INTRODUCTION

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Cell survival largely depends on an innate ability of the cell to rapidly and effectively respond to changes in the external environment. This response can be summarized as perception of the external challenge, elicitation and transmission of an internal signal, and activation of transcription factors leading to alterations in gene expression. The NF-B/Rel family of inducible transcription factors regulates an array of host genes controlling immune activation, inflammation, and the prevention of apoptosis (1, 17, 37, 57). In unstimulated cells, NF-B is sequestered in the cytoplasm through its association with proteins of the IB family of inhibitors (2, 3). Upon exposure to a wide array of stimuli, IB becomes phosphorylated on two N-terminal serines (Ser-32 and Ser-36) (7, 13, 50, 55). This modification targets IB for rapid degradation by the ubiquitin-proteasome pathway (8, 47), unmasking the nuclear localization signal within the p50-p65 NF-B heterodimer and allowing its translocation to the nucleus as an active transcription factor.

Tumor necrosis factor alpha (TNF-), interleukin-1 (IL-1), lipopolysaccharides (LPS), and ligands recognizing the CD3-CD28 costimulatory T-cell receptor complex represent a subset of the diverse physiological inducers of IB phosphorylation and subsequent NF-B activation (53). Several kinases have been implicated as signaling intermediates in the pathway leading to NF-B activation, most notably select members of the mitogen-activated protein kinase kinase kinase (MAP3K) family, including NF-B inducing kinase (NIK), MEKK1, and Cot/Tpl-2 (19, 27, 28, 33, 36). NIK has been proposed as a downstream component of the TNF- signaling pathway (36) which may be activated directly or indirectly by cytoplasmic adaptor proteins like RIP (23, 54) or TRAF2 (20, 45). These proteins are recruited to the cytoplasmic tails of the type 1 TNF- receptor following ligand binding. Overexpression of wild-type NIK potently activates NF-B, while a catalytically inactive NIK mutant dominantly interferes with TNF- and IL-1 induction of NF-B (36, 49). MEKK1 was originally identified as a key participant in the c-Jun activation pathway but more recently has been shown to also participate in the NF-B signaling pathway leading to site-specific phosphorylation of IB and NF-B activation (19, 27, 28, 40-42). Cot/Tpl-2 is a proto-oncogene kinase that appears to play a role in CD3-CD28 activation of NF-B (33). Pathological inducers of NF-B have also been identified, including the human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein (10, 16, 56, 61). Gram-negative bacteria contain LPS, which induces NF-B through interaction with the Toll-like receptor 2, leading to NIK activation (5, 25, 60).

These various MAP3Ks do not directly phosphorylate IB; rather, they activate a second set of kinases termed IB kinase  (IKK) and IKK (14, 27, 39, 44, 58, 64). These IKKs interact with each other and reside in a ~900-kDa multicomponent signaling complex termed the signalsome (14, 27, 39). The predominant IKK-IKK heterodimeric complex also contains NEMO/IKK/IKKAP1, a protein that lacks intrinsic kinase activity but is essential for IKK signaling (38, 46, 59), and a scaffolding protein termed IKAP (11). Although this multimeric complex exhibits virtually no basal activity, it readily responds to TNF- and LPS stimulation (14, 27, 43) as well as to ectopic expression of NIK, Cot, MEKK1, or Tax, but not to functionally defective versions of these inducers (10, 16, 33, 34, 44, 56, 61). Tax induces the sustained nuclear expression of NF-B/Rel through activation of the IKKs mediated through its assembly with IKK/NEMO (9, 18, 22). Recent studies with mice lacking the Ikk gene suggest that IKK is absolutely required for the kinase activity of the IKK complex and subsequent NF-B activation in response to proinflammatory cytokines. In contrast, in mice lacking the Ikk gene, NF-B is normally induced following TNF- signaling (21, 30-32, 51, 52). However, interpretation of these results is complicated by earlier studies showing that coexpression of a catalytically inactive form of IKK (IKKK44M) or addition of antisense IKK (IKK-as) RNA inhibits NF-B activation in response to TNF-, IL-1, HTLV-1 Tax or the intermediate kinases NIK, Cot/Tpl2, and MEKK1 (14-16, 28, 41, 44, 56, 58). It seems possible that the formation of IKK homodimeric signaling complexes, accentuated in the absence of IKK, explains these paradoxical results. In this regard, Mercurio and colleagues have identified low-molecular-weight homodimeric IKK complexes; however, these particular complexes exhibit diminished IB kinase activity in response to TNF- (38). It seems likely, as in the case of the IKK^/ mice, that fully functional IKK homodimeric signalsomes can also form, although the heterodimeric IKK- complex is clearly the most favored and abundant complex formed under normal conditions.

In this study, we explore the biochemical basis for regulation of the heterodimeric IKK-IKK complex resident within the physiologically relevant signalsome. In unstimulated cells, we find that the assembly of IKK with IKK into a heterodimeric complex inhibits the high intrinsic activity of IKK. In cells stimulated with such agonists as TNF-, NIK, Cot, MEKK1, or HTLV-1 Tax, we find that IKK activation is a prerequisite for stimulation of IKK activity. Conversely, IKK activation is not required for induction of IKK by agonists like TNF- and NIK. In contrast, protein kinase C  (PKC) appears to directly target IKK homodimeric complexes. Together these studies demonstrate that signal-coupled activation of the IKK-IKK heterodimeric complex present in signalsomes proceeds in a directional manner through IKK to IKK.

	MATERIALS AND METHODS

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Expression vectors, biological reagents, and cell cultures. Wild-type and kinase-deficient constructs of IKK, IKK, NIK, and Cot/Tpl-2 have been described elsewhere (16, 33, 34). Plasmids pCDNA-IKK(K44M)-HA, pCDNA-IKK(S176A)-HA, and pCDNA-IKK(K44ASTS/AAA) were generated by site-directed mutagenesis using PCR. Mutated residues were confirmed by sequencing. The expression vector encoding MEKK1 was a gift from G. Johnson (National Jewish Medical and Research Center, Denver, Colo.), the IKK-as construct was kindly provided by Michael Karin (University of California, San Diego), and the PKC(A148E) construct was a gift from Amnon Altman (La Jolla Institute for Allergy and Immunology, San Diego, Calif.). Plasmids pCMV4Tax and pCMV4TaxM22 have also been described elsewhere (6, 48). Recombinant human TNF- was purchased from Endogen (Cambridge, Mass.). The following epitope-specific reagents were used: anti-Flag M2 antibodies conjugated to agarose beads (Sigma, St. Louis, Mo.), polyclonal anti-Flag epitope-specific antibodies, IKK-, IKK-, and c-Myc-specific antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.), hemagglutinin (HA)-conjugated Sepharose beads, and polyclonal anti-HA antibodies (BabCo, Richmond, Calif.). The 293 human embryonic kidney cell line and HeLa epithelial cell line were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics.

IKK kinase assays. 293 cells were transfected with IKK-Flag and either IKK-HA or IKKK44M-HA expression vectors; 24 h posttransfection, cells were resuspended in lysis buffer (1% Nonidet P-40, 250 mM NaCl, 50 mM HEPES [pH 7.4], 1 mM EDTA) supplemented with a cocktail of protease inhibitors (Roche Biochemicals, Indianapolis, Ind.), 1 mM phenylmethylsulfonyl fluoride, 50 µM dithiothreitol, and 50 µM Na₃VO₄, freshly prepared before use. Lysates were immunoprecipitated with anti-Flag M2 antibody conjugated to agarose beads. The immunoprecipitates were then incubated with 1 µCi of [-³²P]ATP and 1 µg of recombinant glutathione S-transferase (GST)-IB substrate at 30°C for 30 min. Reactions were stopped by adding 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiling for 5 min. Products were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and exposed to Hyperfilm MP (Amersham Life Sciences, Piscataway, N.J.). The membranes were subsequently probed with Flag-specific antibodies to determine the amount of IKK-Flag present. Cell lysates were similarly examined to confirm the expression of each protein.

IKK complex phosphorylation assays. To assess the role of IKK in regulating the activation of IKK under both unstimulated and NIK-stimulated conditions, expression vectors encoding IKKK44A-Flag or IKKK44A-STS/AAA-Flag were transfected into HeLa cells in the presence or absence of various IKK constructs as indicated. After 48 h, cells were lysed as described above. Lysates were immunoprecipitated with either anti-Flag M2 antibody-conjugated agarose beads or anti-IKK antibodies and protein A-conjugated agarose beads, washed three times in lysis buffer, equilibrated in kinase buffer (10 mM HEPES [pH 7.4], 1 mM MnCl₂, 5 mM MgCl₂, 12.5 mM -glycero-2-phosphate, 50 µM Na₃VO₄, 2 mM NaF, 50 µM dithiothreitol, and resuspended in 20 µl of kinase buffer. The immunoprecipitates were then incubated with 2 µCi of [-³²P]ATP at 30°C for 30 min. Reactions were stopped and separated as described above. The membranes were subsequently probed with epitope-specific antibodies to determine the amount of IKK present.

