Abstract
Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation. IRAK3 inhibits signalling cascades downstream of myddosome complexes associated with toll like receptors. IRAK3 contains a death domain that interacts with other IRAK family members, a pseudokinase domain and a C-terminus domain involved with tumour necrosis factor receptor associated factor 6 (TRAF6). Previous bioinformatic studies revealed that IRAK3 contained a guanylate cyclase centre in its pseudokinase domain but its role in IRAK3 action is unresolved. We demonstrate that wildtype IRAK3 is capable of producing cGMP. Furthermore, we show that a specific point mutation in the guanylate cyclase centre reduced cGMP production. Cells containing toll like receptor 4 and a nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) reporter system were transfected with IRAK3 or mutant IRAK3 proteins. Cell-permeable cGMP treatment of untransfected control cells suppresses downstream signalling through modulation of the NFĸB in the presence of lipopolysaccharides. Cells transfected with wildtype IRAK3 also suppress lipopolysaccharide induced NFĸB activity in the absence of exogenous cGMP. Lipopolysaccharide induced NFĸB activity was not suppressed in cells transfected with the IRAK3 mutant with reduced cGMP-generating capacity. Whereas in the presence of exogenously applied cell-permeable cGMP the IRAK3 mutant was able to retain its function by suppressing lipopolysaccharide induced NFĸB activity. Furthermore, increasing the amount of membrane permeable cGMP did not affect IRAK3’s ability to reduce NFĸB activity. These results suggest that cGMP generated by IRAK3 may be involved in regulatory function of the protein where the presence of cGMP may selectively affect downstream signalling pathway(s) by modulating binding and/or activity of nearby proteins that interact in the inflammatory signalling cascade.
Highlights
Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation
An alternative possibility is that IRAK3 directly interacts with the myeloid differentiation primary response 88 (MyD88)-IRAK myddosome and IRAK3 forms a complex with mitogen activated protein kinase kinase kinase 3 (MEKK3) and tumour necrosis factor receptor associated factor 6 (TRAF6) that stimulates MEKK3 dependent NFκB activation promoting expression of anti-inflammatory molecules[16,17,18]
Since IRAK3 expressing HEK BLUE hTLR4 cells suppressed lipopolysaccharide-induced NFĸB activity (Fig. 3A), we explored whether excess 8-bromo-cGMP enhanced the effect of IRAK3 on the suppression of NFĸB activity
Summary
Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation. Increasing the amount of membrane permeable cGMP did not affect IRAK3’s ability to reduce NFĸB activity These results suggest that cGMP generated by IRAK3 may be involved in regulatory function of the protein where the presence of cGMP may selectively affect downstream signalling pathway(s) by modulating binding and/or activity of nearby proteins that interact in the inflammatory signalling cascade. We reported that the mammalian protein interleukin 1 receptor associated kinase 3 (IRAK3) contained a guanylate cyclase centre in its pseudokinase domain and the recombinant protein generated cGMP11. Tumour necrosis factor receptor associated factor 6 (TRAF6) interacts with IRAK1/2 causing dissociation of the myddosome complex from the TLR and downstream this eventually stimulates transforming growth factor beta kinase 1 (TAK1) dependent activation of NFκB13,14. The molecular pharmacological mechanisms underpinning the role of cGMP in inflammation are still only partly understood[34,35,36,37,38]
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