Since many NF-kappa B activating pathways converge at the level of IKK2, molecular interactions of this kinase are pivotal for regulation of NF-kappa B signaling.*Methodology/Principal Findings*We searched for proteins interacting with IKK2 using the C-terminal part (amino acids 466-756) as bait in a yeast two-hybrid system and identified the N-terminal part (amino acids 1-228) of the TNF-receptor associated factor TRAF1 as putative interaction partner. I-kappa B kinase 2 (IKK2 or IKK-beta) is one of the most crucial signaling kinases for activation of NF-kappa B, a transcription factor that is important for inflammation, cell survival and differentiation. This systematic analysis provided valuable information about the diverse roles of TRAFs in the innate immune response to pathogenic bacterial infection in teleost fish and will contribute to the functional characterization of CsTRAF genes in further research. Functional analysis of CsTRAF3, CsTRAF4 and CsTRAF6 revealed that only CsTRAF6 could strongly activate the NF-кB pathway after overexpression of CsTRAF3, CsTRAF4 and CsTRAF6 in HEK-293T cells. The results indicate that only CsTRAF2a is relatively highly expressed in the brain and that the other CsTRAFs are highly expressed in immune-related tissues and may participate in the immune response after infection with pathogenic bacteria. Real-time quantitative PCR was used to investigate the expression patterns of CsTRAF genes in tissues of healthy fish and in the gills, livers and spleens of fish after bacterial infection with Vibrio harveyi. Analysis of the gene structures and motifs showed the diversity and distribution of exon-intron structures and conserved motifs in Chinese tongue sole and several other teleost species. Phylogenetic analysis indicated the evolutionary relationships of TRAF family members in teleost fish and revealed an absence of TRAF1 in most species and TRAF5 in some species of teleosts. Sequence analysis revealed various genomic structures of the CsTRAFs and showed that they contain typical conserved domains compared with mammalian TRAFs. In the present study, we identified and characterized seven TRAF genes, namely, CsTRAF2a, CsTRAF2b, CsTRAF3, CsTRAF4, CsTRAF5, CsTRAF6 and CsTRAF7, in Chinese tongue sole, and the complete ORFs of the CsTRAFs were cloned. However, systematic identification of the TRAF gene family in teleost fish has not yet been reported, and there is little available information about its roles in innate immunity in Chinese tongue sole (Cynoglossus semilaevis), an aquaculture fish of high economic value. TRAFs collectively play important roles in multiple biological processes and organismal immunity. Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) play crucial roles as signaling mediators for the TNF receptor (TNFR) superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily.

Traf protein scaffold