c-Src-dependent tyrosine phosphorylation of IKKbeta is involved in tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression.
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 IkappaBalpha kinase (IKK) activation was also blocked by these inhibitors, which slightly reversed TNF-alpha-induced but completely reversed TPA-induced IkappaBalpha 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 PKC 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-kappaB-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-alpha or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKbeta, were identified as being important for NF-kappaB 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-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate ICAM-1 expression.
Mesh Terms:
Amino Acid Sequence, Blotting, Western, Cell Nucleus, Enzyme Inhibitors, Genes, Dominant, Humans, I-kappa B Kinase, I-kappa B Proteins, Intercellular Adhesion Molecule-1, Kinetics, Luciferases, Models, Biological, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Phosphorylation, Plasmids, Precipitin Tests, Protein Binding, Protein Kinase C, Protein Kinase C-alpha, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Signal Transduction, Time Factors, Transcriptional Activation, Transfection, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha, Tyrosine
Amino Acid Sequence, Blotting, Western, Cell Nucleus, Enzyme Inhibitors, Genes, Dominant, Humans, I-kappa B Kinase, I-kappa B Proteins, Intercellular Adhesion Molecule-1, Kinetics, Luciferases, Models, Biological, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Phosphorylation, Plasmids, Precipitin Tests, Protein Binding, Protein Kinase C, Protein Kinase C-alpha, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Signal Transduction, Time Factors, Transcriptional Activation, Transfection, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha, Tyrosine
J. Biol. Chem.
Date: Mar. 14, 2003
PubMed ID: 12645577
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