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IκB-related kinase ε (IKKε) plays a crucial role in the activation of nuclear factor κB (NF-κB) by phosphorylating inhibitor of NF-κB (IκB) and in the regulation of interferon (IFN) gene expression by phosphorylating IFN regulatory factors (IRFs). In this study, we cloned an IKKε homologue cDNA (designated as CgIKKε-like) from the Pacific oyster, Crassostrea gigas. The full 2896-bp cDNA sequence comprised a 2163-bp open reading frame (ORF) encoding 720 amino acids. CgIKKε-like is ubiquitously expressed, and its mRNA levels in hemocytes after poly I:C, V. alginolyticus , or OsHV-1 μVar challenge were analyzed by real-time PCR. Compared to that in the control, CgIKKε-like mRNA expression levels were significantly increased at 3 h and peaked at 6 h after OsHV-1 μVar challenge; no obvious changes were observed in expression levels until 24 h after either V. alginolyticus or poly I:C challenge, reaching a maximum at 24 h (p < 0.01) and then rapidly decreasing. CgIKKε-like transfection into human cell lines induced NF-κB and ISRE activation, while transfection with CgIKKε-like deletion mutants abolished NF-κB and ISRE reporter gene activation. Additionally, CgIKKε-like could interact with CgTBK1 and could form homodimers strongly, which may be critical for the immune signaling transduction. Last but not least, we found that CgIKKε-like may increase CgIκBs phosphorylation and could interact with CgIRF8. Together, these results suggest that CgIKKε-like could respond to pathogenic infection, participate in the immune signal transduction and activate NF-κB and ISRE reporter genes. Thus, CgIKKε-like could play an important role in the oyster immune system. • An IKKε-like gene was identified in the Pacific oyster Crassostrea gigas. • CgIKKε-like mRNA expression was induced after viral and bacterial challenge. • CgIKKε-like could induce NF-κB and ISRE activation in human cell lines. • CgIKKε-like interacts with CgTBK1 and forms homodimers strongly. • CgIKKε-like interacts with CgIRF8 and may phosphorylate CgIκBs. [ABSTRACT FROM AUTHOR]

Interleukin-1 receptor-associated kinases (IRAKs) play important roles in MyD88-dependent TLR signaling, the crucial innate immune pathway in molluscs. In this study, we examined the full-length IRAK4 genetic sequence in the Pacific oyster ( Crassostrea gigas ) by molecular cloning. Phylogenetic analysis revealed that CgIRAK4 is most closely related to Mytilus edulis , and forms a clade with other molluscs. CgIRAK4 transcripts are widely expressed in all tissues, with the highest expression observed in the hemocytes and gill. Moreover, CgIRAK4 is significantly upregulated after Oyster herpesvirus-1 microvariant (OsHV-1 μvar), Vibrio alginolyticus , and poly I:C challenge. Yeast two-hybrid and co-immunoprecipitation assays reveal that the CgIRAK4 death domain is necessary to mediate interaction between CgIRAK4 and two CgMyD88 isoforms. In addition, CgIRAK4 overexpression cannot induce NF-κB transcriptional activity, but blocks that induced by CgMyD88 in HEK293T cells. These findings elucidate the mechanisms of MyD88-dependent TLR signaling in molluscs, and the differences in IRAK-mediated pathway activation between invertebrates and vertebrates. [ABSTRACT FROM AUTHOR]