4), which contained IKK, IKK, and IKK or solely IKK and IKK, respectively, and examined proteolysis of endogenous IB protein. The overexpression of the different proteins was monitored by Western blot analyses (Fig. 6C, data not shown). Densitometric analyses were calculated following normalization to actin. In the presence of both reconstituted exogenous complexes a significant proteolytic removal of IB was detected (Fig. 6C). Remarkably, in the presence of exogenous IKK-IKK, a low but clearly detectable level of endogenous IB remained (15 or 4% of control, dependent on the amount of transfected DNA), whereas almost no IB appeared to be left in the presence of the reconstituted classical IKK-IKK-IKK complexes (Fig. 6C). This demonstrates a reduced proteolysis of endogenous IB in the presence of overexpressed IKK-IKK compared with the situation when IKK-containing complexes are reconstituted and indicates differences between the two complexes in mediation of IB proteolysis. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-IKK complexes in vivo in monocytic cells. Consistent with our data, earlier experiments indicated that heterogenous IKK complexes exist in HeLa cells (24), whereas in the SLB T-cell line no IKK-only pool was found, which suggests cell type-specific differences. Recombinant IKK and IKK are known to form homo- or heterodimers, which also implies that similar IKK complexes may indeed exist in vivo (34). Complete recombinant reconstitution of the IKK in yeast reveals that the stoichiometric architecture of the core IKK complex is either 112 or, remarkably, 22 (37).
Our data show that these monocytic IKK-IKK complexes can be activated by TNF, a potent stimulus for NF-B (3, 28). In our experiments, following IKK immunodepletion, a significant TNF-induced increase of IKK activity was detected in the anti-IKK precipitate, albeit with a lower absolute basal and peak kinase activity as well as a reduced fold induction value when compared with non-depleted cells. These data agree with a previous experiment, which demonstrates that after sequential immunoprecipitation with an IKK antibody there is some kinase activity remaining in the HeLa extract (24). The levels of IKK or IKK were not changed by TNF treatment in our study suggesting that no dynamic rearrangement occurs during this stimulation. In earlier studies using IKK–/– mice, the fibroblast cells display significant IKK activation in phosphorylating IB and IB in vitro in response to the proinflammatory stimuli TNF or IL-1, measured by kinase assays with antibodies to either IKK or IKK (19–22). The latter effect is presumably due to the presence of endogenous or, alternatively, formation of new (since IKK is missing) but still functional IKK-IKK complexes (21). In accordance with functional IKK-IKK complexes, several previous data including ours demonstrate that, when wild-type or dominant negative mutant forms of IKK are expressed or fibroblasts of IKKAA/AA knock in mice were stimulated, the TNF- or LPS-induced NF-B activation or B-dependent transcription in monocytic cells is at most partly affected whereas a dominant negative mutant of IKK is much more effective (4, 18, 42). Taken together, these data suggest that IKK-IKK complexes participate in NF-B-associated signaling.
When we overexpressed IKK together with IKK we were able to demonstrate that these proteins associate with each other as was expected from earlier studies (26, 37). This appears to be independent of IKK, since in these newly formed complexes no endogenous IKK could be detected. Most importantly, we showed that these exogenous IKK-IKK complexes can be activated by TNF. This indicates that the IKK-IKK complex formed by overexpression itself fulfills the requirement to be connected upstream to regular signaling pathways.
It has been recently demonstrated that IKK is a specific target for NIK and that once activated it may lead to phosphorylation-dependent processing of NF-B/p100 (1, 32, 33). Furthermore, it has been suggested that the classical IKK complex is not involved in this pathway that rather depends on a different IKK-containing complex (1). Therefore, to evaluate the participation of IKK-IKK we tested if the removal of IKK-containing complexes would affect the kinase activity induced by overexpression of the NIK protein. Remarkably, following IKK immunodepletion, no NIK-induced kinase activity could be detected in the remaining IKK-IKK complexes, which implies that the latter complexes do not participate in NIK signaling. It should also be mentioned that these experiments may serve as an indirect functional approach to demonstrate the efficiency of the IKK immunodepletion procedure applied in our studies.