IKK and IKK phosphorylation assays. A kinase-inactive mutant of either IKKK44A-Flag or IKKK44M-HA was transfected into HeLa or 293 cells in combination with plasmids encoding either Myc-NIK, Myc-NIK(KK429/430AA), or other agonists including HA-MEKK1, Myc-Cot, PKC(A148E), Tax, or Tax M22. IKK, IKKK44M, IKKS176A, IKK-as, or IKKK44A constructs were also cotransfected as indicated. At 24 or 48 h posttransfection, IKK complexes were immunoprecipitated as described above. Reactions were carried out in ATP-free kinase buffer containing 2 µCi of [-³²P]ATP. After 30 min, reactions were halted by addition of an equal volume of dissociation buffer (50 mM Tris-Cl [pH 7.4], 20 mM -mercaptoethanol, 10% SDS) and boiled for 15 min to completely dissociate the immunoprecipitated complex. The dissociated tagged proteins and beads were then washed in 1 ml of lysis buffer and centrifuged for 2 min at maximum speed. The supernatant was collected and incubated for a second immunoprecipitation with antibodies specific for the IKK or IKK epitope tag conjugated to agarose beads. After at least 4 h, the immunoprecipitates were collected, washed with lysis buffer, and resuspended in SDS-PAGE buffer. Products were analyzed as described above.

IKK signalsome purification. Unstimulated and TNF--stimulated HeLa cells (6 × 10⁶ cells) were harvested and resuspended in 400 µl of lysis buffer, spun twice for 10 min each time at 12,000 rpm, and loaded on a phenyl-Superose 6 column (Amersham-Pharmacia, Piscataway, N.J.) equilibrated with lysis buffer containing 10% glycerol. Fractions were collected, boiled in sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose. Membranes were immunoblotted with anti-IKK antibodies to identify the high-molecular-weight fractions containing the endogenous signalsome. HeLa cells were transfected with IKK-K44A and NIK in the presence of either IKK or IKK-K44M. After 48 h, lysates were collected and fractionated on a size exclusion column by fast protein liquid chromatography (FPLC). Fractions corresponding to those that contained the endogenous signalsomes, as shown with anti-IKK immunoblotting, and the transfected Flag-tagged IKK-K44A were collected. These fractions were pooled in pairs and immunoprecipitated with anti-Flag agarose. These immunoprecipitates were then subjected to an in vitro kinase assay followed by heat dissociation and reimmunoprecipitation as described above. Immunoprecipitates were boiled in sample buffer, separated by SDS-PAGE, transferred to nitrocellulose, and exposed to film. The amount of IKKK44A-Flag in each sample was assessed by immunoblotting.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

IKK negatively regulates the constitutive activity of IKK. Since IKK exhibits high constitutive activity and appears to be a much more potent IB kinase than IKK (29), we investigated the possibility that IKK functions within the heterodimeric complex as a negative regulator of IKK activity. To evaluate this possibility, we coexpressed IKK with either kinase-proficient or kinase-deficient IKK in 293 cells. In agreement with prior studies (62), overexpressed IKK alone induced significant phosphorylation of IB in the absence of other stimuli (Fig. 1, lanes 1 and 5). As shown in Fig. 1, titration of either kinase-active or -inactive IKK produced a dose-related inhibition of IKK basal activity. These studies confirm and extend previous reports (58, 62) demonstrating that IKK negatively regulates the high constitutive activity of IKK observed under basal conditions. The levels of IKK and IKK present in each sample are shown in the lower panels of Fig. 1.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   IKK regulates the basal IB kinase activity of IKK. 293 cells were transfected with 0.6 µg of IKK-Flag expression vector alone or with increasing doses of either IKKK44M-HA or IKK-HA expression plasmids (0.6 µg, 1.2 µg, and 2.4 µg or 0.6 µg, 1.2 µg, 2.4 µg, and 3.6 µg, respectively). After 24 or 48 h, cell lysates were immunoprecipitated with anti-Flag M2-agarose. Immunoprecipitated complexes were assayed for kinase activity by incubation with 0.5 µg of GST-IB and [-32P]ATP. The resultant products were separated by SDS-PAGE (7.5% gels), transferred to nitrocellulose membranes, and subjected to autoradiography. The levels of IKK and IKK in each lysate were determined by immunoblotting with Flag-specific or HA-specific antibodies (lower panels).

