Grass carp (Ctenopharyngodon idella) TAK1 promotes NF-κB activation via interaction with TAB1 in response to potential vaccine antigen of Vibrio mimicus

Abstract

In mammals, transforming growth factor β-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) are known as two crucial upstream mediators in the NF-κB signaling pathway. TAK1 interacts with TAB1 to form the TAK1–TAB1 complex, which triggers NF-κB activation through a series of cascade reactions. However, the interaction of TAK1 and TAB1 in grass carp (Ctenopharyngodon idella) and the effects of their interaction on the NF-κB activation remain largely unknown. Here, the transcriptional expression of the CiTAK1 and CiTAB1 genes was firstly detected by RT-qPCR, and both genes showed ubiquitous transcriptional expression in all the examined tissues of healthy grass carp. And the expression levels of both genes were significantly up-regulated in the different tissues of grass carp and C. idella kidney (CIK) cells after stimulation with the potential vaccine antigen of Vibrio mimicus (named OVepis), implying that both proteins are involved in the OVepis-induced immune response. Subsequently, an analysis of the subcellular localization showed that CiTAK1 was a cytoplasmic protein, whereas CiTAB1 was distributed throughout the HEK293T and CIK cells. Co-immunoprecipitation and pull-down assays proved that CiTAK1 could directly interact with CiTAB1 in vitro, which was further confirmed by co-localization assay, where these two proteins co-located in the cytoplasm. Finally, the effects of CiTAK1 and CiTAB1 co-overexpression on the NF-κB activation and transcriptional expression of downstream pro-inflammatory cytokines were investigated. The results showed that the CiNF-κB promoter activity and transcriptional expression levels of IL-1β, IL-8 and TNF-α enhanced significantly in the CiTAK1 and CiTAB1 co-overexpressed cells compared with non-overexpression control or overexpression of CiTAK1 or CiTAB1 alone, especially under rOVepis stimulation. Taken together, the data generated in this study indicate that the direct interaction between CiTAK1 and CiTAB1 might induce CiNF-κB signaling pathway activation in response to vaccine antigen of V. mimicus.