Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB

Tobias Kensche,Fuminori Tokunaga,Fumiyo Ikeda,Eiji Goto,Kazuhiro Iwai,Ivan Dikic

doi:10.1074/jbc.m112.347195

Abstract

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.

Highlights

Binding of NEMO to ubiquitin chains is essential for the activation of Nuclear factor-␬B (NF-␬B)
Because direct NEMO-ubiquitin interactions have only been determined by using isolated NEMO fragments, we examined how full-length NEMO interacts with different types of ubiquitin chain linkages
By applying competition assays using fulllength NEMO, we found that NEMO preferably binds linear ubiquitin chains compared with Lys-63- or Lys-11-linked chains, even when lysine-linked ubiquitin chains were longer than the linear chains (Fig. 1)

Summary

Background

Binding of NEMO to ubiquitin chains is essential for the activation of NF-␬B. Results: Full-length NEMO preferentially binds linear ubiquitin chains in competition with lysine-linked ubiquitin chains. More recent studies have shown that an E3 ligase, linear ubiquitin chain assembly complex (LUBAC), regulates the TNF signaling pathway by linearly ubiquitylating NEMO leading to the activation of IKK kinases [11,12,13]. More recent structural and functional studies indicate that the isolated NEMO UBAN domain preferentially binds to linear ubiquitin chains, but it can bind with lower affinity to other types of ubiquitin chains including Lys-11, Lys-48, and Lys-63, presumably when they form longer chains [10, 14] This led to the hypothesis that in addition to the linkage type the length of the chains might be a critical determinant in NF-␬B activation [19]. By utilizing engineered NEMO chimeras that recognize exclusively Lys-63-linked ubiquitin chains as well as NEMO that is permanently linearly ubiquitylated we have tested the contribution of the linkage and length of specific ubiquitin chains in the activation of the NF-␬B pathway upon TNF␣ stimulation in vivo

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION