Abstract
NF-κB signaling is a central pathway of immunity and integrates signal transduction upon a wide array of inflammatory stimuli. Noncanonical NF-κB signaling is activated by a small subset of TNF family receptors and characterized by NF-κB2/p52 transcriptional activity. The medical relevance of this pathway has recently re-emerged from the discovery of primary immunodeficiency patients that have loss-of-function mutations in the MAP3K14 gene encoding NIK. Nevertheless, knowledge of protein interactions that regulate noncanonical NF-κB signaling is sparse. Here we report a detailed state-of-the-art mass spectrometry-based protein–protein interaction network including the noncanonical NF-κB signaling nodes TRAF2, TRAF3, IKKα, NIK, and NF-κB2/p100. The value of the data set was confirmed by the identification of interactions already known to regulate this pathway. In addition, a remarkable number of novel interactors were identified. We provide validation of the novel NIK and IKKα interactor FKBP8, which may regulate processes downstream of noncanonical NF-κB signaling. To understand perturbed noncanonical NF-κB signaling in the context of misregulated NIK in disease, we also provide a differential interactome of NIK mutants that cause immunodeficiency. Altogether, this data set not only provides critical insight into how protein–protein interactions can regulate immune signaling but also offers a novel resource on noncanonical NF-κB signaling.
Highlights
NF-κB signaling is one of the most important signaling pathways in immunity with two major branches, termed canonical and noncanonical
Noncanonical NF-κB signaling is characterized by the induction of proteolytic processing of NF-κB2/p100 to p522 (Figure 1A)
Downstream of noncanonical receptor activation, signals are relayed through the TNF receptorassociated factor (TRAF) proteins TRAF2 and TRAF3 via regulation of the serine/threonine kinase NF-κB inducing kinase (NIK, MAP3K14).[4]
Summary
NF-κB signaling is one of the most important signaling pathways in immunity with two major branches, termed canonical and noncanonical. Because noncanonical NF-κB signaling is required for healthy lymphoid tissue survival, the pathway is commonly activated in cancers, for example, B-cell neoplasms[14] or multiple myelomas.[15,16] Fine-tuned regulation of the noncanonical pathway and cross-talk with other immune and cellular pathways is still not fully understood This fact is supported by the recent discovery of additional novel regulators.[17,18] For example, the TRAF3 interactor and deubiquitinase OTUD7B acts via protein−protein interaction to regulate the pathway.[18] Because the NF-κB pathway is of such essential interest, it has been extensively explored in largescale proteomic studies;[19] a detailed and technologically up-to-date interactome analysis of noncanonical NF-κB signaling has not yet been reported. As a proofof-principle, we compare the interactome of wild-type NIK to previously reported single gene-defect causing mutants, namely, the recently identified novel kinase inactive mutant observed in human patients (NIKP565R),[8] a second catalytically inactive mutant (NIKK429A/K430A),[9] and the human aly equivalent mutant (NIKG860R).[11]
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