Characterization of the hemin-sensitive eukaryotic initiation factor 2α kinase from mouse nonerythroid cells
Characterization of the hemin-sensitive eukaryotic initiation factor 2α kinase from mouse nonerythroid cells
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DOI：
10.1074/jbc.273.48.32340
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发表时间：
1998-11-27
影响因子：
4.8
通讯作者：
de Haro, C
中科院分区：
文献类型：
--
作者：
Berlanga, JJ;Herrero, S;de Haro, C
血红素调节的真核起始因子2α（eIF2α）激酶（血红素调节抑制剂（HRI））在网织红细胞中因血红素缺乏而被激活，在这些细胞的翻译调控中起重要作用。此前，HRI从兔网织红细胞和大鼠脑中被克隆出来，但血红素调节的eIF2α激酶活性仅从红细胞系细胞中被纯化出来。在这项研究中，我们报道了从小鼠肝脏和NIH 3T3细胞提取物中纯化出一种对血红素敏感的eIF2α激酶活性。此外，我们已经克隆并鉴定了这种小鼠肝脏eIF2α激酶（mHRI），它与兔和大鼠的HRI分别具有83%和94%的同一性。纯化的酶和重组mHRI都表现出自身激酶和eIF2α激酶活性，并且这两种活性在体外都被氯化血红素抑制。此外，当野生型mHRI在293细胞中表达时，它在体内会发生自身磷酸化，而无活性的mHRI - K196R突变体则不会。通过逆转录 - 聚合酶链反应对小鼠各种组织中mHRI mRNA表达的定量分析显示，其在肝脏、肾脏和睾丸中的水平相对较高。这些结果提供了有力的证据，表明mHRI是哺乳动物细胞中一种普遍存在的eIF2α激酶，这意味着它可能在非红细胞组织的翻译调控中起重要作用。
The heme-regulated eukaryotic initiation factor 2 alpha (eIF2 alpha) kinase (heme-regulated inhibitor (HRI)) is activated by heme deficiency in reticulocytes and plays an important role in translational control in these cells. Previously, HRI was cloned from rabbit reticulocytes and rat brain, but a heme-regulated eIF2 alpha kinase activity has only been purified from erythroid cells. In this study, we report the purification of a heme-sensitive eIF2 alpha kinase activity from both mouse liver and NIH 3T3 cell extracts. Furthermore, we have cloned and characterized this mouse Liver eIF2 alpha kinase (mHRI), which exhibits 83 and 94% identities to rabbit and rat HRIs, respectively. Both the purified enzyme and recombinant mHRI exhibited an autokinase and an eIF2 alpha kinase activity, and both activities were inhibited in vitro by hemin. In addition, wild-type mHRI, but not the inactive mHRI-K196R mutant, was autophosphorylated in vivo when it was expressed in 293 cells. Quantitation of mHRI mRNA expression in various mouse tissues by reverse transcription-polymerase chain reaction revealed relatively high levels in liver, kidney, and testis. These results provide strong evidence that mHRI is a ubiquitous eIF2 alpha kinase of mammalian cells, suggesting that it could play important roles in the translational regulation of nonerythroid tissues.