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.
Huang
Wei-Chien
WC
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan.
Chen
Jun-Jie
JJ
Chen
Ching-Chow
CC
D000595:Amino Acid Sequence; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002467:Cell Nucleus / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005799:Genes, Dominant; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis* / Q000737:chemistry; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*; D019061:src-Family Kinases
D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D014443:Tyrosine; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha