Ctenopharyngodon idella PERK (EIF2AK3) decreases cell viability by phosphorylating eIF2a under ER stress
Ctenopharyngodon idella PERK (EIF2AK3) decreases cell viability by phosphorylating eIF2a under ER stress
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草鱼 PERK (EIF2AK3) 在内质网应激下通过磷酸化 eIF2a 来降低细胞活力
DOI：
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发表时间：
2017
影响因子：
4.7
通讯作者：
Tao Zhang
中科院分区：
文献类型：
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作者：
Tao Zhang
蛋白激酶RNA样ER（内质网）激酶（PERK）是eIF2a的上游激酶，是位于真核细胞ER中的I型跨膜蛋白。 PERK主要由两个结构域组成，BIP蛋白结合的腔内结构域和解离的C末端区域。含有典型的丝氨酸/苏氨酸激酶结构域，促进eIF2a的磷酸化。本研究中，我们从草鱼（Ctenopharyngodon idella）中克隆了一个PERK（又名EIF2AK3）基因。草鱼PERK（CiPERK）全长cDNA为5192 bp，包括176 bp的50个非翻译区、1719 bp的30个非翻译区和3297 bp的最长开放阅读框(ORF)编码1098个氨基酸。系统发育分析表明，CiPERK 与其他硬骨鱼中的对应物具有高度的序列同源性。 RT-PCR 表明，TM（衣霉素）刺激后 CiPERK 表达显着上调。为了研究CiPERK的功能，将CiPERK的N端序列和CiGRP78序列分别亚克隆到表达载体中。pCMV-HA和pCMV-Flag用于免疫共沉淀和GST-Pulldown测定。分析表明，CiPERK 和 CiGRP78 在正常条件下可以相互结合。然而，在内质网应激（TM刺激）下，CiPERK可以提高eIF2a磷酸化水平。此外，CCK测定显示CIK细胞中CiPERK的过度表达降低了细胞活力。
As an upstream kinase of eIF2a, 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 eIF2a. 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 50 untranslated region, a.1719 bp of 30 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 upregulated 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 eIF2a phosphorylation level. In addition, CCK assay showed the.overexpression of CiPERK in CIK cells decreases the cell viability.