Ctenopharyngodon idellaPERK (EIF2AK3) decreases cell viability by phosphorylating eIF2α under ER stress

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Abstract

As an upstream kinase of eIF2α, 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 domains, the intracavitary domain for BIP protein combination and the dissociative C-terminal region containing a typical serine/threonine kinase domain which promotes the phosphorylation of eIF2α. In this study, we cloned a PERK (also known as EIF2AK3) gene from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp PERK (CiPERK) is 5192 bp including a 176 bp of 5′ untranslated region, a 1719 bp of 3′ untranslated region and a 3297 bp of the longest open reading frame (ORF) 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 was significantly up-regulated following the 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 eIF2α phosphorylation level. In addition, CCK assay showed the overexpression of CiPERK in CIK cells decreases the cell viability.

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