The NF-κB protein p105 acts in a dual fashion as a cytoplasmic IκB molecule, able to associate with other NF-κB subunits, and as the precursor for the p50 subunit. We have shown previously that p105 is subject to signal-dependent proteolysis, which gives rise to induced release of p50 homodimers and is mediated by the IKK complex (13
). In this work we have investigated specificity-determining residues for the action of IKK and provide evidence for IKK-dependent ubiquitination and complete degradation of p105. By using purified recombinant IKKβ, we have shown first that IKK directly phosphorylates p105. This experiment is important, since it excludes the formal possibility that unknown kinases downstream of IKK could account for the observed p105 phosphorylation. Using this purified kinase, we have determined that serines 923 and 927 are the major substrate sites. Serines 923 and 927 are in the same spacing (SXXXS) as the phosphorylation sites in IκBα, -β, and -
and are both preceded by acidic amino acids (Fig. ). The p105 sequence in fact bears extended similarity with the sequences surrounding IKK sites in the small IκBs. Serine 927 is part of a conserved DSGΨ (where Ψ is a hydrophobic residue) motif. All small IκBs and p105 share at the −5 position of this serine an acidic amino acid and at −4 an acidic residue or serine. The −2 and +3 positions bear further IκB-specific preferences, cysteine and acidic residues, respectively. However, compared to the small IκBs, the phosphorylated residues in p105 (Fig. , underlined) are in nonequivalent positions, −4 and +1 versus +1 and +5 relative to the generally conserved central DSGψ motif (Fig. ). The sequence conservation suggests that IKK may utilize solely serines in the +4 spacing, preferentially when both are preceded by acidic residues. This, however, is not sufficient to create a bona fide IKK substrate site, since Vpu is not phosphorylated by IKK (D. Krappmann, not shown). Perhaps the −4 and −5 positions of the IκBs are specificity-determining residues.
FIG. 8 Phylogenetic conservation (h, human; m, murine; r, rat; ch, chicken; p, porcine) of destruction boxes in p105, in IκBα,-β, and -, in β-catenin, and in HIV-1 Vpu. Position numbers of the critical serine residues (more ...)
Functionally, the conserved serines differ in the various IκBs. Unlike the situation in IκBα, where single mutation of either serine 32 or 36 did not fully abrogate inducible phosphorylation (6
), mutation of S927 almost completely abrogated phosphorylation of p105. In IκB
, any mutations of serines in the conserved motif had little or no effect on phosphorylation (45
), although degradation was abolished by mutation of serines 18 and 22 to alanine (52
). Perhaps the relatively high number of serines close to the conserved substrate site in IκB
provide alternative phosphorylation sites for the IKK complex in the mutant proteins.
Our analysis of physical interaction of IKKβ with p105 revealed that the kinase binds to a region that is nonoverlapping with the destruction box and that mutation or deletion of the substrate serines does not affect interaction strength. The docking site was delineated to the N-terminal half of a death domain and is separated by 70 amino acids from the destruction box (Fig. A). The interaction of IKK with a docking site may contribute to substrate recognition in addition to specificity-determining residues flanking the substrate serines.
The death domain confers homo- and heterotypic protein interactions and is mostly found in receptors or adaptors that signal cell death. It is also conserved in signaling molecules which are not associated with apoptotic pathways but regulate NF-κB activation, such as Drosophila
Pelle and Tube, which are part of the Toll-Dorsal pathway (9
). The fact that the death domain is phylogenetically conserved in p105 and in p100 (reference 44
and data not shown) indicates an essential function for these molecules. The physical IKK-death domain interaction may indicate that IKKs also interact with death domains of other molecules and that this interaction could be relevant for recruitment of IKKs to activated receptor-adapter complexes. Similarly, the death domain could engage the precursors into heterotypic complexes with other signaling molecules.
We have also analyzed the physical interaction of IKKs with IκBα and IκBβ, which do not contain a death domain. Compared to the robust IKK-p105 interaction, IκBα was only weakly bound by IKKβ (V. Heissmeyer, unpublished data). However, human IκBβ revealed a stronger interaction with IKKβ, which was conferred by a C-terminal PEST sequence shared by the IκBβ1 and IκBβ2 splicing isoforms (V. Heissmeyer, unpublished data).
In contrast to p105, p100 is not phosphorylated by IKKs (13
), consistent with the fact that the carboxy-terminal amino acids of p100, downstream of the death domain, show no conservation with p105. Accordingly, p100 was not degraded upon coexpression with IKKβ and βTrCP (Fig. B and A). Thus, p100 is the only cytoplasmic IκB protein not directly phosphorylated by IKKs. However, IKKβ binds to p100 (V. Heissmeyer, data not shown). It is thus possible that IKK, once bound to the death domain of p100, activates a further, unknown kinase to phosphorylate p100.
