A broad range of organisms and tissues contain 14-3-3 proteins, which have been associated with many diverse functions including critical roles in signal transduction pathways, exocytosis and cell cycle regulation. We report here the crystal structure of the human T-cell 14-3-3 isoform (tau) dimer at 2.6 A resolution. Each monomer (Mr 28K) is composed of an unusual arrangement of nine antiparallel alpha-helices organized as two structural domains. The dimer creates a large, negatively charged channel approximately 35 A broad, 35 A wide and 20 A deep. Overall, invariant residues line the interior of this channel whereas the more variable residues are distributed on the outer surface. At the base of this channel is a 16-residue segment of 14-3-3 which has been implicated in the binding of 14-3-3 to protein kinase C.

The p105 precursor protein of NF-B1 acts as an NF-B inhibitory protein, retaining associated Rel subunits in the cytoplasm of unstimulated cells. Tumor necrosis factor ␣ (TNF␣) and interleukin-1␣ (IL-1␣) stimulate p105 degradation, releasing associated Rel subunits to translocate into the nucleus. By using knockout embryonic fibroblasts, it was first established that the IB kinase (IKK) complex is essential for these pro-inflammatory cytokines to trigger efficiently p105 degradation. The p105 PEST domain contains a motif (AspSer 927 -Gly-Val-Glu-Thr), related to the IKK target sequence in IB␣, which is conserved between human, mouse, rat, and chicken p105. Analysis of a panel of human p105 mutants in which serine/threonine residues within and adjacent to this motif were individually changed to alanine established that only serine 927 is essential for p105 proteolysis triggered by IKK2 overexpression. This residue is also required for TNF␣ and IL-1␣ to stimulate p105 degradation. By using a specific anti-phosphopeptide antibody, it was confirmed that IKK2 overexpression induces serine 927 phosphorylation of co-transfected p105 and that endogenous p105 is also rapidly phosphorylated on this residue after TNF␣ or IL-1␣ stimulation. In vitro kinase assays with purified proteins demonstrated that both IKK1 and IKK2 can directly phosphorylate p105 on serine 927. Together these experiments indicate that the IKK complex regulates the signal-induced proteolysis of NF-B1 p105 by direct phosphorylation of serine 927 in its PEST domain.

NF-B1 p105 functions both as a precursor of NF-B1 p50 and as a cytoplasmic inhibitor of NF-B.Following the stimulation of cells with tumor necrosis factor alpha (TNF-␣), the IB kinase (IKK) complex rapidly phosphorylates NF-B1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-␣ to trigger p105 degradation, which releases the associated Rel/NF-B subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser 927 -Gly-ValGlu-Thr-Ser 932 ) homologous to the IKK target sequence in IB␣. In this study, TNF-␣-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1؊/؊ and IKK2 ؊/؊ double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-␣ to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105 758-967 fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for ␤TrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of ␤TrCP by RNA interference blocks TNF-␣-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that ␤TrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, ␤TrCP affinity for the IKKphosphorylated sequence on p105 is substantially lower than that on IB␣. Thus, it appears that reduced p105 recruitment of ␤TrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-␣ stimulation relative to that for IB␣.NF-B transcription factors play an important role in the regulation of genes involved in immune and inflammatory responses, apoptosis, and development (7, 11). Mammals express five NF-B proteins that bind DNA as homo-and heterodimers: RelA, RelB, c-Rel, NF-B1 p50, and NF-B2 p52 (22). NF-B1 and NF-B2 are synthesized as large precursors of 105 kDa (p105) and 100 kDa (p100), respectively, that require proteolytic processing by the proteasome to produce their respective p50 and p52 NF-B subunits (1,22).Mature NF-B dimers are retained in the cytoplasm of unstimulated cells through their interaction with a family of inhibitory proteins, termed IBs (7). A number of agonists activate NF-B, including proinflammatory cytokines, lipopolysaccharide, double-stranded RNA, and the viral transactivator Tax. These stimuli induce IB phosphorylation via the IB kinase (IKK) complex, which contains two related kinases, IKK1 (IKK␣) and IKK2 (IKK␤), and a structural subunit, NEMO (IKK␥). This triggers IB ubiquitination and subsequent proteolysis by the proteasome (11). NF-B dimers are thereby released to translocate into the nucleus, where their transcriptional activity is also regulated by phosphorylation (7).The NF-B1 precursor p105 functions as an IB due to the presence of ankyrin repeats in its C terminus and retains associated p50, c-Rel, and RelA in th...

NF-κB1 p105 forms a high-affinity, stoichiometric interaction with TPL-2, a MEK kinase essential for TLR4 activation of the ERK mitogen-activated protein kinase cascade in lipopolysaccharide (LPS)-stimulated macrophages. Interaction with p105 is required to maintain TPL-2 metabolic stability and also negatively regulates TPL-2 MEK kinase activity. Here, affinity purification identified A20-binding inhibitor of NF-κB 2 (ABIN-2) as a novel p105-associated protein. Cotransfection experiments demonstrated that ABIN-2 could interact with TPL-2 in addition to p105 but preferentially formed a ternary complex with both proteins. Consistently, in unstimulated bone marrow-derived macrophages (BMDMs), a substantial fraction of endogenous ABIN-2 was associated with both p105 and TPL-2. Although the majority of TPL-2 in these cells was complexed with ABIN-2, the pool of TPL-2 which could activate MEK after LPS stimulation was not, and LPS activation of TPL-2 was found to correlate with its release from ABIN-2. Depletion of ABIN-2 by RNA interference dramatically reduced steady-state levels of TPL-2 protein without affecting levels of TPL-2 mRNA or p105 protein. In addition, ABIN-2 increased the half-life of cotransfected TPL-2. Thus, optimal TPL-2 stability in vivo requires interaction with ABIN-2 as well as p105. Together, these data raise the possibility that ABIN-2 functions in the TLR4 signaling pathway which regulates TPL-2 activation.

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