1 Center for Environmental Medicine and Lung Biology and 2 Department of Pharmacology, University of North Carolina, Chapel Hill 27599; and 3 Human Studies Division, National Health Effects and Environmental Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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We showed previously that epithelial
growth factor (EGF) receptor (EGFR) signaling is triggered by
metallic compounds associated with ambient air particles. Specifically,
we demonstrated that As, Zn, and V activated the EGFR
tyrosine kinase and the downstream kinases MEK1/2 and ERK1/2. In this
study, we examined the role of Ras in EGFR signaling and the nuclear
factor-B (NF-
B) activation pathway and the possible
interaction between these two signaling pathways in a human airway
epithelial cell line (BEAS-2B) exposed to As, V, or Zn ions. Each metal
significantly increased Ras activity, and this effect was inhibited by
the EGFR tyrosine kinase activity inhibitor PD-153035.
Adenoviral-mediated overexpression of a dominant-negative mutant form
of Ras(N17) significantly blocked MEK1/2 or ERK1/2 phosphorylation in
As-, Zn-, or V-exposed BEAS-2B cells but caused little inhibition of
V-, Zn- or EGF-induced EGFR tyrosine phosphorylation. This
confirmed Ras as an important intermediate effector in EGFR signaling. Interestingly, V, but not As, Zn, or EGF, induced I
B
serine phosphorylation, I
B
breakdown, and NF-
B DNA binding. Moreover, PD-153035 and overexpression of Ras(N17) each significantly blocked V-induced I
B
breakdown and NF-
B activation, while
inhibition of MEK activity with PD-98059 failed to do so. In
summary, exposure to As, Zn, and V initiated EGFR signaling and
Ras-dependent activation of MEK1/2 and ERK1/2, but only V induced
Ras-dependent NF-
B nuclear translocation. EGFR signaling
appears to cross talk with NF-
B signaling at the level of Ras, but
additional signals appear necessary for NF-
B activation. Together,
these data suggest that, in V-treated BEAS-2B cells, Ras-dependent
signaling is essential, but not sufficient, for activation of NF-
B.
G proteins; epidermal growth factor receptor; nuclear factor-B; mitogen-activated protein kinases; air pollution
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INTRODUCTION |
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THE RAS PROTEINS are the 21-kDa products of the Ras family of genes, which were first identified in retroviruses that trigger sarcoma-type tumors in rats (37). The importance of Ras proteins in growth regulation was recognized in the early 1980s, when it was discovered that ~30% of all human tumors contain an activating mutation in Ras (69). Ras activity is regulated through a GDP-GTP binding cycle, in which guanine nucleotide exchange factors (GEFs) promote the formation of active, GTP-bound Ras, while GTPase-activating proteins accelerate the formation of inactive, GDP-bound Ras (8). Increasing evidence has shown that Ras proteins act as a crucial switching station in intracellular signaling networks that regulate cell growth and differentiation. Ras activation is initiated by activated receptors with intrinsic or associated tyrosine kinase activity. Ras can also be activated through G protein-coupled receptors. Once activated, Ras proteins transmit signals to multiple downstream effectors, which mediate gene expression, remodeling of actin cytoskeleton, cell proliferation, survival, and transformation (69).
A major intracellular signaling pathway mediated by Ras is that initiated by the epidermal growth factor (EGF) receptor (EGFR). Altered regulation of EGFR signaling can lead to pleiotropic cellular responses, including mitogenesis or apoptosis, proliferation, oncogenic transformation, enhanced motility, protein secretion, and differentiation or dedifferentiation (62, 71). Generally, EGFR signaling begins with ligand binding, which leads to dimerization of EGFR monomers, activation of EGFR tyrosine kinase activity, autophosphorylation of EGFR tyrosine residues, and phosphorylation of downstream effectors. The best understood effector pathway is activation of Ras by the recruitment of son-of-sevenless (SOS) to the cell membrane via the adapter proteins Shc and/or Grb2. GTP-bound Ras is capable of interacting with downstream effectors such as the kinase Raf, which ultimately leads to the activation of mitogen-activated protein kinase (MAPK) and the corresponding cellular responses mentioned above (25). Our previous studies demonstrated that certain metals (As, Cu, V, or Zn) are able to activate the EGFR tyrosine kinase, MAPK kinase (MEK), and MAPK [extracellular signal-related kinase (ERK)] in human airway epithelial cells (58, 74). In preliminary studies, overexpression of dominant-negative Ras(N17) was observed to suppress the expression of interleukin (IL)-8 , IL-6, and cyclooxygenase-2 genes in BEAS-2B cells exposed to sodium metavanadate (+5) or vanadyl sulfate (+4) (unpublished observation). Because GTP-Ras is the putative activator of Raf-1 in the presence of certain cofactors (25), we have examined the role of active Ras in EGFR signaling induced by these metals.
As with signaling of Ras, nuclear factor-B (NF-
B) has been found
to play an important role in metal-induced gene expression of
proinflammatory cytokines such as IL-6 and IL-8 in human airway epithelial cells (34, 51). NF-
B is a mammalian
transcriptional activator that mediates the inducible expression of a
wide variety of genes, including products involved in proinflammatory
responses (15, 63). In most unstimulated cells, NF-
B
exists in the cytoplasm mainly as a heterodimer composed of two
subunits of 50 and 65 kDa and stays in an inactive form associated with
inhibitory proteins termed I
Bs, with I
B
being the predominant
form (4, 50, 52, 72). NF-
B activation is induced by a
variety of extracellular stimuli, which activate signal transduction
pathways that target the NF-
B-I
B complex for disruption
(28, 50). Degradation of I
B by the proteasome allows
NF-
B to translocate into the nucleus, where it binds to specific
response elements on the promoter region of certain genes and regulates
their transcription (5, 28, 46). Reactive oxygen species
(ROS) have been shown to activate NF-
B in rat lung epithelial cells
in a Ras-dependent manner (32).