Activation of IKK phosphorylation by NIK depends on catalytically active IKK. To explore a potential complementary role for IKK in regulating IKK under stimulated conditions, we examined NIK-induced phosphorylation of IKK in the presence of functionally active or inactive forms of IKK. Since wild-type IKK exhibits potent autophosphorylation, we used the kinase-deficient mutant IKKK44A as a substrate in these experiments. As expected, expression of IKKK44A alone or in combination with IKKK44M did not result in significant phosphorylation of either IKK (Fig. 2A, lanes 1 and 2). A slight degree of autophosphorylation of kinase-proficient IKK was detected (Fig. 2A, lane 3). However, in the presence of NIK, phosphorylation of both IKK and IKKK44M was significantly enhanced (Fig. 2A, lanes 5 and 6 versus lanes 2 and 3). Conversely, IKKK44A was not phosphorylated when coexpressed with NIK alone or with combinations of NIK and kinase-deficient IKKK44M (Fig. 2A, lanes 4 and 5). Notably, a significant level of IKK phosphorylation occurred when kinase-proficient IKK was present with NIK (Fig. 2A, lane 6).

View larger version (42K): [in this window] [in a new window]   FIG. 2.   NIK-induced phosphorylation of IKKs. (A) HeLa cells were transfected with 1 µg of IKKK44A-Flag expression vector alone or in combination with 1 µg of IKK-HA or IKKK44M-HA with and without 1 µg of Myc-NIK expression plasmids. (B) HeLa cells were transfected with 1 µg of IKKK44A-Flag or IKKK44A-STS/AAA-Flag and NIK expression vectors alone or in combination with 1 µg of each IKK-HA construct as indicated (2 µg of IKK was used in lanes 6 and 12). Cells were harvested 48 h after transfection, and IKKK44A was immunoprecipitated with anti-Flag M2-agarose (A) or anti-IKK/NEMO (B) antibodies. Immunoprecipitated complexes were subjected to in vitro kinase assay in the presence of [-32P]ATP. The products were separated by SDS-PAGE (7.5% gels), transferred to nitrocellulose membranes, and subjected to autoradiography. The level of IKK in each lysate was detected by immunoblotting with Flag-specific antibodies (lower panel).

NIK-induced activation of the heterodimeric IKK- signalsome is directional. As shown in Fig. 2B, coexpression of kinase-inactive IKK did not inhibit the ability of NIK to phosphorylate IKK, suggesting that the IKK heterodimeric complex was activated in a directional manner from IKK to IKK (Fig. 2B, lanes 2 and 6). To confirm this directionality in a more sensitive manner, we selectively isolated either IKKK44M or the activation loop mutant IKKS176A from the other signalsome components. Specifically, anti-IKK/NEMO-immunoprecipitated signalsomes were subjected to an in vitro kinase assay. The IKK substrates were then separated from the other reaction products by heat dissociation followed by reimmunoprecipitation with HA-specific antibodies. As shown in Fig. 3, neither IKKK44M nor IKKS176A was phosphorylated when expressed with IKKK44A (lanes 1 to 3). However, IKKK44M was robustly phosphorylated by NIK (lane 4), and this phosphorylation was not affected by coexpression of kinase-inactive IKK when normalized for the amounts of IKK present (lane 5). In contrast, IKKS176A was not phosphorylated by NIK (lane 6), thereby confirming that this activation loop residue serves as the target for NIK in the directional activation of the IKK heterodimeric complex.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   NIK-induced phosphorylation of IKK is not blocked by catalytically inactive IKK. 293 cells were transfected with 1 µg of IKKK44M-HA or IKKS176A alone or in combination with IKKK44-Flag and Myc-NIK as indicated. Each transfection was supplemented with empty vector to a final total of 4 µg of DNA. Cells were harvested, and signalsomes were immunoprecipitated with anti-IKK/NEMO antibodies. Following an in vitro kinase assay and heat dissociation, the tagged IKK constructs were reimmunoprecipitated with anti-HA-Sepharose. The products were separated by SDS-PAGE (7.5% gels), transferred to nitrocellulose membranes, and subjected to autoradiography. The level of IKK in each lysate was detected by immunoblotting with HA-specific antibodies (lower panel).

