Hierarchical phosphorylation of the translation inhibitor 4E-BP1.
Hierarchical phosphorylation of the translation inhibitor 4E-BP1.
复制标题
DOI：
10.1101/gad.912401
复制
发表时间：
2001-11
影响因子：
10.5
通讯作者：
A. Gingras;B. Raught;S. Gygi;A. Niedźwiecka;M. Miron;S. Burley;R. Polakiewicz;A. Wysłouch-Cieszyńska-A.-Wysłouch-Cieszyń
中科院分区：
文献类型：
--
作者：
A. Gingras;B. Raught;S. Gygi;A. Niedźwiecka;M. Miron;S. Burley;R. Polakiewicz;A. Wysłouch-Cieszyńska-A.-Wysłouch-Cieszyń
在大多数情况下，翻译在起始阶段受到调节，此时核糖体被募集到mRNA的5'末端。eIF 4 E结合蛋白（4 E-BP）通过与翻译因子eIF 4 E结合阻断翻译起始，并阻止翻译机制向mRNA的募集。4 E-BP以可逆的方式抑制翻译。低磷酸化的4 E-BP与eIF 4 E强烈相互作用，而通过用激素、细胞因子或生长因子刺激细胞引起的4 E-BP过度磷酸化导致eIF 4 E结合活性的消除。我们以前报道过4 E-BP 1在Thr 37和Thr 46上的磷酸化对血清剥夺和雷帕霉素治疗相对不敏感，并且这些残基的磷酸化是随后一组未鉴定的血清响应位点的磷酸化所必需的。在这里，使用质谱法，我们确定血清响应，雷帕霉素敏感的网站作为丝氨酸65和苏氨酸70。利用二维等电聚焦/SDS-PAGE和蛋白质印迹与磷酸化特异性抗体的新组合，我们还建立了4 E-BP 1磷酸化在体内的顺序; Thr 37/Thr 46的磷酸化之后是Thr 70的磷酸化，Ser 65最后被磷酸化。最后，我们表明，丝氨酸65和苏氨酸70单独磷酸化是不足以阻止结合eIF 4 E，这表明磷酸化事件的组合是必要的解离4 E-BP 1从eIF 4 E。
In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.