The signaling pathway involved in TNF-α-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-α-or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-α-or TPA-induced I-κB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-κBα 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-α or TPA. Overexpression of the constitutively active PKCα (PKCα 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-κB-inducing kinase (NIK)(KA), or IKKβ (KM) mutant. The dominant-negative PKCα (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, IKKα/β was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-α or TPA treatment. Two tyrosine residues, Tyr 188 and Tyr 199, near the activation loop of IKKβ, were identified to be crucial for NF-κB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKβ induced by TNF-α or TPA. These data suggest that, in addition to activating NIK, TNF-α also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKα/β, and go on to activate NF-κB, via serine phosphorylation and degradation of IκB-α, and, finally, to initiate COX-2 expression.