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Molecular and Cellular Biology, March 1999, p. 2180-2188, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Activation of Ikappa B Kinase beta  by Protein Kinase C Isoforms

Maria-José Lallena,1 María T. Diaz-Meco,1 Gary Bren,2 Carlos V. Payá,2 and Jorge Moscat1,*

Laboratorio Glaxo Wellcome-CSIC de Biología Molecular y Celular, Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, 28049 Madrid, Spain,1 and Department of Immunology, Mayo Clinic, Rochester, Minnesota 559052

Received 29 June 1998/Returned for modification 26 August 1998/Accepted 12 November 1998


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The atypical protein kinase C (PKC) isotypes (lambda /iota PKC and zeta PKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha ) and are required for the activation of NF-kappa B by this cytokine through a mechanism that most probably involves the phosphorylation of Ikappa B. The inability of these PKC isotypes to directly phosphorylate Ikappa B led to the hypothesis that zeta PKC may use a putative Ikappa B kinase to functionally inactivate Ikappa B. Recently several groups have molecularly characterized and cloned two Ikappa B kinases (IKKalpha and IKKbeta ) which phosphorylate the residues in the Ikappa B molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-kappa B through the activation of the IKKs. We report here that alpha PKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zeta PKC positively modulates IKKbeta activity but not that of IKKalpha , whereas the transfection of a zeta PKC dominant negative mutant severely impairs the activation of IKKbeta but not IKKalpha in TNF-alpha -stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKbeta , which is entirely dependent on the activity of alpha PKC but not that of the atypical isoforms. In contrast, the inhibition of alpha PKC does not affect the activation of IKKbeta by TNF-alpha . Interestingly, recombinant active zeta PKC and alpha PKC are able to stimulate in vitro the activity of IKKbeta but not that of IKKalpha . In addition, evidence is presented here that recombinant zeta PKC directly phosphorylates IKKbeta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappa B pathway at the level of IKKbeta activation and Ikappa B degradation.


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The transcription factor NF-kappa B plays a critical role in a number of cell functions, including key inflammatory and immune responses (2, 16). NF-kappa B is composed of dimers of different members of the Rel protein family (1, 2, 30). The most classical form of NF-kappa B is a heterodimer of p50 and p65 (RelA) (1, 2, 30) that is sequestered in the cytosol by Ikappa B, which prevents its nuclear translocation and activity (30, 31). Upon cell stimulation by inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha ) or interleukin 1 (IL-1), Ikappa Balpha is phosphorylated in residues 32 and 36, which trigger the ubiquitination and subsequent degradation of Ikappa B through the proteasome pathway (31). These events release NF-kappa B which translocates to the nucleus, where it activates several genes (1, 2, 30, 31). The identification of the kinase responsible for the signal-induced phosphorylation of Ikappa B has been the subject of intense research. Recently, several groups have succeeded in the identification and molecular cloning of two Ikappa B kinase (IKK) activities (IKKalpha and IKKbeta ) that phosphorylate residues 32 and 36 of Ikappa Balpha and whose activity is potently stimulated by TNF-alpha and IL-1 (9, 22, 25, 33, 34). The IKKs bind NF-kappa B-inducing kinase (NIK) (25, 33), a member of the mitogen-activated protein (MAP) kinase kinase kinase family that interacts with TNF receptor-associated factor 2 (20), linking Ikappa B degradation and NF-kappa B activation to the TNF receptor complex. TNF-alpha and interleukin 1 are potent activators of protein kinase C zeta  (zeta PKC) in vivo (19, 23, 26). Interestingly, we and others have previously shown that the atypical PKC isoforms zeta  and lambda /iota play a critical role during NF-kappa B activation (4-6, 8, 10, 11, 19, 28). Thus, the blockade of the atypical PKCs with either microinjected pseudosubstrate peptide inhibitors (10), antisense oligonucleotides (10, 11), or the transfection of kinase-dead dominant negative mutants of zeta PKC or lambda /iota PKC (4-6, 8, 11, 19, 28) dramatically impairs NF-kappa B activation. However, the mechanisms whereby the atypical PKCs participate in this pathway have not yet been elucidated. Because zeta PKC is unable to directly phosphorylate Ikappa B (7), it is possible that the signals generated by the stimulation of the atypical PKCs could be mediated by the novel IKKs.

We report here that the atypical PKCs bind to the IKKs in vitro and in vivo. Importantly, overexpression of zeta PKC positively modulates IKKbeta activity but not that of IKKalpha whereas the transfection of a zeta PKC dominant negative mutant severely impairs the activation of IKKbeta but not that of IKKalpha in TNF-alpha -stimulated cells. In addition, recombinant active zeta PKC dramatically stimulates in vitro IKKbeta activity but not that of IKKalpha from unstimulated cells. Collectively these results demonstrate a critical role for the atypical PKCs in the NF-kappa B pathway through the regulation of IKKbeta activity.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Plasmids, cell culture, and transfections. The hemagglutinin (HA)-tagged expression plasmids for zeta PKC, lambda /iota PKC, Raf, zeta PKCCAT, zeta PKCMUT, and lambda /iota PKCMUT have previously been described (4, 8). The HA-alpha PKC was made by inserting an EcoRI-EcoRV fragment encompassing the full-length bovine alpha PKC into pCDNA3. The Flag-IKKbeta and IKKalpha constructs were provided by D. Goeddel (Tularik, Inc.) and A. Israel, respectively. The Flag-Ikappa Balpha and the p65 constructs were generously provided by D. Ballard (Vanderbilt University). The Flag-IKKalpha plasmid was made by inserting the EcoRI fragment containing the rat IKKalpha cDNA into pCDNA3-Flag. Flag-tagged constructs encompassing the kinase or the regulatory domains of IKKalpha or IKKbeta were generated by PCR. The Flag-IKKbeta KD and Flag-IKKbeta AA (S177A S181A) constructs were obtained by site-directed mutagenesis (Stratagene). The glutathione S-transferase (GST)-Ikappa BDelta C and GST-Ikappa BDelta CA32/36 were transformed into Escherichia coli JM101, and expression of GST fusion proteins and their purification on glutathione-Sepharose were carried out according to the manufacturer's procedures. Cultures of 293 cells were maintained in high-glucose Dulbecco's modified Eagle's medium containing 10% fetal calf serum, penicillin G (100 µg/ml), and streptomycin (100 µg/ml) (Flow). Subconfluent cells were transfected by the calcium phosphate method (Clontech, Inc.).

In vitro translation and immunoprecipitation. For in vitro translation studies, zeta PKC, zeta PKCCAT, lambda /iota PKC, alpha PKC, or Raf were in vitro translated in rabbit reticulocyte lysates, either alone or together with Flag-IKKalpha , Flag-IKKbeta , or their respective catalytic and regulatory domains, exactly as described in the manufacturer's protocol (Promega), and the Flag-tagged proteins were immunoprecipitated with the monoclonal M2 anti-Flag antibody (Kodak) as described previously (8). Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography in an InstantImager (Packard). For coimmunoprecipitation experiments, subconfluent 293 cells plated on 10-cm-diameter dishes were transfected with 10 µg of expression plasmid. After transfection (36 h), cells were or were not stimulated with 20 ng of TNF-alpha (Promega) per ml or 5 µM phorbol 12-myristate 13-acetate (PMA) (Sigma) for different times. In some experiments, cells were incubated with 10 nM GF109203X (Calbiochem) for 10 min prior to the stimulation. Cells were then harvested and lysed in buffer A (40 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM beta -glycerophosphate, 10 mM NaF, 10 mM PNPP [para-nitrophenyl-phosphate], 300 µM Na3VO4, 1 mM benzamidine, 2 M PMSF [phenylmethylsulfonyl fluoride], aprotinin [10 µg/ml], leupeptin [1 µg/ml], pepstatin [1 µg/ml], 1 mM dithiothreitol [DTT]). The IKK proteins were precipitated with 3 µg of M2 monoclonal antibody to the Flag epitope (Kodak) and 10 µl of protein G-agarose and then immunoblotted with a polyclonal antiserum to the HA-tagged PKCs or to the endogenous PKCs (Santa Cruz Biotechnology, Inc.). The immunocomplexes were washed in a high-salt buffer (500 mM NaCl). Proteins were detected with ECL reagent (Amersham). In another set of experiments, cell extracts prepared as described above were immunoprecipitated with a polyclonal anti-MKP-1 (MAP kinase phosphatase 1) antibody (Santa Cruz Biotechnology, Inc.), and the extensively washed immunocomplexes were analyzed by immunoblotting with monoclonal anti-lambda /iota PKC antibody (Transduction Laboratories). For the detection of endogenous IKK a polyclonal anti-IKKalpha antibody (H-744; Santa Cruz Biotechnology) was used.

IKK kinase assay. Kinase activity was assayed in a solution consisting of 20 mM HEPES (pH 7.7), 10 mM beta -glycerophosphate, 2 mM MgCl2, 2 mM MnCl2, 10 mM PNPP, 300 µM Na3VO4, 1 mM dithiothreitol, 10 µM ATP, 1 mM benzamidine, 2 M PMSF, aprotinin (10 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), and 2 µCi of [gamma -32P]ATP at 30°C for 30 min. Ikappa B substrate proteins were expressed and purified from E. coli. Flag-tagged IKK immune complexes were isolated as described above and washed in kinase buffer before the level of kinase activity was determined. The kinase reaction was stopped by the addition of 5× SDS-PAGE sample buffer, subjected to SDS-PAGE analysis, and visualized in an InstantImager. In some experiments, the immunoprecipitates of Flag-IKKs, either untreated or inactivated with FSBA [5'-(4-fluorosulfonylbenzoyl)adenine] as described previously (7), were or were not incubated with recombinant preparations of either alpha PKC (maximally activated by phosphatidylserine plus diacylglycerol according to the manufacturer's instructions) or a permanently active mutant of zeta PKC, both produced from baculovirus in Sf9 insect cells. The recombinant baculovirus alpha PKC was obtained from Panvera. The recombinant zeta PKCCAT was prepared by using the Bac-to-Bac baculovirus expression system (Life Technologies).

