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首页> 外文期刊>Molecular and Cellular Biology >Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 α-Subunit Kinase, PEK, Involved in Translational Control
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Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 α-Subunit Kinase, PEK, Involved in Translational Control

机译：涉及转化控制的胰腺真核起始因子2α-亚基激酶PEK的鉴定与表征

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In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In mammals, the phosphorylation was shown to be carried out by eIF-2α kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2α kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2α kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced inEscherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2α on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In theSaccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2α kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2α. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2α kinase plays an important role in translational control from nematodes to mammals.
机译：响应各种环境压力，真核细胞通过真核翻译起始因子2（eIF-2α）的α亚基的磷酸化来下调蛋白质合成。在哺乳动物中，磷酸化显示是通过eIF-2α激酶PKR和HRI进行的。我们报道了从大鼠胰岛细胞中编码一种新的相关激酶（我们称之为胰腺eIF-2α激酶或PEK）的cDNA的鉴定和表征。除了具有在eIF-2α激酶中具有序列和结构特征的催化结构域外，PEK还包含一个独特的氨基末端区域，长度为550个残基。使用在大肠杆菌或Sf-9昆虫细胞中产生的重组PEK，我们证明PEK在丝氨酸和苏氨酸残基上均被自身磷酸化，并且重组酶可以特异性磷酸化丝氨酸51上的eIF-2α。 Northern印迹分析表明，PEK mRNA在所有检查的组织中都有表达，在胰腺细胞中表达最高。与我们的mRNA分析一致，PEK活性主要在胰腺和胰岛细胞中检测到。在体外和体内均证实了PEK在蛋白质合成中的调节作用。向网状细胞裂解物中添加重组PEK导致翻译的剂量依赖性抑制。在酿酒酵母模型系统中，PEK通过要求eIF-2α中的丝氨酸51磷酸化位点的过程功能性地取代了内源酵母eIF-2α激酶GCN2。我们还从秀丽隐杆线虫和河豚鱼Fugu rubripes 中鉴定了PEK同源物，表明该eIF-2α激酶在从线虫到哺乳动物的翻译控制中起着重要作用。

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