COMMUNICATION
Mitogen-activated Protein Kinase/ERK Kinase Kinases 2 and 3 Activate Nuclear Factor-kappa B through Ikappa B Kinase-alpha and Ikappa B Kinase-beta *

Quan Zhao and Frank S. LeeDagger

From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recent evidence indicates that nuclear factor-kappa B (NF-kappa B), a transcription factor critically important for immune and inflammatory responses, is activated by a protein kinase cascade. The essential features of this cascade are that a mitogen-activated protein kinase kinase kinase (MAP3K) activates an Ikappa B kinase (IKK) that site-specifically phosphorylates Ikappa B. The Ikappa B protein, which ordinarily sequesters NF-kappa B in the cytoplasm, is subsequently degraded by the ubiquitin-proteasome pathway, thereby allowing the nuclear translocation of NF-kappa B. Thus far, only two MAP3Ks, NIK and MEKK1, have been identified that can activate this pathway. We now show that MEKK2 and MEKK3 can in vivo activate IKK-alpha and IKK-beta , induce site-specific Ikappa Balpha phosphorylation, and, relatively modestly, activate an NF-kappa B reporter gene. In addition, dominant negative versions of either IKK-alpha or IKK-beta abolish NF-kappa B activation induced by MEKK2 or MEKK3, thereby providing evidence that these IKKs mediate the NF-kappa B-inducing activities of these MEKKs. In contrast, other MAP3Ks, including MEKK4, ASK1, and MLK3, fail to show evidence of activation of the NF-kappa B pathway. We conclude that a distinct subset of MAP3Ks can activate NF-kappa B.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The transcription factor nuclear factor-kappa B (NF-kappa B)1 plays a critical role in immune and inflammatory responses (1, 2). NF-kappa B, prototypically a heterodimer of p50 and p65 subunits, is sequestered in the cytoplasm of most cell types by virtue of its association with a family of inhibitor molecules, the Ikappa Bs. Upon exposure to a wide variety of agents, including the proinflammatory cytokine TNF-alpha , lipopolysaccharide, oxidative stress, and the HTLV-I Tax protein, the Ikappa B protein is phosphorylated at its N terminus. In the case of Ikappa Balpha , the most extensively studied Ikappa B isoform, this phosphorylation occurs at Ser-32 and Ser-36 (3, 4). This phosphorylation event targets Ikappa B for degradation by the ubiquitin-proteasome pathway (5), allowing the subsequent nuclear translocation of NF-kappa B.

An Ikappa B kinase (IKK) complex with a native molecular mass of 700 kDa was originally identified in cytoplasmic extracts of HeLa cells and shown to perform the site-specific phosphorylation of Ikappa Balpha (6, 7). A significant advance was the subsequent cloning of the cDNAs for the catalytic, protein kinase subunits of this complex, IKK-alpha and IKK-beta (8-12). Several lines of evidence now indicate that IKK-alpha and IKK-beta can be regulated by phosphorylation. The initial indications were that the IKK complex can be activated in vitro by the MAP3K MEKK1 (MAPK/ERK kinase kinase 1) (7) and that the complex, activated either in vitro by MEKK1 or in vivo by exposure of cells to TNF-alpha can be inactivated by phosphatase treatment (7, 8). Additional work then demonstrated that (i) mutation of potential phosphoacceptor residues to alanine in the activation loop of IKK-alpha or IKK-beta abrogated activity (12), (ii) mutation of these same residues in IKK-beta to the phosphoresidue mimetic glutamic acid results in its constitutive activation (12), (iii) both IKK-alpha and IKK-beta can be activated in vivo when overexpressed with MEKK1 or the related MAP3K NF-kappa B-inducing kinase (NIK) (10, 11, 13-15), and (iv) immunoprecipitated NIK can phosphorylate immunoprecipitated IKK-alpha (16). Therefore, an important conceptual advance in our understanding of NF-kappa B regulation is that it can be activated by protein kinase cascade, the core elements of this cascade being a MAP3K and an IKK (7, 10, 17).

