The Mechanism Of STING-mediated Activation Of IFN-? Regulated By Chicken TBK1 Gene

TANK binding kinase 1(TANK binding kinase-1,TBK1),belongs to noncanonical I?B kinase family,is a serine/threonine kinase.It can activate many related regulatory signaling pathways by phosphorylation of IRF3/7(Interferon regulatory Factor 3/7)or NF-kappa B(Nuclear factor-kappa B,NF-?B),inducing a series of antiviral factors such as interferon I and pro-inflammatory cytokines which plays an important role in innate immunity which mediated by TLRs,TLRs and dsDNA receptors.Although the mechanism by which TBK1 activate IRF3/7 by phosphorylation to regulate IFN production has been more thorough,it is not clear whether TBK1 can regulate the production of IFN in other ways.Our previous study found that chTBK1(chicken TBK1,chTBK1)can interacted with chSTING(chicken STING,chSTING)in chicken cells,and may be involved in chSTING-induced IFN production.The purpose of this study is to investigate the role and regulation mechanism of chTBK1 in regulating chSTING to activate IFN-?signaling pathway.The chTBK1 gene was cloned.Multiple sequence alignment showed that the amino acid sequence of chTBK1 owes at least 80%homology to mammalian TBK1,chTBK1 protein contained an N-terminal protein kinases catalytic domain(aa 9-306),an Ubiquitin-like domain(aa309-384),and a C terminal coiled-coil domain(aa 627-660),which are highly conserved among chicken TBK1 and other mammalian TBK1.A fast and effective real-time fluorescent quantitative PCR(qRT-PCR)detection method for chTBK1 gene was then established.With this method,the distribution of chTBK1 in healthy chicken tissues and the expression changes of chTBK1 in DF-1 cells which infected H9N2 avian influenza virus(AIV)was detected.It was found that chTBK1 was widely distributed in different tissues and organs of healthy chickens and the highest expression is spleen and thymus;The mRNA level of chTBK1 was significantly up-regulated after infected with H9N2 AIV.The Co-Immunoprecipitation assay confirmed that chTBK1 could interact with chSTING,suggesting that chTBK1 may be involved in the regulation of chSTING function.Through gene overexpression and RNA interference experiments showed that chTBK1 could inhibit the activation of chSTING to IFN-?signaling pathway.Further dual luciferase reporter assay found that the inhibitory effect of chTBK1 kinasedead mutant(chTBK1-S172A)on chSTING was relieved,while the overexpression of serine/threonine protein kinase chNEK7 had no significant effect on chSTING,which indicates that the negative regulation of chTBK1 on chSTING depends on its kinase activity and has kinase specificity.Through Western bloting and Mn2~+-Phos-tag-SDS-PAGE experiments,chTBK1 can induce phosphorylation of chSTING and inhibit the formation of chSTING dimers.We further analyzed the possible phosphorylation sites on chSTING and constructed the corresponding mutants,combined with Mn2+-Phos-tag-SDS-PAGE which is a phosphorylated protein detection method,we found that chTBK1 could catalyze the phosphorylation of five serine loci of chSTING,which are Ser285?Ser299?Ser327?Ser331 and Ser362 respectively;Futher study about the effect of chTBK1 on the function of different serine mutants of chSTING,which finally determined that Ser327 and Ser331 on chSTING are the catalytic targets for chTBK1to negatively regulate chSTING.Further studies have found that chSTING completely loses the activation function to IFN?in chTBK1-deficient DF-1 cell line,indicating that although chTBK1 negatively regulate chSTING,chTBK1 is indispensable for chSTING's activation to IFN.In conclusion,this study reveals a new mechanism of chSTING-mediated type I interferon pathway regulated by chTBK1:chTBK1 relies on its kinase activity to specifically phosphorylate chSTING at Ser327 and Ser331 sites,thereby inhibiting the formation of chSTING dimers,than negatively regulate the activation of type I interferon pathway.The results of the study can provide a theoretical guidance for the prevention and treatment of viral diseases in poultry.