Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-B activation

Chuan-Jin Wu, Dietrich B. Conze, Tao Li, Srinivasa M. Srinivasula & Jonathan D. Ashwell

Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

NF-BIKKIKKIKKNEMORIPTNF-TNF-R1Ubc13UbiquitinThe transcription factor NF-B is sequestered in the cytoplasm in a complex with IB¹. Almost all NF-B activation pathways converge on IB kinase (IKK), which phosphorylates IB resulting in Lys 48-linked polyubiquitination of IB and its degradation. This allows migration of NF-B to the nucleus where it regulates gene expression². IKK has two catalytic subunits, IKK and IKK, and a regulatory subunit, IKK or NEMO. NEMO is essential for NF-B activation, and NEMO dysfunction in humans is the cause of incontinentia pigmenti and hypohidrotic ectodermal dysplasia and immunodeficiency (HED–ID)³. 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)--stimulated cells, the recruitment of IKK to TNF receptor (TNF-R) 1, and the activation of IKK and NF-B. RIP is also destabilized in the absence of NEMO binding and undergoes proteasomal degradation in TNF--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-B activation.

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