In this study, we explored the role of Ras in EGFR signaling and
activation of NF-B and the possible interaction between these two
signaling pathways during exposure of human airway epithelial cells to
the combustion-derived metals As, V, and Zn. We observed that all three
metals significantly increased Ras activity and that Ras proved to be a
crucial intermediate in signaling from the EGFR tyrosine kinase to the
downstream kinases MEK1/2 and ERK1/2. Interestingly, of these three
metals, only V caused I
B
degradation and NF-
B DNA binding,
which was significantly suppressed by the EGFR tyrosine kinase activity
inhibitor PD-153035 and overexpression of dominant-negative Ras(N17)
(11). However, inhibition of MEK activity with PD-98059
failed to inhibit V-induced I
B
degradation and NF-
B
activation, suggesting that V-induced activation of NF-
B is EGFR
tyrosine kinase and Ras dependent, but MEK independent.
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MATERIALS AND METHODS |
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Materials.
American Chemical Society-grade metal salts were obtained from Alfa
(Ward Hill, MA) or Sigma (St. Louis, MO); tissue culture medium,
supplements, and supplies from Clonetics (San Diego, CA); SDS-PAGE
supplies, such as molecular-mass standards, polyacrylamide, ready gels,
and buffers, from Bio-Rad (Richmond, CA); [-32P]ATP
(6,000 Ci/mmol) from Amersham Pharmacia Biotech; specific anti-phospho
MEK1/2 (Ser217/221) and anti-I
B
(Ser32)
antibody from New England Biolabs (Boston, MA); protein A-Sepharose from Pharmacia Biotech (Uppsala, Sweden); specific anti-phospho ERK
(Tyr204) antibody, anti-I
B
antibody,
agarose-conjugated anti-EGFR antibody, horseradish peroxidase
(HRP)-conjugated goat anti-rabbit secondary antibody p-Tyr HRP antibody
from Santa Cruz Biotechnology (Santa Cruz, CA); and MEK activity
inhibitor PD-98059 (3), EGFR kinase inhibitor PD-153035
(27, 29), and proteasome inhibitor MG-132 (17) from Calbiochem-Novabiochem (La Jolla, CA).
Cell culture and exposure. BEAS-2B (subclone S6) cells were obtained from Drs. Curtis Harris and John Lechner (National Institutes of Health). The BEAS-2B cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 construct (53). BEAS-2B cells (passages 70-80) were grown to 90-100% confluence on tissue culture-treated Costar 6- or 12-well plates in keratinocyte basal medium (KBM) supplemented with 30 µg/ml bovine pituitary extract, 5 ng/ml human EGF, 500 ng/ml hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/ml insulin, as described previously (18, 49). Cells were placed in KBM (without supplements) for 20-22 h before treatment with metals. In some experiments, BEAS-2B cells were pretreated with 1 µM PD-153035 for 2 h or 20 µM MG-132 for 30 min before metal exposure.
A suspension of 100 mM sodium arsenite, vanadyl sulfate, and zinc sulfate was prepared in water and used as a stock for dilution into KBM, as described previously (58).Western blotting.
BEAS-2B cells treated with metals were lysed in RIPA buffer (1%
Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS in PBS, pH 7.4) containing 0.1 mM vanadyl sulfate and protease inhibitors (0.5 mg/ml
aprotinin, 0.5 mg/ml E-64, 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 mg/ml leupeptin) (58). After
normalization for protein content, cell extracts were subjected to
SDS-PAGE on 11% gradient polyacrylamide gels or 4-15%
Tris · HCl ready gels (Bio-Rad) with a Tris-glycine-SDS buffer.
Proteins were electroblotted onto a nitrocellulose membrane. The blots were blocked with 3% nonfat milk, washed briefly, incubated with phospho-specific MEK1/2, ERK1/2, or IB
antibodies in 3% BSA at
4°C overnight, and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Bands were detected using chemiluminescence reagents and hypersensitive films, as described previously (58).
Immunoprecipitation.
Confluent BEAS-2B cells were challenged with metals and lysed with RIPA
buffer. The lysate supernatants were precleared with protein
A-Sepharose and immunoprecipitated by incubation with agarose-conjugated anti-EGFR antibody for 1 h at 4°C. Immune
complexes were washed twice with RIPA buffer and once with cold PBS.
The immunoprecipitates were suspended with 25 µl of 4°C sample
loading buffer (62.5 mM Tris · HCl, pH 6.8, 10% glycerol, 2%
SDS, 0.7 M -mercaptoethanol, and 0.05% bromphenol blue) and boiled
for 5 min before separation on 4-15% Tris · HCl ready
gels. The EGFR tyrosine phosphorylation bands were detected as
described previously (74).
Ras activation assay. The Ras activation assay kit was purchased from Upstate Biotechnology (Lake Placid, NY). Ras activity was determined according to the supplier's instruction. Briefly, BEAS-2B cells treated with metals were lysed with 5× Mg2+ lysis/wash buffer (MLB: 125 mM HEPES, pH 7.5, 750 mM NaCl, 5% Igepal CA-630, 50 mM MgCl2, 5 mM EDTA, and 10% glycerol). The lysates were precleared with glutathione agarose. Five microliters of a 50% slurry of Raf-1 Ras binding domain-agarose were incubated with 500-1,000 µg of cell lysate at 4°C for 30 min. The agarose was collected by centrifugation and washed with MLB three times and once with cold PBS and then boiled in 25 µl of reducing sample loading buffer. GTP-bound Ras protein was resolved by electrophoresis, transferred to nitrocellulose, and probed with a mouse monoclonal anti-Ras (clone Ras-10) antibody (1 µg/ml). Protein bands were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a enhanced chemiluminescence detection system (74).
