Cloning And Function Analysis Of TAK1 And Its Binding Proteins TAB1 And TAB2 In Large Yellow Croaker

Based on the transcriptome database of Large yellow croaker(Larimichthys crocea)in our lab,the cDNA sequences of Transforming growth factor-β-activating kinase 1(LcTAK1),TAK1-binding protein 1(LcTAB1)and TAK1-binding protein 2(LcTAB2)were identified from large yellow croaker.Their molecular characterization and structures were analyzed.The tissue expression profiles of LcTAK1,LcTAB1 and LcTAB2 were analyzed,as well as their spatiotemporal expression in Large yellow croaker kidney(LCK)cells were detected after the pathogen-related molecular patterns(PAMPs)stimulation.The subcellular localization of LcTAK1,LcTAB1 and LcTAB2 were analyzed.The over-expression plasmids of LcTAK1,LcTAB1 and LcTAB2 were constructed.TAK1 and its binding protein TAB1/TAB2 were separately or co-transfected into HEK293 T cell lines,and the activation of NF-κB as well as the transcription level expression of downstream cytokine such as tumor necrosis factor-alpha(TNF-alpha),and interleukin(IL).IL-6,IL-1β,IL-8 and type I interferon(IFN-I)was analyzed after stimulation with different PAMPs.The obtained results can described as follows:(1)The gene clone and expression analysis of Lc TAK1 and its binding proteinsThe open reading frame(ORF)of LcTAK1 was 1725 bp in length,encoding 574 amino acids with predicted molecular weight(Mw)of 64.2 kDa and theoretical isoelectric point(pI)of 6.69.LcTAK1 protein contained a protein kinase domain S_TKc and belonged to conservative TAK1 family.The expression of LcTAK1 were widely distributed in all determined tissues,with the most predominant expression in brain,followed by spleen and quite weak in muscle.LcTAK1 transcripts increased significantly in LCK cells after flagellin,LPS and poly I:C stimulation(p < 0.05).Subcellular localization revealed that LcTAK1 expressed in the cytoplasm.The ORF of LcTAB1 was 1518 bp encoding 505 amino acids with predicted Mw of 64.2 kDa and p I of 6.69.A typical PP2 Cc domain and a conserved sequence motif(PYVDFSQFYLLWGSDH)at C-terminal were identified.LcTAB1 was belonged to conservative TAB1 family and were broadly expressed in all examined tissues,with The greatest value transcripts in brain,followed by spleen and quite weak in muscle.LcTAB1 transcripts enhanced greatly after LPS and poly I:C challenge(p < 0.05).Subcellular localization showed that LcTAB1 expressed in the cytoplasm.The ORF of LcTAB2 was 2271 bp encoding 756 amino acids with predicted Mw of 81.7 kDa and p I of 8.26.LcTAB2 protein contained an N-terminal CUE domain and a C-terminal ZnF domain.LcTAB2 was belonged to conserved TAB2 family and were widely distributed in all determined tissues,with the high expression in brain and blood,followed by spleen and the lowest expression in liver.LcTAB2 transcripts increased significantly in LCK cells after flagellin,LPS and poly I:C stimulation(p < 0.05).Subcellular localization revealed that LcTAB2 expressed in the cytoplasm.(2)The regulatory mechanism of LcTAK1 and LcTAB1 in NF-κB signaling pathwayOur results revealed that NF-κB luciferase promoter expression could not be induced by overexpression of LcTAK1 or LcTAB1 alone.however,The activation of NF-κB and expression level of IL-8 could be induced by co-overexpression of LcTAK1 and LcTAB1(p < 0.01).Moreover,the roles of LcTAK1 and LcTAB1 in immune response analysis showed that NF-κB activation enhanced significantly in co-overexpressed HEK293 T cells following LPS and poly I:C stimulation(p < 0.05).However,the transcriptional expression levels of TNF-α,IL-6 and IL-8 were induced only after LPS challenge(p < 0.01).These findings suggested that the TAK1-TAB1 complex of large yellow croaker might play an important role in proinflammatory cytokines and chemokine release after LPS stimulation via inducing NF-κB activation.(3)The regulatory mechanism of LcTAK1 and LcTAB2 in NF-κB signaling pathwayThe NF-κB,TNF-α and IL-1β promoter activation of large yellow croaker could be induced by overexpression of LcTAB1 alone(p < 0.05);Overexpression of LcTAK1 alone could up-regulated the activation of NF-κB-P65,TNF-α,IL-1β and IFN I promoters of large yellow croaker(p < 0.05).Moreover,the results of TNF-α promoters in the large yellow croaker were different from those of mammalian TNF-α expression level in HEK293 T cells,indicating that TAK1 may play different kinase functions in the NF-κB signaling pathway in different species.However,the co-overexpression of LcTAK1 and LcTAB2 significantly inhibited the activities of NF-κB,TNF-α,IL-1β,and IFN I promoters(p < 0.05).It was showed that LcTAK1 and LcTAB2 participate in NF-κB mediated inflammatory and antiviral immune response and LcTAB2 may negatively regulate LcTAK1.