Abstract
TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification–mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I∶C) stimulation, we found that TBK1 activation was strictly dependent on the integrity of the TBK1/TANK interaction.
Highlights
Antiviral immune responses depend on the rapid production of proinflammatory cytokines and type-I interferons (IFNs)
Tax1bp1 which we find associated with TANK-binding kinase 1 (TBK1), I-kB kinases (IKKs)-i, Sintbad and NAP1, is a ubiquitinbinding protein that dampens TNFR1 signaling via the E3 ligase Itch [25]
Because of the large scope of the endeavor, we focused here on three main aspects: (i) the structure-function analysis of the central node TBK1 to understand the structural requirements for TBK1 activity, (ii) the relationship of the TBK1 adaptors with TBK1 and with one another and (iii) the functional consequences of disrupting the TBK1 complex architecture
Summary
Antiviral immune responses depend on the rapid production of proinflammatory cytokines and type-I interferons (IFNs). Analyses of cells that are single knockouts for TBK1 and IKK-i suggest a certain level of redundancy between TBK1 and IKK-i [3], loss of TBK1 alone seems to have a more profound impact on type-I interferon induction than loss of IKK-i alone [3]. This is highlighted by the more recent observation that the interferon response to doublestranded DNA depends exclusively on TBK1 and not on IKK-i [5]
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