From the Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan
Received for publication, August 20, 2002, and in revised form, January 2, 2003
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ABSTRACT |
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The signaling pathway involved in tumor necrosis
factor- Extravasation of leukocytes from the microvasculature at sites of
inflammation or injury is a critical event in inflammation-mediated diseases, such as rheumatoid arthritis, psoriasis, bronchial asthma, atopic dermatitis, and allograft rejection (1-3). The process of
leukocyte migration includes several steps (4, 5). The first of these
is adhesion of the leukocyte to the endothelial cell. The initial
interaction between the leukocyte and the endothelium is transient,
resulting in the leukocyte rolling along the vessel wall. The rolling
leukocyte then becomes activated by local factors generated by the
endothelium, resulting in its arrest and firm adhesion to the vessel
wall. Finally, the leukocyte squeezes between the endothelial cells and
migrates to the inflammation site. These complex processes are
regulated, in part, by specific adhesion molecules and their counter
ligands on both circulating leukocytes and vascular endothelial cells;
these include E-selectin (endothelial-leukocyte adhesion molecule-1,
CD62E) and immunoglobulin superfamily members, such as intercellular
adhesion molecule-1 (ICAM-1,
CD54)1 and vascular cell
adhesion molecular-1 (6, 7). In a number of inflammation and immune
responses, ICAM-1 binds to two integrins belonging to the
Basal levels of ICAM-1 are low, but high expression can be induced in a
number of cell types by a wide range of ligands, including bacterial
lipopolysaccharide, phorbol esters, or inflammatory cytokines, such as
tumor necrosis factor (TNF)- Materials--
Rabbit polyclonal antibodies specific for
I Cell Culture--
A549, a human alveolar epithelial cell
carcinoma, were obtained from the American Type Culture Collection
(Manassas, VA) and cultured in DMEM supplemented with 10% fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in
six-well plates for transfection experiments, in 6-cm dishes for IKK,
c-Src, or Lyn kinase activity measurements and Western blot analysis,
or in 10-cm dishes for electrophoretic mobility shift assay and
co-immunoprecipitation tests.
Plasmids--
The ICAM-1 promoter construct (pIC339) was a gift
from Dr. van der Saag (Hubrecht Laboratory, Utrecht, Netherlands). The
Immunoprecipitation and Kinase Activity Assay--
Following
treatment with TNF- Western Blot Analysis--
Following treatment with TNF- Preparation of Nuclear Extracts and the Electrophoretic Mobility
Shift Assay--
Control cells or cells pretreated with various
inhibitors for 30 min were treated with TNF-
Oligonucleotides corresponding to the downstream NF- Site-directed Mutagenesis--
Using a QuikChangeTM
site-directed mutagenesis kit according to the manufacturer's manual,
lysine 295 in the mouse c-Src cloned in the pBluescript vector
was substituted with methionine by changing the triplets from AAG to
ATG. Tyrosine 199, tyrosine 188, or both sites in the human IKK Transient Transfection and Luciferase Assay--
A549 cells,
grown to 50% confluency in six-well plates, were transfected with the
human ICAM-1(pIC-339/0)/firefly luciferase (Luc) or Co-immunoprecipitation Assay--
Cell lysates containing 1 mg
of protein were incubated for 1 h at 4 °C with 2 µg of
anti-IKK Effect of Inhibitors of PKC, Tyrosine Kinase, or Src Kinase on the
Induction of ICAM-1 Promoter Activity by TNF- Induction of IKK Activation, I Induction of c-Src and Lyn Activation by TNF- Induction of ICAM-1 Promoter Activity by Overexpression of PKC Induction by TNF- Inhibitory Effect of the Dominant-negative Mutants IKK
To further confirm the involvement of tyrosine phosphorylation in the
PKC
Because Tyr188 and Tyr199 in IKK The PKC-dependent tyrosine kinase activation pathway
is involved in TNF- In nonstimulated cells, NF- To elucidate the relationship between the PKC/c-Src/IKK c-Src is involved in NF- Because involvement of the PKC/c-Src/IKK In summary, the signaling pathways involved in TNF- (TNF-
)-induced intercellular adhesion molecule-1 (ICAM-1)
expression was further studied in human A549 epithelial cells. TNF-
-
or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced
ICAM-1 promoter activity was inhibited by a protein kinase C (PKC)
inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and
herbimycin A), or an Src-specific tyrosine kinase inhibitor (PP2).
TNF-
- or TPA-induced I
B kinase (IKK) activation was also blocked
by these inhibitors, which slightly reversed TNF-
-induced but
completely reversed TPA-induced I
B
degradation. c-Src and Lyn,
two members of the Src kinase family, were abundantly expressed in A549
cells, and their activation by TNF-
or TPA was inhibited by the same
inhibitors. Furthermore, the dominant-negative c-Src (KM) mutant
inhibited induction of ICAM-1 promoter activity by TNF-
or TPA.
Overexpression of the constitutively active PKC
or wild-type c-Src
plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c-Src (KM) or IKK
(KM) mutant but not by
the nuclear factor-
B-inducing kinase (NIK) (KA) mutant. The c-Src
(KM) mutant failed to block induction of ICAM-1 promoter activity
caused by overexpression of wild-type NIK. In
co-immunoprecipitation and immunoblot experiments, IKK
was found
to be associated with c-Src and to be phosphorylated on tyrosine
residues after TNF-
or TPA treatment. Two tyrosine residues,
Tyr188 and Tyr199, near the activation loop of
IKK
, were identified as being important for NF-
B activation.
Substitution of these residues with phenylalanines abolished ICAM-1
promoter activity and c-Src-dependent phosphorylation of
IKK
induced by TNF-
or TPA. These data suggest that, in addition to activating NIK, TNF-
also activates PKC-dependent
c-Src. These two pathways converge at IKK
and go on to activate
NF-
B, via serine phosphorylation and degradation of I
B-
, and,
finally, to initiate ICAM-1 expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 subfamily, LFA-1 and Mac-1, both expressed by
leukocytes and both promoting the adhesion and transendothelial migration of leukocytes (7-9). Similar processes govern the
adhesion of leukocytes to lung airway epithelial cells and contribute
to the damage to these cells seen in asthma (10).
