Double-stranded RNA-activated protein kinase (PKR) fused to green fluorescent protein induces apoptosis of human embryonic kidney cells: Possible role in the Fas signaling pathway

Abstract

金沢大学医薬保健研究域薬学系PKR is an interferon-inducible, double-stranded (ds) RNA-activated serine/threonine protein kinase, and has been shown to play roles in viral pathogenesis, cell growth and apoptosis. We expressed PKR as a fusion protein with enhanced jellyfish green fluorescence protein (EGFP) in human embryonic kidney 293 cells to visualize the effect of PKR transfection. The EGFP-fusion construct with wild-type PKR showed both auto- and substrate-phosphorylation activities independent of dsRNA, indicating EGFP-PKR is constitutively active. The EGFP-construct with a mutant PKR with the first RNA binding domain deleted still possessed kinase activities. On the other hand, the EGFP-fusion with a catalytically inactive mutant of PKR with the substitution of K at 296 with R, which has been shown to have tumorigenic properties, did not possess kinase activities. Transfection of the constitutive active forms of EGFP-PKR constructs induced apoptosis in 293 cells without dsRNA, whereas the EGFP-fusion with the catalytically inactive mutant did not cause apoptosis but rather protected cells from Fas-induced cell death. In addition, Fas-stimulation increased endogenous PKR activities. These results constitute evidence that PKR is sufficient to induce apoptosis, and plays a role in Fas-mediated apoptosis