Grass Carp (Ctenopharyngodon Idellus) PKZ Regulate Autophagy Under Z-DNA Stimulation

Signal transducer and activator transcription 3(STAT3)is an important signal transducer in cells.After being activated by cytokines,STAT3 can regulate downstream genes and play an important role in signal transduction and transcriptional regulation.Protein kinase Z(PKZ)is a new member of e IF2α kinase family.As a protein kinase unique to fish,its function has been less studied.In mammals,STAT3 regulates autophagy by binding to protein kinase R(PKR).PKZ and PKR are very similar in structure and function.However,as a DNA-binding protein,the regulatory mechanism of activation and inactivation of PKZ remains unclear,and the relationship between PKZ and autophagy under Z-DNA stimulation has not been reported.In this study,we used a Z-DNA/Poly(d G:d C)to stimμlate grass carp cells,and detected the expression of STAT3(Ci STAT3)and PKZ(Ci PKZ)by fluorescence quantitative measurement.We found that Ci STAT3 and Ci PKZ were up-regμlated in response to the stimμlation.Poly(d G:d C)was found to induce autophagy and enhance Ci PKZ phosphorylation by immunofluorescence,transmission electron microscopy and immunocoprecipitation.After knockdown of Ci PKZ,autophagy spots in cells were reduced,indicating that PKZ promoted autophagy in cells.STAT3 is a new autophagy regulatory factor.In this paper,we selected sttatic,an inhibitor of STAT3,and verified that sttatic can effectively promote autophagy.In order to verify whether Ci STAT3 inhibits autophagy by inhibiting Ci PKZ activity,we first found that Ci STAT3 and Ci PKZ overlap in cytoplasm by immunofluorescence,laying the foundation for their interaction.The interaction between Ci STAT3 and Ci PKZ was confirmed by co-immunoprecipitation,and overexpression of Ci STAT3 significantly reduced PKZ phosphorylation and e IF2α phosphorylation.Conversely,after knocking down STAT3 in grass carp cells or stimμlation with sttatic,e IF2α phosphorylation was enhanced and autophagy was enhanced.Subsequently,through PKZ interference experiments,it was proved that STAT3 inhibited e IF2α phosphorylation by inhibiting PKZ activity,thus inhibiting autophagy of grass carp cells.This study demonstrated the interaction between PKZ and STAT3 in grass carp,and clarified the molecular mechanism of PKZ regulated by STAT3 in fish and autophagy stimulated by Z-DNA.