These findings have already begun to provide a framework for understanding how NF-kappa B can be activated by diverse stimuli. For example, compelling evidence has been presented to show that NIK mediates the NF-kappa B-inducing activity of TNF-alpha (17, 18), whereas MEKK1 mediates the NF-kappa B-inducing activity of Tax (15). Thus, different stimuli can activate NF-kappa B by targeting different MAP3Ks.

These findings moreover raise the possibility that yet other MAP3Ks might activate NF-kappa B. MAP3Ks were originally identified as components of signaling cascades in which a MAP3K phosphorylates and activates a MAP2K, which in turn phosphorylates and activates a MAPK; the latter include the mitogen-activated ERK and the stress-activated c-Jun N-terminal kinase (JNK, also known as stress-activated protein kinase) and p38 families (19). Here we show that MEKK2 and MEKK3, but not certain other MAP3Ks, can activate NF-kappa B, and show that this activation occurs by their activation of IKK-alpha and IKK-beta .

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- pCMV5-HA-MEKK2 (20), pCMV5-HA-MEKK3 (20), and pCMV5-Delta MEKK4 (21) were gifts of Dr. Gary Johnson (National Jewish Medical and Research Center). pcDNA3-ASK1 (22) was a gift of Dr. Hidenori Ichijo (The Cancer Institute, Tokyo). pcDNA3-MLK3 was constructed by subcloning into the BamHI (blunt)/EcoRI site of pcDNA3 the 2.7-kilobase pair NcoI (blunt)/EcoRI coding sequence fragment of pPTK1-3.2 (23); the latter was a gift of Dr. Richard Spritz (University of Wisconsin-Madison). (PRDII)3E1bCAT, a reporter gene that contains three copies of the NF-kappa B binding site from the interferon-beta enhancer, an E1b promoter, and the CAT gene, was a gift of Dr. Tom Maniatis (Harvard University). The sources of (PRDII)2CAT, which contains two copies of the NF-kappa B binding site from the interferon-beta enhancer, pCMV5-MEKK1, which encodes for a C-terminal 672-residue fragment of MEKK1 (24), and all other plasmids have been described (7, 14).

Tissue Culture and Transfection-- HeLa cells were maintained as described (7). Transfections performed in 3.5-cm-diameter wells were conducted by calcium phosphate precipitation (25) or by using Fugene 6 according to the manufacturer's instructions (Boehringer Mannheim). CAT and protein measurements were performed as described (7, 26).

Immunoprecipitations-- Cells were washed once with Dulbecco's phosphate-buffered saline containing 1 mM EDTA and then lysed by the addition of 1 ml of buffer B (14) containing 10 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride. After centrifugation of the whole cell lysate at 16,000 × g for 10 min at 4 °C, the supernatant was incubated with 10 µl of M2-agarose with end over end rotation for 1 h at 4 °C. The resin was then washed three times with buffer B and eluted by the addition of 20 µl of 2× SDS-PAGE loading buffer.

Western Blotting-- Immunoprecipates were subjected to SDS-PAGE and then transferred to Immobilon-P membranes (Millipore). Membranes were blocked and then incubated with anti-Ikappa Balpha (C-21, Santa Cruz Biotechnology), anti-phospho-Ser-32 Ikappa Balpha (New England Biolabs), anti-Flag (D-8, Santa Cruz Biotechnology), or anti-JNK1 (C-17, Santa Cruz Biotechnology) polyclonal rabbit antibodies. After washing, the membranes were incubated with anti-rabbit IgG-horseradish peroxidase conjugates, washed, and then developed using SuperSignal substrate (Pierce).

