Grass Carp (Ctenopharyngodon Idella) TNK1 Modulates JAK-STAT Signaling Through Phosphorylating STAT1

TNK1(thirty-eight-negative kinase 1),as a member of the ACK family of cytoplasmic non receptor tyrosine kinases,is usually coupled with receptors to exert its biological functions,that is,it plays a pivotal role in cytokine receptor-mediated intracellular signal transduction pathway.Mammalian TNK1 has been shown to participate in antiviral immunity and regulate cell apoptosis.However,the role of TNK1 has rarely been studied in fish.In the classic JAK-STAT signaling pathway,STAT acts as the core to regulate cell growth,differentiation and immune response.Signal transducer and activator of transcription(STAT)is an important class of cytoplasmic proteins,which can be used as transcription factors to regulate gene expression,transmit extracellular signals to the nucleus and control the basic functions of cells,such as cell proliferation,apoptosis and immune response.In mammals,as the main regulator of IFN signal transduction,STAT1 has been widely studied in antiviral immunity.In recent years,the role of STAT1 in fish innate immunity has been confirmed,but the molecular mechanism of STAT1 immune activation has not been fully revealed.In this study,we found that grass carp TNK1 was involved in activating STAT1 to initiate antiviral innate immunity.To evaluate the mechanism of TNK1 regulating innate immunity of grass carp through JAK-STAT signaling pathway,we cloned the full-length c DNA sequence of grass carp(Ctenopharyngodon idella)TNK1(CiTNK1).CiTNK1 protein consists of N-terminal Tyrkc(tyrosine kinase)domain,C-terminal SH3(Src homology 3)domain and Pro-rich domain.Phylogenetic analysis showed that CiTNK1 has a closer relationship with Danio rerio TNK1.The results of tissue expression analysis showed that CiTNK1 was highly expressed in the liver and intestine of grass carp,and had obvious response to poly(I:C)stimulation for 6h.The autophosphorylation of CiTNK1 was found under poly(I:C)stimulation,indicating that poly(I:C)activated the activity of TNK1.In grass carp cells,the expression of CiTNK1 significantly up-regulated the expression of IFN,and co-transfection with CiSTAT1 synergistically facilitated IFN1 expression.Meanwhile,specific small interfering RNA(si RNA)was used to interfere with CiTNK1,and the expression of IFN1 was decreased.These results indicate that grass carp TNK1 is involved in the regulation of IFN.Subcellular localization and coimmunoprecipitation indicated that CiTNK1 is targeted in the cytoplasm and interacts with grass carp STAT1(CiSTAT1).It shows that grass carp TNK1 and STAT1 are related in signal transduction.This is because that the presence of CiTNK1 enhanced the phosphorylation of CiSTAT1 and causes activation of CiSTAT1.This indicates that grass carp TNK1 regulates JAK-STAT pathway in a STAT1-dependent manner to trigger the expression of IFN1,thereby affecting antiviral innate immunity.