Abstract
IκB kinases (IKKs) play critical roles in innate immunity through signal-induced activation of the key transcription factors nuclear factor-κB (NF-κB) 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 IKKα/β-2 gene. We found that oyster IKKα/β-2, a homolog of human IKKα/IKKβ, responded to challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)]. As a versatile immune molecule, IKKα/β-2 activated the promoters of NF-κB, TNFα, and IFNβ, 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 IKKα/β-1, we investigated the signal transduction pathways mediated by these two IKKα/β proteins. Our results showed that IKKα/β-1 and IKKα/β-2 could interact with the oyster TNF receptor-associated factor 6 (TRAF6) and that IKKα/β-2 could also bind to the oyster myeloid differentiation factor 88 (MyD88) protein directly, suggesting that oyster IKKα/βs participate in both RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling for the reception of upstream immune signals. The fact that IKKα/β-1 and IKKα/β-2 formed homodimers by interacting with themselves and heterodimers by interacting with each other, along with the fact that both oyster IKKα/β proteins interacted with NEMO protein, indicates that oyster IKKα/βs and the scaffold protein NEMO form an IKK complex, which may be a key step in phosphorylating IκB proteins and activating NF-κB. Moreover, we found that oyster IKKα/βs 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 IKKα/β-1 and IKKα/β-2 may induce the phosphorylation of IκB proteins to activate NF-κB. These results reveal the immune function of oyster IKKα/β-2 and establish the existence of mollusk TLR and RLR signaling mediated by IKKα/β 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.
Highlights
The innate immune response represents the first line of defense of eukaryotic organisms against microbial infections [1]
In higher vertebrates such as mammals, innate immune signaling begins with pattern recognition receptors (PRRs), such as Tolllike receptors (TLRs) and RIG-I-like receptors (RLRs), which recognize pathogen-associated molecule patterns (PAMPs) [2, 3]
We found that all IKKs or IKKrelated kinases in oyster could be classified into two groups: an IKKα/IKKβ group and a TANK-binding kinase 1 (TBK1)/IKKε group
Summary
The innate immune response represents the first line of defense of eukaryotic organisms against microbial infections [1] In higher vertebrates such as mammals, innate immune signaling begins with pattern recognition receptors (PRRs), such as Tolllike receptors (TLRs) and RIG-I-like receptors (RLRs), which recognize pathogen-associated molecule patterns (PAMPs) [2, 3]. This recognition triggers immune signaling cascades, leading to the activation of the key transcription factors NF-κB and interferon regulatory factors (IRFs) and initiating antiviral or antibacterial immune responses [4,5,6]. The phosphorylation of IRF3 and IRF7 by TBK1 and IKKε promotes IRF3 and IRF7 homodimerization and their subsequent nuclear import, followed by the induction of type I IFN gene expression
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