The N-terminal lysine residue-rich domain II and the 340-430 amino acid segment of eukaryotic initiation factor 2-associated glycoprotein p67 are the binding sites for the γ-subunit of eIF2
The N-terminal lysine residue-rich domain II and the 340-430 amino acid segment of eukaryotic initiation factor 2-associated glycoprotein p67 are the binding sites for the γ-subunit of eIF2
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DOI：
10.1016/j.yexcr.2006.03.034
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发表时间：
2006-10-01
影响因子：
3.7
通讯作者：
Datta, Bansidhar
中科院分区：
文献类型：
--
作者：
Ghosh, Arnab;Datta, Rekha;Datta, Bansidhar
真核起始因子 2 (eIF2) 相关糖蛋白 p67 在保护 eIF2 α 免受 eIF2 α 特异性激酶磷酸化方面发挥着重要作用。为了了解 p67 和 eIF2 亚基之间相互作用的分子细节，我们应用了多种生化和突变分析来识别 p67 和 eIF2 gamma 内的相互作用域。这些研究与体内和体外功能测定相结合，以解决 p67 和 eIF2 γ 之间相互作用在 eIF2 α 磷酸化中的重要性。酵母双杂交检测的研究表明，p67 与 eIF2 γ 相互作用强烈，与 eIF2 α 相互作用相对较弱，与 eIF2 β 没有相互作用。进一步的突变分析提供的证据表明，p67 的 N 端富含赖氨酸的结构域 II 和 340-430 氨基酸片段与 eIF2 gamma 的 C 端 409-472 氨基酸片段强烈相互作用。 GST Pull-down 测定表明 p67 和 eIF2 gamma 之间的相互作用是直接的。通过免疫共沉淀研究，我们发现p67和eIF2γ之间的相互作用不仅可以在生长培养基中生长的哺乳动物细胞中检测到，还可以在用编码p67和eIF2γ的表达质粒瞬时转染的细胞中检测到。然而，在缺乏富含赖氨酸的结构域 II 和 340-430 个氨基酸片段的 p67 突变体中无法检测到这种相互作用。我们还发现 p67 与 eIF2 γ 的结合与保护 eIF2 α 免受磷酸化之间存在非常好的相关性。总而言之，我们的数据为 p67 和 eIF2 gamma 之间的相互作用提供了遗传证据，并且这种相互作用调节 eIF2 α 的磷酸化。 (c) 2006 Elsevier Inc. 保留所有权利。
Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67, plays an important role in protecting eIF2 alpha from phosphorylation by eIF2 alpha-specific kinases. To understand the molecular details of interaction between p67 and the subunits of eIF2, we applied several biochemical and mutational analyses to identify interacting domains within p67 and eIF2 gamma. These studies were combined with functional in vivo and in vitro assays to address the importance of the interactions between p67 and eIF2 gamma in eIF2 alpha phosphorylation. Studies from yeast two-hybrid assays show that p67 interacts strongly with eIF2 gamma, relatively weakly with eIF2 alpha, and no interaction with eIF2 beta. Further mutational analyses provided evidence that the N-terminal lysine-rich domain II and the 340-430 amino acid segment of p67 interact strongly with the C-terminal 409-472 amino acid segment of eIF2 gamma. GST pull-down assays show that the interaction between p67 and eIF2 gamma is direct. From co-immunoprecipitation studies, we find that the interaction between p67 and eIF2 gamma could not only be detected in mammalian cells growing in growth medium, it could also be detected in transiently transfected cells with expression plasmids encoding p67 and eIF2 gamma. However, this interaction could not be detected in p67 mutants lacking lysine-rich domain II and the 340-430 amino acid segment. We also find a very good correlation between p67 binding to eIF2 gamma and the protection of eIF2 alpha from phosphorylation. Altogether, our data provide genetic evidence for the interaction between p67 and eIF2 gamma and that this interaction modulates the phosphorylation of eIF2 alpha. (c) 2006 Elsevier Inc. All rights reserved.