IKK-dependent NIK-induced phosphorylation of IKK occurs in the signalsome. To investigate the directional phosphorylation of IKK within the heterodimeric IKK complex in the presence and absence of NIK, we selectively isolated the Flag-tagged IKKK44A substrate from the other signalsome components as described above. Briefly, the anti-IKK/NEMO immunoprecipitates were subjected to an in vitro kinase assay followed by heat dissociation and reimmunoprecipitation with Flag-specific antibodies. As shown in Fig. 4A, the IKKK44A substrate was not phosphorylated in the presence of kinase-inactive (lane 2) or kinase-proficient (lane 3) IKK but was slightly phosphorylated in the presence of NIK (lane 4). However, the combination of NIK and IKK induced robust phosphorylation of the IKKK44A substrate (lane 6). This phosphorylation of IKKK44A was dependent on the kinase activity of IKK, as addition of the IKKK44M mutant failed to support the NIK-induced response (lane 5). In contrast, a kinase-inactive form of NIK failed to induce IKK phosphorylation even in the presence of kinase-proficient IKK (lanes 7 to 9). Consequently, despite the presence of equivalent levels of IKK in the anti-IKK immunoprecipitates (Fig. 4A, lower panel), only those signalsomes that contained functional IKK were able to transmit an activation signal from NIK to IKK.

View larger version (45K): [in this window] [in a new window]   FIG. 4.   NIK-induced phosphorylation of IKK requires catalytically active IKK. (A) HeLa cells were transfected with IKKK44A alone or with either wild-type or kinase-inactive NIK in combination with wild-type or kinase-inactive IKK. Cells were lysed 48 h posttransfection. Signalsomes were immunoprecipitated with anti-IKK/NEMO antibodies and subjected to an in vitro kinase assay followed by heat dissociation in 10% SDS. IKKK44A substrates were selectively immunoprecipitated from the disrupted complexes by a second immunoprecipitation with anti-Flag M2-agarose. (B) Unstimulated and TNF--stimulated (5 min) HeLa cell lysates were subjected to FPLC size fractionation on a Superose 6 column. Fractions were collected, separated by SDS-PAGE, and immunoblotted with anti-IKK antibodies to identify fractions containing the endogenous signalsome (fractions 12 to 17, ~900 kDa). (C) HeLa cells, transfected with Flag-tagged, kinase-inactive IKK, NIK, and either kinase-proficient or kinase-defective IKK, were lysed and size fractionated by FPLC. Fractions were separated by SDS-PAGE followed by immunoblotting with anti-Flag or anti-IKK antibodies. (D) Fractions corresponding to those containing the endogenous IKK signalsome, as identified by anti-IKK and anti-IKK antibodies, were collected, pooled, immunoprecipitated, and subjected to an in vitro kinase assay as described for Fig. 2. The level of phosphorylated IKK-K44A is shown in the upper panel; the levels of protein as determined by anti-Flag immunoblotting are shown in the lower panel.

IKK mediates phosphorylation of IKK induced by TNF- and HTLV-1 Tax. Since overexpression of a MAP3kinase such as NIK represents a somewhat artificial stimulation condition, we tested whether the heterodimeric IKK complex is directionally activated in response to TNF-, a physiological inducer of NF-B. HeLa cells were transfected with IKKK44A alone (Fig. 5A, lanes 1, 4, 7, and 10) or in combination with either kinase-deficient IKKK44M (lanes 2, 5, 8, and 11) or kinase-proficient IKK (lanes 3, 6, 9, and 12) and stimulated with TNF- (20 ng/ml) for 0, 1, 5, or 10 min. Under basal conditions, no phosphorylation on IKKK44A was observed when this mutant was expressed alone or with either kinase-inactive or kinase-proficient IKK (Fig. 5A, lanes 1, 2 and 3). In response to addition of TNF-, coexpression of kinase-proficient IKK resulted in a marked phosphorylation of IKKK44A (Fig. 5A, lanes 6, 9, and 12). In contrast, TNF- induced only minimal phosphorylation of IKKK44A expressed either alone (lanes 4, 7, and 10) or with kinase-inactive IKKK44M (Fig. 5A, lanes 5, 8, and 11). The slightly higher levels of IKKK44A protein (lower panel) probably account for the modestly higher levels of IKKK44A phosphorylation observed in the presence of IKKK44M. In addition, disruption of endogenous IKK protein expression by transfection of an IKK antisense construct also resulted in a dose-dependent inhibition of IKK phosphorylation in response to TNF- stimulation in the anti-IKK/NEMO-immunoprecipitated complexes (Fig. 5B). Thus, IKK phosphorylation in response to TNF- stimulation is dependent on IKK in the context of the physiological signalsome.