, interleukin (IL)-1
, and
interferon-
(11-13). Induction of ICAM-1 expression requires
de novo mRNA and protein synthesis (8, 14), indicating regulation at the transcriptional level. The promoter region of the
human ICAM-1 gene has been cloned and sequenced and shown to contain
putative recognition sequences for a variety of transcriptional factors, including nuclear factor-
B (NF-
B), activator protein-1 (AP-1), AP-2, and the interferon-stimulated response element (15). Of
these, NF-
B family proteins are essential for the enhanced ICAM-1
gene expression seen in human alveolar epithelial cells on exposure to
cytokines (16, 17). The intracellular signaling pathways by which
TNF-
and IL-1
cause ICAM-1 expression in A549 human alveolar
epithelial cells have been explored and found to involve the sequential
activation of protein kinase C
(PKC
), protein-tyrosine
kinase, nuclear factor-
B-inducing kinase (NIK), and I
B
kinase
(IKK
) (16, 17). The role of protein-tyrosine kinase has
been further investigated in the present study. Using an immunocomplex
kinase assay and site-directed mutagenesis, we have demonstrated that
c-Src is involved in TNF-
-inducing NF-
B transcriptional
activation and that, in addition to serine phosphorylation of IKK
by
NIK, Tyr188 and Tyr199 phosphorylations by
PKC-dependent c-Src activation also contribute to
TNF-
-induced ICAM-1 expression in human alveolar epithelial cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
, IKK
, c-Src, Lyn, Lck, and Fyn were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA), and rabbit polyclonal
anti-phosphotyrosine antibody was purchased from Upstate Biotechnology
(Lake Placid, NY). Human recombinant TNF-
was purchased from R&D
Systems (Minneapolis, MN). TPA was purchased from L.C. Service Corp.
(Woburn, MA). Dulbecco's modified Eagle medium (DMEM), fetal calf
serum, penicillin, and streptomycin were obtained from Invitrogen
(Gaithersburg, MD). Staurosporine, GST-agarose beads, and protein
A-Sepharose were obtained from Sigma (St. Louis, MO). Herbimycin A and
PP2 were obtained from Calbiochem (San Diego, CA). Horseradish
peroxidase-labeled donkey anti-rabbit second antibody and the enhanced
chemiluminescence (ECL) detecting reagent were obtained from Amersham
Biosciences (Uppsala, Sweden). [
-32P]ATP (3000 Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA).
Tfx-50 and the luciferase assay kit were obtained from Promega
(Madison, MA). Plasmid purification and DNA recovery kits were obtained
from Qiagen (Chatsworth, CA). The QuikChangeTM mutagenesis
kit was obtained from Stratagene (La Jolla, CA). EcoRI,
XboI, and SalI restriction enzymes and T4 DNA
ligase were obtained from New England BioLabs (Beverly, MA).
B-luc plasmid was from Stratagene. The PKC-
constitutively active
(PKC-
/AE) or dominant-negative mutant (PKC
/KR) were provided by
Dr. A. Altman (La Jolla Institute for Allergy and Immunology, San
Diego, CA). The wild-type (wt) and dominant-negative mutants of NIK and IKK
(NIK wt and mutant KA; IKK
wt and mutant KM) were gifts from
Signal Pharmaceuticals (San Diego, CA). The dominant negative mutant of
IKK
(AA) was from Dr. Karin (University of California, San Diego,
CA). pGEX-I
B
-(1-100) was a gift from Dr. Nakano
(University of Juntendo, Tokyo). pGEX-IKK
-(132-206) was a gift from
Dr. Nakanishi (University of Nagoya, Nagoya).
or TPA, with or without 30-min pretreatment with
PKC, tyrosine kinase, or Src kinase inhibitors, the cells were rapidly
washed with phosphate-buffered saline and lysed with ice-cold lysis
buffer (50 mM Tris-HCl, pH 7.4, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 5 µg/ml
leupeptin, 20 µg/ml aprotinin, 1 mM NaF, and 1 mM Na3VO4), then IKK, c-Src, or Lyn
was immunoprecipitated. For the in vitro kinase assay, 100 µg of total cell extract was incubated for 1 h at 4 °C with 0.5 µg of rabbit anti-IKK
, anti-c-Src, or anti-Lyn antibody, then
protein A-Sepharose CL-4B beads (Sigma) were added to the mixture and
incubation was continued for 4 h at 4 °C. The
immunoprecipitates were collected by centrifugation, washed three times
with lysis buffer without Triton X-100, then incubated for 30 m
30 °C in 20 µl of kinase reaction mixture (20 mM
HEPES, pH 7.4, 5 mM MgCl2, 5 mM
MnCl2, 0.1 mM Na3VO4, 1 mM DTT) containing 10 µM
[
-32P]ATP and either 1 µg of bacterially expressed
GST-I
B
-(1-100) as IKK substrate, 1 µg of acidic denatured
enolase as c-Src or Lyn substrate, or 6 µg of bacterially expressed
GST-IKK
-(132-206), GST-IKK
-(132-206) (Y188F),
GST-IKK
-(132-206) (Y199F), or GST-IKK
-(132-206) (Y188F; Y199F)
as c-Src substrate. The reaction was stopped by addition of an equal
volume of Laemmli buffer, the proteins were separated by
electrophoresis on 10% SDS-polyacrylamide gels, and phosphorylated
GST-I
B
-(1-100), phosphorylated GST-IKK
-(132-206), or
phosphorylated enolase was visualized by autoradiography. Quantitative data were obtained using a computing densitometer and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).
or
TPA, total or immunoprecipitated cell lysates were prepared and
subjected to SDS-PAGE using 7.5% running gels, as described previously
(17). The proteins were transferred to a nitrocellulose membrane, which
was then incubated successively at room temperature for 1 h with
0.1% milk in TTBS (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20), for 1 h with rabbit
antibody specific for IKK
, I
B
, c-Src, Lyn, Lck, or Fyn, and
for 30 min with horseradish peroxidase-labeled anti-rabbit antibody.
After each incubation, the membrane was washed extensively with TTBS.
The immunoreactive bands were detected using ECL detection reagent and
Hyperfilm-ECL (Amersham Biosciences).
for 10 min or with TPA
for 30 min, then nuclear extracts were isolated as described previously (17). Briefly, cells were washed with ice-cold phosphate-buffered saline and pelleted, then the cell pellet was resuspended in a hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM
KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
DTT, 0.5 mM PMSF, 1 mM NaF, and 1 mM Na3VO4) and incubated for 15 min
on ice, then lysed by the addition of 0.5% Nonidet P-40 followed by
vigorous vortexing for 10 s. The nuclei were pelleted and
resuspended in extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 mM NaF, and 1 mM Na3VO4), and the tube was
vigorously shaken at 4 °C for 15 min on a shaking platform. The
nuclear extracts were then centrifuged, and the supernatants were
aliquoted and stored at
80 °C.