Infection with adenovirus. Ras(N17), a GDP-bound dominant-negative mutant, is used widely as an interfering mutant to assess Ras function in vivo (11, 66). Confluent BEAS-2B cells were infected with a multiplicity of infection of 100 plaque-forming units/cell nonrecombinant control adenovirus ad5CMV3 or recombinant inactive Ras(N17) (obtained from Dr. Craig Logsdon, University of Michigan) for 24 h (48). The infection medium was removed and replaced with KBM for another 24 h before challenge with metals. BEAS-2B cells were lysed, and the lysates were subjected to Western blotting or electrophoretic mobility shift assay (EMSA).
Separation of cytoplasmic and nuclear proteins.
BEAS-2B cells treated with metals were lysed with cytoplasmic extract
buffer [10 mM Tris · HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT)] containing protease inhibitors
(34). Nonidet P-40 (0.1%) was added to the cell lysate,
and the lysate was vortexed for 15 s. After centrifugation at
14,000 rpm for 30 s, the supernatant (cytoplasmic fraction) was
transferred to another tube and boiled with sample loading buffer to
detect the degradation of IB
through Western blotting using
rabbit
B
antibody (Santa Cruz Biotechnology). The nuclear pellets
were resuspended with 100 µl of nuclear extract buffer (20 mM
Tris · HCl, pH 8.0, 400 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 25% glycerol, and 1 mM DTT) containing protease inhibitors.
After brief centrifugation, the supernatants containing the nuclear
fraction were subjected to EMSA.
EMSA.
EMSA was conducted as described previously (34). Briefly,
an oligonucleotide probe (sequence from 5' to 3': GGCTGGGGATTCCCCATCT) for NF-B binding site on myosin heavy chain class II gene was labeled by incubating 15 U T4 polynucleotide kinase (New England Biolabs, Beverly, MA), 100 ng double-stranded probe, and 100 µCi [
-32P]ATP at 37°C for 30 min. Unincorporated
32P was removed using a desalting column (Nuc Trap,
Stratagene, San Diego, CA), and DNA-protein binding reactions were
performed for 10 min at room temperature in a mixture containing 2 µg
nuclear extract, 1 µl labeled probe, 10 µl running buffer (10 mM
Tris · HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA, 1 mM DTT, and 5%
glycerol), and 2 µg poly(dI-dC) (Roche Molecular Biochemicals).
Samples were separated by electrophoresis through 4.5% nondenaturing
polyacrylamide gels containing 0.5× Tris-borate-EDTA. Gels were dried,
and radiolabeled species were autoradiographed using a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
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Activation of Ras in BEAS-2B cells exposed to metals. Ras has been reported to mediate its effects in part through activation of a cascade of kinases: Raf (c-Raf-1, A-Raf, and B-Raf), MEK (MAPK/ERK kinases 1 and 2), and ERK1/2 (21, 22). We previously showed that exposure to As, V, and Zn ions activates the MAPK cascade (MEK/ERK) in BEAS-2B cells (74). We therefore examined whether Ras plays a role in metal-induced activation of the MAPK cascade in BEAS-2B cells.
BEAS-2B cells grown to confluence were deprived of growth factors for 20-22 h and then challenged with 100 µM As, V, or Zn for 1 h. Ras activity was determined using a commercially available Ras activation assay kit, which is based on affinity precipitation of GTP-bound Ras with agarose-conjugated Raf-1-glutathione S-transferase. Western blotting using specific anti-Ras antibody showed that As, V, or Zn exposure induced a marked increase in levels of GTP-bound Ras compared with controls. As expected, EGF strongly activated Ras in BEAS-2B cells (Fig. 1).
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Ras mediates metal-induced signaling from EGFR tyrosine kinase to
MEK1/2.
To determine whether Ras activation by metal exposure is dependent on
EGFR signaling, we pretreated BEAS-2B cells for 2 h with vehicle
alone or 1 µM PD-153035, a specific EGFR tyrosine kinase inhibitor
(40), before exposure to As, V, or Zn and measured levels
of GTP-bound Ras. As, V, Zn, and EGF each induced an increase in the
active GTP-bound Ras formation in BEAS-2B cells (Fig.
2). PD-150335 markedly suppressed Ras
activation in BEAS-2B cells exposed to As, V, or Zn, with no detectable
change in Ras protein expression. PD-153035 also significantly blocked
EGF-induced Ras activation. This implicated EGFR tyrosine kinase
activity as a requirement for metal-induced Ras activation in BEAS-2B
cells.
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Ras(N17) blocked V-induced NF-B DNA binding activity and
I
B
degradation.
Because previous studies showed that some metal ions can activate
NF-
B, we next studied the role of Ras in NF-
B DNA binding activity in cells exposed to As, V, or Zn ions. The possible role of
active Ras in metal-induced NF-
B DNA binding activity was tested in
this study. BEAS-2B cells were infected with control or Ras(N17)
adenovirus and challenged with metals. The cells were lysed, and
nuclear proteins were extracted for EMSA. Interestingly, among As, V,
and Zn, only V induced NF-
B DNA binding in BEAS-2B cells, while As,
Zn, or EGF treatment did not induce an increase of NF-
B DNA binding
activity relative to the vehicle control (Fig.
4A), which is consistent with
our previous observations in primary human bronchial cells
(34). Moreover, V-induced NF-
B DNA binding activity was
suppressed by the Ras(N17) overexpression (Fig. 4A). The
positive control tumor necrosis factor significantly enhanced NF-
B
DNA binding activity (data not shown). Similar to our previous
observations in primary human airway epithelial cells
(33), supershift and lysis using specific antibodies
against the p50, p65, and c-Rel compounds of the NF-
B binding
complex identified the V-induced NF-
B DNA binding complex in BEAS-2B cells as the p65/p50 heterodimer (Fig. 4B). This suggested
that Ras is required for V-induced NF-
B nuclear translocation in
BEAS-2B cells.
