Regulation of NF-kappa B activity by I kappa B alpha and I kappa B beta stability.

Transcription factor NF-kappa B must be released from cytoplasmic inhibitory molecules (I kappa Bs) in order to move to the nucleus and to activate its target genes. Little is known about the mechanisms regulating the maintenance of constitutive nuclear NF-kappa B in some cell-types and of sustained nuclear NF-kappa B activity after stimulation. Increased turnover has been implicated in the regulation of constitutive NF-kappa B activity in mature B cells. We therefore compared the turnover of I kappa B alpha and I kappa B beta in mature B cells and HeLa cells. Both proteins display a high turnover in B cells although I kappa B beta is considerably more stable than I kappa B alpha. The half-life of both inhibitors is increased in HeLa cells. In contrast, all other NF-kappa B/I kappa B molecules tested are relatively stable in both cell-types. The elevated turnover of endogenous I kappa B alpha in Namalwa cells is inhibited by a proteasome inhibitor and thus seems to be driven by the same degradation machinery as the slower turnover in non-B cells. Furthermore, we investigated the processes involved in persistent activation of NF-kappa B. TNF-alpha signaling leads to a rapid depletion of cellular I kappa B beta pools. I kappa B alpha is efficiently resynthesized whereas I kappa B beta levels stay low for a prolonged time. NF-kappa B binding activity can be detected for several hours after stimulation. We found that removal of the TNF-alpha containing medium causes a rapid decrease in nuclear NF-kappa B. A phosphoform of newly synthesized I kappa B alpha is visible when degradation by the proteasome is inhibited and new I kappa B alpha displays the same properties regarding phosphorylation and degradation in response to a second inducer. There is no significant difference in the turnover of pre- and post-inductive I kappa B alpha. These observations suggest that resynthesis of I kappa B alpha and removal of the stimulus are obligatory steps for the inactivation of nuclear NF kappa B.