Protein Kinase Assays-- Immunocomplex kinase assays for IKK and JNK were performed essentially as described (14), except that 10 µCi of [gamma -32P]ATP was employed per assay, 1 µg of GST-Ikappa Balpha (5-55) was used in the IKK assays, and the ATP concentration employed to initiate the IKK reactions was 50 µM instead of 200 µM. Kinase activities were quantitated using a Molecular Dynamics Storm 860 PhosphorImager.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MEKK2 and MEKK3 Induce NF-kappa B Activity and Site-specific Phosphorylation of Ikappa Balpha -- To examine the possibility that MAP3Ks other than MEKK1 or NIK might activate NF-kappa B HeLa cells were cotransfected with a reporter gene that contains two NF-kappa B binding sites and expression constructs for a series of MAP3Ks, including MEKK2 (20), MEKK3 (20), the catalytic domain of MEKK4 (Delta MEKK4) (21), apoptosis signal-regulating kinase 1 (ASK1) (22), and mixed-lineage kinase 3 (MLK3, also known as protein-tyrosine kinase 1 or SH3 domain-containing proline-rich kinase (23, 27, 28). All can activate the JNK pathway (20-22, 28). In addition, MEKK3, MEKK4, and ASK1 can activate the p38 pathway (22, 29, 30), whereas MEKK2 and MEKK3 can activate the ERK pathway (20). As shown in Fig. 1A, under conditions where overexpression of the positive controls MEKK1 and NIK induces activation of the NF-kappa B reporter gene, overexpression of MEKK3 (as reported previously; Ref. 31) and, to a lesser extent, MEKK2 induces activation as well. Delta MEKK4, ASK1, and MLK3 did not induce activation in either these cells (Fig. 1A) or the murine fibroblast cell line L929 (data not shown) but as expected did induce robust activation of coexpressed JNK1 in HeLa cells (data not shown).


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Fig. 1.   Activation of NF-kappa B and site-specific phosphorylation of Ikappa Balpha induced by MEKK2 and MEKK3. A, HeLa cells were cotransfected by calcium phosphate precipitation with 3 µg of (PRDII)2CAT and 6 µg of pCMV5-MEKK1, pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, pCMV5-Delta MEKK4, pcDNA3-NIK, pcDNA3-ASK1, pcDNA3-MLK3, or pCMV5. Cells were harvested 41 h post-transfection. CAT activities were normalized to protein concentrations of extracts. Shown is a representative result, performed in duplicate with standard deviations, from three independent experiments. B, HeLa cells were cotransfected using Fugene 6 with 0.5 µg of expression vectors for wild-type (WT) (pCMV4-FlagIkappa Balpha ) or mutant (M) (pCMV4-FlagIkappa Balpha (S32A/S36A)) Ikappa Balpha , and 4 µg of pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, or pCMV5. 24 h post-transfection, the epitope-tagged Ikappa Balpha was immunoprecipitated with M2-agarose and subjected to 12% SDS-PAGE. Top, immunoblotting (IB) was first performed with anti-Ikappa Balpha antibodies (C-21, Santa Cruz Biotechnology). The positions of unphosphorylated (Ikappa Balpha ) and phosphorylated (P-Ikappa Balpha ) are indicated to the right, and those of molecular mass markers (in kDa) are shown on the left. Bottom, the immunoblot was then stripped and reprobed with anti-phospho-Ser-32 Ikappa Balpha antibodies (New England Biolabs). Shown are representative results from three independent experiments.

NIK and MEKK1 activate NF-kappa B by inducing the site-specific phosphorylation of Ikappa B. In the case of Ikappa Balpha , this phosphorylation, which occurs at Ser-32 and Ser-36, is manifested by slower mobility when Ikappa Balpha is examined by SDS-PAGE (3, 4). To examine whether MEKK2 and MEKK3 might act through the same mechanism, HeLa cells were cotransfected with expression vectors for Flag-tagged wild-type or phosphorylation-defective (S32A/S36A) Ikappa Balpha and expression vectors for MEKK2, MEKK3, or empty expression vector. The Flag-tagged Ikappa Balpha was then immunoprecipitated with anti-Flag antibodies and examined by Western blotting with anti-Ikappa Balpha antibodies. As shown in Fig. 1B, MEKK2 and MEKK3 both induce the appearance of a more slowly migrating Ikappa Balpha species (top panel, lanes 3 and 5, upper bands) that is abolished when an S32A/S36A Ikappa Balpha mutant is examined (lanes 4 and 6), consistent with this species being N-terminally phosphorylated Ikappa Balpha . Delta MEKK4, ASK1, and MLK3 did not induce the appearance of this more slowly migrating species (data not shown). This Ikappa Balpha species was examined further by reprobing this blot with antibodies specific for phospho-Ser-32 Ikappa Balpha . As shown in Fig. 1B (bottom panel, lanes 3 and 5), the slower migrating Ikappa Balpha species induced by MEKK2 or MEKK3 is immunoreactive with these antibodies. We conclude that MEKK2 and MEKK3 can induce site-specific, N-terminal phosphorylation of Ikappa Balpha in vivo.