B consensus
sequence (5'-AGCTTGGAAATTCCGGA-3') in the human ICAM-1 promoter were synthesized, annealed, and end-labeled with
[
-32P]ATP using T4 polynucleotide kinase. The nuclear
extract (6-10 µg) was incubated at 30 °C for 20 min with 1 ng of
32P-labeled NF-
B probe (40,000-60,000 cpm) in 10 µl
of binding buffer containing 1 µg of poly(dI-dC), 15 mM
HEPES, pH 7.6, 80 mM NaCl, 1 mM EGTA, 1 mM DTT, and 10% glycerol as described previously (17).
DNA·nuclear protein complexes were separated from the DNA probe by
electrophoresis on a native 6% polyacrylamide gel, then the gel was
vacuum-dried and subjected to autoradiography using an intensifying
screen at
80 °C.
cloned in the pcDNA3.1 vector or in the human GST-IKK
(132-206)
cloned in the pGEX vector were substituted with phenylalanine by
changing the triplet from TAC to TTC. The mutated primers used were as
follows: primer 1 (5'-CGAGGGTTGCCATCATGACTCTGAAGCCAGGCA-3') and primer
2 (3'-GCTCCCAACGGTAGTACTGAGACTTCGGTCCGT-5') for c-Src
(K295M) mutation, primer 3 (5'-GGGGACCCTGCAGTTCCTGGCCCCAGAGC-3') and primer 4 (3'-CCCCTGGGACGTCAAGGACCGGGGTCTCG-5') for IKK
(Y188F)
mutation, and primer 5 (5'-GGAGCAGCAGAAGTTCACAGTGACCGTCG-3') and primer 6 (3'-CCTCGTCGTCTTCAAGTGTCACTGGCAGC-5') for IKK
(Y199F) mutation. DNA prepared from overnight cultures of picked
colonies using Miniprep (Qiagen) was subjected to restriction digest
analysis, and the nucleotide changes were confirmed by DNA sequencing.
The mutated c-Src plasmid containing the point mutation was digested
with EcoRI and XhoI and inserted into the pcDNA3(+) vector.
B-luc plasmid
using Tfx-50 (Promega) according to the manufacturer's
recommendations. Briefly, reporter DNA (0.4 µg) and
-galactosidase DNA (0.2 µg) were mixed with 0.6 µl of Tfx-50 in
1 ml of serum-free DMEM. After 10- to 15-min incubation at room
temperature, the mixture was applied to the cells, then, 1 h
later, 1 ml of complete growth medium was added. On the
following day, the medium was replaced with fresh medium. Forty-eight
hours after transfection, the cells were treated with inhibitors (as indicated) for 30 min, then TNF-
or TPA was added for 6 h. Cell extracts were then prepared and luciferase and
-galactosidase activities were measured, the luciferase activity being normalized to
the
-galactosidase activity. In experiments using dominant-negative mutants, cells were co-transfected with reporter (0.2 µg) and
-galactosidase (0.1 µg) and either the dominant-negative NIK, IKK
, or c-Src mutant or the respective empty vector (0.4 µg). In experiments using wt plasmids, cells were
co-transfected with the following mixture: 0.2 µg of reporter
plasmid; 0.1 µg of
-galactosidase plasmid; 0.4 µg of the
constitutively active PKC
(A/E) plasmid, wt c-Src or NIK plasmid (or
the respective empty vector); and 0.4 µg of the dominant-negative
NIK, IKK
, or c-Src mutant (or the respective empty vector).
or anti-c-Src antibody or with 4 µg of
anti-phosphotyrosine antibody, then 50 µl of 50% protein A-agarose
beads were added and mixed for 16 h at 4 °C. The
immunoprecipitates were collected and washed three times with lysis
buffer without Triton X-100, then Laemmli buffer was added and the
samples were subjected to electrophoresis on 10% SDS-polyacrylamide
gels. Western blot analysis was performed as described above using
antibodies against phosphotyrosine, IKK
, or c-Src.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or TPA in A549
Cells--
In our previous study (17), we found that PKC and tyrosine
kinase were involved in TNF-
-induced ICAM-1 expression. Transient transfection using the ICAM-1 promoter-luciferase construct, pIC-339 (
339/0) was performed to elucidate the signaling pathway mediated by
these kinases. The pIC-339 construct contains the downstream NF-
B
site (
189/
174) responsible for mediating the induction of ICAM-1
promoter activity by TNF-
or TPA (17). As shown in Fig.
1, TNF-
led to a 2.9-fold increase in
ICAM-1 promoter activity. When cells were pretreated with inhibitors of
PKC (staurosporine), tyrosine kinases (genistein or herbimycin A), or
Src kinases (PP2), the TNF-
-induced increase was inhibited by 69%,
84%, 65%, or 66%, respectively. TPA treatment, a direct PKC
activator, resulted in a 3.5-fold increase in ICAM-1 promoter activity,
and this effect was inhibited by genistein, herbimycin A, or PP2 by
74%, 60%, or 87%, respectively. None of these inhibitors alone
affected the basal luciferase activity (data not shown).
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Fig. 1.
Effect of various inhibitors on
TNF- - or TPA-induced ICAM-1 promoter activity
in epithelial cells. A549 cells were transfected with the pIC339
luciferase expression vector as described under "Experimental
Procedures" then pretreated for 30 min with vehicle, 300 nM staurosporine, 30 µM genistein, 1 µM herbimycin A, or 10 µM PP2 before
incubation for 6 h with 10 ng/ml TNF-
or 1 µM
TPA. Luciferase activity was then measured as described under
"Experimental Procedures," normalized to the
-galactosidase
activity and expressed as the mean ± S.E. for three independent
experiments performed in triplicate. *, p < 0.05, compared with TNF-
or TPA alone.
B
Degradation, and
NF-
B-specific DNA-Protein Complex Formation by TNF-
and TPA, and
the Inhibitory Effect of Inhibitors of PKC, Tyrosine Kinase, or Src
Kinase--
Because TNF-
- and TPA-induced ICAM-1 promoter activity
in A549 cells is inhibited by the dominant-negative IKK
mutant (17), endogenous IKK activity was measured by immunoprecipitation with anti-IKK
antibody. When cells were treated with 10 ng/ml TNF-
for
5, 10, 30, or 60 min, maximal IKK activity was seen after 5 min (Fig.
