NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain

Abstract

An important property of NEMO, the core element of the IKK complex involved in NF-jB activation, resides in its ability to specifically recognize poly-ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the Cterminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63-linked chains, we demonstrate that together they form a bipartite high-affinity K63-specific ubiquitin-binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C-terminal half of NEMO is to specifically bind K63-linked poly-ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly-ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.

Nuclear initiated NF-κB signaling: NEMO and ATM take center stage

“…Cordier et al [96] demonstrated that the NEMO ZF is a ubiquitin-binding module. Moreover, the ZF helps to establish the specificity of the NOA/NUB/ UBAN domain toward K63-linked polyubiquitin chains [97]. Whether the role of the ZF in promoting NEMO SUMOylation is mechanistically related to the regulation of monomer-dimer states of free NEMO and/or its ubiquitin-binding activity needs to be investigated.…”

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

“…However, it was proposed that the C terminus of NEMO, comprising the UBAN and the ZnF, has a higher affinity toward Lys-63-and Lys-48-linked ubiquitin chains consisting of three or more ubiquitin moieties (25). To elucidate the ubiquitin binding preferences of full-length NEMO we performed in vitro binding studies with bacterially purified recombinant fulllength NEMO and ubiquitin chains of different linkage types and lengths.…”

Toward an integrative view of Optineurin functions

“…18 Interestingly, a constitutively active GTP-bound mutant of Rab8 (Q67L) but not a with its activity. 5,8 Zhu and colleagues have proposed that, following TNFα stimulation, Optn would compete with NEMO for binding to polyubiquitinated substrates (such as RIP1, Table 1), therefore downregulating NFκB activation. 9 Accordingly, a point mutation Asp 474 →Asn (D474N) in the UBD of Optn abolished its binding activity to Lys63-linked polyUb and prevents its interaction with RIP1.…”