Parameters that define Suppressor of IKK epsilon (SIKE): cytoskeletal protein interactions revealed through determination of binding affinities and SIKE's dimer interface

Abstract

Upon pathogen challenge multiple receptors both inside and on the surface of the cell, recognize pathogen associated molecular patterns (PAMPs) and initiate the production of proinflammatory, antiviral, and apoptotic responses. Pathways converge at key hubs that serve to amplify and regulate the signals, and are often responsible for determining the downstream response. TANK Binding Kinase 1 (TBK1) serves as a catalytic hub in the antiviral TLR3 mediated innate immune pathway. Suppressor of IKK epsilon (SIKE) is a recently identified high affinity alternative substrate of TBK1. It was initially found to inhibit TBK1 activation of type 1 interferon production. Upon subsequent study, it was found that SIKE was phosphorylated at six serine residues by TBK1. This phosphorylation of SIKE corresponds to the activation of the antiviral response, and releases SIKE from the SIKE:TBK1 interaction. The primary function of SIKE remains unknown. Examination of SIKE's interaction network has established direct interactions with cytoskeletal proteins including tubulin and α‐actinin. Migration assays have shown that chronic myelogenous leukemia (CML) cells in which SIKE has been knocked out migrate at a slower rate. Together, these studies suggest that SIKE plays a role in cytoskeletal rearrangements associated with innate immune responses such as migration and phagocytosis. The goal of this study is to define the interaction surface of SIKE as well as the binding affinities associated with these interactions. A quartz crystal microbalance with dissipation (QCM‐D) assay was developed to obtain binding affinities for the SIKE:cytoskeletal protein complexes. A gold sensor was utilized and functionalized with protein G and α‐His antibody to which 6xHis‐SIKE was immobilized. Increasing concentrations of binding partner were flowed over the chip to develop a binding curve. Prior to examining SIKE:cytoskeletal protein interactions, SIKE's oligomeric state was defined by crosslinking studies. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) shows that SIKE is a dimer. Chemical crosslinking followed by tandem mass spectrometry was employed to determine the dimeric interface of the SIKE dimer. BS3 was used in an excess of 20 and 100X. Data were analyzed with Mascot using xComb strategy. Residues 13, 66, 68, 110, and 169 were found to be accessible to BS3 modification, while in the dimer only residues 13, 110, 119, and 120 were found to be accessible. Residues 110 and 119 are found in multiple crosslinked peptides in both dimer and monomer samples. Residues 178 and 195 were found in crosslinked peptides unique to the dimer. These data have been applied to SIKE dimer models, generated by computational docking experiments to select for models consistent with the crosslinking restraints. These data define the dimer interface of SIKE as well as the surfaces available for SIKE cytoskeletal protein interactions. Together, these studies begin to reveal parameters that determine SIKE's interactions and the protein surfaces available to mediate these interactions.Support or Funding InformationStudies were funded by USD SURE, Beckman Foundation, NIH‐NIAID R21.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.