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-[kappa]B follows the degradation of its inhibitor, l[kappa]B[alpha], an event coupled with stimulation-dependent inhibitor phosphorylation. Prevention of the stimulation-dependent phosphorylation of I[kappa]B[alpha], either by treating cells with various reagents or by mutagenesis of certain putative I[kappa]B[alpha] phosphorylation sites, abolishes the inducible degradation of I[kappa]B[alpha]. Yet, the mechanism coupling the stimulation-induced phosphorylation with the degradation has not been resolved. Recent reports suggest a role 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 forms of I[kappa]B[alpha] recovered from stimulated cells in a complex with RelA and p50, only the newly phosphorylated form, pI[kappa]B[alpha], is a substrate for an in vitro reconstituted ubiquitin-proteasome system. Proteolysis requires ATP, ubiquitin, a specific ubiquitin-conjugating enzyme, and other ubiquitin-proteasome components. In vivo, inducible I[kappa]B[alpha] degradation requires a functional ubiquitin-activating enzyme and is associated with the appearance of high molecular weight adducts of I[kappa]B[alpha]. Ubiquitin-mediated protein degradation may, therefore, constitute an integral step of a signal transduction process.