2A), whereas maximal
degradation of I
B-
was seen after 10 min, I
B-
levels being
restored to the resting level after 1 h of treatment (Fig.
2B). In TPA-treated cells, maximal IKK activity was seen
after 30 min of treatment (Fig. 2A), whereas maximal
I
B-
degradation was seen after 60 min (Fig. 2B). The TNF-
-induced IKK activation was inhibited by a PKC, tyrosine kinase,
or Src kinase inhibitor by 56%, 49%, or 50%, respectively, whereas
these same inhibitors suppressed TPA-induced IKK activation by 71%,
91%, or 90%, respectively (Fig.
3A). The I
B
degradation induced by TPA was reversed by PKC, tyrosine kinase, and Src kinase inhibitors, but that induced by TNF-
was only slightly affected by
these inhibitors (Fig. 3B). The effect of these inhibitors on TNF-
- or TPA-induced NF-
B-specific DNA·protein
binding was examined. As shown in Fig. 3C, when cells were
treated with TNF-
for 10 min, increased NF-
B-specific
DNA·protein binding was seen, and this effect was inhibited by PKC,
tyrosine kinase, and Src kinase inhibitors by 20%, 51%, and 48%,
respectively. TPA treatment for 30 min also increased NF-
B-specific
DNA·protein binding, and this was more effectively suppressed by
these inhibitors (75%, 74%, and 87%, respectively; Fig.
3C).
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Fig. 2.
Kinetics of
TNF- -induced IKK activation and
I
B-
degradation.
A549 cells were treated with 10 ng/ml TNF-
or 1 µM TPA
for 5, 10, 30, or 60 min, then cell lysates were prepared. In
A, cell lysates were immunoprecipitated with anti-IKK
antibody, then the kinase assay (KA) and autoradiography for
phosphorylated GST-I
B
(1-100) were performed on the precipitates
as described under "Experimental Procedures." Levels of
immunoprecipitated IKK
protein were estimated by Western blotting
(WB) using anti-IKK
antibody. In B, cytosolic
levels of I
B-
were measured using anti-I
B-
antibody as
described under "Experimental Procedures."
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Fig. 3.
Effect of various inhibitors on
TNF- - or TPA-induced IKK activity,
I
B
degradation, and
NF-
B-specific DNA· protein complex
formation in epithelial cells. A549 cells were pretreated for 30 min with 300 nM staurosporine, 1 µM
herbimycin A, or 10 µM PP2 before incubation with 10 ng/ml TNF-
for 10 min or 1 µM TPA for 30 min, then
whole cell lysates or nuclear extracts were prepared. In A,
whole cell lysates were immunoprecipitated with anti-IKK
antibody,
and the kinase assay (KA) and autoradiography for
phosphorylated GST-I
B
-(1-100) were performed on the precipitates
as described under "Experimental Procedures." Levels of
immunoprecipitated IKK
were estimated by Western blotting
(WB) using anti-IKK
antibody. In B,
cytosolic levels of I
B-
were measured by Western blotting using
anti-I
B-
antibody as described under "Experimental
Procedures." In C, the NF-
B-specific DNA·protein
activity in nuclear extracts was determined by electrophoretic mobility
shift assay as described under "Experimental Procedures."
and TPA, and the
Inhibitory Effect of Inhibitors of PKC, Tyrosine Kinase, or Src
Kinase--
TNF-
- or TPA-induced IKK activation was inhibited by
PKC, tyrosine kinase, and Src kinase inhibitors, indicating the
involvement of tyrosine kinase, or at least the Src family, downstream
of PKC in the induction of IKK activation. To further characterize the
tyrosine kinase involved, Western blot analysis using antibodies against the Src family members, c-Src, Lck, Lyn, and Fyn, was performed. Because c-Src is reported to be expressed in platelets and
neuronal tissues, Lck in T lymphocytes, Lyn at high levels in
platelets, and Fyn in the brain and T lymphocytes, we used the Jurkat T
cell line, the HL-60 promyelocytic cell line, and brain as positive
controls. As shown in Fig. 4A,
c-Src was abundantly expressed in brain, in Jurkat and HL-60 cells, and
in the human alveolar epithelial cell lines NCI-H292 and A549. Lck was
abundantly expressed in brain and Jurkat cells, but only weakly
expressed in NCI-H292 and A549 cells. Lyn was abundantly expressed in
brain and in Jurkat, HL-60, NCI-H292, and A549 cells, whereas Fyn was only expressed in brain and in Jurkat and HL-60 cells. c-Src and Lyn in
A549 cells were therefore isolated by immunoprecipitation using
anti-c-Src or anti-Lyn antibody, and their in vitro kinase activity was measured using enolase as substrate. As shown in Fig.
4B, when A549 cells were treated with 10 ng/ml TNF-
for 10, 30, or 60 min, maximal c-Src and Lyn activity (enolase
phosphorylation) was seen after 10 min and was maintained to 60 min. In
addition, marked autophosphorylation of c-Src and Lyn was seen over the same time period. TPA (1 µM) also induced c-Src and Lyn
activation after 30-min treatment of A549 cells (Fig.
5). The TNF-
- and TPA-induced
activation of c-Src and Lyn was inhibited by staurosporine, herbimycin
A, and PP2 (Fig. 5).
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Fig. 4.
Src family expression and
time-dependent activation of c-Src or Lyn by
TNF- in A549 cells. In A,
Jurkat, HL-60, NCI-H292, or A549 cells and brain lysates were prepared
and subjected to Western blotting using antibodies against c-Src, Lck,
Lyn, or Fyn as described under "Experimental Procedures." In
B, A549 cells were treated with 10 ng/ml TNF-
for 10, 30, or 60 min, then whole cell lysates were prepared and immunoprecipitated
with anti-c-Src or anti-Lyn antibody. The kinase assay (KA)
and autoradiography for phosphorylated enolase were performed on the
precipitates as described under "Experimental Procedures." Levels
of immunoprecipitated c-Src or Lyn were estimated by Western blotting
(WB) using anti-c-Src or anti-Lyn antibody,
respectively.
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Fig. 5.
