The transcription factor NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. Almost all NF-kappaB activation pathways converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in Lys 48-linked polyubiquitination of IkappaB and its degradation. This allows migration of NF-kappaB to the nucleus where it regulates gene expression. IKK has two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO dysfunction in humans is the cause of incontinentia pigmenti and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The recruitment of IKK to occupied cytokine receptors, and its subsequent activation, are dependent on the attachment of Lys 63-linked polyubiquitin chains to signalling intermediates such as receptor-interacting protein (RIP). Here, we show that NEMO binds to Lys 63- but not Lys 48-linked polyubiquitin, and that single point mutations in NEMO that prevent binding to Lys 63-linked polyubiquitin also abrogates the binding of NEMO to RIP in tumour necrosis factor (TNF)-alpha-stimulated cells, the recruitment of IKK to TNF receptor (TNF-R) 1, and the activation of IKK and NF-kappaB. RIP is also destabilized in the absence of NEMO binding and undergoes proteasomal degradation in TNF-alpha-treated cells. These results provide a mechanism for NEMO's critical role in IKK activation, and a key to understanding the link between cytokine-receptor proximal signalling and IKK and NF-kappaB activation.

Precursor CD4+ T cells develop into effector Th1 and Th2 cells that play a central role in the immune response. We show that the JNK MAP kinase pathway is induced in Th1 but not in Th2 effector cells upon antigen stimulation. Further, the differentiation of precursor CD4+ T cells into effector Th1 but not Th2 cells is impaired in JNK2-deficient mice. The inability of IL-12 to differentiate JNK2-deficient CD4+ T cells fully into effector Th1 cells is caused by a defect in IFNgamma production during the early stages of differentiation. The addition of exogenous IFNgamma during differentiation restores IL-12-mediated Th1 polarization in the JNK2-deficient mice. The JNK MAP kinase signaling pathway, therefore, plays an important role in the balance of Th1 and Th2 immune responses.

SUMMARY NEMO is the regulatory subunit of the IκB kinase (IKK) in NF-κB activation and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch while the distal ubiquitin mostly employs the C-terminal arm including the K63-linkage residue. These studies uncover the energetics and the common U-shaped geometry for mutual recognition of NEMO and diubiquitins.