Tyrosine Phosphorylation of I-kappaB Kinase alpha/beta by protein Kinase C-Dependent C-Src Activation Is Involved in TNF-alpha-Induced Cyclooxygenase-2 Expression~1

摘要

The signaling pathway involved in TNF-alpha-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells.A protein dinase 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 KINADE (IKK) activation was also blocked by these inhibitors,which revesed I-kappaBalpha degradation.Activation of c-Src and Lyn kinaseswto src family members,was inibited by the PKC,tyrosine inase or src kinase inhibitors.The dominate-negative c-Src (km) mutant inhibited induction of COX-2 promoter activity by TNF-alpha or TPA.Overexpression of the constitutivel active PKC alpha (PKC alpha A/E) or wild-type c-Src plamida induced COX-2 promoter activity,and these effects werelihibited by the cominant-negative C-sRC (KM),NF-kappaB-inducing kinase (NIK) (KA),or IKKBETA (km) mutant.The dominant-negative PKC alpha(K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression.In coimmunoprectipitation exsperiments,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 redidues,Tyr~188 and Try~199,near the activation loop of IKKbeta,were identified to be dcrucial for NF-kappa B activation.Substitution of these residuce with phenylalanines attenuated COX-2 promoter activity and C-sRE-Dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA.These date suggets 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 phophorylation and dtgradation of IkappaB-alpha,and finally,to initiate COX-2 expression.