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.
Huang
Wei-Chien
WC
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chen
Jun-Jie
JJ
Inoue
Hiroyasu
H
Chen
Ching-Chow
CC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000081247:CSK Tyrosine-Protein Kinase; D051546:Cyclooxygenase 2; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D051550:I-kappa B Kinase; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000096:biosynthesis; D010649:Phenylalanine / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014443:Tyrosine / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
C412373:AG 1879; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D011743:Pyrimidines; D014409:Tumor Necrosis Factor-alpha; D014443:Tyrosine; D010649:Phenylalanine; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C060902:lyn protein-tyrosine 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; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate