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TNK1 (thirty-eight-negative kinase 1) belongs to the ACK (Activated Cdc42 Kinases) family of intracellular non-receptor tyrosine kinases that usually acts as an important regulator in cytokine receptor-mediated intracellular signal transduction pathways. JAK-STAT signal pathway acts as a key point in cellular proliferation, differentiation and immunomodulatory. Mammalian TNK1 is involved in antiviral immunity and activation of growth factors. However, TNK1 has rarely been studied in fish. To evaluate the role of fish TNK1 in JAK-STAT pathway, we cloned the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) TNK1 (CiTNK1). CiTNK1 protein consists of N-terminal Tyrkc (tyrosine kinase) domain, C-terminal SH3 (Src homology 3) domain and Pro-rich domain. Phylogenetic analysis showed that CiTNK1 has a closer relationship with Danio rerio TNK1. The expression and phosphorylation of CiTNK1 in grass carp tissues and cells was increased under poly(I:C) stimulation. Subcellular localization and co-immunoprecipitation indicated that CiTNK1 is targeted in the cytoplasm and interacts with grass carp STAT1 (CiSTAT1). Co-transfection of CiTNK1 and CiSTAT1 into cells facilitated the expression of IFN I. This is because that the presence of CiTNK1 enhanced the phosphorylation of CiSTAT1 and causes activation of CiSTAT1. Our results revealed that TNK1 can potentiate the phosphorylation of STAT1 and then regulates JAK-STAT pathway to trigger IFN I expression in fish. • CiTNK1 can respond to poly (I:C) stimulation. • poly(I:C) induces the auto-phosphorylation of CiTNK1. • CiTNK1 and CiSTAT1 trigger IFN I expression. • CiTNK1 can interact with CiSTAT1. • The presence of CiTNK1 enhances CiSTAT1 phosphorylation. [ABSTRACT FROM AUTHOR]

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3β at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2 , MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3β in a non-contact manner. Meanwhile CiGSK3β interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3β, and then it depressed the expression of IFN I via GSK3β-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3β and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3β-TBK1 signal axis. • CiSHP2 responded to poly I:C stimulation and down-regulated CiIFN I expression. • CiSHP2 didn't bind to CiGSK3β but could regulate its phosphorylation at Tyr216. • CiGSK3β physically bound to CiTBK1 in the absence of poly I: C. • Knocked-down of CiTBK1 can block the regulation of CiGSK3β on CiIFN I. [ABSTRACT FROM AUTHOR]

In vertebrates, TANK Binding Kinase 1 (TBK1) plays an important role in innate immunity, mainly because it can mediate production of interferon to resist the invasion of pathogens. In mammals, cell division cycle-25a (Cdc25a) is a member of the Cdc25 family of cell division cycle proteins. It is a phosphatase that plays an important role in cell cycle regulation by dephosphorylating its substrate proteins. Currently, many phosphatases are reported to play a role in innate immunity. This is because the phosphatases can shut down or reduce immune signaling pathways by down-regulating phosphorylation signals. However, there are no reports on fish Cdc25a in innate immunity. In this paper, we conducted a preliminary study on the involvement of grass carp Cdc25a in innate immunity. First, we cloned the full-length cDNA of grass carp Cdc25a (CiCdc25a), and found that it shares the highest genetic relationship with that of Anabarilius grahami through phylogenetic tree comparison. In grass carp tissues and CIK cells, the expression of CiCdc25a mRNA was up-regulated under poly (I:C) stimulation. Therefore, CiCdc25a can respond to poly (I:C). The subcellular localization results showed that CiCdc25a is distributed both in the cytoplasm and nucleus. We also found that CiCdc25a can down-regulate the expression of IFN 1 with or without poly (I:C) stimulation. In other words, the down-regulation of IFN1 by CiCdc25a is independent of poly (I:C) stimulation. Further functional studies have shown that the inhibition of IFN1 expression by CiCdc25a may be related to decrease of TBK1 activity. We also confirmed that the phosphorylation of TBK1 at Ser172 is essential for production of IFN 1. In short, CiCdc25a can interact with TBK1 and subsequently inhibits the phosphorylation of TBK1, thereby weakens TBK1 activity. These results indicated that grass carp Cdc25a down-regulates IFN 1 expression by reducing TBK1 phosphorylation. • CiCdc25a can respond to poly (I:C) stimulation. • The phosphorylation at Ser172of CiTBK1 is particularly important for the production of CiIFN 1. • CiCdc25a can physically interact with CiTBK1. • CiCdc25a down-regulates CiIFN1 via CiTBK1. • CiCdc25a can attenuate the phosphorylation of CiTBK1 to inhibit its activity. [ABSTRACT FROM AUTHOR]

Mre11A is considered as a cytosolic DNA receptor in mammals. However, it is rarely known about Mre11A in other vertebrates. Recently, a mammalian ortholog of Mre11A has been identified in grass carp (Ctenopharyngodon idellus) in our lab. Phylogenetic-tree analysis provided evidence for a close genetic relationship between C.idellus Mre11A and Carassius auratus Mre11A. The tissue expression profile of CiMre11A was detected, with a relatively higher level of expression in kidney, intestines, liver and spleen than that in other tissues after grass carp reovirus (GCRV) infection. Similarly, CiMre11A was also up-regulated in CIK cells after treatment with GCRV. Q-PCR and dual-luciferase assays indicated that the transcription levels of IFN1 and ISG15 were inhibited by Ci Mre11A knockdown, but were gradually augmented after CIK cells were transfected with increasing amounts of Ci Mre11A. Subcellular localization assays showed that a part of Ci Mre11A was translocated from the nucleus to the cytoplasm. Co-immunoprecipitation and co-localization assays demonstrated that Ci Mre11A interacts with Ci STING in response to GCRV infection. In CIK cells, the expressions of both IFN1 and ISG15 were acutely up-regulated by Ci Mre11A overexpression, as well as by co-overexpression of Ci Mre11A and Ci STING. Ci Mre11A and Ci STING induced the phosphorylation and cytoplasmic-to-nuclear translocation of IRF7 in CIK cells. The multiplication of GCRV in CIK cells was inhibited by the overexpression of Ci Mre11A and Ci STING. • A Mre11A orthologous gene from grass carp (Ctenopharyngodon idella) is cloned and characterized. • GCRV induces the nuclear-to-cytoplasm translocation of Mre11A. • Mre11A interacts with STING and then initiates IFN 1 and ISG15 expression. • Mre11A and STING promote the phosphorylation and nuclear translocation of IRF7. • Mre11A and STING protect CIK cells against GCRV infection. [ABSTRACT FROM AUTHOR]