Pathogen challenge elicits an immediate response by the innate immune system. Multiple innate immune signaling pathways converge to activate TANK binding kinase 1 (TBK1), a ubiquitously expressed kinase. Substrates of TBK1 have proven to be critical players in coordinating host defenses, dictating the shape of the downstream innate immune response. We have defined a new TBK1 substrate, Suppressor of IKK? (SIKE). SIKE was first identified as a TBK1 inhibitor. In our studies to define SIKE-mediated TBK1 inhibition, we discovered that SIKE was phosphorylated by TBK1 and the TBK1:SIKE interaction was modulated by SIKE phosphorylation. With respect to other TBK1 substrates, SIKE acted as a nanomolar, mixed-type inhibitor of TBK1. Although SIKE influenced TBK1 activity as a high affinity substrate, SIKE's primary function remains unknown. To address this gap in knowledge, we examined SIKE's localization pattern and interaction network. Gross localization studies indicated that GFP-SIKE was cytosolic, accumulated in cytoskeletal structures, and dense cytosolic granules. Tandem MS/MS data from immunoprecipitated SIKE complexes revealed that SIKE interacted with the cytoskeleton, translation machinery, and mRNA transport proteins associated with ribonucleoprotein complexes (RNPs). RNPs are formed following viral infection to reduce the availability of translation machinery for viral replication. This relationship suggests that RNPs form part of the host's antiviral defenses, yet the linkage between innate immune signaling and these RNA-mediated defenses is poorly defined. Based upon our preliminary data, we hypothesize that SIKE functions as a bridge between the innate immune response and post-transcriptional mRNA pools in cytosolic RNPs to mediate a rapid host response at the level of translation.
In Aim 1, we will establish SIKE interactions in epithelial cells that form a defensive barrier to invading pathogen and where rapid RNP formation may represent a key defense mechanism, and phagocytic immune cells responsible for sequestering/removing pathogen where rapid cytoskeletal rearrangement and cell migration are essential functions. We will assess pathogen-specific SIKE interactions using dsRNA in the form of polyinosinic-polycytidylic acid, which mimics key aspects of viral infection, an inducer of RNPs, and S. typhimurium, which is taken up by phagocytic cells and activates TBK1-dependent pathways.
In Aim 2, we will examine the impact of SIKE's phosphorylation state on its localization with respect to cell-type and pathogen stimuli. We will investigate SIKE' function at the cell periphery in association with the cytoskeleton versus its role in cytosolic granules through functional assays including cell migration, phagocytosis, formation of pathogen containing vacuoles, and RNP formation and resolution. These studies to determine SIKE's function in bridging TBK1-mediated innate immune responses and RNA granules will provide critical new insight into how TBK1 shapes our innate immune defenses and establish a foundation for building long-term studies on the function, regulation and viral subversion of TBK1-mediated innate immune responses.
A critical barrier to developing effective modulating agents for our immune response is our incomplete understanding of how protein interactions elicit key events downstream of innate immune sensors. This research seeks to characterize the function of a newly identified TBK1 substrate - Suppressor of IKK?, a downstream component of the innate immune response. These studies will explore a previously unrecognized connection between the innate immune response and cytosolic RNA granules providing a new paradigm for coordinating host defenses to stem pathogen invasion and a mechanistic model to examine viral subversion of this host defense mechanism.
| Sonnenschein, Halie A; Lawrence, Kenneth F; Wittenberg, Karli A et al. (2018) Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and ?-actinin, linking innate immunity to the cytoskeleton. FEBS Open Bio 8:1064-1082 |