View larger version (35K): [in this window] [in a new window]   FIG. 5.   IKK phosphorylation induced by TNF- in the presence and absence of IKK. (A) HeLa cells were transfected with 2 µg of IKKK44A-Flag and 2 µg of IKK-HA or 2 µg of IKKK44M-HA expression vector. Forty-eight hours after transfection, cells were stimulated with TNF- (20 ng/ml) for 1, 5, and 10 min and lysed. Lysates were immunoprecipitated with anti-Flag M2-agarose and analyzed as for Fig. 4. Levels of IKKK44A were evaluated by immunoblotting (lower panel). (B) HeLa cells were transfected with 0.5 µg of IKKK44A-Flag and 1 µg IKK-HA with increasing amounts of IKK-as (0.5, 1, 2, and 4 µg). Forty-eight hours after transfection, cells were stimulated with TNF- (20 ng/ml) for 10 min and lysed. Lysates were immunoprecipitated with anti-IKK/NEMO antibodies and analyzed as for Fig. 4. Levels of IKKK44A-Flag and IKK-HA were evaluated by immunoblotting (lower panel).

View larger version (21K): [in this window] [in a new window]   FIG. 6.   IKK phosphorylation induced by HTLV-1 Tax. (A) Approximately 3 × 105 293 cells were transfected with 1 µg of kinase-deficient IKK (IKKK44A-Flag) in combination with 1 µg of IKK-HA or IKKK44M-HA expression construct in the presence of wild-type Tax (1 µg) or the M22 Tax mutant (2 µg) as indicated. Cell lysates were then immunoprecipitated with anti-Flag M2-agarose and subjected to an in vitro kinase assay with [-32P]ATP. The reaction products were separated by SDS-PAGE (7.5% gel), transferred to a nitrocellulose membrane, and analyzed by autoradiography. The amount of IKKK44A-Flag in each reaction is shown in the lower panel. (B) The levels of wild-type amd mutant Tax proteins in the cell lysates were assessed by immunoblotting with Tax-specific antiserum.

IKK is required for phosphorylation of IKK by Cot/Tpl-2 and MEKK1 but not by PKC. We next investigated whether a similar directional activation of the heterodimeric IKK complex occurs during stimulation by other MAP3Ks like Cot/Tpl-2, MEKK1, and PKC (X. Lin, A. O'Mahony, Y. Mu, R. Geleziunas, and W. C. Greene, unpublished data), which represent known inducers of NF-B. As with NIK, in HeLa cells, IKKK44A was not phosphorylated when coexpressed with IKK or Cot alone (Fig. 7A, lanes 3 and 4). However, the combination of Cot and kinase-active IKK induced potent phosphorylation of IKKK44A (Fig. 7A, lane 6). This activation failed to occur in the presence of IKKK44M (Fig. 7A, lane 5). The level of IKK phosphorylation did not result from a reduced expression of the IKKK44A substrate as determined by immunoblotting (Fig. 7A, lower panel). Similarly, MEKK1 coexpressed with wild-type IKK potently induced phosphorylation of IKKK44A in HeLa cells (Fig. 7C, lane 6) but failed to do so when expressed either alone or with IKKK44M (Fig. 7C, lanes 4 and 5). In sharp contrast, a constitutively active PKC(A/E) mutant induced phosphorylation of IKKK44A when expressed alone (Fig. 7E, lane 9). Interestingly, this phosphorylation was inhibited when either wild-type, kinase-inactive, or T-loop mutant IKK was coexpressed (Fig. 7E, lanes 10 to 12). This pattern of phosphorylation suggests that PKC may specifically target signalsomes containing homodimeric IKK complexes whereas Cot and MEKK1 operate through the heterodimeric complex in a directional manner.

View larger version (44K): [in this window] [in a new window]   FIG. 7.   Kinase-deficient IKK blocks MEKK1- and Cot/Tpl-2-induced, but not PKC-induced, phosphorylation of IKK. HeLa cells and 293 cells were transfected with 1 µg of IKKK44A-Flag expression plasmid and 1 µg of HA-tagged wild-type or kinase-deficient IKK in the presence or absence of the Myc-Cot (A and B), HA-MEKK1 (C and D), and NIK and PKC(A/E) (E) expression vectors. After 24 h (293) and 48 h (HeLa), cells were harvested and lysates were immunoprecipitated with anti-Flag M2-agarose (A to D) or with IKK-specific antibodies (E). The immunoprecipitated complexes were subjected to an in vitro kinase assay and analyzed as for Fig. 4. The levels of phosphate incorporated into Flag-tagged, kinase-deficient IKK are shown in the upper panel, and the levels of Flag-tagged IKK are shown in the lower panels.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

