Both IRAK3 and IRAK1 Activate the MyD88–TRAF6 Pathway in Zebrafish

IL-1R-associated kinases (IRAKs) are signal transducers of the TLR/IL-1R-MyD88-TRAF6 pathways. Vertebrates possess two IRAK lineages, IRAK1/2/3 and IRAK4. In mammals, IRAK4/IRAK1 and IRAK4/IRAK2 are pathway enhancers, whereas IRAK3 is a repressor. However, in bony fish, IRAK2 is absent, and it remains elusive how fish IRAK1/3/4 functionally differ from their mammalian counterparts. In this study, we explored this using the zebrafish model. First, we showed that in human 293T cells, zebrafish IRAK1 and IRAK4 were components of the Myddosome (MyD88-IRAK4-IRAK1) complex, with IRAK1 serving as a potent pathway enhancer. Then, we discovered two zebrafish IRAK3 variants: one (IRAK3a) contains an N-terminal Death domain, a middle pseudokinase domain, and a C-terminal TRAF6-binding domain, whereas the other (IRAK3b) lost both the kinase and TRAF6-binding domains. This truncation of IRAK3 variants could be a conserved phenomenon in fish, because it is also observed in trout and grass carp. We proceeded to show that zebrafish IRAK3a acts as a pathway enhancer by binding with MyD88 and TRAF6, but its activity is milder than IRAK1, possibly because it has no kinase activity. Zebrafish IRAK3b, however, plays a sheer negative role, apparently because of its lack of kinase and TRAF6-binding domains. Moreover, zebrafish IRAK3a/3b inhibit the activity of IRAK1/4, not by interacting with IRAK1/4 but possibly by competing for MyD88 and TRAF6. Finally, we have verified the essential activities of zebrafish IRAK1/3a/3b/4 in zebrafish cells and embryos. In summary, to our knowledge, our findings provide new insights into the molecular functions of fish IRAKs and the evolution of the IRAK functional modes in vertebrates.