The NF-κB family of transcriptional regulators plays important roles in regulating a broad array of basic pathophysiological processes. Among them are the immune and inflammatory responses, development and differentiation, malignant transformation, and apoptosis (9
). The p50 and p52 subunits of NF-κB are generated from the inactive precursors p105 and p100, respectively, following limited, ubiquitin- and proteasome-mediated processing. In both cases the C-terminal domain of the molecule is destroyed while the remaining N-terminal part becomes the active subunit (7
). Typically, the processed subunits homodimerize or heterodimerize with other members of the rel family of regulators, such as p65 (RelA), RelB, or c-Rel to generate the active homo- or heterodimeric transcription factor. Binding of a member of the IκB family of inhibitors to the dimer generates an inactive trimeric complex that is sequestered in the cytosol. Following stimulation, specific IκB kinases (IKKs) are activated and phosphorylate IκB on specific Ser residues. Phosphorylation leads to recruitment of the SCFβ-TrCP
ubiquitin ligase complex, rapid polyubiquitination, and subsequent degradation of the inhibitor by the 26S proteasome. Consequently, active NF-κB can be translocated into the nucleus and initiates specific transcription (19
The ubiquitin pathway is involved, via specific degradation of short-lived regulatory proteins, in regulation of a broad array of cellular processes. Among these processes are cell cycle progression, differentiation and development, apoptosis, and the immune and inflammatory responses. Degradation of a protein via the ubiquitin system involves two successive steps: (i) formation of a polyubiquitin chain that is covalently anchored to the target substrate, and (ii) degradation of the tagged protein by the 26S proteasome. Conjugation involves activation of ubiquitin by the ubiquitin-activating enzyme, E1, followed by its transfer to a member of the ubiquitin-carrier protein (E2) family of enzymes. In most cases, E2 transfers the activated ubiquitin moiety to a
group of an internal lysine residue in the substrate that is specifically bound to an E3, a member of the ubiquitin-protein ligases family of proteins. Subsequent conjugation of additional activated ubiquitin molecules to previously attached moieties generates the polyubiquitin chain that serves as a degradation signal for the 26S proteasome.
The mechanisms involved in limited processing of p105 have been elucidated only partially. Lin and Ghosh have demonstrated that a glycine repeat (also termed the glycine-rich region [GRR]) that spans residues 376 to 404 in human p105 is essential for processing (23
) and probably serves as a processing stop signal for the 26S proteasome (30
). Several single residues that reside upstream of the GRR and are involved in proper folding of p50 are also essential for processing, most probably via inhibiting unfolding and entry into the proteasome (21
). These findings suggest that processing requires at least two motifs, a physical stop signal(s) and a ubiquitination-E3 recognition site. As for regulation of generation of p50, processing appears to proceed via two independent mechanisms: (i) basal-constitutive and (ii) signal induced. Fan and Maniatis (7
) have shown that a truncated form of p105, p60, can still be processed to p50. Lin and colleagues (22
) have shown that p105 can be processed cotranslationally, and synthesis of the complete molecule is not required for generation of p50. Because the signal-induced E3 recognition motif resides within the C-terminal domain of the molecule, these studies imply that all the motifs that are required for processing in the resting cell are contained within the first ~550 amino acid residues. Other studies have suggested a role for phosphorylation of the C-terminal domain of p105 in regulated, signal-induced processing of the molecule (10
), though here it appears that some of the p105 molecules are completely destroyed. Heissmeyer and colleagues (15
) have shown that IKK-mediated phosphorylation of Ser residues that reside in a sequence that spans amino acid residues 922 to 933 leads to rapid degradation of p105. It was recently shown that this IKK-mediated phosphorylation leads to recruitment of the SCFβ-TrCP
ubiquitin ligase. Consequently the molecule is ubiquitinated and rapidly processed, with a certain proportion being completely degraded (29
). Heissmeyer and colleagues later reported similar findings (14
). While the structural motives and ubiquitin system enzymes involved in basic-constitutive processing are not known, this process appears to require an additional adjacent downstream domain that contains lysine residues 440 and 441 (which are probably important for ubiquitination) and an acidic region (residues 445 to 453) that may function as an E3 recognition motif (30
Interestingly, processing of p100, the gene product of nfκb2
, to yield the p52 subunit is mediated by a similar mechanism. Like p105, part of it may occur cotranslationally and requires the GRR (18
). A recent study has demonstrated that phosphorylation of Ser residues 867 and 870 that is mediated by IKKα is required for processing (35
). It is interesting that the phosphorylation sites of IKK in p105 and p100 are similar to those of IκBα, β-catenin, and human immunodeficiency virus Vpu, where the two critical serines are interspaced by three residues.
The C-terminal segment of p105 that resides between the GRR and the IKK/TrCP motif contains seven ankyrin repeats. Active NF-κB subunits, such as p50 and p65, dock to this region and inhibit further processing of the precursor. This results in sequestration of the docked subunits as an inactive form in the cytosol. Thus, the ankyrin repeat domain serves as an inhibitor of NF-κB activity (13
). In agreement with these findings, Harhaj and colleagues have shown that constitutive processing of newly synthesized p105 molecules, to which p50 and p65 are not yet docked, is more efficient than that of older molecules that are already associated with p50 and p65 (12
). Accelerated, signal-induced processing or degradation of p105 leads to release of the docked active factors (5
) with their subsequent translocation to the nucleus.
However, as noted, IKKβ and β-TrCP expression also lead to degradation of a certain proportion of the p105 precursor molecules (5
), and it was important to identify the role of the kinase and ligase in regulating the two distinct processes. Here we demonstrate that IKKβ mediates both processing and degradation of p105, and the two functions require the C-terminal phosphorylation domain. In contrast, β-TrCP is involved only in degradation of the molecule. However, processing still requires an intact ubiquitin system and probably ubiquitin ligase E3.