Grass Carp (Ctenopharyngodon Idella) STAT3 Regulates The EIF2? Phosphorylation Through Interaction With PKR

Signal transduction and activator of transcription(STAT)is important protein signal molecule.It can be activated by various cytokine receptors and plays an important role in signal transduction and transcription regulation.In mammals,STAT3 plays an important role in signal transduction pathways such as JAK2/STAT3,and also plays an important role in cell physiology such as growth,proliferation,differentiation and participates in inflammation,tumorigenesis,metabolic disorders and immune responses.In mammals,STAT3 is a protein that shuttles between the nucleus and the cytoplasm.Compared with STAT3 in the nucleus,there has been little research on the function of STAT3 in the cytoplasm for a long time.In mammals,some recent studies have shown that STAT3 in the cytoplasm regulates autophagy through interaction with double-stranded RNA-activated protein kinase R(PKR)and plays an important role in cellular antiviral responses.As a freshwater cultured fish,grass carp(Ctenopharyngodon idella)is a well-studied biological material for development and immunology.In this study,the open reading frames of grass carp STAT3(CiSTAT3 2223 bp)and PKR(CiPKR 2067 bp)was cloned.Then,through quantitative real-time PCR experiments,we analyzed the expression characteristics of STAT3 in different tissues of grass carp.we found that the expression of STAT3 was ubiquitous in all tissues of grass carp,and the highest expression was in liver tissue.Moreover,the expression level of STAT3 was up-regulated under the stimulation of Poly I:C in the tissues of grass carp.At the same time,we found that the expression of STAT3 was also up-regulated under the stimulation of Poly I:C in grass carp kidney cells(CIK cells).To explore the potential function of grass carp cytoplasmic STAT3 in antiviral signaling pathways,we analyzed the relationship between Ci STAT3 and CiPKR.Coimmunoprecipitation(Co-IP)and GST pull-down experiments confirmed that CiSTAT3 can bind directly to Ci PKR to form a complex.The SH2 domain of CiSTAT3 and the C-terminus of Ci PKR play an important role in this process.Under the induction of Poly I:C,Ci STAT3 dissociated from CiPKR,and engaging in the cell's antiviral response.Through Western Blot experiments,we also found that overexpression of CiSTAT3 in CIK cells can significantly reduce the phosphorylation of eIF2?.In contrast,siRNA-mediated knockdown of Ci STAT3 upregulates the phosphorylation of e IF2?.To further understand whether the phosphorylation levels of CiSTAT3 and eIF2? were affected by PKR,we performed a knockdown of CiPKR.The results showed that the knockdown of PKR significantly decreased the phosphorylation of eIF2?.In summary,we conclude that grass carp STAT3 can responds to the induction of Poly I:C,indicating that it is involved in the anti-viral immune response.Under normal conditions,grass carp STAT3 is associated with PKR,and this combination depolymerizes when the virus is infected.Grass carp STAT3 can reduce the phosphorylation of eIF2?,and this process is mainly accomplished by combining PKR.In addition,we confirmed that overexpression of grass carp STAT3 can significantly increase cell viability through a cell viability assay.These results reveal that the molecular mechanism of regulation of eIF2? phosphorylation and cellular activity of grass carp cytoplasmic STAT3 is similar to that of mammals.