When first identified, IKK and IKK were viewed as functionally interchangeable IB kinases that coexist within a macromolecular IKK signaling complex termed the signalsome. In the wake of targeted gene disruption studies, it is clear that these kinases play significantly different roles within the heterodimeric signalsome, IKK being the principal IB kinase while the function of IKK is less clear. We now demonstrate that activation of signalsomes containing heterodimeric IKK-IKK complexes proceeds in a directional manner. Specifically, we show that a wide variety of NF-B inducing MAP3Ks act through IKK to induce phosphorylation of the activation loop residues of IKK in various cell lines. In contrast, kinase-deficient IKK exerts no inhibitory effects on NIK-induced phosphorylation of IKK, underscoring the directional nature of this activation process. Our studies further indicate that phosphorylation of IKK induced by the physiological agonist TNF- or the pathological stimulant HTLV-1 Tax similarly proceeds through IKK to IKK. Interestingly, not all agonists require IKK for induction of IKK phosphorylation. For example, we found that PKC is able to induce phosphorylation of IKK in the absence of IKK. The addition of wild-type IKK inhibits this PKC response, suggesting that expression of IKK may disrupt IKK homodimeric complexes that may be selectively activated by PKC. These findings raise the intriguing possibility that different upstream activators couple preferentially to heterodimeric or homodimeric complexes, increasing signalling specificity.

Functional asymmetry within the heterodimeric signalsome was first suggested by the observation that IKK is a significantly more potent IB kinase than IKK. While both kinases are capable of phosphorylating IB in vitro, they do so with dramatically different efficiencies, with IKK exhibiting 50- to 60-fold greater activity than IKK (28, 29, 38, 58). Additional support for disparate roles in NF-B activation has come from the targeted inactivation of the IKK and IKK genes in mice. Disruption of the Ikk locus results in embryonic lethality at ~14 days of gestation due to massive hepatic cell apoptosis leading to liver degeneration, a phenotype remarkably similar to that seen in mice deficient in the RelA/p65 subunit of NF-B (4, 31, 32, 52). This enhanced hepatocyte death is likely due to the loss of the antiapoptotic effects of NF-B since IKK-deficient embryonic fibroblasts have severely depressed IB kinase activity and diminished NF-B activation in response to either TNF- or IL-1 (31, 52). Indeed, IKK-deficient cells were 30-fold more sensitive to TNF--induced apoptosis than their wild-type counterparts (52). The amount of IKK protein was greater in homozygous IKK-deficient embryos than in wild-type embryos, suggesting that there is a selective pressure to enhance IKK expression in IKK-deficient cells, although this up-regulation of IKK does not fully compensate for the loss of IKK activity and therefore is unable to counteract the extensive cell death (52). Of interest is the observation that IKK continued to assemble into a minimally responsive ~900-kDa signalsome in these IKK-deficient cells (31, 52).

IKK-defective animals survive to birth but die within 1 to 4 h of birth and exhibit a range of morphogenic abnormalities including a thickened, undifferentiated epidermis that appears to restrict extension of the limbs and a number of skeletal malformations (21, 30, 51). Intriguingly, skin abnormalities, although not identical, have also been reported for mice deficient for IB, a negative regulator of NF-B (26). In this study we, like others, have shown that IKK can similarly function as a negative regulator of basal IKK activity (29, 62). It is interesting to speculate whether these skin abnormalities may emerge as a consequence of disrupting the normal negative regulators of IKK activity and NF-B activation.

Disruption of the Ikk locus surprisingly does not impair TNF- induction of NF-B, a finding confirmed in three independent studies. Of note, there is a quantitative decrease in the total level of NF-B binding in these IKK-deficient animals (21, 30, 51). This result seems at odds with the abundance of IKK expression in the wild-type animals, its tight association with IKK expression, and the high degree of sequence similarity shared by these genes. Indeed, the widespread assembly of IKK with IKK in signalsomes in many tissues argues that IKK plays a broader function than regulating epidermal development (63). Moreover, previous studies with kinase-inactive or activation loop mutants of IKK (15, 35) as well as transfection of IKK-as constructs (14) have all reported a negative impact on IKK activity underlying the conditional importance of IKK expression. In view of our described findings, we propose that the IKK-deficient animals have likely compensated for the loss of the IKK regulator by assembling functional homodimeric IKK signalsomes (21). These homodimeric IKK signalsomes (38) may be positively selected for during embryogenesis in the IKK-deficient animals to prevent the extensive apoptosis that would result from a loss of IKK activity. In view of the dramatic difference in the IB-phosphorylating activities of these two kinases, we would argue that IKK has mainly evolved to negatively regulate the high constitutive activity of IKK under basal conditions and to couple its activation in stimulated conditions to many upstream agonists. Likewise, a proportion of complexes consisting of IKK homodimers have evolved with an alternative regulatory mechanism, perhaps IKK, which also plays a role in coupling of the signalsome to different upstream activators. Therefore, loss of a regulating kinase like IKK may be compensated for, but loss of the functional kinase, IKK, cannot be tolerated. The generation of IKK and IKK conditional knockout and knock-in animals will no doubt clarify the nature of the physiological interplay between these two kinases in the regulation of NF-B induction.

