Structural motifs involved in ubiquitin-mediated processing of the NF-kappa B precursor p105: Roles of the glycine-rich region and a downstream ubiquitination domain

The ubiquitin proteolytic system plays a major role in a variety of basic c ellular processes. In the majority of these processes, the target proteins are completely! degraded. In one exceptional case, generation of the p50 su bunit of the transcriptional regulator NF-kappa B, the precursor protein p1 05 is processed in a limited manner: the N-terminal domain yields the p50 s ubunit, whereas the C-terminal domain is degraded. The identity of the mech anisms involved in this unique process have remained elusive. It has been s hown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an imp ortant processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the proc essing of the ubiquitin-tagged precursor by the 26S proteasome, Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the pr esence of the GRR appears to stop further degradation of p50 and to stabili ze the molecule. It appears that the localization of the GRR within p105 pl ays an important role in directing processing: transfer of the GRR within p 105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to t he requirement fur an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino add residues 441 to 454 are im portant for processing. In particular, both Lys 441 and Lys 442 appear to s erve as major ubiquitination targets, while residues 446 to 454 are indepen dently important for processing and may serve as the ubiquitin ligase recog nition motif.