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As a dsRNA-dependent and interferon-induced protein kinase, PKR is involved in antiviral immune response and apoptosis mediated by various cytokines. In mammalian cells, PKR can also be activated in the absence of dsRNA. A PKR activator, PACT (PKR activating protein), also referred to as RAX (PKR-associated protein X) plays an important role. In recent years, with the increasing recognition of fish interferon system, PKR and PACT have been gradually revealed in fish. However, the function of fish PACT is unclear. In our previous work, we suggested that grass carp (Ctenopharyngodon idella) PACT must be involved in IRF2 and ATF4-mediated stress response pathways. In the present study, we found that the expression of C. idella PACT (CiPACT) and CiPKR were significantly up-regulated under the stimulation of LPS. It indicated that CiPACT and CiPKR may play an important role in response to LPS stimulation. In addition, the response time of CiPACT to LPS is earlier than that of CiPKR. It has also shown that overexpression of CiPACT in CIK cells can significantly enhance the level of p-eIF2α, induces apoptosis and translocation of Cip65 to nucleus from cytoplasm. To further understand the mechanism, we carried out the co-immunoprecipitation assay. It proved that the interaction of CiPACT and CiPKR made the phosphorylation of CiPKR. Overexpression of CiPACT induced the down-regulation of intracellular expression of bcl-2 and up-regulation of bax. However, in CiPKR knocked-down cells the expression of bcl-2 and bax were just the opposite. Therefore, the mechanism of fish PACT induces apoptosis and activates NF-кB is dependent on PKR., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

PKR plays a significant role in IFN antiviral responses and in mediating apoptosis. Its activity is crucial for cellular antiviral and subsequent recovery. In mammalian cells, Protein Activator of the Interferon-induced Protein Kinase (PACT) and Trans-Activation-Responsive RNA-Binding Protein 2 (TARBP2) have the opposite effect on PKR activity in a dsRNA independent manner. There are some corresponding regulators of PKR in fish, too. In previous studies, we found that grass carp PACT can activate PKR in dsRNA independent manner. In this study, we tried to find out the effect of grass carp TARBP2 on PKR regulation. Grass carp TARBP2 expression is significantly increased at 6h post-poly I:C stimulation in CIK cells and grass carp tissues, indicating that it may play a role in poly I:C-mediated immune response. Then, we found that CiTARBP2 interacts with CiPKR and CiPACT, suggesting that it may regulate PKR activity by direct interaction with PKR or its regulators. Further, poly I:C promotes the phosphorylation of CiTARBP2 and enhances the interaction of CiTARBP2 and CiPKR. Finally, over-expression of CiTARBP2 decreases CiPKR phosphorylation and inhibits PKR-induced apoptosis. Therefore, our study reveals that CiTARBP2 can bind to CiPKR, CiPACT and CiTARBP2. The phosphorylated TARBP2 has stronger affinity to PKR, which results in the decrease of PKR phosphorylation and inhibition of cell apoptosis. • Grass carp TARBP2 is upregulated by poly I:C. • CiTARBP2 can form heterodimers with CiPKR or CiPACT. • Poly I: C promotes the phosphorylation of CiTARBP2 and enhances the interaction of CiTARBP2 and CiPKR. • CiTARBP2 decreases the phosphorylation of CiPKR and inhibits PKR-induced apoptosis. [ABSTRACT FROM AUTHOR]

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53. • The expression of CiDYRK2 was up-regulated by GCRV and poly (I:C) stimulation. • CiDYRK2 can physically bind to Cip53. • CiDYRK2 promoted apoptosis by increasing the phosphorylation level of p53. [ABSTRACT FROM AUTHOR]