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Effect of an EGFR tyrosine kinase inhibitor, PD-153035, or an MEK
activity inhibitor, PD-98059, on V-induced NF-B activation in
BEAS-2B cells.
NF-
B DNA binding activity and I
B
phosphorylation and
degradation suggested that only V induced NF-
B activation in BEAS-2B cells. Moreover, Ras is necessary for V-induced NF-
B activation. The
EGFR is required for V-induced Ras activation. Therefore, the EGFR
tyrosine kinase activity should also be necessary in V-induced NF-
B
activation. We observed that the selective EGFR tyrosine kinase
inhibitor PD-153035 suppressed V-induced I
B
degradation and
NF-
B DNA binding activity in BEAS-2B cells (Fig. 5, top panels). Next, we
tested whether MEK, a downstream kinase of Ras, could also contribute
to V-induced NF-
B nuclear translocation in BEAS-2B cells. Confluent
BEAS-2B cells were pretreated with the MEK activity inhibitor PD-98059
and then stimulated with V. Extracted nuclear proteins were then
subjected to EMSA for NF-
B activation. V induced I
B
degradation and NF-
B DNA binding. PD-98059 had no inhibitory effect
on V-induced I
B
degradation and NF-
B DNA binding (Fig. 5,
bottom panels), suggesting that MEK1/2 does not participate
in V-induced NF-
B activation in BEAS-2B cells.
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DISCUSSION |
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Ras is an important component of multiple signal transduction
cascades (69). The data shown here demonstrate that
combustion-derived metals such as As, V, and Zn induce Ras activation
in human airway epithelial cells. Active Ras not only mediates
signaling from the EGFR to the MAPK cascade in response to exposure to
As, V, or Zn, but it also plays a pivotal role in V-induced NF-B DNA binding. Although As, V, and Zn can each induce the activation of Ras,
only V increases NF-
B activity, and it does so in a Ras-dependent manner, suggesting that, in BEAS-2B cells, Ras may be essential, but
not sufficient, to induce NF-
B activity. Moreover, the MEK activity
inhibitor PD-98059 failed to attenuate V-induced NF-
B DNA binding
activity. These data suggest different mechanisms for signaling
initiated by As, V, and Zn in human airway epithelial cells. The
proposed mechanisms for metallic ion-induced intracellular signaling in
BEAS-2B cells are depicted in Fig. 6.
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V ions are known inhibitors of protein tyrosine phosphatases, including
those that dephosphorylate signaling kinases (23). Zn
(10, 70) and, to a lesser extent, As (14)
ions are also reported inhibitors of certain tyrosine phosphatases.
Part of the impetus for the work undertaken in this study is the aim to identify the initiating event(s) in metal ion-induced signaling. Our
previous studies have suggested that dysregulation of protein tyrosine
phosphate metabolism and inhibition of tyrosine phosphatases may be
pivotal effects in this process (59, 60). We have shown that exposure to V and Zn, but not As, can cause profound inhibition of
multiple tyrosine phosphatases in human airway epithelial cells (59). Thus the possibility exists that the effects of As,
V, and Zn ions on signaling to NF-B and/or MEK/ERK are secondary to
an inhibitory effect on phosphatases that keeps these proteins in an
inactive state in resting cells. In principle, the effect of
phosphatase inhibition could occur at any or all levels in a signaling
cascade. However, the data we present in this study, showing that As,
V, and Zn each signal to MEK/ERK in a Ras-dependent manner, suggest
that inhibition to tyrosine phosphatases, if required in the signaling
induced by these metal ions, occurs upstream of Ras.
An EGFR tyrosine kinase inhibitor significantly blocked metal-induced
Ras activation in human airway epithelial cells, and dominant-negative Ras(N17) posed no detectable effect on EGFR tyrosine
kinase activity. These data suggest that EGFR tyrosine kinase is
required for Ras activation in BEAS-2B cells exposed to
combustion-derived metals. The events involved in the signaling from
EGFR to Ras were not examined in this study. It has been documented
that Ras activation by the EGFR occurs through adapter proteins and
GEFs (8). Furthermore, activation of Ras is responsible for metal-induced MEK and ERK phosphorylation in BEAS-2B cells exposed
to metals. One question raised by these findings concerns the mechanism
through which Ras transmits the signal from the EGFR to MEK and ERK.
Our previous study demonstrated that MEK/ERK phosphorylation induced by
As, V, or Zn occurred in a Raf-1-independent manner (74).
Observations from mammalian cells indicate that the events downstream
of Ras are more complex than simply activating the Raf kinases.
Multiple Ras effectors have been recognized, such as
p120Ras GAP, PI3KRIN1, AF6, and RalGDS (24, 30, 38,
55, 76). Additionally, MEK kinase (MEKK1), a large (196-kDa)
protein, has been shown to be activated by low-molecular-weight
GTP-binding proteins (Ras, Rac, and Cdc42) and activates MEK/ERK, JNK,
and IB kinases (44, 57, 78).
Interestingly, in addition to the pivotal role of Ras in the EGFR
signaling in BEAS-2B cells exposed to combustion-derived metals, this
study also shows that active Ras is necessary for V-induced NF-kB
activation. As described previously (33), the crucial step
in NF-B activation is the signal-induced proteolytic degradation of
I
B in the cytoplasm (47). I
B breakdown is initiated after the phosphorylation of its NH2-terminal serine
residues by I
B kinases such as IKK1 and IKK2 (7, 41,
47). V has been shown to induce IKK activity and increase
I
B
phosphorylation and degradation and NF-
B nuclear
translocation in different cell types, including human airway
epithelial cells (16, 31, 33, 34). Several studies have
suggested that IKK1 and IKK2 are themselves phosphorylated and
activated by one or more upstream activating kinases such as MEKK1 and
NF-
B-inducing kinase (6, 19, 42, 54, 67, 73, 79, 80),
with NF-
B-inducing kinase displaying a preference for IKK1 and MEKK1
a preference for IKK2 (47). In addition, IKK2 activity was
also found to be significantly elevated by V in mouse macrophages
(16). Therefore, MEKK1, one of the downstream kinases of
Ras, could be involved in V-induced IKK2 activation (16),
further leading to I
B
breakdown and NF-
B nuclear
translocation. Moreover, the MEK activity inhibitor PD-98059 had no
detectable inhibitory effect on V-induced NF-
B activation in BEAS-2B
cells, which implies that the upstream kinases of MEK, but not MEK
itself, are responsible for V-induced NF-
B nuclear translocation.
