Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain

Abstract

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-B, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing 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 presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.The NF-B proteins are a group of dimeric ubiquitous eukaryotic transcription factors belonging to the Rel family. They play key roles in basic processes such as regulation of the immune and inflammatory responses, development, differentiation, malignant transformation, and apoptosis (3,4,17). All members of the Rel family contain a Rel homology domain within the N-terminal domain of the protein, while some members, like p105, p100, and Relish, contain ankyrin repeats at the C-terminal domain. The precursor molecules p105 and probably p100 undergo ubiquitin-and proteasome-mediated limited proteolytic processing to yield the corresponding active subunits p50 and p52 (32, 33). These subunits are derived from the N-terminal domain of the molecule. The C-terminal domain is degraded (6, 14). These subunits typically heterodimerize with members of the Rel family that do not contain ankyrin repeats such as p65, RelB, and c-Rel. In the resting cell, the heterodimer generates a ternary complex with a member of the I B family of inhibitory proteins. I B binding sterically hinders a nuclear localization site, and consequently, the complex is retained in the cytosol (20,24). Following cellular stimulation by a wide array of activators, such as cytokines (tumor necrosis factor alpha and interleukin 1, for example), viral and ba...

Ceramide triggers an NF-κB-dependent survival pathway through calpain

“…It is therefore tempting to speculate that this region is involved not only in proteasome-mediated, 54,55 but also in calpain-mediated degradation. Interestingly, previous studies demonstrated that p105 could be phosphorylated in vitro by casein kinase II (CK2).…”

“…Interestingly, previous studies demonstrated that p105 could be phosphorylated in vitro by casein kinase II (CK2). 55 CK2 phosphorylation was recently shown to prime i-kBa for calpain-mediated degradation. 56 In addition, CK2 is a C-terminal i-kB kinase responsible for NFkB activation during UV response 57 and promotes aberrant activation of NF-kB, transformation and survival of breast cancer cells.…”

Glycogen Synthase Kinase-3β Regulates NF-κB1/p105 Stability

“…Three structural motives are known to be involved in the processing of the NF-B precursor p105. Namely a ubiquitination domain between amino acids 441 and 450, an upstream glycine-rich region (amino acids 376 -404) required to stop further degradation of p50 and to stabilize the molecule (6), and a death domain located at the carboxyl terminus of the protein (7). Stimulation of cells with tumor necrosis factor-␣ (TNFϪ␣) triggers phosphorylation of serine 927 within the p105 PEST region by I B kinase (IKK) (8,9).…”

A p105-based Inhibitor Broadly Represses NF-κB Activities

“…Second, although both require the proteasome activity, p50 biogenesis and p105 degradation are separate processes (23,(25)(26)(27)(28). The middle region of p105, which contains a glycine-rich region (29) and a putative degradation signal for the proteasome (30), is likely to be responsible for p50 generation. The C terminus of p105, which harbors the consensus sequences for the I B kinases, the ubiquitin ligase ␤-transducin repeat-containing protein, and a death domain (DD), is required for signal-induced p105 degradation (26 -28).…”