Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

Sunhee Lee,Madhavi Challa-Malladi,Shawn B Bratton,Casey W Wright

doi:10.1074/jbc.m114.587808

Abstract

Activation of the noncanonical NF-κB pathway hinges on the stability of the NF-κB-inducing kinase (NIK), which is kept at low levels basally by a protein complex consisting of the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1/2) proteins and the tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2/3). NIK is brought into close proximity to the c-IAPs through a TRAF2-TRAF3 bridge where TRAF2 recruits c-IAP1/2 and TRAF3 binds to NIK. However, it is not clear how the c-IAPs specifically recognize and ubiquitylate NIK in the complex. We have identified an IAP-binding motif (IBM) at the amino terminus of NIK. IBMs are utilized by a number of proapoptotic proteins to antagonize IAP function. Here, we utilize mutational studies to demonstrate that wild-type NIK is destabilized in the presence of c-IAP1, whereas the NIK IBM mutant is stable. NIK interacts with the second baculovirus IAP repeat (BIR2) domain of c-IAP1 via the IBM, and this interaction, in turn, provides substrate recognition for c-IAP1 mediated ubiquitylation and degradation of NIK. Furthermore, in the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-κB target genes. Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-κB signaling is off in the absence of an activating signal.

Highlights

nuclear factor-␬B (NF-␬B)-inducing kinase (NIK) stability is necessary for activation of the noncanonical NF-␬B pathway
We focused our attention on c-IAP1. These results suggested that the NF-␬B-inducing kinase (NIK) IAP-binding motif (IBM) was crucial for directing c-IAP1-mediated NIK degradation but were somewhat surprising since the initiator methionine in NIK would be predicted to block the c-IAP1-NIK IBM interaction
NIK did not interact with X-linked IAP (XIAP), regardless of the IBM status, whereas second mitochondrial activator of caspases (Smac) interacted with XIAP in an IBM-dependent manner (Fig. 3D). These results reveal that the NIK IBM recognizes a specific baculovirus IAP repeat (BIR) domain of c-IAP1 and suggests that the modest interaction observed between NIK (A2G/ V3G) and c-IAP1 (H588A) (Fig. 2B) is a consequence of indirect binding mediated through the TRAF bridge

Summary

Background

NIK stability is necessary for activation of the noncanonical NF-␬B pathway. Results: The N terminus of NIK harbors an IBM that binds to c-IAP1 and promotes NIK degradation. In the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-␬B target genes Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-␬B signaling is off in the absence of an activating signal. NF-␬B-inducing kinase (NIK) is required for stimulation of the noncanonical NF-␬B pathway and, under basal conditions, is continuously targeted for proteasomal degradation by an E3 ubiquitin ligase complex consisting of the cellular inhibitor of apoptosis 1 and 2 (c-IAP1 and c-IAP2) proteins and the TNF receptor-associated factors 2 and 3 (TRAF2 and TRAF3) [8, 9]. These findings are significant as they define a key regulatory element of the c-IAP1⁄7TRAF21⁄7TRAF31⁄7NIK complex

EXPERIMENTAL PROCEDURES

RESULTS

E V W T H588A DE A DEHA E V W T H588A DEA DEHA

DISCUSSION