Transfection of transformed rat lung epithelial cells with a
constitutively active MEKK1 construct or dominant-negative MEKK1 can
significantly modulate oxidant-induced NF-
B activation (32). It is possible that MEKK1 was involved in the
V-induced NF-
B activation in BEAS-2B cells. MEKK1 may therefore act
as a bifurcation point in V-induced MEK/ERK activation and NF-
B nuclear translocation.
Ras-dependent NF-B activation induced by diverse stimuli has been
reported (32, 33, 40). It is intriguing that, although As,
V, and Zn are able to induce Ras activation in BEAS-2B cells, only V
induces NF-
B activation. In one study, Ras(N17) failed to block
tumor necrosis factor-
-induced NF-
B activation in rat lung
epithelial cells (32). Together, these observations
suggest that active Ras is not sufficient for V-induced NF-
B DNA
binding activity in BEAS-2B cells and that input from other signaling cascades or cofactors is necessary to activate NF-
B. In preliminary work, As and Zn were unable to induce oxidative stress in BEAS-2B cells. However, vanadyl sulfate (+4)- or vanadate (+5)-induced NF-
B
nuclear translocation could be blocked by overexpression of catalase or
the antioxidant N-acetyl-L-cysteine,
respectively (33, 77), suggesting a crucial role for ROS
in V-induced NF-
B activation. Additional evidence has shown that
oxidant-induced NF-
B activation is Ras dependent in transformed rat
lung epithelial cells (32). Therefore, it is possible that
generation of ROS, which are induced by V, but not by As or Zn,
contributes to the additional signals required for NF-
B activation
(7). The mechanisms for synergism of Ras with ROS in
V-induced NF-
B DNA binding in BEAS cells are not known. However, it
has recently been demonstrated that the role of ROS in NF-
B
activation is cell type or stimulus specific, and its role may be
facilitatory, rather than causal (9).
In addition to its ability to generate ROS, other factors may also
explain the effect of V on NF-B DNA binding relative to that of As
or Zn exposure. Arsenite has been reported to directly inhibit the
activity of IKK and, thereby, limit the phosphorylation and degradation
of I
B
(56). This is consistent with our data showing
that As exposure of BEAS-2B cells does not lead to I
B degradation
(34). Furthermore, As and other thiol-reactive metals, including Zn, have been found to inhibit NF-
B DNA binding through interactions with critical protein sulfhydryls (64).
In addition to Ras, members of the Ras superfamily of GTP-binding
proteins such as Rho and Rac, also regulate a variety of signal
transduction pathways in eukaryotic cells (20). It has recently been reported that, in certain circumstances, Rac is required
for NF-B activation (12). However, in our preliminary study, we did not observe Rac activation in BEAS-2B cells exposed to
As, V, or Zn (data not shown).
Metal ions can be abundant in airborne particulate matter (PM)
(1, 2, 45). It has been argued that metals contribute to
PM-induced pulmonary inflammatory gene expression (13, 26, 34-36, 65, 68). The role of Ras in metal-induced EGF
signaling and NF-B activation in human airway epithelial cells may
provide an important link in the elucidation of the mechanism of
toxicity of PM inhalation.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of L. Dailey and advice from Dr. W. Reed.
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FOOTNOTES |
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This work was funded in part by US Environmental Protection Agency Grant 824915.
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names constitute endorsement or recommendation for use.
Address for reprint requests and other correspondence: J. M. Samet, Human Studies Div., National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711 (E-mail: Samet.jim{at}epa.gov).
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.
10.1152/ajplung.00390.2001
Received 13 March 2001; accepted in final form 26 November 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aastrup, P,
Riget F,
Dietz R,
and
Asmund G.
Lead, zinc, cadmium, mercury, selenium and copper in Greenland caribou and reindeer (Rangifer tarandus).
Sci Total Environ
245:
149-159,
2000[ISI][Medline].
2.
Adamson, IY,
Prieditis H,
Hedgecock C,
and
Vincent R.
Zinc is the toxic factor in the lung response to an atmospheric particulate sample.
Toxicol Appl Pharmacol
166:
111-119,
2000[ISI][Medline].
3.
Alessi, DR,
Cuenda A,
Cohen P,
Dudley DT,
and
Saltiel AR.
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem
270:
27489-27494,
1995
4.
Baeuerle, PA,
and
Baltimore D.
NF-B: ten years after.
Cell
87:
13-20,
1996[ISI][Medline].
5.
Baldwin, AS, Jr.
The NF-B and I
B proteins: new discoveries and insights.
Annu Rev Immunol
14:
649-683,
1996[ISI][Medline].
6.
Bhullar, IS,
Li YS,
Miao H,
Zandi E,
Kim M,
Shyy JY,
and
Chien S.
Fluid shear stress activation of IB kinase is integrin-dependent.
J Biol Chem
273:
30544-30549,
1998
7.
Bonizzi, G,
Piette J,
Merville MP,
and
Bours V.
Cell type-specific role for reactive oxygen species in nuclear factor-B activation by interleukin-1.
Biochem Pharmacol
59:
7-11,
2000[ISI][Medline].
