The Fish Specific Protein Kinase Z(PKZ) Initiates Innate Immune Responses

Protein kinase Z(PKZ) is a new member of eIF2? kinases in fish.Although PKZ was identified in the early time,the functions were rarely reported.Lately studies suggest that PKZ can interact with and phosphorylate eIF2?,then induce apoptosis.Furthermore,PKZ is up-regulated in response to various pathogenes,and the antiviral mechanism of PKZ is not clear.Freshwater fisheries occupy a large proportion in aquaculture,which is easy to cause economic losses attribute to infected by pathogens.In this study,grass carp PKZ is taken as the research object.Grass carp PKZ is consist of two Z-DNA binding domains in its N terminus and a eIF2? kinase catalytic domain in its C terminus.Our results: PKZ and critical adapters of innate immune were up-regulated by B-DNA and Z-DNA stimulation at mRNA or protein level,interestingly,PKZ responsed to stimulation at early time,which may attribute to its specific function;It is meaningful to known where PKZ works in cell.Subcellular localizaation indicated that PKZ localized in cytoplasm,which provided possibility for PKZ recongnized viral nucleic acids;DNA-pulldown assay demonstrated that PKZ prefered to interacting with DNA rather than RNA,and N terminus of PKZ was active region for recognizing DNA;PKZ can up-regulate IFN expression at mRNA,protein and promoter activities levels;Co-IP assay indicated that can separately interact with IRF3,STING,ZDHHC1,eIF2?,IRF9 and STAT2.Further investigation revealed that PKZ can activate IRF3 and STAT2,which demonstrated that PKZ transduced signals through IRF3-and ISGF3-like mediated pathways;Moreover,IFN expression was up-regulated in CO cells in which IRF3,STING,ZDHHC1,IRF9 and STAT2 was separately overexpressed.However,the knockdown of PKZ blocked the IFN expression.On the other way,knockdown of IRF3 or IRF9 inhibited the IFN expression through PKZ.These data confirmed that PKZ conducted antiviral signals in IRF3-dependent or ISGF3-like dependent pathways under stimulation with DNA to initiate innate immune responses.In addition,IRF3-dependent pathway was detailedly studied.Two DNA-induced proteins,STING and ZDHHC1,were found through reading literature.Grass carp ZDHHC1 was first identified and analyzed.STING and ZDHHC1 localized in endoplasmic reticulum and were up-regulated upon the stimulation of B-DNA and Z-DNA;Over-expressions of ZDHHC1 and STING in CIK cells up-regulated IFN expression Furthermore,we also found that siRNA-mediated knockdown of STING and ZDHHC1 could inhibit the expressions of IFN in fish cells.In order to investigating the mechanism of STING/ZDHHC1-mediated pathway,co-IP and GST-pulldown assays demonstrated that STING and ZDHHC1 can separately interact with IRF3 and promote the dimerization and nuclear translocation of IRF3,bimolecular fluorescence complementary(BiFC) and co-localization assays indicated that STING can directly interact with ZDHHC1.These data demonstrated that STING and ZDHHC1 formed heterodimers in response to DNA stimulation,then recruited and activated IRF3,finally initiated IFN expression.At last,the mechanism for self-regulation of IRF3 was studied.IRF3 is a critical mediator for innate immune signaling.Promoter sequences of IRF2 and IRF3 were cloned and elements analyzed with previously cloned IRF1 and IRF7 promoter sequences,which indicated that IRF-E elements were found in these promoters.Gel mobility shift assays suggested that IRF3 protein can bind to the promoters of IRF1,IRF2,IRF3,IRF7.Furthermore,IRF3 can up-regulate IRF1,IRF2,IRF3 and IRF7 transcription levels,which resulted in the control of innate immunity in fish.fish ISGF3-like dependent pathway requires further study in future.