Reporter assays. For reporter gene assays, 293 cells were seeded into six-well plates. Cells were transfected the following day by the calcium phosphate precipitation method with 100 ng of kappa B-luciferase reporter gene plasmid and various amounts of each expression construct. The total amount of DNA transfected (5 µg) was kept constant by supplementation with the control vector pCDNA3. After 24 h, cells either were left untreated or were stimulated with TNF-alpha (20 ng/ml) for 6 h prior to harvest. Extracts were prepared, and the level of luciferase activity was determined as described previously (8).


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Interaction of PKC isoforms with the IKKs in vitro. Binding assays were performed with in vitro-translated 35S-labeled Flag-tagged IKKbeta or IKKalpha and HA-tagged lambda /iota PKC, zeta PKC, alpha PKC, or Raf-1. The immunoprecipitation of IKKbeta with an anti-Flag antibody reveals that IKKbeta associates in vitro with both atypical PKCs and alpha PKC but that it is unable to interact with Raf-1 (Fig. 1A). The same results were obtained when the interaction of IKKalpha with all these kinases was investigated (Fig. 1B). In order to map the regions in the IKKs and PKCs that mediate their interaction, in vitro-translated 35S-labeled IKKalpha or IKKbeta , or fragments of these kinases encompassing either their catalytic domain or the regulatory region (leucine zipper plus the helix-loop-helix), were incubated with HA-tagged versions of either full-length zeta PKC or two fragments corresponding to the catalytic domain and the regulatory region of this kinase. Experiments similar to those whose results are shown in Fig. 1A and B were carried out, and the results are shown in Fig. 1C. Interestingly, it seems that both catalytic domains are responsible for the interaction between IKK and PKC.


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  		FIG. 1.   In vitro interaction of PKC with IKK. 35S-labeled Flag-tagged IKKbeta (A) or IKKalpha (B) and HA-tagged lambda /iota PKC, zeta PKC, alpha PKC, or Raf-1 were incubated either alone or in combination as described in Materials and Methods. IKKbeta and IKKalpha were immunoprecipitated (IP) with an anti-Flag antibody, and the immunoprecipitates were fractionated by SDS-PAGE, followed by autoradiography in an InstantImager. An aliquot (one-tenth of the amount of labeled protein used for the in vitro binding reaction) was loaded in parallel (Ext.). Essentially identical results were obtained in two other independent experiments. (C) Summary of results of three independent experiments in which 35S-labeled Flag-tagged versions of either full-length IKKbeta or two fragments of this kinase encompassing the catalytic (black box) or the regulatory domain (leucine zipper [LZ] plus the helix-loop-helix [HLH]) were incubated with either full-length zeta PKC or its catalytic (zeta PKCCAT) and regulatory (zeta PKCREG) regions, after which IKKbeta was immunoprecipitated as described above, and the level of association of the zeta PKC constructs was determined by SDS-PAGE and autoradiography. The numbers at left of panels indicate positions of molecular mass markers in kilodaltons.

Interaction of the atypical PKCs and of alpha PKC with the IKKs in vivo. To determine whether the IKKs bind to the atypical PKCs in vivo, 293 cells were transfected with HA-tagged lambda /iota PKC, zeta PKC, or alpha PKC along with Flag-tagged IKKbeta or IKKalpha . Cell lysates were immunoprecipitated with an anti-HA antibody, and the immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with an anti-Flag antibody. An immunoreactive band corresponding to Flag-IKKbeta was detected only in immunoprecipitates from cells transfected with HA-lambda /iota PKC (Fig. 2A, left panel), HA-alpha PKC (Fig. 2A, right panel), or HA-zeta PKC (Fig. 2B, left panel). Similar data were obtained when cell lysates were immunoprecipitated with an anti-Flag antibody and immunoblotted with the anti-HA antibody (Fig. 2A, left and right panels, and Fig. 2B, left panel). Also, similar results were obtained when the interaction of lambda /iota PKC, zeta PKC, or alpha PKC with IKKalpha was investigated (Fig. 2B, right panel, and both panels of Fig. 2C). In marked contrast, when this experiment was performed with HA-Raf and Flag-IKKbeta or Flag-IKKalpha , no association was detected (data not shown).


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  		FIG. 2.   lambda /iota PKC and alpha PKC interact with IKKbeta in vivo. Subconfluent cultures of 293 cells in 100-mm-diameter plates were transfected with 10 µg of either pCDNA3 or expression vectors for either HA-lambda /iota PKC (A, left panel; B, right panel), HA-alpha PKC (A, right panel; C, left panel), HA-zeta PKC (B, left panel; C, right panel), Flag-IKKbeta (A, both panels; B, left panel), or Flag-IKKalpha (B, right panel; C, both panels) and enough empty vector to give 20 µg of total DNA. Parallel cultures were transfected with 10 µg of Flag-IKKbeta or Flag-IKKalpha plus 10 µg of either HA-lambda /iota PKC, HA-alpha PKC, or HA-zeta PKC. After transfection (36 h), cell extracts were immunoprecipitated with an anti-Flag antibody or an anti-HA antibody. Immunoprecipitates were extensively washed in high-salt buffer (500 mM NaCl), fractionated by SDS-PAGE, and analyzed by immunoblotting with anti-HA or anti-Flag antibodies (IP). An aliquot (one-tenth of the amount of extract [Ext.] used for the immunoprecipitation) was loaded in parallel gels and analyzed by immunoblotting with the corresponding antibodies. Essentially identical results were obtained in two other independent experiments. The numbers at right of panels indicate positions of molecular mass markers in kilodaltons. WB, Western blot.