Effect of various inhibitors on
TNF- - or TPA-induced c-Src or Lyn activation
in epithelial cells. A549 cells were pretreated with 300 nM staurosporine, 1 µM herbimycin A, or 10 µM PP2 for 30 min before incubation with 10 ng/ml TNF-
for 10 min or 1 µM TPA for 30 min. Whole cell lysates
were prepared and immunoprecipitated with anti-c-Src or anti-Lyn
antibody, and the kinase assay (KA) and autoradiography for
phosphorylated enolase were performed on the precipitate as described
under "Experimental Procedures." Levels of immunoprecipitated c-Src
or Lyn were estimated by Western blotting (WB) using
anti-c-Src or anti-Lyn antibody, respectively.
or c-Src, and the Inhibitory Effect of Dominant-negative Mutants of
c-Src or IKK
--
The TNF-
- or TPA-induced activation of c-Src
and Lyn was inhibited by PKC, tyrosine kinase, or Src kinase
inhibitors. This indicated that PKC-dependent c-Src and Lyn
activation was required to induce IKK and NF-
B activation in A549
cells. To further examine the involvement of c-Src, a dominant-negative
mutant was generated by site-directed mutagenesis of mouse c-Src lysine
295 to methionine. Overexpression of c-Src (KM) attenuated the TNF-
-
or TPA-induced ICAM-1 promoter activity (Fig.
6). The TNF-
-induced ICAM-1 promoter activity was also inhibited by the dominant-negative NIK (KA) and
IKK
(KM) mutants, as previously reported (17). To characterize the
relationship between PKC, c-Src, NIK, and IKK
, overexpression of the
constitutively active form of PKC
(A/E) or of wt c-Src, NIK, or
IKK
was performed. Overexpression of PKC
(A/E) or wt c-Src, NIK,
or IKK
significantly increased ICAM-1 promoter activity by 2-, 2.7-, 3.4-, or 2.5-fold, respectively (Fig.
7A). The ICAM-1 promoter
activity induced by overexpression of PKC
(A/E) or c-Src wt was
inhibited by the dominant-negative c-Src (KM) or IKK
(KM) mutant,
but not by the NIK (KA) mutant. In contrast, the dominant-negative IKK
(KM) mutant, but not the c-Src (KM) mutant, attenuated the promoter activity induced by overexpression of NIK wt (Fig.
7B). These results indicate the involvement of both the
PKC/c-Src/IKK
and NIK/IKK
pathways in TNF-
-induced ICAM-1
expression in A549 cells.
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Fig. 6.
Effect of various dominant-negative mutants
on TNF- - or TPA-induced ICAM-1 promoter
activity in A549 cells. A549 cells were co-transfected with pIC339
and the dominant-negative c-Src (K295M), NIK (KA), or IKK
(KM)
mutant, or the respective empty vector, then treated for 6 h with
10 ng/ml TNF-
or 1 µM TPA. Luciferase activity was
then measured as described under "Experimental Procedures," and the
results were normalized to the
-galactosidase activity and expressed
as the mean ± S.E. for three independent experiments performed in
triplicate. *, p < 0.05; **, p < 0.01 compared with TNF-
or TPA alone.
View larger version (33K):
[in a new window]
Fig. 7.
Effect of various dominant-negative mutants
on wild-type plasmid-induced ICAM-1 promoter activity. In
A, A549 cells were co-transfected with pIC339 and the
constitutively active form of PKC (A/E), wild-type c-Src, IKK
, or
NIK, or the respective empty vector. In B, A549 cells were
co-transfected for 24 h with PKC
(A/E), wild-type c-Src, or NIK
and c-Src (K295M), IKK
(KM), or NIK (KA). Luciferase
activity was then assayed as described under "Experimental
Procedures," and the results were normalized to the
-galactosidase
activity and expressed as the mean ± S.E. for three independent
experiments performed in triplicate. *, p < 0.05; **,
p < 0.01 compared with the control vector.
or TPA of Tyrosine Phosphorylation of IKK
and of the c-Src and IKK
Association, and the inhibitory Effect of
PP2--
Because c-Src-dependent IKK activation was shown
to be involved, co-immunoprecipitation of c-Src and IKK
was
performed to examine whether c-Src directly regulates IKK activity
through phosphorylation of tyrosine residues. When cells were treated with TNF-
for 5, 10, or 15 min, IKK
was tyrosine-phosphorylated in a time-dependent manner, the maximal effect being seen
at 10 min; a similar effect was seen after 30-min treatment with TPA (Fig. 8A). Both effects were
inhibited by PP2 (Fig. 8A). To demonstrate that c-Src
associated with IKK
and phosphorylated its tyrosine residues, cell
lysates were immunoprecipitated with anti-IKK
antibodies, then the
immunoprecipitates were separated by SDS-PAGE, transferred to
membranes, and blotted with anti-phosphotyrosine antibodies. As shown
in Fig. 8B, tyrosine phosphorylation of IKK
was seen
after TNF-
or TPA treatment, the effect being maximal at 10 or 30 min, respectively, and inhibited by PP2. When cell lysates were
immunoprecipitated with anti-phosphotyrosine antibody and immunoblotted
with anti-IKK
or anti-c-Src antibody, both IKK
and c-Src were
shown to be tyrosine-phosphorylated after TNF-
or TPA treatment, and
these effects were again inhibited by PP2 (Fig. 8C). These
results indicate that c-Src can associate with IKK
and phosphorylate
its tyrosine residues after TNF-
or TPA stimulation. The association
between c-Src and IKK was further examined. Anti-IKK
antibody was
used to precipitate IKK from A549 cells, then the immunoprecipitated
proteins were subjected to Western blotting using anti-c-Src antibody.
As shown in Fig. 9A, an
increased amount of c-Src co-precipitated with IKK
after TNF-
or
TPA stimulation. In the converse experiment in which c-Src was
precipitated using anti-c-Src antibody, IKK
was shown to be
associated with c-Src in a time-dependent manner after
TNF-
or TPA treatment (Fig. 9B). These results show that
there is an association between c-Src and IKK
and that IKK
tyrosine residues are phosphorylated.
View larger version (32K):
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Fig. 8.
Tyrosine phosphorylation of
IKK induced by TNF-
or TPA and the inhibitory effect of PP2. Control cells or
cells pretreated for 30 min with 10 µM PP2 were
stimulated with TNF-
for 5, 10, or 15 min or with TPA for 10 or 30 min. In A, crude lysates were prepared. In B and
C, equal amounts (1 mg) of cell lysate were
immunoprecipitated (IP) with anti-IKK
(A) or
anti-phosphotyrosine (PY) (B) antibodies. Crude
lysates and immunoprecipitated proteins were separated by SDS-PAGE on a
10% gel and immunoblotted (WB) with anti-phosphotyrosine
(PY) (A and B), anti-IKK
(C), or anti-c-Src (C) antibodies or reprobed
with anti-IKK
(A and B) antibody.
