Investigating the Role of the Pseudokinase IRAK-3 in Signalling by Toll-Like Receptors

Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) are key pattern recognition receptors of the innate immune system. Upon binding of a ligand, these receptors recruit the adaptor protein myeloid differentiation primary response gene 88 (MyD88), which is followed by recruitment of members of the interleukin-1 receptor associate kinase (IRAK) family to form the oligomeric signalling complex known as the myddosome. This formation leads to the activation of several E3 ubiquitin ligases, ultimately generating hybrid ubiquitin chains that allow the TAK1 and IKK complexes to co-localise and trigger numerous downstream signalling pathways, resulting in the production of inflammatory mediators as part of the innate immune response. Of the four IRAK family members, only IRAK-1 and IRAK-4 are true kinases. IRAK-2 and IRAK-3 are pseudokinases and lack the critical aspartate residue for kinase activity, therefore are catalytically inactive. Despite mutation and dysfunction of various pseudokinases being linked to disease, the mechanism of action of many pseudokinases is poorly understood and therefore no drugs targeting pseudokinases have been approved for clinical use. The function and mechanism of action of the pseudokinase IRAK-3 has been subject to conflicting reports since its discovery. Generally considered to be a negative regulator of inflammatory signalling and to do so by interacting with the myddosome in some way, IRAK- 3 has been linked to a number of diseases. While its expression is thought to be favourable in the acute inflammatory phase of allergic asthma, it has also been reported that IRAK-3 knockout mice are more resistant to the pathology of certain diseases, including chronic phase allergic asthma, sepsis and inflammation-driven tumorigenesis. Therefore, I focused my research on elucidating the role of IRAK-3 in macrophages. In Chapter 3, I used an IRAK-3 PROTAC developed by collaborators at AstraZeneca to clarify the role of IRAK-3 in regulating TLR signalling and worked towards identifying a mechanism of action for this. In Chapter 4, I used proteomics to study the role of IRAK-3 in TLR1/2-stimulated macrophages to identify previously unknown functions of IRAK-3. I discovered that IRAK-3 regulates the secretion of the pro-inflammatory cytokines IL6, TNF and IL-12(p40) in MyD88-dependent TLR signalling, while not affecting secretion of the anti-inflammatory cytokine IL-10. IRAK-3 depletion did not affect phosphorylation of the activation loops of various signalling components downstream of the myddosome, including the canonical IKK complex and MAP kinases JNK1/2 and p38a. However, I found that IRAK-3 promoted phosphorylation of the activation loops of the IKK-related kinases, TBK1 and IKKe. The decreased activity of IKKe in IRAK-3 deficient macrophages was sustained for 2 hours, while the decreased activity of TBK1 was sustained for at least 8 hours. Since the IKK-related kinases negatively regulate TLR signalling, their activation being promoted by IRAK-3 may explain how IRAK-3 negatively regulated pro-inflammatory cytokine secretion. I also used mass spectrometry to analyse the proteome of mouse macrophages after TLR1/2 stimulation, in the presence and absence of IRAK-3. This led me to discover that rather than the broad anti-inflammatory effects that IRAK-3 has previously been thought to have, the effects of IRAK-3 are in fact very specific with only a select few proteins affected by IRAK- 3 depletion. Expression of some pro-inflammatory cytokines and chemokines were enhanced in the absence of IRAK-3, while TLR1/2 stimulation-induced expression of two anti- inflammatory proteins, a pattern recognition receptor and a protein regulating the actin cytoskeleton were reduced in the absence of IRAK-3. These findings open several potential new avenues of investigation into the function and mechanism of IRAK-3. Together, this work deepens the understanding of the function of the pseudokinase IRAK-3 and lays the foundation for potential therapeutic targeting in inflammatory diseases.