c-Src-dependent Tyrosine Phosphorylation of IKKbeta Is Involved in Tumor Necrosis Factor-alpha -induced Intercellular Adhesion Molecule-1 Expression*

Wei-Chien Huang, Jun-Jie Chen, and Ching-Chow ChenDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The signaling pathway involved in tumor necrosis factor-alpha (TNF-alpha )-induced intercellular adhesion molecule-1 (ICAM-1) expression was further studied in human A549 epithelial cells. TNF-alpha - 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-alpha - or TPA-induced Ikappa B kinase (IKK) activation was also blocked by these inhibitors, which slightly reversed TNF-alpha -induced but completely reversed TPA-induced Ikappa Balpha degradation. c-Src and Lyn, two members of the Src kinase family, were abundantly expressed in A549 cells, and their activation by TNF-alpha 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-alpha or TPA. Overexpression of the constitutively active PKCalpha or wild-type c-Src plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c-Src (KM) or IKKbeta (KM) mutant but not by the nuclear factor-kappa 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, IKKbeta was found to be associated with c-Src and to be phosphorylated on tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKbeta , were identified as being important for NF-kappa B activation. Substitution of these residues with phenylalanines abolished ICAM-1 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways converge at IKKbeta and go on to activate NF-kappa B, via serine phosphorylation and degradation of Ikappa B-alpha , and, finally, to initiate ICAM-1 expression.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 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).

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)-alpha , interleukin (IL)-1beta , and interferon-gamma (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-kappa B (NF-kappa B), activator protein-1 (AP-1), AP-2, and the interferon-stimulated response element (15). Of these, NF-kappa 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-alpha and IL-1beta cause ICAM-1 expression in A549 human alveolar epithelial cells have been explored and found to involve the sequential activation of protein kinase Calpha (PKCalpha ), protein-tyrosine kinase, nuclear factor-kappa B-inducing kinase (NIK), and Ikappa B kinase beta  (IKKbeta ) (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-alpha -inducing NF-kappa B transcriptional activation and that, in addition to serine phosphorylation of IKKbeta by NIK, Tyr188 and Tyr199 phosphorylations by PKC-dependent c-Src activation also contribute to TNF-alpha -induced ICAM-1 expression in human alveolar epithelial cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Rabbit polyclonal antibodies specific for Ikappa Balpha , IKKbeta , 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-alpha 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). [gamma -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).

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 kappa B-luc plasmid was from Stratagene. The PKC-alpha constitutively active (PKC-alpha /AE) or dominant-negative mutant (PKCalpha /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 IKKbeta (NIK wt and mutant KA; IKKbeta wt and mutant KM) were gifts from Signal Pharmaceuticals (San Diego, CA). The dominant negative mutant of IKKbeta (AA) was from Dr. Karin (University of California, San Diego, CA). pGEX-Ikappa Balpha -(1-100) was a gift from Dr. Nakano (University of Juntendo, Tokyo). pGEX-IKKbeta -(132-206) was a gift from Dr. Nakanishi (University of Nagoya, Nagoya).

Immunoprecipitation and Kinase Activity Assay-- Following treatment with TNF-alpha 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-IKKbeta , 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 [gamma -32P]ATP and either 1 µg of bacterially expressed GST-Ikappa Balpha -(1-100) as IKK substrate, 1 µg of acidic denatured enolase as c-Src or Lyn substrate, or 6 µg of bacterially expressed GST-IKKbeta -(132-206), GST-IKKbeta -(132-206) (Y188F), GST-IKKbeta -(132-206) (Y199F), or GST-IKKbeta -(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-Ikappa Balpha -(1-100), phosphorylated GST-IKKbeta -(132-206), or phosphorylated enolase was visualized by autoradiography. Quantitative data were obtained using a computing densitometer and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).

Western Blot Analysis-- Following treatment with TNF-alpha 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 IKKbeta , Ikappa Balpha , 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).

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-alpha 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.

Oligonucleotides corresponding to the downstream NF-kappa B consensus sequence (5'-AGCTTGGAAATTCCGGA-3') in the human ICAM-1 promoter were synthesized, annealed, and end-labeled with [gamma -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-kappa 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.

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 IKKbeta cloned in the pcDNA3.1 vector or in the human GST-IKKbeta (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 IKKbeta (Y188F) mutation, and primer 5 (5'-GGAGCAGCAGAAGTTCACAGTGACCGTCG-3') and primer 6 (3'-CCTCGTCGTCTTCAAGTGTCACTGGCAGC-5') for IKKbeta (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.

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 kappa B-luc plasmid using Tfx-50 (Promega) according to the manufacturer's recommendations. Briefly, reporter DNA (0.4 µg) and beta -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-alpha or TPA was added for 6 h. Cell extracts were then prepared and luciferase and beta -galactosidase activities were measured, the luciferase activity being normalized to the beta -galactosidase activity. In experiments using dominant-negative mutants, cells were co-transfected with reporter (0.2 µg) and beta -galactosidase (0.1 µg) and either the dominant-negative NIK, IKKbeta , 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 beta -galactosidase plasmid; 0.4 µg of the constitutively active PKCalpha (A/E) plasmid, wt c-Src or NIK plasmid (or the respective empty vector); and 0.4 µg of the dominant-negative NIK, IKKbeta , or c-Src mutant (or the respective empty vector).

Co-immunoprecipitation Assay-- Cell lysates containing 1 mg of protein were incubated for 1 h at 4 °C with 2 µg of anti-IKKbeta 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, IKKbeta , or c-Src.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of Inhibitors of PKC, Tyrosine Kinase, or Src Kinase on the Induction of ICAM-1 Promoter Activity by TNF-alpha or TPA in A549 Cells-- In our previous study (17), we found that PKC and tyrosine kinase were involved in TNF-alpha -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-kappa B site (-189/-174) responsible for mediating the induction of ICAM-1 promoter activity by TNF-alpha or TPA (17). As shown in Fig. 1, TNF-alpha 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-alpha -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-alpha - 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-alpha or 1 µM TPA. Luciferase activity was then measured as described under "Experimental Procedures," normalized to the beta -galactosidase activity and expressed as the mean ± S.E. for three independent experiments performed in triplicate. *, p < 0.05, compared with TNF-alpha or TPA alone.

Induction of IKK Activation, Ikappa Balpha Degradation, and NF-kappa B-specific DNA-Protein Complex Formation by TNF-alpha and TPA, and the Inhibitory Effect of Inhibitors of PKC, Tyrosine Kinase, or Src Kinase-- Because TNF-alpha - and TPA-induced ICAM-1 promoter activity in A549 cells is inhibited by the dominant-negative IKKbeta mutant (17), endogenous IKK activity was measured by immunoprecipitation with anti-IKKbeta antibody. When cells were treated with 10 ng/ml TNF-alpha for 5, 10, 30, or 60 min, maximal IKK activity was seen after 5 min (Fig. 2A), whereas maximal degradation of Ikappa B-alpha was seen after 10 min, Ikappa B-alpha 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 Ikappa B-alpha degradation was seen after 60 min (Fig. 2B). The TNF-alpha -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 Ikappa Balpha degradation induced by TPA was reversed by PKC, tyrosine kinase, and Src kinase inhibitors, but that induced by TNF-alpha was only slightly affected by these inhibitors (Fig. 3B). The effect of these inhibitors on TNF-alpha - or TPA-induced NF-kappa B-specific DNA·protein binding was examined. As shown in Fig. 3C, when cells were treated with TNF-alpha for 10 min, increased NF-kappa 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-kappa 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-alpha -induced IKK activation and Ikappa B-alpha degradation. A549 cells were treated with 10 ng/ml TNF-alpha or 1 µM TPA for 5, 10, 30, or 60 min, then cell lysates were prepared. In A, cell lysates were immunoprecipitated with anti-IKKbeta antibody, then the kinase assay (KA) and autoradiography for phosphorylated GST-Ikappa Balpha (1-100) were performed on the precipitates as described under "Experimental Procedures." Levels of immunoprecipitated IKKbeta protein were estimated by Western blotting (WB) using anti-IKKbeta antibody. In B, cytosolic levels of Ikappa B-alpha were measured using anti-Ikappa B-alpha antibody as described under "Experimental Procedures."


