Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 a -Subunit Kinase, PEK, Involved in Translational Control
Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 a -Subunit Kinase, PEK, Involved in Translational Control
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在各种环境胁迫下，真核细胞通过磷酸化真核翻译起始因子2 （eif - 2a）的a亚基来下调蛋白质合成。在哺乳动物中，磷酸化被证明是由eif - 2a激酶PKR和HRI进行的。我们报道了从大鼠胰岛细胞中鉴定和鉴定的cDNA，该cDNA编码一种新的相关激酶，我们称之为胰腺eIF-2 a激酶，或PEK。除了在eIF- 2a激酶中具有保守的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。利用在大肠杆菌或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都是自磷酸化的，并且重组酶可以特异性地磷酸化丝氨酸-51上的eif - 2a。Northern blot分析表明PEK mRNA在所有组织中均有表达，在胰腺细胞中表达水平最高。与我们的mRNA检测结果一致，PEK活性主要在胰腺和胰岛细胞中检测到。体外和体内实验均证实了PEK对蛋白质合成的调节作用。将重组PEK添加到网状细胞裂解物中引起了剂量依赖性的翻译抑制。在Saccharomyces模型系统中，PEK通过一个需要eif - 2a中丝氨酸-51磷酸化位点的过程，在功能上取代了内源性酵母eif - 2a激酶GCN2。我们还从秀丽隐杆线虫和河豚中发现了PEK同源物，这表明这种eIF-2激酶在线虫到哺乳动物的翻译控制中起着重要作用。实验使用从酵母中纯化的活化版人类PKR(51)，发现[35 S]蛋氨酸的掺入减少了60%（数据）。相反，添加催化活性不高的PEK- D 785-1108只导致网状细胞裂解物中蛋白质合成的适度减少。这些研究结果表明，PEK可以减少哺乳动物的翻译。
In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphor- ylation of the a subunit of eukaryotic translation initiation factor 2 (eIF-2 a ). In mammals, the phosphorylation was shown to be carried out by eIF-2 a 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-2 a kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF- 2 a kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia 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-2 a 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 the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2 a kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2 a . We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes , suggesting that this eIF-2 a kinase plays an important role in translational control from nematodes to mammals. experiment using an activated version of human PKR purified from yeast (51) and found a 60% reduction in the incorporation of [ 35 S]methionine (data In contrast, the addition of the catalytically inactive PEK- D 785-1108 caused only a modest reduction in protein synthesis in the reticulocyte lysates. Results from these studies suggest that PEK can function to reduce mammalian translation.