8.
Bourne, HR,
Sanders DA,
and
McCormick F.
The GTPase superfamily: conserved structure and molecular mechanism.
Nature
349:
117-127,
1991[ISI][Medline].
9.
Bowie, A,
and
O'Neill LA.
Oxidative stress and nuclear factor-B activation: a reassessment of the evidence in the light of recent discoveries.
Biochem Pharmacol
59:
13-23,
2000[ISI][Medline].
10.
Brautigan, DL,
Bornstein P,
and
Gallis B.
Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn.
J Biol Chem
256:
6519-6522,
1981
11.
Cai, H,
and
Cooper GM.
Inducible expression of Ras N17 dominant inhibitory protein.
Methods Enzymol
255:
230-237,
1995[ISI][Medline].
12.
Cammarano, MS,
and
Minden A.
Dbl and the Rho GTPases activate NFB by I
B kinase (IKK)-dependent and IKK-independent pathways.
J Biol Chem
276:
25876-25882,
2001
13.
Carter, JD,
Ghio AJ,
Samet JM,
and
Devlin RB.
Cytokine production by human airway epithelial cells after exposure to an air pollution particle is metal-dependent.
Toxicol Appl Pharmacol
146:
180-188,
1997[ISI][Medline].
14.
Cavigelli, M,
Li WW,
Lin A,
Su B,
Yoshioka K,
and
Karin M.
The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase.
EMBO J
15:
6269-6279,
1996[Abstract].
15.
Chabot-Fletcher, M.
A role for transcription factor NF-B in inflammation.
Inflamm Res
46:
1-2,
1997[ISI][Medline].
16.
Chen, F,
Demers LM,
Vallyathan V,
Ding M,
Lu Y,
Castranova V,
and
Shi X.
Vanadate induction of NF-B involves I
B kinase-
and SAPK/ERK kinase 1 in macrophages.
J Biol Chem
274:
20307-20312,
1999
17.
Chen, Z,
Hagler J,
Palombella VJ,
Melandri F,
Scherer D,
Ballard D,
and
Maniatis T.
Signal-induced site-specific phosphorylation targets IB
to the ubiquitin-proteasome pathway.
Genes Dev
9:
1586-1597,
1995[Abstract].
18.
Devlin, RB,
McKinnon KP,
Noah T,
Becker S,
and
Koren HS.
Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
266:
L612-L619,
1994
19.
DiDonato, JA,
Hayakawa M,
Rothwarf DM,
Zandi E,
and
Karin M.
A cytokine-responsive IB kinase that activates the transcription factor NF-
B.
Nature
388:
548-554,
1997[ISI][Medline].
20.
Diekmann, D,
Abo A,
Johnston C,
Segal AW,
and
Hall A.
Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity.
Science
265:
531-533,
1994[ISI][Medline].
21.
Egan, SE,
Giddings BW,
Brooks MW,
Buday L,
Sizeland AM,
and
Weinberg RA.
Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation.
Nature
363:
45-51,
1993[ISI][Medline].
22.
Egan, SE,
and
Weinberg RA.
The pathway to signal achievement.
Nature
365:
781-783,
1993[ISI][Medline].
23.
Fauman, EB,
and
Saper MA.
Structure and function of the protein tyrosine phosphatases.
Trends Biochem Sci
21:
413-417,
1996[ISI][Medline].
24.
Feig, LA,
Urano T,
and
Cantor S.
Evidence for a Ras/Ral signaling cascade.
Trends Biochem Sci
21:
438-441,
1996[ISI][Medline].
25.
Force, T,
and
Bonventre JV.
Growth factors and mitogen-activated protein kinases.
Hypertension
31:
152-161,
1998
26.
Frampton, MW,
Ghio AJ,
Samet JM,
Carson JL,
Carter JD,
and
Devlin RB.
Effects of aqueous extracts of PM10 filters from the Utah valley on human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
277:
L960-L967,
1999
27.
Fry, DW,
Kraker AJ,
McMichael A,
Ambroso LA,
Nelson JM,
Leopold WR,
Connors RW,
and
Bridges AJ.
A specific inhibitor of the epidermal growth factor receptor tyrosine kinase.
Science
265:
1093-1095,
1994[ISI][Medline].
28.
Ghosh, S,
May MJ,
and
Kopp EB.
NF-B and Rel proteins: evolutionarily conserved mediators of immune responses.
Annu Rev Immunol
16:
225-260,
1998[ISI][Medline].
29.
Hamilton, M,
and
Wolfman A.
Oncogenic Ha-Ras-dependent mitogen-activated protein kinase activity requires signaling through the epidermal growth factor receptor.
J Biol Chem
273:
28155-28162,
1998
30.
Han, J,
Luby-Phelps K,
Das B,
Shu X,
Xia Y,
Mosteller RD,
Krishna UM,
Falck JR,
White MA,
and
Broek D.
Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav.
Science
279:
558-560,
1998
31.
Huang, C,
Ding M,
Li J,
Leonard SS,
Rojanasakul Y,
Castranova V,
Vallyathan V,
Ju G,
and
Shi X.
Vanadium-induced NFAT activation through hydrogen peroxide.
J Biol Chem
5:
5,
2001.
32.
Janssen-Heininger, YM,
Macara I,
and
Mossman BT.
Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-B: requirement of Ras/mitogen-activated protein kinases in the activation of NF-
B by oxidants.
Am J Respir Cell Mol Biol
20:
942-952,
1999
33.
Jaspers, I,
Samet JM,
Erzurum S,
and
Reed W.
Vanadium-induced B-dependent transcription depends upon peroxide-induced activation of the p38 mitogen-activated protein kinase.
Am J Respir Cell Mol Biol
23:
95-102,
2000
34.
Jaspers, I,
Samet JM,
and
Reed W.