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Fig. 3.   Effect of various inhibitors on TNF-alpha - or TPA-induced IKK activity, Ikappa Balpha degradation, and NF-kappa 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-alpha 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-IKKbeta antibody, and the kinase assay (KA) and autoradiography for phosphorylated GST-Ikappa Balpha -(1-100) were performed on the precipitates as described under "Experimental Procedures." Levels of immunoprecipitated IKKbeta were estimated by Western blotting (WB) using anti-IKKbeta antibody. In B, cytosolic levels of Ikappa B-alpha were measured by Western blotting using anti-Ikappa B-alpha antibody as described under "Experimental Procedures." In C, the NF-kappa B-specific DNA·protein activity in nuclear extracts was determined by electrophoretic mobility shift assay as described under "Experimental Procedures."

Induction of c-Src and Lyn Activation by TNF-alpha and TPA, and the Inhibitory Effect of Inhibitors of PKC, Tyrosine Kinase, or Src Kinase-- TNF-alpha - 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-alpha 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-alpha - 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-alpha 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-alpha 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-alpha - 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-alpha 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.

Induction of ICAM-1 Promoter Activity by Overexpression of PKCalpha or c-Src, and the Inhibitory Effect of Dominant-negative Mutants of c-Src or IKKbeta -- The TNF-alpha - 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-kappa 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-alpha - or TPA-induced ICAM-1 promoter activity (Fig. 6). The TNF-alpha -induced ICAM-1 promoter activity was also inhibited by the dominant-negative NIK (KA) and IKKbeta (KM) mutants, as previously reported (17). To characterize the relationship between PKC, c-Src, NIK, and IKKbeta , overexpression of the constitutively active form of PKCalpha (A/E) or of wt c-Src, NIK, or IKKbeta was performed. Overexpression of PKCalpha (A/E) or wt c-Src, NIK, or IKKbeta 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 PKCalpha (A/E) or c-Src wt was inhibited by the dominant-negative c-Src (KM) or IKKbeta (KM) mutant, but not by the NIK (KA) mutant. In contrast, the dominant-negative IKKbeta (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/IKKbeta and NIK/IKKbeta pathways in TNF-alpha -induced ICAM-1 expression in A549 cells.


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Fig. 6.   Effect of various dominant-negative mutants on TNF-alpha - 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 IKKbeta (KM) mutant, or the respective empty vector, then treated for 6 h with 10 ng/ml TNF-alpha or 1 µM TPA. Luciferase activity was then measured as described under "Experimental Procedures," and the results were normalized to the beta -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-alpha or TPA alone.


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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 PKCalpha (A/E), wild-type c-Src, IKKbeta , or NIK, or the respective empty vector. In B, A549 cells were co-transfected for 24 h with PKCalpha (A/E), wild-type c-Src, or NIK and c-Src (K295M), IKKbeta (KM), or NIK (KA). Luciferase activity was then assayed as described under "Experimental Procedures," and the results were normalized to the beta -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.

Induction by TNF-alpha or TPA of Tyrosine Phosphorylation of IKKbeta and of the c-Src and IKKbeta Association, and the inhibitory Effect of PP2-- Because c-Src-dependent IKK activation was shown to be involved, co-immunoprecipitation of c-Src and IKKbeta was performed to examine whether c-Src directly regulates IKK activity through phosphorylation of tyrosine residues. When cells were treated with TNF-alpha for 5, 10, or 15 min, IKKbeta 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 IKKbeta and phosphorylated its tyrosine residues, cell lysates were immunoprecipitated with anti-IKKbeta 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 IKKbeta was seen after TNF-alpha 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-IKKbeta or anti-c-Src antibody, both IKKbeta and c-Src were shown to be tyrosine-phosphorylated after TNF-alpha or TPA treatment, and these effects were again inhibited by PP2 (Fig. 8C). These results indicate that c-Src can associate with IKKbeta and phosphorylate its tyrosine residues after TNF-alpha or TPA stimulation. The association between c-Src and IKK was further examined. Anti-IKKbeta 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 IKKbeta after TNF-alpha or TPA stimulation. In the converse experiment in which c-Src was precipitated using anti-c-Src antibody, IKKbeta was shown to be associated with c-Src in a time-dependent manner after TNF-alpha or TPA treatment (Fig. 9B). These results show that there is an association between c-Src and IKKbeta and that IKKbeta tyrosine residues are phosphorylated.


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Fig. 8.   Tyrosine phosphorylation of IKKbeta induced by TNF-alpha or TPA and the inhibitory effect of PP2. Control cells or cells pretreated for 30 min with 10 µM PP2 were stimulated with TNF-alpha 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-IKKbeta (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-IKKbeta (C), or anti-c-Src (C) antibodies or reprobed with anti-IKKbeta (A and B) antibody.