Taken together the data suggest that the atypical PKCs, as well as alpha PKC, can interact with the IKKs when ectopically expressed in 293 cells.

TNF-alpha -dependent interaction of endogenous lambda /iota PKC with the IKKs and the signalosome. To further analyze these interactions, 293 cells transfected with either Flag-IKKbeta or Flag-IKKalpha were or were not stimulated with TNF-alpha or PMA. Cell lysates were immunoprecipitated with a monoclonal anti-Flag antibody, and the immunoprecipitates were resolved by SDS-PAGE and analyzed with a polyclonal anti-lambda /iota PKC antibody. Interestingly, the addition of TNF-alpha but not that of PMA promotes the interaction of endogenous lambda /iota PKC with IKKbeta (Fig. 3A) and IKKalpha (Fig. 3B). Similar results were obtained when the immunoprecipitates were analyzed with a zeta PKC polyclonal antibody that also cross-reacts with lambda /iota PKC (data not shown). The lack of a reliable antibody with an absolute specificity for zeta PKC precludes the definitive identification of this atypical PKC isoform in the IKK complex. However, the evidence presented in Fig. 1 and 2, in conjunction with the functional data shown below, strongly indicates that most probably native zeta PKC, like lambda /iota PKC, will associate with the IKKs in TNF-alpha -activated cells. Of note, alpha PKC is the only other PKC isotype detectable in 293 cells (14a). When the IKK immunoprecipitates were analyzed by immunoblotting with an antibody selective for alpha PKC, no association of endogenous alpha PKC with IKKalpha or IKKbeta was observed in unstimulated cells or in PMA- or TNF-alpha -treated cells (data not shown).


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  		FIG. 3.   Interaction of endogenous lambda /iota PKC with IKKbeta and with the signalosome. (A and B) Subconfluent cultures of 293 cells in 100-mm-diameter plates transfected with 10 µg of either Flag-IKKbeta (A) or Flag-IKKalpha (B) were stimulated with 20 ng of TNF-alpha per ml or PMA (5 µM) for 5 min. Afterward, cell extracts (200 µg) were immunoprecipitated (IP) with a monoclonal anti-Flag antibody, and the immunoprecipitates were analyzed by immunoblotting (WB) with a polyclonal anti-lambda /iota PKC antibody. The immunoprecipitates were analyzed in parallel gels by immunoblotting with an anti-Flag antibody. The extract (Ext.) lane contained 20 µg of cell protein. Essentially identical results were obtained in two other independent experiments. (C) Subconfluent cultures of 293 cells in 100-mm-diameter plates were stimulated with 20 ng of TNF-alpha per ml or PMA (5 µM) for different times. Afterward, cell extracts (200 µg) were immunoprecipitated (IP) with a polyclonal anti-MKP-1 antibody, and immunoprecipitates were analyzed by immunoblotting (WB) with a monoclonal anti-lambda /iota PKC antibody. The immunoprecipitates were analyzed in parallel gels by immunoblotting with an anti-IKK antibody. The extract (Ext.) lane contained 20 µg of cell protein. Essentially identical results were obtained in two other independent experiments.

Recent evidence indicates that the IKKs are part of a large complex termed the signalosome that can be immunoprecipitated with an antibody raised against MKP-1 (22). Therefore, it was of interest to determine if the atypical PKCs could be recruited to the signalosome upon cell stimulation. To address this possibility, 293 cells were stimulated either with PMA or TNF-alpha for different times and cell lysates were immunoprecipitated with a polyclonal anti-MKP-1 antibody and analyzed by immunoblotting with a monoclonal anti-lambda /iota PKC antibody. The upper panel of Fig. 3C shows that the stimulation with TNF-alpha but not that with PMA promotes the recruitment of lambda /iota PKC to the signalosome complex. Analysis with an anti-IKK antibody reveals that the anti-MKP-1 immunoprecipitates contained similar amounts of IKK (Fig. 3C, lower panel). However, no association of alpha PKC with the signalosome complex was detected in these experiments (data not shown). This observation and the lack of any association of endogenous alpha PKC with the transfected IKKbeta or IKKalpha suggest that alpha PKC, in contrast to the atypical isoforms, does not stably associate with the IKKs in vivo unless it is overexpressed in cotransfection experiments.