View larger version (33K):
[in a new window]
Fig. 9.
c-Src co-immunoprecipitates with
IKK after TNF-
or TPA
treatment. A549 cells were treated with TNF-
for 5, 10, or 15 min or with TPA for 10 or 30 min. Equal amounts (1 mg) of cell lysate
were immunoprecipitated (IP) with anti-IKK
(A)
or anti-c-Src (B) antibodies. Immunoprecipitated proteins
were separated by SDS-PAGE on a 10% gel and immunoblotted
(WB) with anti-IKK
or anti-c-Src antibodies.
C, alignment of subdomains VII and VIII of the kinase
domains of PKC
, Akt1, and IKK
/
.
(Y188F),
IKK
(Y199F), or IKK
(FF) on TNF-
- and TPA-induced ICAM-1
Promoter Activity and on the PKC
- and c-Src-induced, but not the
NIK-induced, Increase in NF-
B Activity--
The above experiments
demonstrate that c-Src directly interacts with IKK
and
phosphorylates its tyrosine residues after TNF-
or TPA stimulation.
When the amino sequences of subdomains VII and VIII in the kinase
domain of PKC
, AKT1, and IKK
/
were aligned, the tyrosine
residues were found to be conserved (Fig. 9C). Hypothesizing that Tyr188 and/or Tyr199 of IKK
were the
targets for c-Src phosphorylation after TNF-
or TPA stimulation, we
used site-directed mutagenesis to generate the dominant-negative
tyrosine mutants, IKK
(Y188F), IKK
(Y199F), and IKK
(Y188F,
Y199F). Overexpression of these mutants attenuated the TNF-
- or
TPA-induced ICAM-1 promoter activity, the double mutant having a
greater inhibitory effect than either of the single mutants (Fig.
10A). The dominant-negative
IKK
(KM) mutant, with Lys44 mutated to methionine, had a
similar inhibitory effect to that of IKK
(Y188F) or IKK
(Y199F)
on TNF-
- and TPA-induced ICAM-1 promoter activity, whereas IKK
(AA), with Ser177 and Ser181 mutated to
alanine, was as effective as IKK
(Y188F) or IKK
(Y199F) in
inhibiting TNF-
-induced ICMA-1 promoter activity but had no effect
on TPA-induced ICAM-1 promoter activity (Fig. 10A).
View larger version (49K):
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Fig. 10.
Effect of the dominant-negative tyrosine
mutants, IKK (Y188F), IKK
(Y199F), and IKK
(FF), on
TNF-
- or TPA-induced ICAM-1 promoter activity
and on wild-type plasmid-induced NF-
B
activity. In A, A549 cells were co-transfected with
pIC339 plus one of the dominant-negative tyrosine mutants (IKK
(188F), IKK
(Y199F), or IKK
(FF)), dominant-negative mutant
(IKK
(KM)), or dominant-negative serine mutant (IKK
(AA)), or the
respective empty vector, then treated with 10 ng/ml TNF-
or 1 µM TPA for 6 h. In B, A549 cells were
co-transfected with
B-luc and the constitutively active form of
PKC
(A/E), wild-type c-Src, or wild-type NIK, plus the
dominant-negative mutants, IKK
(Y188F), IKK
(Y199F), IKK
(FF),
or IKK
(AA), or the respective empty vector. Luciferase activity was
then measured as described under "Experimental Procedures," and the
results were normalized to the
-galactosidase activity and expressed
as the mean ± S.E. for three independent experiments performed in
triplicate. *, p < 0.05; **, p < 0.01 compared with TNF-
or TPA alone (A) or wild-type alone
(B).
/c-Src/IKK
pathway and serine phosphorylation in the
NIK/IKK
pathway, the dominant-negative IKK
mutants with either a
tyrosine or serine mutation were co-transfected with PKC
(A/E), wt
c-Src, or wt NIK to examine their inhibitory effects on the
constitutively active or wt plasmid-induced NF-
B activity. As shown
in Fig. 10B, PKC
(A/E)- or wt c-Src-induced NF-
B
activity was inhibited by all three tyrosine mutants but not by the
double-serine mutant, whereas the converse was true for NIK-induced
NF-
B activity.
were found
to be critical for the PKC
/c-Src/IKK
pathway to elicit NF-
B
activation, leading to induction of TNF-
- or TPA-stimulated ICAM-1
promoter activity (Fig. 10), endogenous c-Src phosphorylation of
Tyr188 and Tyr199 in IKK
was further
examined. c-Src was immunoprecipitated using anti-c-Src antibody, and
its ability to phosphorylate IKK
was measured using
GST-IKK
-(132-206) as an in vitro substrate. When cells
were treated with TNF-
or TPA, IKK
was phosphorylated by c-Src in
a time-dependent manner. The maximal effect was seen at
10-min treatment with TNF-
or 30-min treatment with TPA (Fig. 11A), and both effects were
inhibited by PP2 (Fig. 11B). The c-Src-dependent IKK
phosphorylation was specific for
Tyr188/Tyr199, because it was not seen when
either or both tyrosine residues were substituted with phenylalanines
(Fig. 11C).
View larger version (27K):
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Fig. 11.
c-Src-dependent phosphorylation
of IKK at Y188 and
Y199 is induced by TNF-
or TPA and
inhibited by PP2. A549 cells were treated with 10 ng/ml TNF-
or
1 µM TPA for 5, 10, 30, or 60 min (A) or
pretreated with 10 µM PP2 for 30 min before stimulation
with TNF-
for 10 min or TPA for 30 min (B). Whole cell
lysates were prepared and immunoprecipitated with anti-c-Src antibody,
then a kinase assay (KA) and autoradiography for
phosphorylated GST-IKK
(132-206) were performed as described under
"Experimental Procedures." The amount of immunoprecipitated c-Src
was detected by Western blotting (WB) using anti-c-Src
antibody. In C, cells were treated with 10 ng/ml TNF-
for
10 min or 1 µM TPA for 30 min, and the whole cell lysates
were immunoprecipitated with anti-c-Src antibody followed by kinase
assay (KA) and autoradiography for phosphorylated wt
GST-IKK
-(132-206), GST-IKK
-(132-206) (Y188F),
GST-IKK
-(132-206) (Y199F), or GST-IKK
-(132-206) (Y188F; Y199F).
