Examination of NAK‐associated protein‐1 homo and hetero‐interactions

Abstract

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.