To further establish the interaction of the atypical PKCs with IKK under physiological conditions, 293 cells either were left untreated or were stimulated with TNF-alpha or PMA for 5 min, after which the native IKK complex was immunoprecipitated with an anti-IKKalpha antibody that also cross-reacts with IKKbeta , and the association of the atypical PKCs was analyzed with an anti-lambda /iota PKC antibody. Interestingly, treatment with TNF-alpha but not that with PMA provokes a reproducible interaction of native lambda /iota PKC with native IKK (Fig. 4). Similar results were obtained when the immunoprecipitates were analyzed with a zeta PKC polyclonal antibody that also cross-reacts with lambda /iota PKC (data not shown). Again, when the immunoprecipitates were analyzed with an anti-alpha PKC antibody, no association of this PKC with the IKK complex was observed (data not shown).


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  		FIG. 4.   Interaction of endogenous lambda /iota PKC with endogenous IKK. Subconfluent cultures of 293 cells in 100-mm-diameter plates were stimulated with 20 ng of TNF-alpha per ml or PMA (5 µM) for 5 min. Afterward, cell extracts (200 µg) were immunoprecipitated (IP) with a polyclonal anti-IKK antibody, and immunoprecipitates were analyzed by immunoblotting (WB) with a monoclonal anti-lambda /iota PKC antibody. The immunoprecipitates were analyzed in parallel gels by immunoblotting with an anti-IKK antibody. The extract (Ext.) lane contained 20 µg of cell protein. Essentially identical results were obtained in two other independent experiments.

Role for the PKCs in the activation of IKKbeta and IKKalpha in response to TNF-alpha and PMA. Collectively the above findings suggest that PKCs may be critically involved in the regulation of IKK activity in vivo. To begin analyzing this possibility, 293 cells were transfected with Flag-tagged IKKbeta , and 36 h post-transfection they either were left untreated or were stimulated with TNF-alpha or PMA. Afterward, cell extracts were immunoprecipitated with an anti-Flag antibody, and the ability of IKKbeta to phosphorylate a GST-Ikappa B construct containing the first 250 amino acids of Ikappa Balpha (25) was determined. Cell stimulation with TNF-alpha or PMA activates the ability of IKKbeta to phosphorylate GST-Ikappa B (Fig. 5A) but not a mutant in which Ser32 and Ser36 were replaced by Ala (data not shown). The PMA effect is most likely accounted for by the activation of alpha PKC. Consistent with this notion, the incubation with GF109203X completely abrogated the activation of IKKbeta by PMA but not that by TNF-alpha (Fig. 5A). This strongly suggests that alpha PKC mediates the activation of IKKbeta by PMA but not that by TNF-alpha , which is entirely consistent with previous observations demonstrating that the PMA-sensitive PKC isoforms are not involved in the activation of NF-kappa B by TNF-alpha but are responsible for the PMA effects (6, 10, 11, and references therein). In order to determine whether the atypical PKCs could be involved in the activation of IKKbeta by TNF-alpha , 293 cells were transfected with Flag-IKKbeta along with either a plasmid control or expression vectors for wild-type or dominant negative zeta PKC. Thirty-six hours posttransfection, cells were stimulated with either TNF-alpha or PMA for 7 min and the level of activity of IKKbeta was determined as described above. Interestingly, Fig. 5B shows that the simple overexpression of wild-type zeta PKC was sufficient to stimulate IKKbeta and synergistically increase its activation by TNF-alpha (Fig. 5B).


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  		FIG. 5.   Role for alpha PKC and zeta PKC in the activation of IKKbeta by PMA and TNF-alpha . (A) Subconfluent cultures of 293 cells in 100-mm-diameter plates were transfected with Flag-IKKbeta (10 µg), and 36 h posttransfection, cells either were left untreated or were incubated with GF109203X (10 nM) 15 min prior to stimulation with TNF-alpha (20 ng/ml) or PMA (5 µM) for 7 min. Afterward, Flag-IKKbeta was immunoprecipitated, and the level of its activity was determined by using recombinant GST-Ikappa B as the substrate as described in Materials and Methods. (B) Subconfluent cultures of 293 cells in 100-mm-diameter plates were transfected with Flag-IKKbeta (10 µg) along with 10 µg of empty plasmid or expression vectors for HA-tagged versions of wild-type (zeta PKCWT) or dominant negative (zeta PKCMUT) zeta PKC. Thirty-six hours posttransfection, cells either were left untreated or were stimulated with TNF-alpha (20 ng/ml) for 7 min. Afterward, Flag-IKKbeta was immunoprecipitated, and the level of its activity was determined as described above. (C) Subconfluent cultures of 293 cells in 100-mm-diameter plates were transfected with Flag-IKKbeta (10 µg) along with 10 µg of empty plasmid or an expression vector for the HA-tagged dominant negative zeta PKC (zeta PKCMUT). Thirty-six hours posttransfection, cells either were left untreated or were stimulated with TNF-alpha (20 ng/ml) or PMA (5 µM) for 7 min. Afterward, Flag-IKKbeta was immunoprecipitated, and the level of its activity was determined as described above. (D) Subconfluent cultures of 293 cells in 100-mm-diameter plates were transfected with Flag-IKKbeta (10 µg) along with 10 µg of empty plasmid or expression vectors for HA-tagged dominant negative (lambda /iota PKCMUT) lambda /iota PKC. Thirty-six hours posttransfection, cells either were left untreated or were stimulated with TNF-alpha (20 ng/ml) for 7 min. Afterward, Flag-IKKbeta was immunoprecipitated, and the level of its activity was determined as described above. (E) Subconfluent cultures of 293 cells in 100-mm-diameter plates were transfected with Flag-IKKalpha (10 µg), and 36 h posttransfection, cells either were left untreated or were stimulated with TNF-alpha (20 ng/ml) for 7 min. Afterward, Flag-IKKalpha was immunoprecipitated, and the level of its activity was determined as described above. The expression levels of the different constructs were determined by using the corresponding antitag antibodies. Essentially identical results were obtained in two other independent experiments. P, phosphorylated protein.

