Background: Suppressor of IB kinase ⑀ (SIKE) inhibits a key innate immune effector molecule, TANK-binding kinase 1 (TBK1), through an undefined mechanism. Results: SIKE is a TBK1 substrate. Conclusion: SIKE controls TBK1 activity by acting as a high affinity substrate. Significance: SIKE attenuates phosphorylation of interferon regulatory factor 3 (IRF3) by serving as an alternative, high affinity substrate for TBK1.

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Protein‐protein interactions form the communication link from membrane bound receptors to their downstream cellular targets.ObjectiveOur long‐term goal is to examine the protein‐protein interactions responsible for the activation and regulation of the Toll‐like receptor 3 (TLR3) signaling cascade that leads to type I interferon production. In the current study we have targeted the kinase scaffold proteins, TRAF family member‐associated NF‐κB activator (TANK) and NAK‐associated protein 1 (NAP1).MethodsBacterial expression systems have been used to produce recombinant target protein and refolding methods developed for insoluble targets. Initial biochemical characterization of recombinant targets was completed using circular dichroism (CD), gel filtration (GF) and analytical ultracentrifugation (AUC).ResultsThe protein domain comprising the N‐terminal region of NAP1 can be successfully refolded as assessed by CD, while TANK is soluble. Both constructs form homo‐oligomers as determined by GF. AUC experiments show that the NAP1 N‐term behaves as an elongated dimer in vitro. Gel filtration studies of TANK's kinase interaction site show that it elutes as a dimer species.ConclusionsThe scaffold proteins, TANK and NAP1, form stable homo‐oligomers via their N‐terminal regions. Funding from the Am. Cancer Society (RJC) and supported in part by the NIH intramural research program, NIDDK (RG).

Protein‐protein interactions form the communication link from membrane‐bound receptors to their downstream cellular targets.ObjectiveOur long‐term goal is to examine the protein‐protein interactions responsible for the activation of the Toll‐like receptor 3 (TLR3) signaling cascade that leads to type I interferon production. In the current study, we have targeted the kinase scaffold protein, NAK‐associated protein 1 (NAP1).MethodsCFP/YFP/Alexa Fluor 546 fusion proteins of the NAP1 kinase binding domain (KBD) and scaffold binding motif (SBM) of the kinases, TANK Binding Kinase‐1 (TBK‐1) and IκB kinase epsilon (IKKε), were generated to assess interactions using fluorescence resonance energy transfer (FRET). Biochemical characterization of recombinant targets was completed using gel filtration (GF).ResultsBy gel filtration studies, NAP1 KBD elutes as a dimeric species. NAP1 KBD directly interacts with TBK1 and IKKε, with low micromolar affinity in vitro. Mutagenesis studies to identify the residues 1ecessary for the kinase recognition sequence are ongoing.ConclusionsNAP1 KBD forms stable homo‐oligomers and directly interacts with TBK1 and IKKε. Funding was provided by the American Cancer Society.