Grass Carp(Ctenopharyngodon Idella) Tyrosine Kinase SRC Up-Regulates IFN1 Expression By Activating TBK1

Innate immunity is the first defense line of against pathogenic microorganisms.When the virus infects the organism,the invading virus can be recognized by PRR.Viral RNA is mainly double stranded RNA,which can be recognized by three PRR:Toll like receptor 3(TLR3),retinoic-acid-inducible gene I(RIG-I)-like receptors(RLR),and nucleotide-binding domain and leucine-rich repeat containing molecules(NLR).As the center of these receptor responses,TANK binding kinase 1(TBK1),a serine / threonine protein kinase with 729 amino acids,contains an N-terminal kinase domain,a ubiquitin like domain,a dimerization domain and a C-terminal domain that can interact with TANK.These domains ensure the functional diversity of TBK1.TBK1 can phosphorylate interferon regulatory factor 3(IRF3),and then dimerize and translocate IRF3 to the nucleus and to regulate IFN 1 expression.SRC is a member of Src family kinase(SFK),which is involved in many cellular processes,including inflammation,apoptosis and innate immunity.In mammals,SRC from N-terminal to C-terminal includes a uniqueSRC homologous(SH4)domain,a specific proline rich(PRM)-binding SH3 domain,a SH2 kinase linker domain and a C-terminal Tyr kinase domain(CSK).SRC participates in the IFN 1 signaling pathway mediated by TBK1,and its immunological function depends on its interaction with TBK1.So far,SRC has not been studied in grass carp,in order to explore the function ofSRC in grass carp innate immunity.In this study,we cloned the ORF of grass carp(Ctenopharyngodon idella)SRC(CiSRC),which is 1599 bp in length and encodes a532 amino acid polypeptide.Phylogenetic tree analysis showed that CiSRC was closely related to Sinocyclooheilus rhinocerousSRC(SrSRC).Protein structure analysis showed that grass carpSRC contained a SH3,a SH2 and a Tyrkc domain.When we stimulated grass carp tissues and cells with poly(I:C),we found that the m RNA expression ofSRC in brain,intestine,gill,skin,liver,spleen and kidney reached the peak after 6 hours of poly(I:C)stimulation,and then gradually decreased;In eyes,the peak value was 12 hours after poly(I:C)stimulation,and it also decreased gradually after reaching the peak value;At the cellular level,CiSRC m RNA expression was significantly up-regulated by exogenous stimulation,peaked at 12 h,then gradually decreased and returned to the background level;However,CiSRC m RNA expression was also up-regulated under endogenous stimulation,peaked at 6 h,and then gradually decreased to the background level.In order to further study the function ofSRC,we studied the subcellular localization of CiSRC and Ci TBK1,the results of subcellular localization showed that CiSRC was located in the cytoplasm and nucleus of CIK cells,while Ci TBK1 was only located in the cytoplasm of CIK cells.We co-transfected GFP-CiSRC and FLAGCi TBK1 into CIK cells and found that they co-located in the cytoplasm,which indicated that CiSRC and Ci TBK1 could interact.Then,GST pull-down and Coimmunoprecipitation experiments showed that CiSRC and CiSRC tyrosine kinase domain deletion mutants(SRC-ΔTyrkc)can interact with Ci TBK1 respectively.Phosphorylation analysis showed that CiSRC promoted the phosphorylation of Ci TBK1.In addition,CiSRC could also promote the phosphorylation of Ci TBK1 with poly(I:C)stimulation.We also demonstrated that CiSRC can up-regulate the expression of Ci IFN 1,but CiSRC tyrosine kinase domain deletion mutant(SRC-ΔTyrkc)had no effect on the expression of Ci IFN 1.These results suggest that CiSRC initiates innate immune response by binding to and up-regulating the phosphorylation of Ci TBK1.