Tyrosine phosphorylation of I-kappa B kinase alpha/beta by protein kinase C-dependent c-Src activation is involved in TNF-alpha-induced cyclooxygenase-2 expression.
The signaling pathway involved in TNF-alpha-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-alpha- or TPA-induced I-kappaB kinase ... (IKK) activation was also blocked by these inhibitors, which reversed I-kappaBalpha degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKCalpha (PKCalpha A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-kappaB-inducing kinase (NIK) (KA), or IKKbeta (KM) mutant. The dominant-negative PKCalpha (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKalpha/beta was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr(188) and Tyr(199), near the activation loop of IKKbeta, were identified to be crucial for NF-kappaB activation. Substitution of these residues with phenylalanines attenuated COX-2 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 cross-link between c-Src and NIK and converge at IKKalpha/beta, and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate COX-2 expression.
Mesh Terms:
Amino Acid Sequence, Amino Acid Substitution, Cyclooxygenase 2, Enzyme Activation, Enzyme Induction, Enzyme Inhibitors, Gene Expression Regulation, Genetic Vectors, Humans, I-kappa B Kinase, Isoenzymes, Membrane Proteins, Molecular Sequence Data, NF-kappa B, Phenylalanine, Phosphorylation, Promoter Regions, Genetic, Prostaglandin-Endoperoxide Synthases, Protein Kinase C, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Pyrimidines, Signal Transduction, Tetradecanoylphorbol Acetate, Transfection, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha, Tyrosine, src-Family Kinases
Amino Acid Sequence, Amino Acid Substitution, Cyclooxygenase 2, Enzyme Activation, Enzyme Induction, Enzyme Inhibitors, Gene Expression Regulation, Genetic Vectors, Humans, I-kappa B Kinase, Isoenzymes, Membrane Proteins, Molecular Sequence Data, NF-kappa B, Phenylalanine, Phosphorylation, Promoter Regions, Genetic, Prostaglandin-Endoperoxide Synthases, Protein Kinase C, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, Pyrimidines, Signal Transduction, Tetradecanoylphorbol Acetate, Transfection, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha, Tyrosine, src-Family Kinases
J. Immunol.
Date: May. 01, 2003
PubMed ID: 12707358
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