Tyrosine phosphorylation-dependent activation of NF-κB -: Requirement for p56 LCK and ZAP-70 protein tyrosine kinases

Phosphorylation of the N-terminal domain of I kappaB inhibitory subunits induces activation of the transcription factor NF-kappaB. Although serine phosphorylation has been shown to induce ubiquitination and subsequent proteasome-mediated degradation of I kappaB-alpha, little is known about the mechanisms that lead to release of active NF-kappaB in T cells as a consequence of tyrosine phosphorylation of I kappaB-alpha [Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L., Traenckner, B.M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P. & Peyron, J.F. (1996) Cell 86, 787-798]. The involvement of the tyrosine kinases p56(lck) and ZAP-70 in this reaction is demonstrated here using specific pharmacological inhibitors and Jurkat mutants unable to express these kinases. Although the inhibitors prevented both pervanadate-induced phosphorylation of I kappaB-alpha on Tyr42 and NF-kappaB activation, we observed that, in p56(lck)-deficient Jurkat mutants, NF-kappaB could still associate with I kappaB-alpha despite phosphorylation on Tyr42. Furthermore, the SH2 domain of p56(lck) appeared to be required for pervanadate-induced NF-kappaB activation but not for Tyr42 phosphorylation. These results show that p56(lck) and ZAP-70 are key components of the signaling pathway that leads to phosphotyrosine-dependent NF-kappaB activation in T cells and confirm that tyrosine kinases must control at least two different steps to induce activation of NF-kappaB. Finally, we show that H2O2, which stimulates p56(lck) and ZAP-70 in T cells, is an activator of NF-kappaB through tyrosine phosphorylation of I kappaB-alpha.