IN-VIVO STIMULATION OF I-KAPPA-B PHOSPHORYLATION IS NOT SUFFICIENT TOACTIVATE NF-KAPPA-B

NF-KB is a major inducible transcription factor in many immune and inf lammatory reactions. Its activation involves the dissociation of the i nhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, whe re they bind to DNA and activate transcription. Phosphorylation of I k appa B in cell-free experiments results in its inactivation and releas e from the Rel complex, but in vivo NF-kappa B activation is associate d with I kappa B degradation. In vivo phosphorylation of I kappa B alp ha was demonstrated in several recent studies, but its role is unknown . Our study shows that the T-cell activation results in rapid phosphor ylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B b locking reagents, suggesting that it plays an essential role in the ac tivation process. However, the in vivo induction of I kappa B alpha ph osphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon c ell stimulation, presumably through inhibition of the cytoplasmic prot easome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extend ed period of time, whereas NF-kappa B activation was abolished. It app ears that activation of NF-kappa B requires degradation of I kappa B a lpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of deg radation.