Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2alpha kinase activity of HRI.
Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2alpha kinase activity of HRI.
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DOI：
10.1021/bi034005v
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发表时间：
2003-05
期刊：
影响因子：
2.9
通讯作者：
M. Rafie-Kolpin;A. Han;Jane-Jane Chen-Jane
中科院分区：
文献类型：
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作者：
M. Rafie-Kolpin;A. Han;Jane-Jane Chen-Jane
在血红素缺乏症中，血红素调节的eif2 α激酶（HRI）通过其多重自磷酸化激活，从而抑制蛋白质合成。鉴定了HRI中的自磷酸化位点，以研究其功能。我们发现在血红素缺乏症中HRI有8个主要的色氨酸磷酸化肽被激活。在本报告中，我们重点研究了Thr483和Thr485位点的自磷酸化在HRI激活环中的作用。破坏Thr485的自磷酸化，而不是Thr483，导致自身激酶活性降低，并将Thr485Ala HRI锁定在低磷酸化状态。最重要的是，Thr485的自磷酸化，而不是Thr483，是获得HRI eif2α激酶活性所必需的。此外，Thr485的自磷酸化对于亚砷酸盐诱导的HRI eIF2alpha激酶活性的激活是必需的，而Thr483的自磷酸化对于亚砷酸盐的激活是不需要的。研究了HRI激活环中另一个保守的苏氨酸残基Thr490的功能。在血红素缺乏或亚砷酸盐处理下，Thr490突变为Ala或Asp导致自身激酶活性降低和eif2 α激酶活性丧失。由于Thr490未被鉴定为自磷酸化位点，因此Thr490本身可能对HRI的催化活性至关重要。重要的是，Thr485在Thr490突变体HRI中磷酸化程度非常低。总的来说，我们的研究结果表明Thr485的自磷酸化对于HRI的过度磷酸化和激活是必不可少的，并且是获得eif2 α激酶活性所必需的。
In heme deficiency, protein synthesis is inhibited by the activation of the heme-regulated eIF2alpha kinase (HRI) through its multiple autophosphorylation. Autophosphorylation sites in HRI were identified in order to investigate their functions. We found that there were eight major tryptic phosphopeptides of HRI activated in heme deficiency. In this report we focused on the role of autophosphorylation at Thr483 and Thr485 in the activation loop of HRI. Disruption of the autophosphorylation of Thr485, but not Thr483, resulted in a lower autokinase activity and locked Thr485Ala HRI in a hypophosphorylated state. Most importantly, autophosphorylation of Thr485, but not Thr483, was essential for attaining eIF2alpha kinase activity of HRI. In addition, autophosphorylation of Thr485 was necessary for arsenite-induced activation of the eIF2alpha kinase activity of HRI, while autophosphorylation at Thr483 was not required for activation by arsenite. The function of Thr490, another conserved Thr residue in the activation loop of HRI, was also investigated. Mutations of Thr490 to either Ala or Asp resulted in reduced autokinase activity and loss of eIF2alpha kinase activity in heme deficiency or upon arsenite treatment. Since Thr490 was not identified as an autophosphorylated site, it is likely that Thr490 itself might be critical for the catalytic activity of HRI. Importantly, Thr485 was very poorly phosphorylated in Thr490 mutant HRI. Collectively, our results demonstrate that autophosphorylation of Thr485 is essential for the hyperphosphorylation and activation of HRI and is required for the acquisition of the eIF2alpha kinase activity.