Proteolytic degradation of MAD3 (I kappa B alpha) and enhanced processing of the NF-kappa B precursor p105 are obligatory steps in the activation of NF-kappa B.

We have studied the role of protein turnover in the induction of NF-kappa B DNA binding activity. Treatment of cells with tumour necrosis factor (TNF), double-stranded RNA (dsRNA), or phorbol esters is shown to be associated with an increase in the rate of p105 to p50 processing, and the loss of immunologically detectable MAD3/I kappa B alpha. Phosphate-labelling experiments indicate that these events are preceded by the phosphorylation of MAD3 and p105. The protease inhibitors TLCK (N alpha-p-Tosyl-L-Lysine Chloromethyl Ketone) and TPCK (N alpha-p-Tosyl-L-Phenylalanine Chloromethyl Ketone) inhibit both p105 to p50 processing and MAD3 degradation, and also cause a complete block to NF-kappa B activation. These data suggest a model for NF-kappa B activation in which phosphorylation destabilises the NF-kappa B/MAD3 complex but that, in vivo, this is insufficient to lead to activation in the absence of an obligatory mechanism that degrades MAD3.