MEKK2 and MEKK3 Activate Both IKK-alpha and IKK-beta -- Both MEKK1 and NIK induce site-specific phosphorylation of Ikappa B by activating IKK-alpha and IKK-beta . To examine whether MEKK2 or MEKK3 acts by the same mechanism, HeLa cells were cotransfected with expression constructs for Flag-tagged IKK-alpha , IKK-beta , or JNK1 and expression constructs for MEKK1, MEKK2, MEKK3, NIK, or MLK3. The IKK or JNK was then immunoprecipitated with anti-Flag antibodies, and the kinase activities of the immunoprecipitated proteins were measured by using as substrates GST fused to the N terminii of Ikappa Balpha (residues 5-55) or c-Jun (residues 1-79), respectively, in the presence of [gamma -32P]ATP.

As shown in Fig. 2, (A and B, top panels), not only do MEKK1 (lane 2) and NIK (lane 5) activate IKK-alpha and IKK-beta activity, as reported previously (10, 11, 13-15), but MEKK2 and MEKK3 do so as well (lanes 3 and 4). Immunoblot analysis reveals comparable IKK expression levels (Fig. 2, A and B, lower panels). Furthermore, the MEKK2- or MEKK3-activated IKK-alpha and IKK-beta display the expected substrate specificity, because a S32A/S36A double mutation in the Ikappa Balpha substrate abolishes phosphorylation of the latter (data not shown). As a negative control, MLK3 does not significantly activate either IKK but does activate JNK (Fig. 2, A, B, and C, upper panels, lane 6) as expected (28). ASK1 and Delta MEKK4 likewise activate JNK but neither IKK-alpha nor IKK-beta (data not shown). NIK, in contrast, activates both IKKs but not JNK (Fig. 2, A, B, and C, upper panels, lane 5) as reported previously (14, 18).


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Fig. 2.   Activation of both IKK-alpha and IKK-beta by MEKK2 and MEKK3. HeLa cells were cotransfected using Fugene 6 with 3 µg of pCMV5-MEKK1, pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, pcDNA3-NIK, pcDNA3-MLK3, or pCMV5, and 0.5 µg of pRK-FlagIKK-alpha (A), pRK-FlagIKK-beta (B), or pcDNA3-FlagJNK1 (C). Whole cell extracts were prepared 24 h post-transfection and divided into two equal aliquots, and IKK or JNK from each aliquot was then immunoprecipitated with M2-agarose. One set of immunoprecipitates was then assayed for kinase activity (KA) toward GST-Ikappa Balpha (5-55) (A and B, top) or GST-cJun (1-79) (C, top) in the presence of [gamma -32P]ATP and then analyzed by 12% SDS-PAGE and autoradiography. The positions of the substrates are indicated to the right. The relative degrees of substrate 32P incorporation are indicated below the gels. The other set of immunoprecipitates was subjected to SDS-PAGE and analyzed by immunoblotting (IB) using anti-Flag (D-8, Santa Cruz Biotechnology) (A and B, bottom) or anti-JNK1 (C-17, Santa Cruz Biotechnology) (C, bottom) antibodies. The positions of IKK or JNK are indicated to the right. Shown are representative results from three to four independent experiments.

