Transforming growth factor ␤-activated kinase 1 (TAK1), a member of the MAPKKK family, was initially described to play an essential role in the transforming growth factor ␤-signaling pathway, but recent evidence has emerged implicating TAK1 in the interleukin (IL)-1 and tumor necrosis factor (TNF) pathways. Notably, two homologous proteins, TAB2 and TAB3, have been identified as adaptors linking TAK1 to the upstream adaptors TRAFs. However, it remains unclear whether the interaction between TAB2/TAB3 and TAK1 is necessary for its kinase activation and subsequent activation of the IKK and MAPK pathways. Here, we characterized the TAB2/TAB3-binding domain in TAK1 and further examined the requirement of this interaction for IL-1, TNF, and RANKL signaling. Through deletion mapping experiments, we demonstrated that the binding motif for TAB2/TAB3 is a non-contiguous region located within the last C-terminal 100 residues of TAK1. However, residues 479 -553 of TAK1 appear to be necessary and sufficient for TAB2/TAB3 interaction. Conversely, residues 574 -693 of TAB2 were shown to interact with TAK1. A green fluorescent protein fusion protein containing the last 100 residues of TAK1 (TAK1-C100) abolished the interaction of endogenous TAB2/TAB3 with TAK1, the phosphorylation of TAK1, and prevented the activation of IKK and MAPK induced by IL-1, TNF, and RANKL. Furthermore, TAK1-C100 blocked RANKL-induced nuclear accumulation of NFATc1 and consequently osteoclast differentiation consistent with the ability of a catalytically inactive TAK1 to block RANKL-mediated signaling. Significantly, our study provides evidence that the TAB2/TAB3 interaction with TAK1 is crucial for the activation of signaling cascades mediated by IL-1, TNF, and RANKL.

The Ser/Thr-specific IκB kinase (IKK), which comprises IKKα or IKKβ and the regulatory protein NEMO, is at the bottleneck for NF-κB activation. IKK activity relies on interaction between NEMO and IKKα or IKKβ. A conserved region in the C-terminal tail of IKKβ or IKKα (NEMO-binding domain, NBD, residues 734-745 of IKKβ) is important for interaction with NEMO. Here we show that the NBD peptide of IKKβ is not sufficient for interaction with NEMO. Instead, a longer region of the IKKβ C-terminal region provides high affinity for NEMO. Quantitative measurements using surface plasmon resonance and isothermal titration calorimetry confirm the differential affinities of these interactions and provide insight into the kinetic and thermodynamic behaviors of the interactions. Biochemical characterization using multiangle light scattering (MALS) coupled with refractive index shows that the longer IKKβ C-terminal region forms a 2:2 stoichiometirc complex with NEMO.NF-κB proteins (NF-κBs) are evolutionarily conserved master regulators of immune and inflammatory responses (1, 2). They play critical roles in a wide array of biological processes, including innate and adaptive immunity, oncogenesis, and development. They are activated in response to ligation of many receptors, including T-cell receptors, B-cell receptors, members of the tumor necrosis factor (TNF) receptor superfamily, and the Tolllike receptor/interleukin-1 receptor (TLR/IL-1R) superfamily.NF-κBs share a highly conserved DNA-binding/dimerization domain called the Rel homology domain (RHD) (2, 3). The mammalian NF-κB family consists of p65 (RelA), RelB, c-Rel, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). While p65, RelB, and c-Rel contain C-terminal transactivation domains, p105 and p100 contain long C-terminal domains that contain multiple ankyrin repeats and act to inhibit these proteins. The activity of NF-κB family members p65, RelB, and c-Rel is tightly regulated by interaction with the inhibitor of κB (IκB) proteins, which also contain ankyrin repeats, like the C-terminal domain of p105 and p100. Thus, in most cells, NF-κBs are held captive in the cytoplasm from translocating to the nucleus by the IκB proteins or IκB-like domains. The Ser/Thr-specific IκB kinase (IKK) is at the bottleneck for NF-κB activation (4). Activated IKK phosphorylates NF-κB-bound IκBs and the IκB-like domains of p100 and p105. This leads to Lys48-linked polyubiquitination and subsequent degradation of IκBs and processing of p100 and p105, respectively, by the proteasome. The freed or processed NF-κB dimers translocate to the nucleus to mediate specific target gene transcription. Recent studies have revealed that IKK activation by cytokines such as TNF and IL-1 and by Tolllike receptors is dependent on Lys63-linked nondegradative polyubiquitination (5). Using biochemical purification and in vitro reconstitution, it was shown that together with a ubiquitin activating enzyme (E1) and a specific dimeric ubiquitin conjugating enzyme Ubc13-Uev1A complex (E2), the RING domain containin...

Cyclin-dependent kinase 5 (Cdk5) is activated on binding of activator proteins p35 and p39. A N-terminally truncated p35, termed p25, is generated through cleavage by the Ca(2+)-dependent protease calpain after induction of ischemia in rat brain. p25 has been shown to accumulate in brains of patients with Alzheimer's disease and may contribute to A-beta peptide-mediated toxicity. Studies from transfected neurons as well as p35 and p25 transgenic mice have indicated that Cdk5, when activated by p25, gains some toxic function compared with p35/Cdk5. It remains unclear, however, whether p25/Cdk5 signaling additionally channels into pathways usually used by p35/Cdk5 and whether p25 is associated with a loss of p35 function. To clarify these issues, we have generated p25-transgenic mice in a p35-null background. We find that low levels of p25 during development induce a partial rescue of the p35-/- phenotype in several brain regions analyzed, including a rescue of cell positioning of a subset of neurons in the neocortex. In accordance with the partial rescue of brain anatomy, phosphorylation of the Cdk5 substrate mouse disabled 1 is partially restored during development. Besides this, p25/Cdk5 fails to phosphorylate other substrates that are normally phosphorylated by p35/Cdk5. Our results show that p25 can substitute for p35/Cdk5 under certain circumstances during development. In addition, they suggest that p25 may have lost some functions of p35.