Importantly, the expression of a dominant negative mutant of zeta PKC severely impaired the activation of IKKbeta by TNF-alpha (Fig. 5B and C) but not that by PMA (Fig. 5C). Similar results were obtained when cells were transfected with a dominant negative mutant of lambda /iota PKC (Fig. 5D). Next, 293 cells were transfected with Flag-IKKalpha along with either a plasmid control or expression vectors for wild-type or dominant negative zeta PKC. Thirty-six hours posttransfection, cells were stimulated with TNF-alpha for 7 min and the level of activity of IKKalpha was determined as described above. Of note, Fig. 5E shows that the overexpression of wild-type zeta PKC produced little or no effect on IKKalpha activity or on its activation by TNF-alpha . Likewise, the expression of a dominant negative mutant of zeta PKC does not significantly affect the activation of IKKalpha by TNF-alpha (Fig. 5E). Collectively these results indicate that the atypical PKCs are critically involved in the activation by TNF-alpha of IKKbeta but not that of IKKalpha , whereas alpha PKC is responsible for the activation of IKKbeta by PMA.

Stimulation of IKKbeta in vitro by recombinant zeta PKC and alpha PKC. To further explore the activation of the IKKs by these PKC isotypes, we carried out an in vitro coupled assay in which immunoprecipitated IKKbeta or IKKalpha from untreated cells was incubated in vitro with recombinant preparations of alpha PKC or a permanently active mutant of zeta PKC, both produced from baculovirus in insect cells. Figures 6A and B show that the presence of catalytically active recombinant zeta PKC dramatically reactivates IKKbeta but not IKKalpha in vitro to an extent comparable to that produced by cell stimulation with TNF-alpha . Likewise, maximally activated alpha PKC was able to activate IKKbeta but not IKKalpha in vitro (Fig. 6A). Control incubations demonstrate that the different PKC isoforms were unable to phosphorylate GST-Ikappa B by themselves in the absence of IKKbeta (data not shown). In addition, the results shown in Fig. 6C demonstrate that recombinant active zeta PKC directly phosphorylates immunopurified IKKbeta . To further establish the direct phosphorylation of IKKbeta by zeta PKC, immunoprecipitates of IKKbeta were treated with FSBA to inactivate its kinase activity as well as that of any hypothetical contaminant associated kinase. Afterward, the level of phosphorylation of the inactivated IKKbeta was determined. The results shown in Fig. 6D demonstrate the capability of recombinant active zeta PKC to phosphorylate inactivated IKKbeta (left panel). Interestingly, the mutation of serines 177 and 181 to alanine substantially inhibits IKKbeta phosphorylation by recombinant zeta PKC (Fig. 6D, right panel).


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  		FIG. 6.   Recombinant active zeta PKC and alpha PKC stimulate IKKbeta but not IKKalpha in vitro. (A) Immunoprecipitates of Flag-IKKbeta (left panel) or Flag-IKKalpha (right panel) expressed in untreated 293 cells were or were not incubated with recombinant preparations of either alpha PKC (maximally activated by phosphatidylserine plus diacylglycerol) or a permanently active mutant of zeta PKC (zeta PKCDEL), both produced from baculovirus in insect cells. Reactions were carried out at 30°C for 30 min in the presence of GST-Ikappa B, after which the level of Ikappa B phosphorylation was determined as described above. As a positive control, the activities of both IKKs from TNF-alpha -activated cells (20 ng/ml; 7 min) were included. (B) The levels of activity of the PKC recombinant preparations used in these experiments were assayed with myelin basic protein (MyBP) as a control. (C) Immunoprecipitates of Flag-IKKbeta expressed in untreated 293 cells were or were not incubated with recombinant zeta PKC at 30°C for 30 min in the absence of GST-Ikappa B, after which the level of direct phosphorylation of IKKbeta was determined. The numbers at left indicate positions of molecular mass markers in kilodaltons. (D, left panel) Immunoprecipitates of wild-type IKKbeta from either 0.5 or 2 mg of protein extracts were inactivated by treatment with FSBA, as described in Materials and Methods. Afterward, they were or were not incubated with recombinant zeta PKC as described above, and the level of phosphorylation of kinase-inactive IKKbeta was determined. (D, right panel) Immunoprecipitates of either wild-type (WT) or activation loop mutant (AA) IKKbeta from 1 mg of protein extracts were inactivated by treatment with FSBA, as described above, after which they were or were not incubated with recombinant zeta PKC and the level of phosphorylation of kinase-inactive IKKbeta was determined. The reaction mixtures in every experiment were analyzed in parallel gels by immunoblotting with an anti-Flag antibody. Essentially identical results were obtained in three other experiments. P, phosphorylated protein.

