Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa B kinases in NCTC 2544 keratinocytes

In this study we examined the regulation of the stress-activated protein (S AP) kinases and inhibitory KB kinases (IKKs) through stimulation of the nov el G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-a ctivated protein kinase activity. Trypsin also stimulated NF kappaB-DNA bin ding and the activation of the upstream kinases IKK alpha and -beta. Phorbo l 12-myristate 13-acetate also strongly activated both SAP kinases and = is oforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Go6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetat e-mediated c-Jun N-terminal kinase activity, although it only partially inh ibited the response to trypsin. In contrast, Go6983 reduced trypsin-stimula ted p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Add itionally, inhibitors of PKC zeta partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC zeta but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta activity . This study shows for the first time proteinase-activated receptor-2-media ted stimulation of both SAP kinase and = signaling and differing roles for PKC isoforms in the regulation of each pathway.