Poly I:C facilitates the phosphorylation of Ctenopharyngodon idellus type I IFN receptor subunits and JAK kinase

Abstract

Members of the Janus kinase (JAK) family, JAK1 and TYK2 take part in JAK-STAT signaling pathway mediated by interferon in mammalian cells. Similar to the mammalian counterparts, fish JAK1 and TYK2 also perform their potential biological activities by phosphorylating cytokine receptors and STAT. In the present study, Ctenopharyngodon idellus JAK1 (CiJAK1) and TYK2 (CiTYK2) were cloned and identified. The full-length cDNA of CiJAK1 (KT724352.1) is 3829 bp, with an Open Reading Frame (ORF) of 3465 bp encoding a putative protein of 1154 amino acids. The full-length cDNA of CiTYK2 (KT724353.1) is 4337 bp, including an ORF of 3168 bp encoding 1055 amino acids. Structurally, both of them have B41, SH2, TyrKc and TyrKc common domains. CiJAK1 and CiTYK2 share a high degree of homology with their respective counterparts from Danio rerio and Cyprinus carpio by phylogenetic tree analysis. Polyinosinic-polycytidylic acid (Poly I:C), a synthetic dsRNA analogue, can launch the JAK-STAT antiviral signaling pathway. To elucidate the molecular mechanism of Poly I:C initiating the antiviral signaling pathway in fish, C. idellus kidney (CIK) cells were stimulated with Poly I:C and then the cell lysates were separated on 10% SDS-PAGE. 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 at 12 h post stimulation of Poly I:C. As a basic mechnism of feedback regulation of JAK-STAT signaling pathway, overexpression of CiCRFB1 and CiCRFB5 in CIK cells facilitated the phosphorylation of CiJAK1 and CiTYK2.