The amount of immunoprecipitated c-Src was detected by Western blotting
(WB) using anti-c-Src antibody. The amounts of
GST-IKK
-(132-206) were detected by Coomassie Brilliant Blue
staining.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced NF-
B activation and ICAM-1 expression
in A549 alveolar epithelial cells and in causing monocytes to adhere to
these cells (17). The role and molecular identity of the tyrosine
kinase involved have been further characterized in the present study.
TNF-
- and TPA-induced ICAM-1 promoter activity were both inhibited
by PKC, tyrosine kinase, and Src kinase inhibitors, indicating the
possible involvement of the Src tyrosine kinase family downstream of
PKC activation in the induction of ICAM-1 expression. IKK
, but not
IKK
, is involved in the TNF-
- and TPA-induced ICAM-1 promoter
activity (17), and TNF-
- or TPA-induced stimulation of IKK activity
and parallel degradation of I
B-
was seen in the present study.
The TNF-
- and TPA-induced IKK activity and NF-
B-specific
DNA·protein binding were attenuated by PKC, tyrosine kinase, and Src
kinase inhibitors, indicating that the Src tyrosine kinase family is
involved downstream of PKC in the induction of IKK
activation
leading to NF-
B activation and ICAM-1 expression in A549 cells.
Western blot analysis showed that c-Src and Lyn were abundantly
expressed in A549 cells and that TNF-
and TPA induced activation of
these two Src tyrosine kinases. The c-Src and Lyn activation induced by
either stimulus was also inhibited by PKC, tyrosine kinase, and Src
kinase inhibitors. Taken together, these results demonstrate that the
tyrosine kinase involved downstream of PKC is c-Src or Lyn. The
involvement of PKC/c-Src/IKK
activation in TNF-
-induced ICAM-1
expression was confirmed by the finding that the dominant-negative
c-Src (KM) mutant attenuated the TNF-
- and TPA-induced ICAM-1
promoter activity.
B dimers are present as cytoplasmic
latent complexes due to the binding of specific inhibitors, the I
Bs,
that mask their nuclear localization signal. Following stimulation by
pro-inflammatory cytokines, the I
Bs are rapidly phosphorylated at
two conserved N-terminal serine residues, and this post-translational
modification is rapidly followed by their polyubiquitination and
proteasomal degradation (18, 19). This leads to the unmasking of the
nuclear localization signal in NF-
B dimers, followed by their
translocation to the nucleus, binding to specific DNA sites (
B
sites), and targeting of gene activation. The protein kinase that
phosphorylates I
Bs in response to pro-inflammatory stimuli has been
identified biochemically and molecularly (20-24). Named IKK, it exists
as a complex, termed the IKK signalsome, which is composed of at least
three subunits, IKK
(IKK1), IKK
(IKK2), and IKK
(25). IKK
and IKK
are very similar protein kinases that act as the catalytic
subunits of the complex (20-24). In mammalian cells, IKK
and IKK
form a stable heterodimer that is tightly associated with IKK
, a
regulatory subunit (26). The IKKs bind NIK (22, 23), a member of the
mitogen-activated protein kinase kinase kinase family, that interacts
with the TRAF6-associated IL-1 receptor complex or TRAF2-associated TNF
receptor complex, thereby linking I
B degradation and NF-
B
activation to IL-1
or TNF-
stimulation (27). The activities of
both IKK
and IKK
are reported to be regulated by NIK (28). Our
results show that the TNF-
-induced increase in ICAM-1 promoter
activity was inhibited by the dominant-negative NIK (KA) and IKK
(KM) but not IKK
(KM) mutants (Fig. 6) (17). The dominant-negative
IKK
(KM) mutant attenuated wt NIK-induced ICAM-1 promoter activity,
indicating the involvement of the NIK/IKK
pathway in TNF-
-induced
ICAM-1 expression.
and
NIK/IKK
pathways in TNF-
-induced ICAM-1 expression,
overexpression of a constitutively active PKC
plasmid and the wt
c-Src, NIK, and IKK
plasmids was used. These plasmids all induced
increase in ICAM-1 promoter activity, and their effects were blocked by the dominant-negative IKK
(KM) mutant. The effect of the
constitutively active PKC
(A/E) was blocked by the dominant-negative
c-Src (KM) mutant, but not by the NIK (KA) mutant. The effect of the
wt c-Src plasmid on ICAM-1 promoter activity was not affected by
the dominant-negative NIK (KA) mutant, and the wt NIK plasmid was not
affected by the dominant-negative c-Src (KM) mutant (Fig.
7B). These results show that the PKC/c-Src/IKK
and
NIK/IKK
pathways function in parallel in the TNF-
-mediated
induction of ICAM-1 expression in A549 cells. The existence of these
two pathways explains why inhibitors of PKC, tyrosine kinases, or Src
kinase could reverse TPA- but not TNF-
-induced I
B-
degradation, because TNF-
could still act via the NIK/IKK
pathway
in the presence of these inhibitors.
B activation in bone marrow macrophages,
U937 cells, and B cells (29-31). In bone marrow macrophages, TNF-
induces activation of c-Src, which associates with I
B-
and
phosphorylates Tyr42 of I
B-
, leading to NF-
B
activation and IL-6 release (29). In contrast to the rapid degradation
of serine-phosphorylated I
B-
(32), tyrosine-phosphorylated
I
B-
is not subject to rapid proteolysis (29, 33). In the present
study of TNF-
-induced ICAM-1 expression, the downstream target of
c-Src was IKK
and rapid degradation of I
B-
was seen (Fig.
