NEMO is a sensor of Lys 63-linked polyubiquitination and functions in NF-κB activation

The transcription factor NF-kappa B is sequestered in the cytoplasm in a complex with I kappa B-1. Almost all NF-kappa B activation pathways converge on I kappa B kinase (IKK), which phosphorylates I kappa B resulting in Lys 48-linked polyubiquitination of I kappa B and its degradation. This allows migration of NF-kappa B to the nucleus where it regulates gene expression(2). IKK has two catalytic subunits, IKK alpha and IKK beta, and a regulatory subunit, IKK gamma or NEMO. NEMO is essential for NF-kappa 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)alpha- stimulated cells, the recruitment of IKK to TNF receptor (TNF-R) 1, and the activation of IKK and NF-kappa B. 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-kappa B activation.