Arsenite exposure of cultured airway epithelial cells activates B-dependent interleukin-8 gene expression in the absence of nuclear factor-
B nuclear translocation.
J Biol Chem
274:
31025-31033,
1999
35.
Jiang, N,
Dreher KL,
Dye JA,
Li Y,
Richards JH,
Martin LD,
and
Adler KB.
Residual oil fly ash induces cytotoxicity and mucin secretion by guinea pig tracheal epithelial cells via an oxidant-mediated mechanism.
Toxicol Appl Pharmacol
163:
221-230,
2000[ISI][Medline].
36.
Kodavanti, UP,
Hauser R,
Christiani DC,
Meng ZH,
McGee J,
Ledbetter A,
Richards J,
and
Costa DL.
Pulmonary responses to oil fly ash particles in the rat differ by virtue of their specific soluble metals.
Toxicol Sci
43:
204-212,
1998[Abstract].
37.
Krauss G. Signal transmission via Ras proteins. In:
Biochemistry of Signal Transduction and Regulation,
translated by Schonbrunnner N and Cooper J. Weinheim, Germany: Wiley,
1999, p. 324-343.
38.
Kuriyama, M,
Harada N,
Kuroda S,
Yamamoto T,
Nakafuku M,
Iwamatsu A,
Yamamoto D,
Prasad R,
Croce C,
Canaani E,
and
Kaibuchi K.
Identification of AF-6 and canoe as putative targets for Ras.
J Biol Chem
271:
607-610,
1996
39.
Laney, JD,
and
Hochstrasser M.
Substrate targeting in the ubiquitin system.
Cell
97:
427-430,
1999[ISI][Medline].
40.
Li, JD,
Feng W,
Gallup M,
Kim JH,
Gum J,
Kim Y,
and
Basbaum C.
Activation of NF-B via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells.
Proc Natl Acad Sci USA
95:
5718-5723,
1998
41.
Ling, L,
Cao Z,
and
Goeddel DV.
NF-B-inducing kinase activates IKK-
by phosphorylation of Ser176.
Proc Natl Acad Sci USA
95:
3792-3797,
1998
42.
Malinin, NL,
Boldin MP,
Kovalenko AV,
and
Wallach D.
MAP3K-related kinase involved in NF-B induction by TNF, CD95 and IL-1.
Nature
385:
540-544,
1997[ISI][Medline].
43.
Maniatis, T.
A ubiquitin ligase complex essential for the NF-B, Wnt/Wingless, and Hedgehog signaling pathways.
Genes Dev
13:
505-510,
1999
44.
Marshall, MS.
Ras target proteins in eukaryotic cells.
FASEB J
9:
1311-1318,
1995
45.
Martin, CJ,
Le XC,
Guidotti TL,
Yalcin S,
Chum E,
Audette RJ,
Liang C,
Yuan B,
Zhang X,
and
Wu J.
Zinc exposure in Chinese foundry workers.
Am J Ind Med
35:
574-580,
1999[ISI][Medline].
46.
May, MJ,
and
Ghosh S.
Signal transduction through NF-B.
Immunol Today
19:
80-88,
1998[ISI][Medline].
47.
Mercurio, F,
and
Manning AM.
Multiple signals converging on NF-B.
Curr Opin Cell Biol
11:
226-232,
1999[ISI][Medline].
48.
Nicke, B,
Tseng MJ,
Fenrich M,
and
Logsdon CD.
Adenovirus-mediated gene transfer of RasN17 inhibits specific CCK actions on pancreatic acinar cells.
Am J Physiol Gastrointest Liver Physiol
276:
G499-G506,
1999
49.
Noah, TL,
Paradiso AM,
Madden MC,
McKinnon KP,
and
Devlin RB.
The response of a human bronchial epithelial cell line to histamine: intracellular calcium changes and extracellular release of inflammatory mediators.
Am J Respir Cell Mol Biol
5:
484-492,
1991[ISI][Medline].
50.
Norris, JL,
and
Baldwin AS, Jr.
Oncogenic Ras enhances NF-B transcriptional activity through Raf-dependent and Raf-independent mitogen-activated protein kinase signaling pathways.
J Biol Chem
274:
13841-13846,
1999
51.
Quay, JL,
Reed W,
Samet J,
and
Devlin RB.
Air pollution particles induce IL-6 gene expression in human airway epithelial cells via NF-B activation.
Am J Respir Cell Mol Biol
19:
98-106,
1998
52.
Rahman, I,
and
MacNee W.
Role of transcription factors in inflammatory lung diseases.
Thorax
53:
601-612,
1998
53.
Reddel, RR,
Ke Y,
Kaighn ME,
Malan-Shibley L,
Lechner JF,
Rhim JS,
and
Harris CC.
Human bronchial epithelial cells neoplastically transformed by v-Ki-ras: altered response to inducers of terminal squamous differentiation.
Oncogene Res
3:
401-408,
1988[Medline].
54.
Regnier, CH,
Song HY,
Gao X,
Goeddel DV,
Cao Z,
and
Rothe M.
Identification and characterization of an IB kinase.
Cell
90:
373-383,
1997[ISI][Medline].
55.
Rodriguez-Viciana, P,
Warne PH,
Dhand R,
Vanhaesebroeck B,
Gout I,
Fry MJ,
Waterfield MD,
and
Downward J.
Phosphatidylinositol-3-OH kinase as a direct target of Ras.
Nature
370:
527-532,
1994[ISI][Medline].
56.
Roussel, RR,
and
Barchowsky A.
Arsenic inhibits NF-B-mediated gene transcription by blocking I
B kinase activity and I
B
phosphorylation and degradation.
Arch Biochem Biophys
377:
204-212,
2000[ISI][Medline].
57.
Russell, M,
Lange-Carter CA,
and
Johnson GL.
Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1).
J Biol Chem
270:
11757-11760,
1995
58.