The overall similarity of the sequence context of IKK phosphorylation sites in p105 and IκBα suggested that p105 should interact with the same type of ubiquitin ligase as IκBα. In fact, the interaction efficiency of βTrCP1 with IκBα and p105 was virtually identical. Furthermore, when comparing the related F-box proteins βTrCP1 and βTrCP2, which bind equally well to phosphorylated IκBα (47
), both also interact with phosphorylated p105 with comparable efficiency. The interaction with βTrCP2 was lost completely when the major IKK sites were mutated (p105AAA), indicating that the phosphorylated minor sites in p105 (between residues 850 and 891) cannot attract the F-box protein. The binding of both βTrCPs again underscores the similarity of the destruction boxes in p105 and the small IκBs and discriminates these proteins from β-catenin, which, upon GSK3β phosphorylation, can attract βTrCP1 but not βTrCP2 (11
). Our data also reveal that the last residue in the DSGΨXS consensus sequence for βTrCP recognition is not maintained for p105, which contains a threonine, a very poor IKK substrate. This is intriguing, since the DSGΨXS motif is strictly conserved in all other proven and potential βTrCP substrates (Fig. ), including armadillo and plakoglobin (not shown). The last serine in the motif is functionally important in IκBα, since single mutation of this residue (serine 36) completely abolishes induced degradation (4
). Yaron et al. (58
) have shown that short IκBα competitor peptides with singly phosphorylated serine 36 or 32 have strongly impaired inhibitory effects on IκBα ubiquitination compared to their doubly phosphorylated counterparts. It is therefore possible that βTrCPs recognize the phosphorylated signal sequences in IκBα and p105 in a slightly different manner.
We have shown that coexpression of IKKβ and βTrCP1 triggers p105 polyubiquitination which results in complete proteasomal degradation but not in enhanced processing of p105 (Fig. and ). This result is in contrast to the conclusions drawn by Orian et al. (38
), who reported that IKK predominantly enhanced processing. We demonstrated that the expression of IKKβ alone led to an increase in p50, but this effect is ascribed to IKKβ-induced expression of p105, resulting in increased amounts of p50 produced by processing and loss of p105 by simultaneous IKKβ-induced degradation. This conclusion is also supported by the observation that IKKβ, which does not phosphorylate p100, enhances p52 production along with p100 expression. Likewise, Cot, a kinase which does not phosphorylate p105, enhanced production of p50 and p52 as well as of p105 and p100, most likely by acting on the expression vector. Importantly, by the use of cycloheximide, we have shown that at the posttranslational level, and thus independent of any effects of the kinase on the expression vector, IKKβ (βTrCP) triggered complete degradation but not processing of p105. The observed degradation was fully dependent on serine 927, in agreement with the pivotal role of this residue as an IKK phosphoacceptor site. We also showed that in mouse pre-B cells, LPS triggered degradation but not processing of endogenous p105. LPS-induced degradation but not basal processing required a functional endogenous IKK complex.
Our data fully support the notion that p105 contains a carboxy-terminal destruction box that, like the N-terminal domain in IκBα, upon IKK phosphorylation, is recognized by an SCFβTrCP E3 ubiquitin ligase which mediates polyubiquitination and complete degradation by the proteasome. Thus, p105 is degraded by the same mechanism as IκBα. It is also interesting to note that both proteins can obviously be degraded when complexed with Rel factors (p50-p65 and the processing product of p105 or other p105-associated Rel factors, respectively).
The basal processing reaction, in contrast, has been shown to require a glycine-rich region (residues 372 to 394) and an acidic domain (residues 446 to 454) (27
), both located at the end of the first half of the precursor. That the basal processing reaction does not require carboxy-terminal sequences containing the destruction box described here is also supported by the fact that deletion or mutation of the IKK phosphorylation sites in p105 does not affect basal processing and that processing was not reduced in cells lacking a functional IKK complex.
IKK-regulated and ubiquitin-mediated p105 degradation is an important bifurcation in NF-κB signaling downstream of the IKK complex. This bifurcation provides a means to regulate p50 homodimers, which may act as inhibitors to limit transcriptional responses of p50-p65 or as activators, depending on the availability of Bcl-3. To dissect the regulation of p50 and Bcl-3 is important for understanding the function of these molecules in the immune response and in oncogenesis.