NF-κB is a dimeric transcription factor consisting of Rel family members, which include Rel-A (also known as p65), c-Rel, Rel-B, p50 and p5214
. p50 and p52 are derived from the larger precursors p105 and p100, respectively, through proteolytic processing by the proteasome. All NF-κB proteins contain a highly conserved Rel-homology domain (RHD) that is responsible for DNA binding, dimerization, nuclear translocation and interaction with the IκB proteins. The IκB proteins, including IκBα, β and
, bind to NF-κB via ankyrin repeats and block its nuclear import and thereby, its transcriptional activity. The C-termini of p105 and p100 also contain the IκB-like ankyrin repeats that must be degraded in order to generate the mature Rel subunits.
The NF-κB activation pathways are broadly classified as the canonical and non-canonical pathways, depending on whether activation involves IκB degradation or p100 processing ()15
. In the canonical pathway, which is the predominant NF-κB signaling pathway, stimulating cells with an agonist such as tumor necrosis factor α (TNFα) or interleukin-1β (IL-1β) activates the IKK complex that is composed of two catalytic subunits IKKα and IKKβ and a regulatory subunit NEMO (also known as IKKγ). Genetic experiments have demonstrated that IKKβ, but not IKKα, phosphorylates IκB proteins at two N-terminal serine residues. This signal-induced phosphorylation targets IκB for polyubiquitination and subsequent degradation by the proteasome7
, thus releasing NF-κB. The non-canonical pathway of NF-κB activation operates mainly in B cells in response to stimulation of a subset of the TNF receptor superfamily, including receptors for BAFF, lymphotoxin-β (LTβ) and CD40 ligand. Stimulation of these receptors activates the protein kinase NIK, which in turn activates IKKα. IKKα then phosphorylates p100 at two C-terminal serine residues leading to the selective degradation of its IκB -like domain by the proteasome16,17
. The mature p52 subunit and its binding partner Rel-B translocate into the nucleus to regulate gene expression.
The ubiquitin-proteasome pathway plays a crucial role in both the canonical and non-canonical pathways of NF-κB activation. Ubiquitination is a reversible covalent modification catalyzed by three enzymatic steps18
. In the first step, ubiquitin is activated by a ubiquitin-activating enzyme (E1) in an ATP-dependent reaction. In the second step, the activated ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2 or Ubc), forming an E2-Ub thioester. Finally, in the presence of a ubiquitin-protein ligase (E3), ubiquitin is attached to a target protein through an isopeptide bond between the carboxyl terminus of ubiquitin and the
-amino group of a lysine residue in the target protein. Ubiquitin contains seven lysines, which can be attached to another ubiquitin in a highly processive reaction to form a polyubiquitin chain. Typically, a polyubiquitin chain that targets a protein for degradation by the proteasome is linked through lysine-48 (K48) of ubiquitin19
. However, ubiquitin chains linked through other lysines of ubiquitin have also been found in cells20
. In particular, K63-linked polyubiquitin chains have recently been found to regulate DNA repair and protein kinase activation through a degradation-independent mechanism (see below).
Ubiquitination of IκB is carried out by an E2 of the Ubc4/5 family7,9,21
and the SCF-βTrCP E3 ligase (Skp1-Cul1-F-box ligase containing the F-box protein βTrCP)22–26
. Two βTrCP proteins, βTrCP1 and βTrCP2, have been found in mammalian cells. Genetic deletion of βTrCP1 in mice leads to only modest retardation of IκBα degradation, suggesting that βTrCP1 and βTrCP2 may have redundant functions27
. Indeed, silencing of both βTrCP1 and βTrCP2 expression by RNAi block IκBα degradation. βTrCP1 and βTrCP2 bind specifically to the phosphorylated form of IκB through their C-terminal WD40 repeats, and to the rest of the SCF complex through the F-box. The SCF complex contains the RING domain protein Roc1/Rbx1, which binds to Ubc4/5, allowing this E2 to ubiquitinate IκB at two conserved N-terminal lysine residues. The polyubiquitinated IκB remains associated with NF-κB, but is selectively degraded by the 26S proteasome, while NF-κB itself is spared7
The ubiquitin-proteasome pathway is also responsible for the processing of p105 and p100 to p50 and p52, respectively. p105 can be processed either co-translationally or post-translationally, both requiring the proteasome6,28
. Co-translational processing of p105 appears to be a constitutive process that does not require phosphorylation or ubiquitination and is likely the major source of p50, which is constitutively present in unstimulated cells. Some agents such as phorbol ester (PMA) and lipopolysaccharides (LPS) can stimulate post-translational processing of p105 by activating IKK, which phosphorylates p105 at a C-terminal domain29
. On the other hand, the processing of p100 is tightly regulated by the non-canonical pathway of NF-κB activation and depends on both phosphorylation and ubiquitination. As discussed above, stimulation of certain receptors of the TNFR family on B cells activates IKKα, which phosphorylates p100 at two C-terminal serine residues (). This phosphorylation relieves the inhibition of p100 processing by a C-terminal death domain, and recruits the SCF-βTrCP ligase to polyubiquitinate p100 at a specific lysine 16,17,30,31
. Subsequently, the C-terminal IκB-like domain is selectively degraded by the proteasome, generating the mature p52 subunit.
How does the proteasome partially degrade a protein? More specifically, what defines where degradation starts and stops? A plausible model for regulated ubiquitin/proteasome-dependent processing has recently been proposed 32
. According to this model (), the ubiquitin tag on a substrate recruits the proteasome to an internal unstructured sequence such as the glycine rich region (GRR) of p105 and p10033
. It is postulated that this region forms a hairpin-like loop that inserts into the proteolytic chamber of the 20S proteasome. Proteolysis begins at the loop and then proceeds in both the N- and C-terminal directions of the target protein. In most cases, the N- and C-terminal domains can be completely unfolded by the 19S subcomplex of the proteasome and threaded into the proteolytic chamber so that the target is completely degraded. In the cases of substrates such as p105 and p100, the RHD domain is a tightly folded structure that may be difficult to unfold. Thus, after initiating proteolysis from the putative GRR loop of p105 or p100, the proteasome chews along the C-terminal polypeptide until it reaches the end, but comes to a halt at the N-terminus when it encounters the tightly folded RHD structure.
A model for the processing of p100 by the proteasome