We have demonstrated directional activation of the heterodimeric IKK complex by a number of MAP3Ks known to play a role in NF-B activation (19, 27, 28, 33, 36, 40-42, 44). This activation occurs through phosphorylation of the serine residues within the activation loops of the IKKs. One recent report suggests that the activation loop serines of IKK are essential for NIK-induced IKK activation (12). We find that these activation loop serines are phosphorylated in the presence of NIK but in an indirect manner dependent on the kinase activity of IKK. In the same study, Delhase and colleagues report that homologous activation loop mutations in IKK do not affect IB phosphorylation (12). This result is at odds with our observations that the activation loop mutant IKKS176A blocks both IKK and IB phosphorylation induced by NIK. In support of our data, NIK was previously shown to phosphorylate IKK on Ser-176 of its activation loop, but it did not phosphorylate IKK (35). These data support a dual regulatory role for IKK leading to the appropriate activation of IKK phosphorylation. As such, IKK could be functionally viewed as a surrogate MAP2-like kinase connecting the upstream MAP3Ks to the downstream MAPK represented by IKK.

The precise nature of the interplay of MEKK1 with IKK or IKK remains unclear. Some studies indicate MEKK1 interacts with, and activates, both IKK and IKK (28, 42). However, other reports show that MEKK1 overexpression in 293 or Jurkat cells preferentially stimulates IKK kinase activity over IKK (24, 41). In addition, Tax has been shown to bind and activate MEKK1, which then directly activates IKK but not IKK (61). However, more recent reports indicate that Tax binds to the signalsome by assembling with NEMO/IKK rather than by binding to IKK directly (9, 18, 22). This interaction may be impaired in the presence of overexpressed upstream kinase-inactive MAP3Ks, which may also interact with IKK. We too find that within the heterodimeric signalsome, both MEKK1 and Tax induce IKK phosphorylation in a manner dependent on the kinase activity of IKK. In agreement with our findings, kinase-inactive forms of both IKK and IKK have been shown to block Tax and MEKK1 induction of IKK activity, clearly implicating both kinases in the pathway (10, 16, 24, 56).

Of interest is our finding that not all signals proceed through IKK. We show that PKC appears to selectively target IKK for activation. Of note, this reaction may involve IKK homodimers since assembly of IKK into the heterodimeric complex inhibits its ability to serve as a target for PKC-mediated activation. These inconsistencies in activation of IKK versus IKK by various upstream kinases may, in part, be reconciled by the existence of a number of distinct IKK complexes (38). The larger ~700-kDa TNF--responsive complex was found to contain IKK, IKK, and IKKAP1 (NEMO/IKK), while a ~300-kDa complex consisting of only IKK and IKKAP1 proved significantly less responsive to TNF--coupled induction (38). It is possible, however, that the higher-molecular-weight complex also contains functional IKK homodimeric complexes. Moreover, the smaller IKK complexes may not respond to TNF- but may couple to different activators. Different cell lines may contain varying amounts of these IKK- heterodimeric versus IKK homodimeric complexes, and these complexes may couple differentially to upstream activating signals. Our studies clearly show that, in the 293 and HeLa cell lines studied, transmission of the NF-B-inducing signal is directional within the heterodimeric IKK signalsome.

In summary, we propose that, when present in the heterodimeric signalsome, IKK exerts a dominant regulating effect on the phosphorylation and activation of IKK kinase activity. This regulatory role of IKK is further underscored by the finding that mutations in the leucine zipper region of IKK disrupts dimerization with IKK, resulting in a strong diminution of IB phosphorylation (38, 58, 62). Interestingly, mutations in the helix-loop-helix motifs of either kinase do not abolish their dimerization but do result in the loss of kinase activity (62), likely reflecting a failure of the IKKs to bind NEMO/IKK/IKKAP1, an essential component of functional signalsomes (38, 46, 59). IKK is thus an essential regulatory component of the IKK heterodimeric signalsome that serves to couple the upstream activating signal to the IKK catalytic component of the complex.