C-Src-Dependent Tyrosine Phosphorylation of Ikk Beta 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 IkappaB 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-Sre (KM) mutant inhibited induction of ICAM-1 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKCalpha or wild-type c-Src plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c- Src (KAI) 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, IKKbeta was found to be associated with c-Src and to be phosphorylated on tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr(188 ) and Tyr(199), 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-Sre. 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