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Fig. 9.   c-Src co-immunoprecipitates with IKKbeta after TNF-alpha or TPA treatment. A549 cells were treated with TNF-alpha 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-IKKbeta (A) or anti-c-Src (B) antibodies. Immunoprecipitated proteins were separated by SDS-PAGE on a 10% gel and immunoblotted (WB) with anti-IKKbeta or anti-c-Src antibodies. C, alignment of subdomains VII and VIII of the kinase domains of PKCdelta , Akt1, and IKKalpha /beta .

Inhibitory Effect of the Dominant-negative Mutants IKKbeta (Y188F), IKKbeta (Y199F), or IKKbeta (FF) on TNF-alpha - and TPA-induced ICAM-1 Promoter Activity and on the PKCalpha - and c-Src-induced, but not the NIK-induced, Increase in NF-kappa B Activity-- The above experiments demonstrate that c-Src directly interacts with IKKbeta and phosphorylates its tyrosine residues after TNF-alpha or TPA stimulation. When the amino sequences of subdomains VII and VIII in the kinase domain of PKCdelta , AKT1, and IKKalpha /beta were aligned, the tyrosine residues were found to be conserved (Fig. 9C). Hypothesizing that Tyr188 and/or Tyr199 of IKKbeta were the targets for c-Src phosphorylation after TNF-alpha or TPA stimulation, we used site-directed mutagenesis to generate the dominant-negative tyrosine mutants, IKKbeta (Y188F), IKKbeta (Y199F), and IKKbeta (Y188F, Y199F). Overexpression of these mutants attenuated the TNF-alpha - 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 IKKbeta (KM) mutant, with Lys44 mutated to methionine, had a similar inhibitory effect to that of IKKbeta (Y188F) or IKKbeta (Y199F) on TNF-alpha - and TPA-induced ICAM-1 promoter activity, whereas IKKbeta (AA), with Ser177 and Ser181 mutated to alanine, was as effective as IKKbeta (Y188F) or IKKbeta (Y199F) in inhibiting TNF-alpha -induced ICMA-1 promoter activity but had no effect on TPA-induced ICAM-1 promoter activity (Fig. 10A).


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Fig. 10.   Effect of the dominant-negative tyrosine mutants, IKKbeta (Y188F), IKKbeta (Y199F), and IKKbeta (FF), on TNF-alpha - or TPA-induced ICAM-1 promoter activity and on wild-type plasmid-induced NF-kappa B activity. In A, A549 cells were co-transfected with pIC339 plus one of the dominant-negative tyrosine mutants (IKKbeta (188F), IKKbeta (Y199F), or IKKbeta (FF)), dominant-negative mutant (IKKbeta (KM)), or dominant-negative serine mutant (IKKbeta (AA)), or the respective empty vector, then treated with 10 ng/ml TNF-alpha or 1 µM TPA for 6 h. In B, A549 cells were co-transfected with kappa B-luc and the constitutively active form of PKCalpha (A/E), wild-type c-Src, or wild-type NIK, plus the dominant-negative mutants, IKKbeta (Y188F), IKKbeta (Y199F), IKKbeta (FF), or IKKbeta (AA), or the respective empty vector. Luciferase activity was then measured as described under "Experimental Procedures," and the results were normalized to the beta -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-alpha or TPA alone (A) or wild-type alone (B).

To further confirm the involvement of tyrosine phosphorylation in the PKCalpha /c-Src/IKKbeta pathway and serine phosphorylation in the NIK/IKKbeta pathway, the dominant-negative IKKbeta mutants with either a tyrosine or serine mutation were co-transfected with PKCalpha (A/E), wt c-Src, or wt NIK to examine their inhibitory effects on the constitutively active or wt plasmid-induced NF-kappa B activity. As shown in Fig. 10B, PKCalpha (A/E)- or wt c-Src-induced NF-kappa 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-kappa B activity.

