Grass Carp Cdc25a Down-regulates IFN1 Expression By Inhibiting TBK1 Phosphorylation

During the fight against pathogenic microorganisms and viruses,eukaryotes have developed a defense against pathogen infection--innate immunity.The occurrence of innate immunity greatly improves the ability of organisms to defend against virus infection.During this process,a variety of cytokines are produced to cause the cells to carry out the corresponding immune response.In the innate immune response,the initiation of IFN expression is one of the important links,because cells produce IFN and then secreted it out of the cell.IFN secreted from the cell transits signals through a specific signaling pathway and then makes a series of antiviral genes express,and finally eliminates the invasion of the virus.TBK1 plays a crucial role in the production pathway of IFN,mainly due to the activation of IRFs by TBK1,which enables them to activate IFN expression.Cdc25a is a member of the Cdc25 family of cyclins and is also a phosphatase.Previous studies have shown that Cdc25a controls cell cycle progression through dephosphorylation of substrate proteins.Recently,many studies have reported that phosphatase can participate in immune response by decreasing or blocking phosphorylation signal transmission.In fish,the function of Cdc25a has been reported,but the function of Cdc25a in innate immune response has not been seen.In order to study the role of Cdc25a in the innate immune signaling pathway of grass carp,we first need to clone the Cdc25a gene of grass carp.The c DNA of Cdc25a gene of grass carp is 2985bp in length,and its Cd S contains 1695bp encoding 564 amino acids.The 5’UTR and 3’UTR are294bp and 996bp,respectively.A phylogenetic tree was constructed using the uploaded Cdc25a sequences of other species and the predicted Cdc25a sequences of grass carp.Further analysis revealed the genetic relationship between Cdc25a of grass carp and Cdc25a of Kanglang white minnow.Then,the expression of Cdc25a in grass carp was detected by q-PCR after Poly I:C stimulation,and the results showed that the expression of Cdc25a in the stimulated tissues（eyes,brain,intestine,gill,liver,kidney,spleen and skin）had a tendency of upregulation.At 6h,the expression level was the highest in liver.In each tissue,the upregulation of Cdc25a eventually returned to normal levels with the extension of stimulation time.When the CIK cells were stimulated by poly I:C,the expression of Cdc25a in grass carp showed a trend of increasing at first and then decreasing,and the expression of Cdc25a was the highest at 6h and tended to rest state after 72h of stimulation.When Cdc25a was overexpressed in CIK cells,the expression of IFN1 was inhibited and down-regulated.On the contrary,when Cdc25a was disturbed,the expression of IFN1 was enhanced.When Cdc25a was overexpressed in CIK cells,IFN1 expression was down-regulated,whereas when Cdc25a was interfered with,IFN1 expression was enhanced.Secondly,Cdc25a was overexpressed in CO cells,and WB detected the expression of IFN1 protein,showing that Cdc25a could also inhibit the expression of IFN1,and this inhibition effect was independent of Poly I:C stimulation.We found that phosphorylation of TBK1 at Ser¹⁷²is extremely important for IFN1 expression.The results of subcellular localization showed that in grass carp cell lines,Cdc25a was distributed in the nucleus and cytoplasm.We overexpressed co-transfected Cdc25a and TBK1 in CIK cells and found that IFN1 expression was still inhibited.When we overexpressed Cdc25a after interference with TBK1,the expression of IFN1 did not further decrease.Meanwhile,the results of Immunofluorescence co-localization and Co-IP assay showed that Cdc25a and TBK1 could interact.Further studies showed that Cdc25a expression significantly inhibited TBK1phosphorylation at Ser¹⁷².So far,our research has found that grass carp Cdc25a responds to poly I:C stimulation and down-regulates the expression of IFN1 by inhibiting the phosphorylation of TBK1 Ser¹⁷².