Mutations in the death domain and GC centre of IRAK3 and the effect on downstream signalling

Galea CA1, Freihat LA1,2, Nguyen HT2 and Irving HR1,2

  1. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
  2. La Trobe Institute for Molecular Science, Latrobe University, Bendigo, VIC, Australia.

Interleukin-1 receptor associated kinase 3 (IRAK3) plays an important role in maintaining homeostasis in the innate immune response and in preventing the development of autoimmune diseases. IRAK3 acts as a negative regulator of inflammation and is involved in inflammation-associated disorders such as lung injury, metabolic syndrome and tumour growth. Prior studies within our group identified IRAK3 as a potential novel guanylate cyclase (GC) catalyzing cyclic guanosine monophosphate (cGMP) synthesis. IRAK3 is predicted to be a mammalian representative of a new class of GCs originally identified in plants, containing a complex domain architecture where the GC centre is encapsulated within the pseudokinase domain. Here we report the effect of mutating amino acid residues of the GC catalytic site and death domain upon IRAK3’s GC activity and anti-inflammatory downstream signalling. HEKBLUE hTLR4 cells containing a SEAP reporter system were transfected with IRAK3 or IRAK3 mutant constructs and NFκB activity was monitored in the presence of lipopolysaccharide (LPS). Overexpression of IRAK3 significantly reduced LPS-induced NFκB activation while IRAK3 mutants possessing reduced GC activity failed to inhibit LPS induced NFκB function. Addition of cell-permeable cGMP restored IRAK3 function and significantly reduced NFκB activity in IRAK3 mutants with reduced cGMP-generating capacity. These findings are providing insight into hidden functions of IRAK3 and may assist in explaining its selectivity and functionality in the inflammatory signalling cascade. Understanding how this novel GC function impacts the anti-inflammatory activity of IRAK3 will aid in the development of novel therapeutics for the treatment of various inflammation-associated diseases.