Because Tyr188 and Tyr199 in IKKbeta were found to be critical for the PKCalpha /c-Src/IKKbeta pathway to elicit NF-kappa B activation, leading to induction of TNF-alpha - or TPA-stimulated ICAM-1 promoter activity (Fig. 10), endogenous c-Src phosphorylation of Tyr188 and Tyr199 in IKKbeta was further examined. c-Src was immunoprecipitated using anti-c-Src antibody, and its ability to phosphorylate IKKbeta was measured using GST-IKKbeta -(132-206) as an in vitro substrate. When cells were treated with TNF-alpha or TPA, IKKbeta was phosphorylated by c-Src in a time-dependent manner. The maximal effect was seen at 10-min treatment with TNF-alpha or 30-min treatment with TPA (Fig. 11A), and both effects were inhibited by PP2 (Fig. 11B). The c-Src-dependent IKKbeta phosphorylation was specific for Tyr188/Tyr199, because it was not seen when either or both tyrosine residues were substituted with phenylalanines (Fig. 11C).


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Fig. 11.   c-Src-dependent phosphorylation of IKKbeta at Y188 and Y199 is induced by TNF-alpha or TPA and inhibited by PP2. A549 cells were treated with 10 ng/ml TNF-alpha 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-alpha 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-IKKbeta (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-alpha 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-IKKbeta -(132-206), GST-IKKbeta -(132-206) (Y188F), GST-IKKbeta -(132-206) (Y199F), or GST-IKKbeta -(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-IKKbeta -(132-206) were detected by Coomassie Brilliant Blue staining.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The PKC-dependent tyrosine kinase activation pathway is involved in TNF-alpha -induced NF-kappa 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-alpha - 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. IKKbeta , but not IKKalpha , is involved in the TNF-alpha - and TPA-induced ICAM-1 promoter activity (17), and TNF-alpha - or TPA-induced stimulation of IKK activity and parallel degradation of Ikappa B-alpha was seen in the present study. The TNF-alpha - and TPA-induced IKK activity and NF-kappa 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 IKKbeta activation leading to NF-kappa 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-alpha 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/IKKbeta activation in TNF-alpha -induced ICAM-1 expression was confirmed by the finding that the dominant-negative c-Src (KM) mutant attenuated the TNF-alpha - and TPA-induced ICAM-1 promoter activity.

In nonstimulated cells, NF-kappa B dimers are present as cytoplasmic latent complexes due to the binding of specific inhibitors, the Ikappa Bs, that mask their nuclear localization signal. Following stimulation by pro-inflammatory cytokines, the Ikappa 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-kappa B dimers, followed by their translocation to the nucleus, binding to specific DNA sites (kappa B sites), and targeting of gene activation. The protein kinase that phosphorylates Ikappa 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, IKKalpha (IKK1), IKKbeta (IKK2), and IKKgamma (25). IKKalpha and IKKbeta are very similar protein kinases that act as the catalytic subunits of the complex (20-24). In mammalian cells, IKKalpha and IKKbeta form a stable heterodimer that is tightly associated with IKKgamma , 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 Ikappa B degradation and NF-kappa B activation to IL-1beta or TNF-alpha stimulation (27). The activities of both IKKalpha and IKKbeta are reported to be regulated by NIK (28). Our results show that the TNF-alpha -induced increase in ICAM-1 promoter activity was inhibited by the dominant-negative NIK (KA) and IKKbeta (KM) but not IKKalpha (KM) mutants (Fig. 6) (17). The dominant-negative IKKbeta (KM) mutant attenuated wt NIK-induced ICAM-1 promoter activity, indicating the involvement of the NIK/IKKbeta pathway in TNF-alpha -induced ICAM-1 expression.

To elucidate the relationship between the PKC/c-Src/IKKbeta and NIK/IKKbeta pathways in TNF-alpha -induced ICAM-1 expression, overexpression of a constitutively active PKCalpha plasmid and the wt c-Src, NIK, and IKKbeta plasmids was used. These plasmids all induced increase in ICAM-1 promoter activity, and their effects were blocked by the dominant-negative IKKbeta (KM) mutant. The effect of the constitutively active PKCalpha (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/IKKbeta and NIK/IKKbeta pathways function in parallel in the TNF-alpha -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-alpha -induced Ikappa B-alpha degradation, because TNF-alpha could still act via the NIK/IKKbeta pathway in the presence of these inhibitors.