Role for the atypical PKCs in the TNF-alpha -induced degradation of Ikappa B and activation of a kappa B-dependent promoter. Consistent with the physiological implications of all these findings are the results of the following experiment. We transfected 293 cells with a Flag-tagged version of Ikappa Balpha and an expression vector for p65 to stabilize the ectopic Ikappa Balpha molecule, according to the protocol described by Chu et al. (5a), and either a control plasmid or expression vectors for wild-type or dominant negative zeta PKC. Afterward, cells were stimulated with TNF-alpha in the presence of cycloheximide, and the ectopically expressed Ikappa B was detected in cell extracts by immunoblot analysis with the anti-Flag antibody. Figure 7 shows that stimulation with TNF-alpha triggers the degradation of Ikappa Balpha , consistent with previously reported data (9, 22, 25, 33, 34). The overexpression of wild-type zeta PKC synergistically increases the ability of TNF-alpha to induce the degradation of Ikappa Balpha (Fig. 7). More importantly, the expression of the dominant negative zeta PKC construct completely abrogates the degradation of Ikappa Balpha in response to TNF-alpha (Fig. 7). Similar results were obtained when cells were transfected with a dominant negative mutant of lambda /iota PKC (data not shown). These results demonstrate that the ability of the atypical PKCs to bind and regulate the IKK activity is critical to the control of Ikappa B degradation in TNF-alpha -activated cells.


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  		FIG. 7.   Role for zeta PKC in the induced degradation of Ikappa B. Subconfluent cultures of 293 cells were transfected with 5 µg of expression plasmid for Flag-tagged Ikappa Balpha along with 5 µg of an expression vector for p65 with 10 µg of either control vector or expression plasmids for HA-tagged versions of wild-type (zeta PKCWT) or dominant negative (zeta PKCMUT) zeta PKC. Thirty-six hours posttransfection cells were incubated with cycloheximide (50 µg/ml) for 1 h in the presence of TNF-alpha (20 ng/ml) for different times. Afterward, cell extracts were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Essentially identical results were obtained in three other experiments.

In addition, 293 cells were transfected with a kappa B-dependent luciferase reporter plasmid along with either a control or an expression vector for a kinase-inactive dominant negative mutant of IKKbeta (either with or without expression plasmids for wild-type zeta PKC or lambda /iota PKC). Cells were stimulated with TNF-alpha for 6 h, and the level of luciferase activity was determined in cell extracts. The results shown in Fig. 8 demonstrate that the simple overexpression of zeta PKC or lambda /iota PKC is sufficient to activate a kappa B-dependent transcription in keeping with previously reported results (8) and to synergize with TNF-alpha . Interestingly, the transfection of a dominant negative mutant of IKKbeta severely impairs not only the TNF-alpha effects but also those of both atypical PKCs.


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  		FIG. 8.   IKKbeta is required for NF-kappa B activation by the atypical PKCs. Subconfluent cultures of 293 cells were transfected with 100 ng of the kappa B-luciferase reporter gene plasmid and 2 µg of each kinase construct. The amount of total DNA transfected (5 µg) was kept constant by supplementation with the control vector PCDNA3. After 24 h, cells either were left untreated or were stimulated with TNF-alpha (20 ng/ml) for 6 h prior to harvest. Extracts were prepared, and the level of luciferase activity was determined as described in Materials and Methods. Results are means ± standard deviations from three independent experiments with incubations in duplicate. C, control; Z, zeta PKC; L, lambda /iota PKC.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The identification and molecular cloning of the IKKs constitute a great advance in the understanding of NF-kappa B activation (9, 22, 25, 33, 34). However, the mechanisms whereby these kinases are regulated are not yet completely understood (13, 29, 32). We show here that the atypical PKCs and alpha PKC seem to be important intermediaries in the activation of IKKbeta by TNF-alpha and PMA, respectively. These findings would be consistent with the reported role played by the atypical PKCs in NF-kappa B activation in TNF-alpha -stimulated cells (4-6, 10, 11, 19) and establish the mechanism whereby the PKC signaling cascades regulate this important transcription factor. We have shown previously that zeta PKC was unable to directly phosphorylate Ikappa B in vitro but that it associated with a putative Ikappa B kinase activity in immunoprecipitates (7). The findings reported in this study suggest that the Ikappa B kinase activity detected in the zeta PKC immunoprecipitates in previous work (7) could be accounted for at least in part by IKKbeta . However, reconstitution experiments, in which recombinant zeta PKC was incubated with cell extracts, demonstrated the association of an Ikappa B kinase activity that in in-gel kinase assays gave a molecular mass of about 50kDa, which is very different from that of the IKKs. This 50-kDa protein has now been identified as casein kinase 2 (24b), which selectively phosphorylates the C terminus of Ikappa B (21). These phosphorylation sites are not involved in the induced degradation of Ikappa B but rather in the control of its stability (21). The inability of the IKKs to renature in the in-gel kinase assays (8a) explains why they remained undetected in previous studies (7).

