Inhibitor of kappaB kinase epsilon (IKKε, IKBKE) and its close homolog TANK-binding kinase I (TBK1) are activated downstream of pattern-recognition receptor activation upon infection (Fitzgerald KA et al., 2003; Hemmi H et al., 2004; Hacker H & Karin M 2006; Taft J et al., 2021; Wegner J et al., 2023). Activity of IKKε (IKBKE), like that of TBK1, is regulated by the phosphorylation of a serine residue 172 (S172) within the activation loop of the N-terminal kinase domain (KD) (Clark et al., 2009). The activation of IKKε, like TBK1, may occur through autophosphorylation or via activity of a distinct protein kinase (Clark et al., 2009).

Structural studies of TBK1 reveal a dimeric assembly which is mediated by several interfaces involving the N-terminal KD, a ubiquitin-like domain (ULD), and an alpha-helical scaffold dimerization domain (SDD) of TBK1, thus supporting a model of trans-autophosphorylation (Larabi A et al., 2013; Tu D et al., 2013). IKKε forms homodimers upon co-expression of tagged monomers in human embryonic kidney 293 (HEK293) cells (Nakatsu Y et al., 2014). The ULDs of TBK1 and IKKε are involved in the control of kinase activation, substrate presentation, and downstream signaling (Ikeda F et al., 2007; Tu D et al., 2013). Upon activation, IKKε (IKBKE) is modified by K63-linked polyubiquitination on lysines 30 and 401 (Zhou AY et al. 2013). The ubiquitination sites and dimer contacts are conserved in IKKε and TBK1 (Tu D et al., 2013; Zhou AY et al., 2013). These findings suggest that both IKKε and TBK1 are regulated through similar activation mechanisms involving dimerization, phosphorylation, and ubiquitination. Activated IKKε (IKBKE) and TBK1 phosphorylate interferon (IFN) regulatory factor 3 (IRF3) and IRF7 leading to IFN production (Fitzgerald KA et al., 2003; Hemmi H et al., 2004; Hacker H & Karin M 2006; Taft J et al., 2021; Wegner J et al., 2023).

In this Reactome reaction, IKKε (IKBKE) is trans-autophosphorylated at S172 within the activated TLR3 complex.

TANK-binding kinase I (TBK1) and its close homolog inhibitor of kappaB kinase epsilon (IKKε or IKBKE) are serine/threonine protein kinases, that are activated by pattern-recognition receptors upon infection. Activity of both TBK1 and IKKε (IKBKE) is regulated by the phosphorylation of a serine residue 172 (S172) within the activation loop of the N-terminal kinase domain (KD) (Clark et al., 2009). The activation of TBK1 and IKKε may occur through autophosphorylation or via activity of a distinct protein kinase (Clark et al., 2009). Structural studies of TBK1 reveal a dimeric assembly which is mediated by several interfaces involving an N-terminal KD, a ubiquitin-like domain (ULD), and an alpha-helical scaffold dimerization domain (SDD) of TBK1 thus supporting a model of trans-autophosphorylation (Larabi A et al., 2013; Tu D et al., 2013). The ULD of TBK1 (and IKBKE) is involved in the control of kinase activation, substrate presentation and downstream signaling (Ikeda F et al., 2007; Tu D et al., 2013). Upon activation, TBK1 is modified by K63-linked polyubiquitination on lysines 30 (K30) and K401 (Tu D et al., 2013). Ubiquitination of TBK1 leads to conformational changes that facilitate activation of the KD while maintaining the overall dimer conformation (Larabi A et al., 2013). The ubiquitination and phosphorylation sites, as well as dimer contacts, are conserved in the close homolog IKKε (IKBKE) suggesting that both kinases are regulated through similar activation mechanisms (Tu D et al., 2013; Zhou AY et al., 2013). Activated TBK1 then phosphorylates IRF3 and IRF7.

TBK1, K63‑polyubiquitinated on K30 and K401, interacts with ubiquitin-binding adaptor protein optineurin (OPTN), which regulates the activity of TBK1 (Pourcelot M et al., 2016).

This Reactome event shows TBK1 phosphorylation within the activated TLR4 complex.

Tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) is a ubiquitin ligase recruited to both MYD88- and TRIF-assembled signalling complexes (Hacker H et al., 2006). However, TRAF3 controls the production of interferon and proinflammatory cytokines in different ways (Tseng PH et al., 2010). Positive or negative type of regulation is dictated by TRAF3 subcellular distribution and its mode of ubiquitination. Thus, TRIF-mediated signaling initiated on endosomes triggers TRAF3 self-ubiquitination through noncanonical (K63-linked) polyubiquitination, which is essential for activation of IRF3/7 and the interferon response. In contrast, during MyD88-dependent signaling initiated from plasma membrane TRAF3 functions as a negative regulator of inflammatory cytokines and mitogen-activated protein kinases (MAPKs), unless it undergoes degradative (K48-linked) polyubiquitination mediated by TRAF6 and a pair of the ubiquitin ligases cIAP1 and cIAP2. The degradation of TRAF3 is essential for MAPK activation via TAK1 and MEKK1 (Tseng PH et al., 2010).

The Toll-like receptor 4 (TLR4) is a membrane-spanning protein distantly related to the IL1 receptor. Both CD14 and members of the Toll family contain multiple leucine-rich repeats. In addition, the latter possess a Toll-homology domain in the cytoplasmic tail, which is important in the generation of a transmembrane signal linked to LPS-induced cell activation. Of all Toll family members, TLR4 is probably the exclusive receptor for LPS from most Gram negative organisms.

Toll-like receptor 4 and lymphocyte antigen 96 (LY96, also known as myeloid differentiation factor 2 (MD2)) form a heterodimer that specifically recognizes structurally diverse LPS molecules. A structural study of TLR4:LY96 complex revealed that LY96 (MD2) interaction with TLR4 relies on hydrogen and electrostatic bonds (Kim HM et al, 2007). LPS binds to the hydrophobic pocket of LY96 and directly mediates the dimerization of the two TLR4:LY96 complexes in a symmetrical manner. Both hydrophobic and hydrophilic interactions contribute to the main dimerization interaction between LY96, LPS and TLR4 multimer components. The phosphate groups of LPS also contribute to the receptor multimerization by forming ionic interactions with positively charged residues of TLR4 and LY96. (Park BS et al, 2009).

The activated TLR4 receptor is composed of two copies of the TLR4:LY96:LPS complex and initiates signal transduction by recruiting intracellular adaptor molecules.

Hemes bind and activate TLR4 signaling at amino acids W23 and Y34 on LY96 (MD-2) (Belcher et al, 2002).

High mobility group box 1 (HMGB1) is an ubiquitous nuclear protein that is actively secreted by innate immune cells and/or released passively by necrotic or damaged cells in response to infection or injury (Andersson U et al. 2000; Scaffidi P et al. 2002; Bonaldi T et al. 2003; Chen G et al. 2004; Beyer C et al. 2012; Yang H et al. 2013). Outside the cell, HMGB1 can serve as an alarmin to activate innate immune responses including chemotaxis and cytokine release in both normal and aberrant immunity (Andersson U et al. 2000; Zetterström CK et al. 2002; Voll RE et al. 2008; Harris HE et al. 2012; Diener KR et al. 2013; Yang H et al. 2013).

HMGB1 can form immunostimulatory complexes with cytokines and other endogenous and exogenous ligands such as bacterial lipopolysaccharide (LPS) to potentiate proinflammatory response (Youn JH et al. 2008, 2011; Wähämaa H et al. 2011; Hreggvidsdottir HS et al. 2009). The activity of HMGB1 depended on the redox state of three cysteines at positions 23, 45 and 106 (C23, C45 and C106) (Urbonaviciute V et al. 2009; Venereau E et al. 2012, 2013; Yang H et al., 2013, 2021). Analysis revealed that the inflammatory activities of HMGB1 required both the formation of an intramolecular disulfide bond between C23 and C45 and the reduced state of C106 (thiol state, C106-SH) (Venereau E et al. 2012; Yang H et al. 2021).

HMGB1 binding to LPS facilitated transfer of LPS to CD14 and enhanced TNFalpha production in human peripheral blood mononuclear cells (PBMCs) (Youn JH et al. 2008). HMGB1 in complex with LPS boosted proinflammatory cytokine- and matrix metalloproteinase (MMP3) production in synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients (Wähämaa H et al. 2011; He ZW et al. 2013).

In addition to its ability to act in a synergy with LPS and other ligands, HMGB1 was shown to stimulate cells by direct interaction with innate immune receptors such as TLR4:LY96 (Yang H et al. 2010; Yang H et al. 2015).