The potencies of MEKK1, MEKK2, MEKK3, and NIK in activating IKK-alpha , IKK-beta , or an NF-kappa B reporter gene were analyzed in more detail (Fig. 3). All four MAP3Ks induce dose-dependent increases in the activities of coexpressed IKK-alpha or IKK-beta . In the case of coexpressed IKK-alpha , the dose response curves are roughly comparable (Fig. 3A; see also Fig. 2A). In the case of coexpressed IKK-beta , NIK is a somewhat less potent activator than the other MAP3Ks (Fig. 3B). In contrast, NIK is a substantially more potent activator of an NF-kappa B reporter gene than the other three MAP3Ks (Fig. 3C). For example, the NF-kappa B reporter gene activity induced by 40 ng of NIK expression vector is comparable or even greater than that induced by 4000 ng of expression vector for either MEKK1, MEKK2, or MEKK3.


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Fig. 3.   Dose response experiments examining IKK and NF-kappa B activation by MAP3Ks. HeLa cells were cotransfected using Fugene 6 with 1 µg of pRK-FlagIKK-alpha (A), 0.5 µg of pRK-FlagIKK-beta (B), or 2 µg of (PRDII)3E1bCAT (C); and 40, 400, or 4000 ng of pCMV5-MEKK1, pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, or pcDNA3-NIK. The total DNA dose was brought up to 5 (A), 4.5 (B), or 6 µg (C) with pCMV5. A and B, IKK was immunoprecipitated with M2-agarose from whole cell extracts prepared 23-24 h post-transfection and assayed for activity toward GST-Ikappa Balpha (5-55) in the presence of [gamma -32P]ATP. IKK expression levels were analyzed by immunoblots of aliquots of the whole cell extracts using anti-Flag antibodies. C, cell extracts prepared 25 h post-transfection were assayed for CAT activity and normalized to the protein concentrations of the extracts. Shown are representative results from two to three independent experiments.

Dominant Negative IKK-alpha and Dominant Negative IKK-beta Inhibit MEKK2- and MEKK3-induced NF-kappa B Activation-- The experiments described above indicate that MEKK2 and MEKK3 can activate both IKK-alpha and IKK-beta in vivo. To examine whether this activation is functionally significant, HeLa cells were cotransfected with expression constructs for MEKK1, MEKK2, MEKK3, or empty expression vector, expression constructs for dominant negative, catalytically inactive IKK-alpha (K44A), IKK-beta (K44A), or empty expression vector, and an NF-kappa B reporter gene. As shown in Fig. 4, under conditions where activation of the NF-kappa B reporter gene induced by MEKK1 is almost completely inhibited by dominant negative IKK-alpha or dominant negative IKK-beta (13, 14, 32), that induced by either MEKK2 and MEKK3 is completely abolished. This therefore provides evidence that IKK-alpha and IKK-beta mediate the NF-kappa B inducing activity of MEKK2 and MEKK3.


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Fig. 4.   Inhibition of the NF-kappa B-inducing activity of MEKK2 and MEKK3 by dominant negative IKK-alpha and dominant negative IKK-beta . HeLa cells were cotransfected using Fugene 6 with 1.5 µg of (PRDII)3E1bCAT, 3 µg of pCMV5-MEKK1, pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, or pCMV5, and 1.5 µg of pRK-FlagIKK-alpha (K44A), pRK-FlagIKK-beta (K44A), or pCMV5. Cells were harvested 25 h post-transfection. CAT activities were normalized to protein concentrations of extracts. Shown is a representative result, performed in duplicate with standard deviations, from two independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we identify two additional, previously cloned MAP3Ks: MEKK2 and MEKK3, which now join NIK and MEKK1 as activators of IKK and NF-kappa B, thereby enlarging our framework for understanding NF-kappa B activation. Such knowledge is essential to providing a foundation for understanding how NF-kappa B can be activated by such a wide variety of stimuli. That a distinct subset of NF-kappa B-inducing MAP3Ks exists is highlighted by the fact that other MAP3Ks identified as activators of the JNK and/or p38 pathways, such as MEKK4, ASK1, and MLK3, fail to activate NF-kappa B.

