STIMULATION-DEPENDENT I-KAPPA-B-ALPHA PHOSPHORYLATION MARKS THE NF-KAPPA-B INHIBITOR FOR DEGRADATION VIA THE UBIQUITIN-PROTEASOME PATHWAY

The nuclear translocation of NF-KB follows the degradation of its inhi bitor, I kappa B alpha, an event coupled with stimulation-dependent in hibitor phosphorylation. Prevention of the stimulation-dependent phosp horylation of I kappa B alpha, either by treating cells with various r eagents or by mutagenesis of certain putative I kappa B alpha phosphor ylation sites, abolishes the inducible degradation of I kappa B alpha, Yet, the mechanism coupling the stimulation-induced phosphorylation w ith the degradation has not been resolved, Recent reports suggest a ro le for the proteasome in I kappa B alpha degradation, but the mode of substrate recognition and the involvement of ubiquitin conjugation as a targeting signal have not been addressed. We show that of the two fo rms of I kappa B alpha recovered from stimulated cells in a complex wi th RelA and p50, only the newly phosphorylated form, pI kappa B alpha, is a substrate for an in vitro reconstituted ubiquitin-proteasome sys tem. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjuga ting enzyme, and other ubiquitin-proteasome components. In vivo, induc ible I kappa B alpha degradation requires a functional ubiquitin-activ ating enzyme and is associated with the appearance of high molecular w eight adducts of I kappa B alpha, Ubiquitin-mediated protein degradati on may, therefore, constitute an integral step of a signal transductio n process.