Understanding NEMO
The connection between tumor-necrosis-factor receptor-1 (TNFR1) signaling, I7 B kinase (IKK)and NF-B activation, as well as the biochemical mechanism underling NEMO's signaling function in IKK activation, have now been revealed.
IB kinase (IKK), a central activator of the transcription factor nuclear factor (NF)-B, is composed of two catalytic subunits and a regulatory subunit — known as NEMO — that is essential for NF-B activation. Tumour-necrosis-factor receptor-1 (TNFR1) activation by TNF results in the recruitment of receptor-interacting protein-1 (RIP1) to TNFR1. RIP1 is polyubiquitylated and the subsequent recruitment of NEMO results in the activation of IKK, which functions to release IB from NF-B and induce NF-B to activate gene expression.
Despite the important role of IKK in NF-B activation, exactly how TNFR1 signalling results in the NEMO-dependent activation of NF-B is unclear. Furthermore, NEMO is essential for IKK activation, but how it regulates IKK is unknown. However, two papers now provide insights — one by Ashwell and colleagues in Nature Cell Biology and another by Chen and co-workers in Molecular Cell.
The Ashwell group sought to identify novel NEMO-interacting partners. They showed that NEMO binds specifically to Lys63-linked polyubiquitin chains, which have been implicated in intracellular signalling, rather than to Lys48-linked chains, which are involved in proteasomal degradation. This interaction was shown to be mediated by the coiled-coil-2 (CC2) and leucine zipper (LZ) motifs of NEMO and is a novel ubiquitin-binding interaction.
Next, these authors studied the role of NEMO binding to Lys63-linked polyubiquitin chains in the TNF-induced activation of IKK. Using NEMO-deficient cells, they showed that IKK activation could be restored by wild-type NEMO, but not by NEMO mutants that could not bind to Lys63-linked polyubiquitin chains. Furthermore, they showed that the in vivo target of NEMO is Lys63-linked polyubiquitylated RIP1.
In NEMO-deficient cells, RIP1 binding to TNFR1 and RIP1 polyubiquitylation still occurred, and the expression of a polyubiquitin-binding-deficient NEMO mutant could not rescue the recruitment of IKK to the TNFR1–RIP1 signalling complex. The Ashwell group therefore concluded that NEMO binding to Lys63-linked polyubiquitylated signalling intermediates such as RIP1 is essential to propagate signals from activated TNFR1 to IKK and NF-B.
The Chen group analysed the process of RIP1 polyubiquitylation and found that TNF stimulation induced the specific attachment of a Lys63-linked polyubiquitin chain to Lys377 of RIP1. They showed that this polyubiquitylation process was required for the activation of IKK and NF-B in response to TNF stimulation; a Lys377Arg RIP1 mutant could not rescue IKK activation in RIP1-deficient cells.
Chen and co-workers then showed that RIP1 polyubiquitylation was required to recruit NEMO to TNFR1, which, in turn, recruited IKK. They therefore analysed the ubiquitin-binding properties of NEMO, and found that it binds preferentially to Lys63-linked chains. They also mapped the ubiquitin-binding site on NEMO to its CC2 and LZ motifs, and showed that mutations that disrupt this site abolish IKK activation.
So, these two important papers have revealed the connection between TNFR1 signalling and IKK and NF-B activation, as well as the biochemical mechanism that underlies the essential signalling function of NEMO in IKK activation.
Rachel Smallridge
References
- Wu, C.-J. et al. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-B activation. Nature Cell Biol. 8, 398–406 (2006) | Article |
- Ea, C.-K. et al. Activation of IKK by TNF requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006) | Article | PubMed |
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