The atypical PKCs can also stimulate the MAP/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling pathway through a still-to-be-defined Raf-independent mechanism (4, 27). This pathway is also relevant for the activation of the kappa B-dependent transcription, since the overexpression of a dominant negative ERK mutant severely impairs the kappa B-dependent promoter activity stimulated by the overexpression of a zeta PKC active mutant or the presence of TNF-alpha (3, 4). However, that mechanism does not involve the actual translocation of NF-kappa B to the nucleus (4) but could be mediated through the action of ERK on the transactivation domain of p65 (3, 4a). This, together with the evidence presented here that the atypical PKCs directly regulate IKKbeta in vitro and in vivo, strongly suggests that the atypical PKCs may control the NF-kappa B pathway at two levels, which would ensure the maximal efficiency in the activation of NF-kappa B-regulated genes.

NIK is another kinase that binds to both IKKalpha and IKKbeta (25, 33). It has recently been demonstrated that NIK activates and phosphorylates IKKalpha in cotransfection experiments but that it is unable to phosphorylate IKKbeta (17, 25). The atypical PKCs also bind to both IKKs but in contrast to NIK activate only IKKbeta and have no effect on IKKalpha . Thus, it seems that there are specific kinase pathways upstream of the different IKKs to control Ikappa B phosphorylation and NF-kappa B activation. In this regard, MEK kinase 1 (MEKK1) has also been shown to selectively activate IKKbeta and to have no effect on IKKalpha (24). However, in contrast with the atypical PKCs or NIK, MEKK1 appears to be unable to stably interact with the IKKs (24). Recent studies demonstrate that Ser176 in the activation loop of IKKalpha is the target of NIK (17) and together with Ser180 is essential for IKKalpha kinase activity (22). In the case of IKKbeta , the mutation of serines 177 and 181 to alanine does not block its enzymatic activity (22); however, its activity is greatly increased when both residues are mutated to glutamic acid (22). This indicates that either or both serines may be important for the activation of IKKbeta by upstream kinases. Actually, Lee et al. (15) demonstrate that a peptide comprising the activation loop of IKKbeta is phosphorylated by MEKK1 on residues corresponding to serines 177 and 181. We show here the direct phosphorylation of IKKbeta by recombinant zeta PKC and the important contribution of those two residues to that phosphorylation. Although serines 177 and 181 do not conform strictly to the PKC consensus site, serine 181 is followed at position +1 by a hydrophobic amino acid which has been shown to be present in all bona fide PKC phosphorylation sites (24a). Another intriguing matter arising from this and other studies (15, 17, 22) is the fact that IKKalpha and IKKbeta are selective for different upstream kinases, despite the fact that the sequence around the phosphorylated residues is highly conserved. This may suggest that other structural determinants in the IKK upstream kinases may be responsible for that specificity. Further studies will be required to answer this question.

A kinase-inactive mutant of IKKalpha blocks the activation by NIK of a kappa B-dependent reporter gene, reinforcing the notion that NIK is upstream of IKKalpha in the NF-kappa B pathway (25). The overexpression of MEKK1 is sufficient to activate a kappa B-dependent reporter gene (15), although the ability of a dominant negative MEKK1 to block NF-kappa B activation by TNF-alpha is still a matter for discussion (18). The observation that the atypical PKCs are critically involved in the regulation of NF-kappa B (4-6, 10, 11, 19) and IKKbeta activity, in conjunction with the fact that they are potently activated by TNF-alpha (19, 23), strongly suggests that these PKCs are among the important players in the NF-kappa B pathway at the level of IKKbeta activation. Recent data indicate that receptor-interacting protein is a critical molecule in the activation of NF-kappa B (12, 14). How the atypical PKCs are connected to receptor-interacting protein in the NF-kappa B signaling cascade is a matter of ongoing research in our laboratory.


    ACKNOWLEDGMENTS

This work was supported by grants SAF96-0216 from CICYT, PM96-0002-C02 from DGICYT, and BIO4-CT97-2071 from the European Union and by funds from Glaxo Wellcome Spain and has benefited from an institutional grant from Fundación Ramón Areces to the CBM.

We are indebted to Esther Garcia, Carmen Ibañez, and Beatriz Ranera for technical assistance and to Gonzalo Paris and Isabel Perez for their help and enthusiasm. We thank Dave Goeddel for a critical reading of the manuscript and for helpful comments during this work.


    FOOTNOTES

* Corresponding author. Mailing address: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain. Phone: 34-913978039. Fax: 34-929690055. E-mail: jmoscat{at}cbm.uam.es.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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Molecular and Cellular Biology, March 1999, p. 2180-2188, Vol. 19, No. 3
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