Poly I:C Facilitates The Phosphorylation Of Ctenopharyngadon Idellus Type ? IFN Receptor Subunits And JAK Kinase

Interferon can play an irreplaceable role in antiviral signaling pathways by binding to the corresponding receptor subunits to exercise their potential physiological functions.Type I interferon receptor blongs to type II cytokine receptor,which has two different subunit components and play a critical signaling in the process of cell resistance to viral infection.In mammalian cells,Interferon mediates JAK-STAT signaling pathways.JAK1 and TYK2,as important members of the JAK family,which take part in this signaling pathway for activating other signal molecules.Mammalian cells are stimulated by viruses and induced type I interferon is secreted to the extracellular,binding to type I IFN receptor of target cells,and activation of IFNAR1 and IFNAR2 can recruit intracellular JAK1 and TYK2,coupled tyrosine kinases phosphorylate each other,Therefore,activated JAK phosphorylate the receptor intracellular domain forming "docking sites",which recruit intracellular signaling STAT1 and STAT2,and then this process activates the type I interferon antiviral signaling pathway.In fish,type I interferon receptor subunits are consisted of CRFB1 and CRFB5.Type I interferon receptor in grass carp was cloned.Its molecular characteristics suggested that CiCRFB1(KF255603)and Ci CRFB5(KF460272)were highly homologous to humans,chickens,zebrafish and puffer fish,respectively,and compared to other subunits of mammals.CiCRFB1 and CiCRFB5 showed higher homology with IFNAR2 and IFNAR1.In this study,we focused on the relationship between CiCRFB1 and CiCRFB5 and CiJAK1 and CiTYK2.We cloned and identified grass carp JAK1(CiJAK1)and TYK2(CiTYK2)by homologous cloning and RACE-PCR.The full-length cDNA of CiJAK1(KT724352.1)is 3829 bp and includes a 3465 bp open reading frame(ORF)encoding a protein with 1154 amino acids.The full-length cDNA of CiTYK2(KT724353.1)is 4337 bp,with a 3168 bp open reading frame(ORF)encoding 1055 amino acids.In the structure,they both have B41,SH2,TyrKc and TyrKc and so on.The results showed that Ci JAK1 and Ci TYK2 had high homology with fish,especially zebrafish and carp.Similar to mammals,fish JAK1 and TYK2 also perform their potential biological functions by activating interferon receptors and STATs.Poly I: C is a synthetic dsRNA analog that initiates cellular antiviral signaling pathways.To elucidate the molecular mechanism about Poly I: C inducing an antiviral signaling pathway in fishcells,we stimulated CIK cells with Poly I: C and then isolated the cell lysates on 10%SDS-PAGE.The prokaryotic expression vectors of CiCRFB1,CiCRFB5,CiJAK1 and CiTYK2 were constructed,and the recombinant protein was expressed and purified,immunizing rabbits for getting polyclonal antibody.Analysising CiJAK1 and CiTYK2 phosphorylation sites,we purchase hosphorylation antibodies of human JAK1 and TYK2 to detect protein phosphorylation levels.The results showed that not only Poly I:C drastically increased the expression level of CiJAK1 and CiTYK2,but also it induced the phosphorylation of CiJAK1 and CiTYK2,as well as C.idellus type I IFN receptor subunits,CiCRFB1 and CiCRFB5.In detail,the levels of p-CiJAK1 and p-CiTYK2 were evidently up-regulated at 3 h post stimulation;however the phosphorylation levels of CiCRFB1 and CiCRFB5 displayed a sharp up-regulation at12 h post stimulation of Poly I:C.As a basic mechnism of feedback regulation of JAK-STAT signaling pathway,the results of cell transfection experiments showed that overexpression of CiCRFB1 and CiCRFB5 in CIK cells facilitated the phosphorylation of CiJAK1 and CiTYK2.