c-Src is involved in NF-kappa B activation in bone marrow macrophages, U937 cells, and B cells (29-31). In bone marrow macrophages, TNF-alpha induces activation of c-Src, which associates with Ikappa B-alpha and phosphorylates Tyr42 of Ikappa B-alpha , leading to NF-kappa B activation and IL-6 release (29). In contrast to the rapid degradation of serine-phosphorylated Ikappa B-alpha (32), tyrosine-phosphorylated Ikappa B-alpha is not subject to rapid proteolysis (29, 33). In the present study of TNF-alpha -induced ICAM-1 expression, the downstream target of c-Src was IKKbeta and rapid degradation of Ikappa B-alpha 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-alpha -, IL-1beta -, or interferon-gamma -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

Because involvement of the PKC/c-Src/IKKbeta pathway had been demonstrated, tyrosine phosphorylation of IKKbeta 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-IKKbeta antibody, IKKbeta was found to be tyrosine-phosphorylated after TNF-alpha or TPA stimulation. Second, in immunoprecipitates formed using anti-phosphotyrosine antibody, both IKKbeta and c-Src were tyrosine-phosphorylated after treatment with TNF-alpha or TPA. Third, all these effects were inhibited by PP2. Fourth, using either immunoprecipitation with anti-IKKbeta antibody followed by blotting with anti-c-Src antibody or immunoprecipitation with anti-c-Src antibody followed by blotting with anti-IKKbeta antibody, an association between c-Src and IKKbeta was demonstrated and shown to be increased after TNF-alpha or TPA treatment. Fifth, an in vitro kinase assay demonstrated that c-Src directly phosphorylated IKKbeta at Tyr188 and Tyr199. IKKbeta 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 IKKbeta activity and leads to reduced Rel A nuclear translocation and NF-kappa 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 IKKbeta was essential for TNF-alpha -induced ICAM-1 expression in A549 cells, because the dominant-negative mutants, IKKbeta (Y188F), IKKbeta (Y199F), or IKKbeta (FF), abrogated the effects of both TNF-alpha 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 PKCdelta is also critical for PKCdelta 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, PKCdelta , and IKKbeta are regulated (41, 42, and present study) (Fig. 9C). The Src tyrosine kinase family therefore directly regulates IKKbeta 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/IKKbeta pathway induces tyrosine phosphorylation, whereas the NIK/IKKbeta pathway induces serine phosphorylation. First, NF-kappa B activity induced by PKCalpha (A/E) or wt c-Src was inhibited by the tyrosine mutants, IKKbeta (Y188F), IKKbeta (Y199F), or IKKbeta (FF), but not by IKKbeta (AA), in which Ser177 and Ser181 are mutated. Second, wt NIK-induced NF-kappa B activity was inhibited by IKKbeta (AA) but not by IKKbeta (Y188F), IKKbeta (Y199F), or IKKbeta (FF) (Fig. 10B). Third, TPA-induced ICAM-1 promoter activity was not affected by IKKbeta (AA) (Fig. 10A). Our data demonstrate, for the first time, that, in addition to phosphorylation of Ser177 and Ser181, Tyr188 and Tyr199 phosphorylation of IKKbeta is required for its full activation and biological functions.

In summary, the signaling pathways involved in TNF-alpha -induced ICAM-1 expression in A549 cells have been further explored. In addition to activating the NIK/IKKbeta pathway, TNF-alpha activates the PKC-dependent c-Src pathway. These two pathways converge at IKKbeta , and are, respectively, responsible for phosphorylation of Ser177/Ser181 and Tyr188/Tyr199 of IKKbeta , then go on to activate NF-kappa B, via serine phosphorylation and degradation of Ikappa B-alpha , then, finally, initiate of ICAM-1 expression. A schematic diagram showing the involvement of these two pathways in TNF-alpha -induced ICAM-1 expression in A549 epithelial cells is shown in Fig. 12.


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Fig. 12.   Schematic representation of the signaling pathways involved in TNF-alpha -induced ICAM-1 expression in A549 epithelial cells. TNF-alpha binds to TNFR1 and activates PC-PLC to induce PKCalpha and c-Src activation, leading to tyrosine phosphorylation of IKKbeta at Tyr188 and Tyr199. TNF-alpha also activates TRAF2 to induce NIK activation, leading to serine phosphorylation of IKKbeta at Ser177 and Ser181. These two pathways converge at IKKbeta , resulting in phosphorylation and degradation of Ikappa B-alpha , stimulation of NF-kappa 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.

Dagger 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, Ikappa B kinase; NF-kappa B, nuclear factor kappa B; TNF, tumor necrosis factor; NIK, nuclear factor-kappa 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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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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