The Molecular Mechanism Of Grass Carp PACT Inducing Apoptosis By Activating PKR

As a dsRNA-dependent and interferon-induced protein kinase,PKR(double-stranded RNA dependent protein kinase)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,also referred to as RAX(PKR-associated protein X)can activate PKR.In recent years,with the increasing recognition of fish interferon system,fish PKR and PACT have been gradually revealed.However,the function of PACT in fish 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 this study,cells were stimulated by LPS,and the expression of C.idella PACT(CiPACT)and PKR(CiPKR)were quantitatively detected by q-PCR.We found that the expression of CiPACT and CiPKR were significantly up-regulated under the stimulation of LPS.It indicated that CiPACT and CiPKR play an important role in innate immunity in response to LPS stimulation.In addition,the response time of CiPACT to LPS is earlier than that of CiPKR.To explore the physiological functions of CiPACT,the recombinant plasmid pcDNA3.1-CiPACT was overexpressed in grass carps ovarian cells(CO cells),then total proteins and nuclear/plasmid proteins of CO cells were extracted,respectively.It has also shown that overexpression of CiPACT in CO cells can significantly enhance the level of p-eIF2α and induces the translocation of Cip65 to nucleus from cytoplasm.Besides,knock down of CiPACT can inhibit eIF2α phosphorylation and the translocation of Cip65 to nucleus from cytoplasm.These results above showed that CiPACT can induce apoptosis and activate NF-κB pathway.Bcl-2 promotes cell survival,while Bax promotes cell death.Therefore,these two genes are the important indicators of cell apoptosis.In this study,the expression of CiBcl-2 and CiBax were quantitatively detected by q-PCR.It shows that over-expression of CiPACT in CIK cells induced the down-regulation of intracellular expression of CiBcl-2 and up-regulation of CiBax.However,in CiPACT knock-down cells the expression of CiBcl-2 and CiBax were just the opposite.To further understand the mechanism of CiPACT,the recombinant plasmid FLAG-tagged CiPACT and GFP-tagged CiPKR were co-transplanted into HEK-293T cells.The immunoprecipition assay(CO-IP)showed that CiPACT can interact with CiPKR.In additon,the recombinant plasmid pcDNA3.1-CiPACT was overexpressed in CO cells to detect phosphorylation of CiPKR by anti-Ser/Thr/Tyr phosphorylated antibody.The result showed that overexpression of CiPACT induced the phosphorylation of CiPKR.The interaction between CiPACT and CiPKR made the phosphorylation of CiPKR.Therefore,the mechanism of fish PACT induces apoptosis and activates NF-κB pathway is dependent on PKR.Fish PACT may induce apoptosis in two ways:on the one hand,PACT activates PKR,which leads to the increased phosphorylation of eIF2α then promotes apoptosis;on the other hand,PACT directly activates apoptosis-related protein Bax to induce apoptosis.In addition,PACT can activate NF-κB,and enable the expression of a series of inflammatory factors to participate in the immune response.PKR is a very important bridge in these processes.