Titration experiments reveal that MEKK2 and MEKK3 are comparable in potency to NIK in activating coexpressed IKK-alpha or IKK-beta but are substantially less potent than NIK in activating an NF-kappa B reporter gene (Fig. 3). Possible factors that might contribute to this apparent discrepancy include the following: (i) MEKK2 and MEKK3 might activate intracellular pathways that inhibit the NF-kappa B pathway and therefore could be relatively less effective in activating an NF-kappa B reporter gene; (ii) NIK might activate other components of the NF-kappa B pathway besides IKK and thus could be relatively more potent in activating an NF-kappa B reporter gene; and (iii) overexpressed IKK might respond less sensitively than endogenous IKK to coexpressed MAP3K and therefore might not accurately reflect activation of the NF-kappa B pathway (33). In any case, relative potencies in transient overexpression assays cannot be used as the sole criterion for assessing physiologic significance. A particularly pertinent example is provided by the fact the HTLV-I protein Tax activates NF-kappa B through MEKK1 (15) despite the fact that this MAP3K, like MEKK2 or MEKK3, is substantially less potent than NIK in activating an NF-kappa B reporter gene (14, 18).

MEKK2 and MEKK3, like other MAP3Ks, contain both catalytic and noncatalytic domains. The catalytic domains of MEKK2 and MEKK3 share 96% homology, consistent with the fact that both can activate NF-kappa B, whereas their noncatalytic domains are 65% homologous (20). MEKK2 is activated by treatment of cells by epidermal growth factor (34). In addition, MEKK2 and MEKK3 bind 14-3-3 proteins, an interaction that is mediated at least in part through their catalytic domains but that does not modulate their JNK-inducing activities (35).

Further experimentation will be required to determine the detailed mechanism by which MEKK2 and MEKK3 activate IKK-alpha and IKK-beta . NIK activates IKK-alpha by inducing phosphorylation of Ser-176 in the activation loop of the latter (16). Phosphorylation of Ser-177 and/or Ser-181 in IKK-beta is essential for its activity, because a double S176A/S181A mutation abolishes activity (12). Therefore, MEKK2 and MEKK3 might directly phosphorylate these IKK residues, particularly because these residues are components of canonical MAP2K activation loop motifs (SXXXS) (12) that might be predicted to be substrates for MAP3Ks. It is worth noting, however, that definitive experimental evidence that either NIK or MEKK1 directly induce IKK-alpha or IKK-beta catalytic activity has yet to be reported.

The fact that many MAP3Ks have the capacity to activate distinct pathways now raises the problem of how specificity in signaling pathways is achieved. One example of this is provided by the observation that Tax activates MEKK1 and induces potent NF-kappa B activity (15) but only modest JNK activity (15, 36), despite the fact that MEKK1 overexpression coordinately activates both (7). Thus, the relative capacities of a MAP3K to activate distinct signaling pathways may be modulated in a manner that is stimulus-specific.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Gary Johnson, Hidenori Ichijo, Richard Spritz, Roger Davis, David Wallach, David Goeddel, Dean Ballard, and Tom Maniatis for gifts of plasmids. We thank Drs. Mark Tykocinski and Leonard Jarett for support and encouragement.

    FOOTNOTES

* This work was supported in part by a Pilot Project Award from the Thomas B. McCabe and Jeannette E. Laws McCabe Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 218 John Morgan Bldg., Philadelphia, PA 19104. Tel.: 215-898-4701; Fax: 215-573-2272; E-mail: franklee{at}mail.med.upenn.edu.

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor-kappa B; ASK1, apoptosis signal-regulating kinase 1; CAT, chloramphenicol acetyl transferase; GST, glutathione S-transferase; HTLV-I, human T-cell leukemia virus, type I; IKK, Ikappa B kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAP2K, mitogen-activated protein kinase kinase; MAP3K, mitogen-activated protein kinase kinase kinase; MEKK, mitogen-activated protein kinase/ERK kinase kinase; MLK3, mixed-lineage kinase 3; NIK, NF-kappa B inducing kinase; PAGE, polyacrylamide gel electrophoresis; TNF-alpha , tumor necrosis factor alpha .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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