Activation of IKK by TNFα Requires Site-Specific Ubiquitination of RIP1 and Polyubiquitin Binding by NEMO

Abstract

The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kappaB (NF-kappaB) by tumor necrosis factor alpha (TNFalpha). Here, we present evidence that TNFalpha induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkappaB kinase (IKK) and NF-kappaB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation.

Ubiquitylated Tax targets and binds the IKK signalosome at the centrosome

“…K63-poly-ubiquitylation of TRAF6 mediates IKK activation by interleukin-1 (Deng et al, 2000). Moreover, the recruitment of IKK-g to occupy TNF-a receptor, and IKK activation, are both dependent on K63-polyubiquitylation of the signaling intermediate RIP1 (Ea et al, 2006;Wu et al, 2006). A lysine to arginine point mutation of RIP1 abolished its poly-ubiquitylation and the recruitment of the IKK complex to the TNF receptor (Ea et al, 2006).…”

“…Moreover, the recruitment of IKK-g to occupy TNF-a receptor, and IKK activation, are both dependent on K63-polyubiquitylation of the signaling intermediate RIP1 (Ea et al, 2006;Wu et al, 2006). A lysine to arginine point mutation of RIP1 abolished its poly-ubiquitylation and the recruitment of the IKK complex to the TNF receptor (Ea et al, 2006). This situation is similar to the K4-8R Tax mutation, which abolishes Tax poly-ubiquitylation, IKK activation (Nasr et al, 2006) and IKK recruitment to the centrosome (this manuscript).…”

“…TNF-a-dependent IKK activation is mediated by K63-linked poly-ubiquitylation of RIP1, whereas K48-linked poly-ubiquitylation of RIP1 mediates degradation by the proteasome (Ea et al, 2006;Wu et al, 2006). To investigate whether Tax is modified by distinct poly-ubiquitylation, we used ubiquitin mutants harboring lysine to arginine mutation on all lysine residues of ubiquitin (Ub-K0), or on all lysine residues except lysine 48 (Ub-K48) or lysine 63 (Ub-K63).…”

Ubiquitin binding mediates the NF-κB inhibitory potential of ABIN proteins

“…Upon stimulation of cells with tumor necrosis factor (TNF) and recruitment of RIP1 to the TNF-receptor complex, RIP1 becomes polyubiquitinated. The K63-linked polyubiquitin chains attached to RIP1 were recently discovered to mediate RIP1 binding to NF-kB essential modulator (NEMO) (Ea et al, 2006;Wu et al, 2006), which is the regulatory subunit of the IkB kinase (IKK) complex. NEMO itself was also reported to be ubiquitinated upon different stimuli including TNF and overexpression of the API2/MALT1 fusion protein that is characteristic for certain MALT lymphomas.…”

The role of K63‐linked polyubiquitination in cardiac hypertrophy

“…Each linkage exhibits different responses. The K63‐linked ubiquitination of RIP1 on lysine 377 by TRAF2 is the response of inflammation stimulated by TNF 55, 56. Although the precise role of TRAF2 in TNFR signalling is currently unclear, the lipid sphingosine‐1‐phosphate (S1P) directly activates TRAF2 ligase activity and both sphingosine kinase 1 (Sphk1) and S1P are required for K63‐linked polyubiquitination of RIP1 and NF‐κB activation 57.…”