To study signaling in the absence of IKK we used the model of IKK–/– fibroblasts (41), which were stimulated with TNF, and the proteolysis pattern of the inhibitory IB proteins was examined. Interestingly, following TNF treatment, IB was degraded more slowly in IKK–/– cells compared with the wild type. This effect could be reverted when IKK was overexpressed in the IKK–/– cells demonstrating that the presence of IKK itself directly affects the degradation of IB. Furthermore, we found that the TNF-induced IKK activity and phosphorylation of IB were reduced in knock out cells compared with wild-type fibroblasts. These findings suggest that the retarded proteolysis of IB in IKK knockout cells is at least partially caused by signaling associated with reduced IKK activity and indicates a slightly different signaling dynamic between IKK-IKK and the IKK-containing core complex. In contrast, the absence of IKK did not affect IB proteolysis since a comparable degree of TNF-induced IB degradation was detected in both knockout and wild-type cells. This may suggest that the reduced but still significant IKK activity in IKK knockout cells is sufficient to mediate degradation of IB (which is known to occur at a slower rate) (7) to a similar extent as wild-type cells.
As already mentioned IKK is not required for most proinflammatory signaling pathways (18–22), and our experiments also indicate that IKK-IKK complexes without IKK function properly in an intact cell. Furthermore, recent studies suggest that IKK plays a role in specific signaling pathways and differentiation processes as well as B-cell-mediated responses (19–22, 42, 43). So one may ask why did IKK remain in the classical IKK core complex assembly at all. Our experiments indicate that in the classical complex IKK may function as a "turbo-like molecule" to accelerate certain signaling pathways such as IB proteolysis. One possibility is that IKK facilitates the association of the core complex with additional proteins such as upstream kinases or adapters that enhance IKK activity and IB phosphorylation (1, 3). Alternatively, IKK could potentially regulate IKK activity by serving itself as an upstream kinase that can phosphorylate other IKK molecules (1, 3, 44). The observation that in our overexpression studies (i.e. without an exogenous stimulus) IB proteolysis was reduced in the presence of IKK-IKK compared with the situation in which IKK-IKK-IKK was reconstituted could indicate that trans-autophosphorylation between the IKK molecules is involved but also that constitutive signaling is affected by the absence of IKK (41, 45). Taken together, the ability of IKK to increase IKK activity is possibly one of the reasons why this kinase, despite its different specific functions, remained a participant of the classical core complex during evolution.
The existence of IKK-IKK besides IKK-IKK-IKK complexes may open attractive therapeutic avenues for inflammation and cancer (9, 10). For example, in sepsis hyperinflammation could be reduced by specifically blocking the latter IKK-containing complexes, which would leave the IKK-IKK subcomplexes still intact to mediate important basic processes as part of a minimal response capacity, e.g. to orchestrate the immune response (1, 8, 46). Alternatively, the blocking of IKK may allow the recovery of the postulated IKK-specific (possibly IKK-independent) pathways (1, 2, 32, 47). An alternate use of both inhibitory strategies during therapy would not allow exhaustion of both sides of the pathways but still effectively reduce inflammation.
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FOOTNOTES |
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Both authors contributed equally to this work.
|| To whom correspondence should be addressed: Institute of Clinical Chemistry and Pathobiochemistry, Technische Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, 81675 München, Germany. Tel.: 49-89-4140-4084; Fax: 49-89-4140-4080; E-mail: brand{at}klinchem.med.tum.de.
1 The abbreviations used are: IKK, IB kinase; TNF, tumor necrosis factor; TNFR, TNF receptor; MEKK, mitogen-activated protein kinase/ERK kinase kinase-1; NIK, NF-B-inducing kinase; Cdc, cell division control; Hsp, heat shock protein; MEF, murine embryonic fibroblast; GST, glutathione S-transferase.
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ACKNOWLEDGMENTS |
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, IKK, and NIK wild-type and mutated form), Prof. Alain Israel, Institut Pasteur, Paris, France, for the IKK (NEMO) overexpression plasmid as well as Prof. Michael Karin (University of California, San Diego, La Jolla, CA) and Dr. Antonio Rossi (Institute of Neurobiology and Molecular Medicine, Rome, Italy) for the wild-type and IKK–/– MEFs. We would also like to thank Dr. Marion Wagner, Andreas Zwergal, Ingmar Ipach, and Bernd Saugel for valuable contributions and interesting discussions as well as Martina Krautkrämer for excellent technical assistance. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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