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

Abstract

Deregulated nuclear factor jB (NF-jB) activation plays an important role in inflammation and tumorigenesis. ABIN proteins have been characterized as negative regulators of NF-jB signaling. However, their mechanism of NF-jB inhibition remained unclear. With the help of a yeast two-hybrid screen, we identified ABIN proteins as novel ubiquitin-interacting proteins. The minimal ubiquitin-binding domain (UBD) corresponds to the ABIN homology domain 2 (AHD2) and is highly conserved in ABIN-1, ABIN-2 and ABIN-3. Moreover, this region is also present in NF-jB essential modulator/IjB kinase c (NEMO/IKKc) and the NEMO-like protein optineurin, and is therefore termed UBD in ABIN proteins and NEMO (UBAN). Nuclear magnetic resonance studies of the UBAN domain identify it as a novel type of UBD, with the binding surface on ubiquitin being significantly different from the binding surface of other UBDs. ABIN-1 specifically binds ubiquitinated NEMO via a bipartite interaction involving its UBAN and NEMO-binding domain. Mutations in the UBAN domain led to a loss of ubiquitin binding and impaired the NF-jB inhibitory potential of ABINs. Taken together, these data illustrate an important role for ubiquitin binding in the negative regulation of NF-jB signaling by ABINs and identify UBAN as a novel UBD.

ABINs: A20 binding inhibitors of NF-κB and apoptosis signaling

“…We therefore hypothesize that also ABIN-1 might compete with NEMO or other ubiquitin-binding proteins for binding to polyubiquitinated signaling proteins in the NF-B pathway (Fig 3B). The latter must be different from RIP1 as we were unable to demonstrate the binding of ABIN-1 to polyubiquitinated RIP1 [12]. ABIN-1 overexpression was recently shown to also prevent the constitutive and EGFinduced NF-B activation in epidermal growth factor (EGF) receptor overexpressing tumor cell lines [42].…”

“…In line with these observations, ABIN-1 was shown to interact with NEMO via a specific NBD that is located C-terminal from AHD2, and which is required for A20-mediated de-ubiquitination of NEMO [27] (Fig 3A). Interestingly, we could demonstrate that ABIN-1 also contains a UBD (referred as UBAN) that overlaps with AHD2 and which is involved in NEMO binding [12]. Altogether, the above mentioned findings led to the suggestion that ABIN-1 physically links A20 to ubiquitinated NEMO, thus facilitating A20-mediated de-ubiquitination of NEMO and NF-B inhibition.…”

“…Page 22 of 38 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 [12,47], but the specific targets still need to be identified. Moreover, it is not unlikely that the ubiquitinbinding potential of ABINs might also provide ABINs the potential to regulate endocytosis and intracellular trafficking of specific receptors and signaling proteins.…”

“…Moreover, A20-binding deficient ABIN-1 mutants (by deletion of AHD1) can still inhibit NF-B [10]. In this context, it is interesting to note that the UBD of ABINs is also present in the C-terminal part of NEMO and the NEMO-related protein optineurin [10,12]. The UBD of NEMO mediates its interaction with K63-polyubiquitinated signaling proteins such as RIP1, which is essential for TNF-induced NF-B signaling [40].…”

Regulation and function of TPL-2, an IκB kinase-regulated MAP kinase kinase kinase

“…ABIN-2 was originally identified in a two-hybrid screen, with the NF-κB inhibitory protein A20 as bait, and overexpression experiments indicate that ABIN-2 may be a downstream effector of A20, mediating its NF-κB inhibitory function [58]. ABIN-2 contains an UBAN (ubiquitin binding in ABIN and NEMO) ubiquitinbinding domain, which is essential for its ability to inhibit NF-κB in overexpression experiments [59]. Binding analyses with recombinant ABIN-2 have indicated interaction with linear ubiquitin chains, and also weak binding to K63-linked ubiquitin chains [60].…”