Protein-binding Function Of PKR Promotes Proliferation Through TRAF2/RIP1/NFκB/c-Myc Pathway In Pancreatic β-cells

Objective: Double-stranded RNA-dependent Protein Kinase(PKR) acts as a core component in the progression of type 2 diabetes. Its kinase activity is involved in insulin resistance in peripheral tissues, and downregulation of pancreatic β-cell function through cell cycle arrest and proliferation inhibition. Apart from kinase activity, PKR has protein-binding function, whose role in pancreatic β-cell growth remains to elusive. The aim of this study was to investigate the mechanism of PKR’s protein-binding function on β-cell proliferation.Methods: Coumermycin/GyrB-PKR-K296 H system was used to establish a rodent model of upregulated protein-binding function of PKR in pancreatic β-cells,including MIN6 cells and primary isolated islets. In the absence of kinase activity,it can get deep insight into protein-binding function of PKR. Co-Immunoprecipitation was performed to examine the physical interaction between PKR and TRAF2,-3,-6,with Western blot to detect PKR and p-eIF2α. MTT, EdU labeling and flow cytometry analysis were used to analyze β-cell viability, proliferation and cell cycle,respectively. Real-time RT-PCR and Western blot were applied to determine the mRNA and protein levels of cell cycle proteins,including cyclin D1,cyclinD2,CDK2,CDK4,c-Myc,p21,p27 and p53. Knockdown of c-Myc,TRAF2,TRAF6 and RIP1 were achieved using specific small interfering RNA(si-c-Myc,si-TRAF2,si-TRAF6, si-RIP1). Bay11-7082 was used to inhibit NFκB activity. The nucleus extraction kit was used to analyze the protein level of p65 in nuclear.Immunofluorescence assay and Luciferase Assay were performed to, respectively,investigate the subcellular distribution of NFκB and its transcriptional activity. MIN6 cells were administered with glucolipotoxity and pro-inflammatory cytokines after transfection and then cell extracts were immunoprecipitated to examine protein-binding function of PKR.Results: By means of Coumermycin/GyrB-PKR-K296 H system, a perfect model lacking kinase activity was built in pancreatic β-cells, with upregulated protein-binding capacity of PKR with TRAF2 and TRAF6. Protein-binding function of PKR was found to promote cell viability and proliferation,and enhance cell cycle progression throughG1 phase to S phase. There were no obvious alteration of cyclin D1,-D2, CDK2,-4, p21, p27 and p53 at mRNA and protein levels,except for c-Myc that was significantly upregulated. The upregulation of c-Myc was attributed to NFκB transcriptional activity. NFκB-dependent reporter activity, c-Myc augmentation and β-cell proliferation were suppressed by TRAF2-siRNA, but not by TRAF6-siRNA. Downstream of TRAF2,RIP1 was induced to be ubiquitinated by protein-binding function of PKR and played a proliferative role in pancreatic β-cells.Additionally, glucolipotoxity and pro-inflammatory cytokines-mediated protein-binding function of PKR was shown to alleviate their deleterious effect on pancreatic β-cell growth.Conclusion: Protein-binding function of PKR can promote cell proliferation through accelerating G1/S phase transition, which is closely associated with c-Myc upregulation. PKR recruits TRAF2 by physically binding and then modulate NFκB transcriptional activity to upregulate the mRNA level of c-Myc. After recruitment,RIP1 is ubiquitinated and plays a critical role in NFκB activation. Taken together,these data suggest indicate a pivotal role for PKR’s protein-binding function on the proliferation of pancreatic β-cell through TRAF2/RIP1/NFκB/c-Myc pathways.Therapeutic opportunities for type 2 diabetes may arise when its kinase catalitic, but not its protein-binding function is downregulated.