Samet, JM,
Graves LM,
Quay J,
Dailey LA,
Devlin RB,
Ghio AJ,
Wu W,
Bromberg PA,
and
Reed W.
Activation of MAPKs in human bronchial epithelial cells exposed to metals.
Am J Physiol Lung Cell Mol Physiol
275:
L551-L558,
1998
59.
Samet, JM,
Silbajoris R,
Wu W,
and
Graves LM.
Tyrosine phosphatases as targets in metal-induced signaling in human airway epithelial cells.
Am J Respir Cell Mol Biol
21:
357-364,
1999
60.
Samet, JM,
Stonehuerner J,
Reed W,
Devlin RB,
Dailey LA,
Kennedy TP,
Bromberg PA,
and
Ghio AJ.
Disruption of protein tyrosine phosphate homeostasis in bronchial epithelial cells exposed to oil fly ash.
Am J Physiol Lung Cell Mol Physiol
272:
L426-L432,
1997
61.
Scherer, DC,
Brockman JA,
Chen Z,
Maniatis T,
and
Ballard DW.
Signal-induced degradation of IB
requires site-specific ubiquitination.
Proc Natl Acad Sci USA
92:
11259-11263,
1995[Abstract].
62.
Seger, R,
and
Krebs EG.
The MAPK signaling cascade.
FASEB J
9:
726-735,
1995
63.
Sha, WC,
Liou HC,
Tuomanen EI,
and
Baltimore D.
Targeted disruption of the p50 subunit of NF-B leads to multifocal defects in immune responses.
Cell
80:
321-330,
1995[ISI][Medline].
64.
Shumilla, JA,
Wetterhahn KE,
and
Barchowsky A.
Inhibition of NF-B binding to DNA by chromium, cadmium, mercury, zinc, and arsenite in vitro: evidence of a thiol mechanism.
Arch Biochem Biophys
349:
356-362,
1998[ISI][Medline].
65.
Silbajoris, R,
Ghio AJ,
Samet JM,
Jaskot R,
Dreher KL,
and
Brighton LE.
In vivo and in vitro correlation of pulmonary MAP kinase activation following metallic exposure.
Inhal Toxicol
12:
453-468,
2000[ISI][Medline].
66.
Stewart, S,
and
Guan KL.
The dominant negative Ras mutant, N17Ras, can inhibit signaling independently of blocking Ras activation.
J Biol Chem
275:
8854-8862,
2000
67.
Uhlik, M,
Good L,
Xiao G,
Harhaj EW,
Zandi E,
Karin M,
and
Sun SC.
NF-B-inducing kinase and I
B kinase participate in human T-cell leukemia virus I Tax-mediated NF-
B activation.
J Biol Chem
273:
21132-21136,
1998
68.
Veronesi, B,
Oortgiesen M,
Carter JD,
and
Devlin RB.
Particulate matter initiates inflammatory cytokine release by activation of capsaicin and acid receptors in a human bronchial epithelial cell line.
Toxicol Appl Pharmacol
154:
106-115,
1999[ISI][Medline].
69.
Vojtek, AB,
and
Der CJ.
Increasing complexity of the Ras signaling pathway.
J Biol Chem
273:
19925-19928,
1998
70.
Wang, Y,
and
Pallen CJ.
Expression and characterization of wild-type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP-.
J Biol Chem
267:
16696-16702,
1992
71.
Wells, A.
EGF receptor.
Int J Biochem Cell Biol
31:
637-643,
1999[ISI][Medline].
72.
Whiteside, ST,
and
Israel A.
IB proteins: structure, function and regulation.
Semin Cancer Biol
8:
75-82,
1997[ISI][Medline].
73.
Woronicz, JD,
Gao X,
Cao Z,
Rothe M,
and
Goeddel DV.
IB kinase-
: NF-
B activation and complex formation with I
B kinase-
and NIK.
Science
278:
866-869,
1997
74.
Wu, W,
Graves LM,
Jaspers I,
Devlin RB,
Reed W,
and
Samet JM.
Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals.
Am J Physiol Lung Cell Mol Physiol
277:
L924-L931,
1999
75.
Yaron, A,
Hatzubai A,
Davis M,
Lavon I,
Amit S,
Manning AM,
Andersen JS,
Mann M,
Mercurio F,
and
Ben-Neriah Y.
Identification of the receptor component of the IB
-ubiquitin ligase.
Nature
396:
590-594,
1998[ISI][Medline].
76.
Yatani, A,
Okabe K,
Polakis P,
Halenbeck R,
McCormick F,
and
Brown AM.
Ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels.
Cell
61:
769-776,
1990[ISI][Medline].
77.
Ye, J,
Ding M,
Zhang X,
Rojanasakul Y,
Nedospasov S,
Vallyathan V,
Castranova V,
and
Shi X.
Induction of TNF- in macrophages by vanadate is dependent on activation of transcription factor NF-
B and free radical reactions.
Mol Cell Biochem
198:
193-200,
1999[ISI][Medline].
78.
Yujiri, T,
Fanger GR,
Garrington TP,
Schlesinger TK,
Gibson S,
and
Johnson GL.
MEK kinase 1 (MEKK1) transduces c-Jun NH2-terminal kinase activation in response to changes in the microtubule cytoskeleton.
J Biol Chem
274:
12605-12610,
1999
79.
Zandi, E,
Chen Y,
and
Karin M.
Direct phosphorylation of IB by IKK
and IKK
: discrimination between free and NF-
B-bound substrate.
Science
281:
1360-1363,
1998
80.
Zandi, E,
Rothwarf DM,
Delhase M,
Hayakawa M,
and
Karin M.
The IB kinase complex (IKK) contains two kinase subunits, IKK
and IKK
, necessary for I
B phosphorylation and NF-
B activation.
Cell
91:
243-252,
1997[ISI][Medline].