Shared Pathways of IκB Kinase-Induced SCF^βTrCP-Mediated Ubiquitination and Degradation for the NF-κB Precursor p105 and IκBα

Abstract

p105 (NFKB1) acts in a dual way as a cytoplasmic IB molecule and as the source of the NF-B p50 subunit upon processing. p105 can form various heterodimers with other NF-B subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKK␤ phosphorylation of p105 is direct and does not require kinases downstream of IKK. p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. The substrate residues were identified as serines 923 and 927, the latter of which was previously assumed to be a threonine. S927 is part of a conserved DSG⌿ motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IB␣, -␤, and -. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules ␤TrCP1 and ␤TrCP2, resulting in polyubiquitination and complete degradation by the proteasome. However, processing of p105 is independent of IKK signaling. In line with this and as a physiologically relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105 and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B cells lacking IKK␥, processing was unaffected, but LPS-induced p105 degradation was abolished. Thus, a functional endogenous IKK complex is required for signal-induced p105 degradation but not for processing.The NF-B transcription factor family plays an evolutionarily conserved role in innate and adaptive immune responses, and its members are essential regulators of proinflammatory processes (12,18,33). NF-B is also an important regulator in cell fate decisions, such as programmed cell death and proliferation control, and is critical in tumorigenesis (32, 42). The vertebrate NF-B/Rel factors p50, p52, p65 (RelA), c-Rel, and RelB form various dimers and are under the control of a coevolved family of cytoplasmic IB molecules, IB␣, -␤, and -ε, and the nuclear IB homologue Bcl-3 (1). p50 and p52 are formed by processing from the precursor molecules p105 and p100, respectively. Prototypic heterodimeric NF-B p50-p65 is rapidly released from cytoplasmic complexes with IBs upon cellular stimulation with diverse agents, including tumor necrosis factor alpha (TNF-␣), interleukin-1␤, bacterial lipopolysaccharides (LPS), and phorbol myristate acetate, following viral infection or exposure to ␥-irradiation (31). All these agents induce an IB kinase (IKK) complex to phosphorylate IB␣, -␤, and -ε. The IKK complex contains the kinases IKK␣ and IKK␤ and the noncatalytic IKK␥ subunit, which is essential for signal responsiveness of the complex (see reference 17 for a review). Once phosphorylated by the IKK complex, the IBs are ubiquitinated and degraded by the 26S proteasome, resulting in nuclear translocation of NF-B. The ubiquitin ligase complex specif...

Regulation and function of TPL-2, an IκB kinase-regulated MAP kinase kinase kinase

“…These phosphorylations induce the binding of SCF βTrCP E3 ligase, which catalyzes the subsequent K48-linked polyubiqitination and proteolysis of p105 by the proteasome [42,44,45]. This predominantly leads to the complete degradation of p105, rather than processing to p50 [42,44]. As a consequence of signal-induced p105 degradation, associated Rel subunits are released to regu- Figure 3 Canonical pathway of NF-κB activation.…”

Effects of dietary protein levels on the disease resistance, immune function and physical barrier function in the gill of grass carp ( Ctenopharyngodon idella ) after challenged with Flavobacterium columnare

“…Correlation analysis indicated that the mRNA levels of pro-inflammatory cytokines TNF-a, IL-1b, IL-8, IFN-g2 and IL-12p40 were positively correlated with the mRNA levels of NF-kB P65 and NF-kB P52 in the gills of grass carp ( Table 5). The nuclear translocation of NF-kB may be inhibited by IkBa in humans [69], and Heissmeyer et al [70] reported that the IKK complex (including IKKa, IKKb and IKKg) catalyses IkBa degradation in 293 cells. In the present study, compared with low or high levels of protein, we found that the optimal levels of dietary protein led to the down-regulation of IKKa, IKKb and IKKg mRNA levels and the up-regulation of IkBa mRNA level in the gills of grass carp.…”

A p105-based Inhibitor Broadly Represses NF-κB Activities

“…However, wild-type p105 cannot be employed as an NF-B super repressor, because expression of the nfkb1 gene always produces both p105 and p50 in the cell, and the latter is a component of the NF-B transcription complex (23,24). In addition, wild-type p105, like I B␣, also undergoes signal-induced complete degradation, releasing the bound NF-B species into the nucleus (25). Compared with I 〉␣, however, p105 possesses important features that make it potentially a broader range NF-B super repressor.…”

“…First, p105 interacts with all Rel proteins, including p50, p52, and RelB, with high affinity through the Rel homology domain (RHD) and I B␥-like ankyrin repeats. 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.…”