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-kappaB 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/IkappaBalpha. Phosphate-labelling experiments indicate that these events are preceded by the phosphorylation of MAD3 and p105. The protease inhibitors TLCK (Nalpha-p-Tosyl-L-Lysine Chloromethyl Ketone) and TPCK (Nalpha-p-Tosyl-L-Phenylalanine Chroromethyl Ketone) inhibit both p105 to p50 processing and MAD3 degradation, and also cause a complete block to NF-kappaB activation. These data suggest a model for NF-kappaB activation in which phosphorylation destabilises the NF-kappaB/MAD3 complex but that, in vivo, this is insufficient to lead to activation in the absence of an obligatory mechanism that degrades MAD3.