Ctenopharyngodon Idella PERK(EIF2AK3)Decreases Cell Viability By Phosphorylating EIF2? Under ER Stress

Endoplasmic reticulum(ER)is very accurate in sensing and responding to cellular stress including stress caused by metabolic and/or protein folding imbalances.Response to stress from the endoplasmic reticulum,the PERK protein kinases,along with other proximal signaling molecules,initiate a program of transcriptional and translational regulation termed the unfolded protein response.As an upstream kinase of eIF2 a,protein kinase RNA-like ER(endoplasmic reticulum)kinase(PERK)is a type I transmembrane protein located in ER in eukaryotic cells.PERK is mainly composed of two structural domains,namely the intracavitary domain of the BIP protein and the dissociative C-terminal region containing a typical serine/threonine kinase domain which promotes the phosphorylation of eIF2 a.In oeder to understand the molecular mechanism of fish PERK.In this study,we cloned a PERK(also known as EIF2AK3)gene from grass carp(Ctenopharyngodon idella).Ci PERK cDNA has a total length of 5192 bp,including 5' non-translation zone 176 bp,3' non-translation area 1719 bp,the longest open reading frame(ORF)3297 bp,encoding 1098 amino acids.Phylogenetic analysis exhibits that CiPERK shares a high degree of sequence homology to the counterparts in other teleosts.RT-PCR indicated that CiPERK expression w significantly up-regulated after stimulation with TM(tunicamycin).To study the function of CiPERK,the N-terminal sequence of CiPERK and CiGRP78 sequence were separately subcloned into the expression vectors pCMV-HA and pCMV-Flag for co-immunoprecipitation and GST-Pulldown assays.The assays indicated that CiPERK and CiGRP78 can combine with each other in normal conditions.However,under ER stress(TM stimulation)CiPERK can improve the eIF2 a phosphorylation level.In addition,CCK assay showed the overexpression of Ci PERK in CIK cells decreases the cell viability.