Oyster Versatile IKKα/βs Are Involved in Toll-Like Receptor and RIG-I-Like Receptor Signaling for Innate Immune Response

I kappa B kinases (IKKs) play critical roles in innate immunity through signal-induced activation of the key transcription factors nuclear factor-kappa B (NF-kappa B) and interferon regulatory factors (IRFs). However, studies of invertebrate IKK functions remain scarce. In this study, we performed phylogenetic analysis of IKKs and IKK-related kinases encoded in the Pacific oyster genome. We then cloned and characterized the oyster IKK alpha/beta-2 gene. We found that oyster IKK alpha/beta-2, a homolog of human IKK alpha/IKK beta, responded to challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)]. As a versatile immune molecule, IKK alpha/beta-2 activated the promoters of NF-kappa B, TNF alpha, and IFN beta, as well as IFN -stimulated response element (ISRE)-containing promoters, initiating an antibacterial or antiviral immune state in mammalian cells. Importantly, together with the cloned oyster IKK alpha/beta-1, we investigated the signal transduction pathways mediated by these two IKK alpha/beta proteins. Our results showed that IKK alpha/beta-1 and IKK alpha/beta-2 could interact with the oyster TNF receptor-associated factor 6 (TRAF6) and that IKK alpha/beta-2 could also bind to the oyster myeloid differentiation factor 88 (MyD88) protein directly, suggesting that oyster IKK alpha/beta s participate in both RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling for the reception of upstream immune signals. The fact that IKK alpha/beta-1 and IKK alpha/beta-2 formed homodimers by interacting with themselves and heterodimers by interacting with each other, along with the fact that both oyster IKK alpha/beta proteins interacted with NEMO protein, indicates that oyster IKK alpha/beta s and the scaffold protein NEMO form an IKK complex, which may be a key step in phosphorylating I kappa B proteins and activating NE-kappa B. Moreover, we found that oyster IKK alpha/beta s could interact with IRF8, and this may be related to the IKK-mediated activation of ISRE promotors and their involvement in the oyster "interferon (IFN)-like" antiviral pathway. Moreover, the expression of oyster IKK alpha/beta-1 and IKK alpha/beta-2 may induce the phosphorylation of I kappa B proteins to activate NF-kappa B. These results reveal the immune function of oyster IKK alpha/beta-2 and establish the existence of mollusk TLR and RLR signaling mediated by IKK alpha/beta proteins for the first time. Our findings should be helpful in deciphering the immune mechanisms of invertebrates and understanding the development of the vertebrate innate immunity network.