2B). Involvement of a tyrosine kinase upstream of IKK
activation has also been reported in CD23 signaling in U937 cells (30)
and in B cell antigen receptor stimulation (31). A similar
PKC-dependent c-Src activation pathway has been found in
human osteoblasts, in which FGF-2 increases N-cadherin expression, in
A7r5 vascular smooth muscle cells, in which TPA induces
Rho-dependent actin reorganization, and in SH-SY5Y neuroblastoma cells, in which TPA induces Cas·Crk complex formation (34-36). Furthermore, the PKC/c-Src/IKK pathway, here shown to be
involved in induction of ICAM-1 expression, might be a common signaling
pathway for inducible gene expression, because TNF-
-, IL-1
-, or
interferon-
-induced COX-2 or ICAM-1 expression in human alveolar
epithelial cells also involves PKC-dependent activation of
c-Src or Lyn (16, 37,
38).2
pathway had been
demonstrated, tyrosine phosphorylation of IKK
by c-Src was further examined. Several lines of evidence show that this occurred. First, in
both crude cell lysates and immunoprecipitates formed using anti-IKK
antibody, IKK
was found to be tyrosine-phosphorylated after TNF-
or TPA stimulation. Second, in immunoprecipitates formed using
anti-phosphotyrosine antibody, both IKK
and c-Src were
tyrosine-phosphorylated after treatment with TNF-
or TPA. Third, all
these effects were inhibited by PP2. Fourth, using either
immunoprecipitation with anti-IKK
antibody followed by blotting with
anti-c-Src antibody or immunoprecipitation with anti-c-Src antibody
followed by blotting with anti-IKK
antibody, an association between
c-Src and IKK
was demonstrated and shown to be increased after
TNF-
or TPA treatment. Fifth, an in vitro kinase assay
demonstrated that c-Src directly phosphorylated IKK
at
Tyr188 and Tyr199. IKK
is a Thr/Ser kinase
and phosphorylation of Ser177 and Ser181 in the
kinase domain is necessary for its activation, because substitution of
these two residues with alanines reduces IKK
activity and leads to
reduced Rel A nuclear translocation and NF-
B-dependent
gene expression (21, 39). MEKK1 and NIK are reported to phosphorylate
these two serine residues (40). The present experiments further
demonstrated Tyr188 and Tyr199 phosphorylation
by c-Src via a PKC-dependent activation pathway. This
tyrosine phosphorylation of IKK
was essential for TNF-
-induced ICAM-1 expression in A549 cells, because the dominant-negative mutants,
IKK
(Y188F), IKK
(Y199F), or IKK
(FF), abrogated the effects
of both TNF-
and TPA. Tyrosine phosphorylation of Thr/Ser kinases,
such as PKCs and Akt, has also been reported to be important for their
activation (41, 42). Akt activation by extracellular stimuli is a
multistep process involving translocation and phosphorylation. Two
phosphorylation sites, Thr308 and Ser473, have
been shown to be critical for growth factor-induced activation of Akt
(43-45). In addition to the phosphorylation of these two sites,
tyrosine phosphorylation plays an important role in regulation of Akt
activity. Both the EGF-induced tyrosine phosphorylation and kinase
activity of Akt are blocked by PP2, and Src phosphorylates Tyr315 and Tyr326 of Akt both in
vivo and in vitro (41). It is noteworthy that these
tyrosine residues are conserved in about 50% of Ser/Thr kinases and
that phosphorylation of the corresponding residues, Tyr512
and Tyr523, in PKC
is also critical for PKC
activation in response to H2O2 (42).
Phosphorylation of the two conserved tyrosine residues in the kinase
domains of Ser/Thr kinases may therefore be a general mechanism by
which Akt, PKC
, and IKK
are regulated (41, 42, and present study)
(Fig. 9C). The Src tyrosine kinase family therefore directly
regulates IKK
activity via phosphorylation at Tyr188 and
Tyr199, rather than solely by NIK-mediated phosphorylation
at Ser177 and Ser181, as previously suggested
(27). Three findings further support the notion that the
PKC/c-Src/IKK
pathway induces tyrosine phosphorylation, whereas the
NIK/IKK
pathway induces serine phosphorylation. First, NF-
B
activity induced by PKC
(A/E) or wt c-Src was inhibited by the
tyrosine mutants, IKK
(Y188F), IKK
(Y199F), or IKK
(FF), but
not by IKK
(AA), in which Ser177 and Ser181
are mutated. Second, wt NIK-induced NF-
B activity was inhibited by
IKK
(AA) but not by IKK
(Y188F), IKK
(Y199F), or IKK
(FF) (Fig. 10B). Third, TPA-induced ICAM-1 promoter activity was
not affected by IKK
(AA) (Fig. 10A). Our data
demonstrate, for the first time, that, in addition to phosphorylation
of Ser177 and Ser181, Tyr188 and
Tyr199 phosphorylation of IKK
is required for its full
activation and biological functions.
-induced ICAM-1
expression in A549 cells have been further explored. In addition to
activating the NIK/IKK
pathway, TNF-
activates the PKC-dependent c-Src pathway. These two pathways converge at
IKK
, and are, respectively, responsible for phosphorylation of
Ser177/Ser181 and
Tyr188/Tyr199 of IKK
, then go on to activate
NF-
B, via serine phosphorylation and degradation of I
B-
, then,
finally, initiate of ICAM-1 expression. A schematic diagram showing the
involvement of these two pathways in TNF-
-induced ICAM-1 expression
in A549 epithelial cells is shown in Fig.
12.
View larger version (14K):
[in a new window]
Fig. 12.
Schematic representation of the signaling
pathways involved in TNF- -induced ICAM-1
expression in A549 epithelial cells. TNF-
binds to TNFR1 and
activates PC-PLC to induce PKC
and c-Src activation, leading to
tyrosine phosphorylation of IKK
at Tyr188 and
Tyr199. TNF-
also activates TRAF2 to induce NIK
activation, leading to serine phosphorylation of IKK
at
Ser177 and Ser181. These two pathways converge
at IKK
, resulting in phosphorylation and degradation of I
B-
,
stimulation of NF-
B in the ICAM-1 promoter, and, finally, initiation
of ICAM-1 expression.
![]() |
FOOTNOTES |
---|
* This work was supported by a research grant from the National Science Council of Taiwan.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.
To whom correspondence should be addressed: Dept. of Pharmacology,
College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd., 1st
Section, Taipei 10018, Taiwan. Tel.: 886-2-2312-3456 (ext. 8321); Fax:
886-2-2394-7833; E-mail: ccchen@ha.mc.ntu.edu.tw.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M208521200
2 W.-C. Huang, J.-J. Chen, and C.-C. Chen, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ICAM-1, intercellular adhesion molecule-1;
IKK, IB kinase;
NF-
B, nuclear
factor
B;
TNF, tumor necrosis factor;
NIK, nuclear
factor-
B-inducing kinase;
GST, glutathione S-transferase;
IL, interleukin;
PKC, protein kinase C;
DMEM, Dulbecco's modified
Eagle's medium;
wt, wild-type;
PMSF, phenylmethylsulfonyl fluoride;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
DTT, dithiothreitol;
TRAF, TNF receptor-associated factor;
MEKK, mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase kinase;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine.
![]() |
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